|
1
|
Jung M, Nicholas N, Grindrod S, Dritschilo A. Dual-targeting class I HDAC inhibitor and ATM activator, SP-1-303, preferentially inhibits estrogen receptor positive breast cancer cell growth. PLoS One 2024; 19:e0306168. [PMID: 39008483 PMCID: PMC11249239 DOI: 10.1371/journal.pone.0306168] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2024] [Accepted: 06/12/2024] [Indexed: 07/17/2024] Open
Abstract
Dual-targeting chromatin regulation and DNA damage repair signaling presents a promising avenue for cancer therapy. Applying rational drug design, we synthesized a potent dual-targeting small molecule, SP-1-303. Here, we report SP-1-303 as a class I isoform selective histone deacetylase (HDAC) inhibitor and an activator of the ataxia-telangiectasia mutated protein (ATM). In vitro enzymatic assays demonstrated selective inhibition of HDAC1 and HDAC3. Cellular growth inhibition studies show that SP-1-303 differentially inhibits growth of estrogen receptor positive breast cancer (ER+ BC) cells with effective growth inhibition concentrations (EC50) for MCF-7 and T47D cells ranging from 0.32 to 0.34 μM, compared to 1.2-2.5 μM for triple negative breast cancer cells, and ~12 μM for normal breast epithelial cells. Western analysis reveals that SP-1-303 decreases estrogen receptor alpha (ER-α) expression and increases p53 protein expression, while inducing the phosphorylation of ATM and its substrates, BRCA1 and p53, in a time-dependent manner in ER+ BC cells. Pharmacokinetic evaluation demonstrates an area under the curve (AUC) of 5227.55 ng/ml × h with an elimination half-life of 1.26 h following intravenous administration in a rat model. Collectively, SP-1-303 emerges as a novel second generation class I (HDAC1 and HDAC3) selective HDAC inhibitor, and ATM activator, capable of modulating ER expression, and inhibiting growth of ER+ BC cells. Combined targeting of class I HDACs and ATM by SP-1-303 offers a promising therapeutic approach for treating ER+ breast cancers and supports further preclinical evaluation.
Collapse
Affiliation(s)
- Mira Jung
- Department of Radiation Medicine, Georgetown University School of Medicine, Washington, DC, United States of America
| | - Nicole Nicholas
- Department of Biochemistry & Molecular & Cellular Biology, Georgetown University School of Medicine, Washington, DC, United States of America
| | - Scott Grindrod
- Shuttle Pharmaceuticals, Inc., Rockville, Maryland, United States of America
| | - Anatoly Dritschilo
- Department of Radiation Medicine, Georgetown University School of Medicine, Washington, DC, United States of America
- Shuttle Pharmaceuticals, Inc., Rockville, Maryland, United States of America
| |
Collapse
|
|
2
|
Scheidemann ER, Demas DM, Hou C, Ma J, He W, Sharma G, Schultz E, Weilbaecher KN, Shajahan-Haq AN. Resistance to abemaciclib is associated with increased metastatic potential and lysosomal protein deregulation in breast cancer cells. Mol Carcinog 2024; 63:209-223. [PMID: 37818798 DOI: 10.1002/mc.23646] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2023] [Revised: 09/15/2023] [Accepted: 09/19/2023] [Indexed: 10/13/2023]
Abstract
Cyclin dependent kinase 4 and 6 inhibitors such as abemaciclib are routinely used to treat metastatic estrogen receptor positive (ER+) breast cancer. However, adaptive mechanisms inhibit their effectiveness and allow for disease progression. Using ER+ breast cancer cell models, we show that acquired resistance to abemaciclib is accompanied by increase in metastatic potential. Mass spectrometry-based proteomics from abemaciclib sensitive and resistant cells showed that lysosomal proteins including CTSD (cathepsin D), cathepsin A and CD68 were significantly increased in resistant cells. Combination of abemaciclib and a lysosomal destabilizer, such as hydroxychloroquine (HCQ) or bafilomycin A1, resensitized resistant cells to abemaciclib. Also, combination of abemaciclib and HCQ decreased migration and invasive potential and increased lysosomal membrane permeability in resistant cells. Prosurvival B cell lymphoma 2 (BCL2) protein levels were elevated in resistant cells, and a triple treatment with abemaciclib, HCQ, and BCL2 inhibitor, venetoclax, significantly inhibited cell growth compared to treatment with abemaciclib and HCQ. Furthermore, resistant cells showed increased levels of Transcription Factor EB (TFEB), a master regulator of lysosomal-autophagy genes, and siRNA mediated knockdown of TFEB decreased invasion in resistant cells. TFEB was found to be mutated in a subset of invasive human breast cancer samples, and overall survival analysis in ER+, lymph node-positive breast cancer showed that increased TFEB expression correlated with decreased survival. Collectively, we show that acquired resistance to abemaciclib leads to increased metastatic potential and increased levels of protumorigenic lysosomal proteins. Therefore, the lysosomal pathway could be a therapeutic target in advanced ER+ breast cancer.
Collapse
Affiliation(s)
- Erin R Scheidemann
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA
| | - Diane M Demas
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA
| | - Chunyan Hou
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA
| | - Junfeng Ma
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA
| | - Wei He
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA
| | | | - Eric Schultz
- Ocean Genomics Inc., Pittsburgh, Pennsylvania, USA
| | | | - Ayesha N Shajahan-Haq
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA
| |
Collapse
|
|
3
|
Corver WE, Ter Haar NT. High-Resolution Multiparameter DNA Flow Cytometry for Accurate Ploidy Assessment and the Detection and Sorting of Tumor and Stromal Subpopulations from Paraffin-Embedded Tissues. Curr Protoc 2023; 3:e825. [PMID: 37428889 DOI: 10.1002/cpz1.825] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/12/2023]
Abstract
This article contains detailed protocols for the simultaneous flow cytometric identification of tumor cells and stromal cells and measurement of DNA content of formalin-fixed, paraffin-embedded (FFPE) tissues. The vimentin-positive stromal cell fraction can be used as an internal reference for accurate DNA content assessments of FFPE carcinoma tissues. This allows clear detection of keratin-positive tumor cells with a DNA index lower than 1.0 (near-haploidy) and of keratin-positive tumor cells with a DNA index close to 1.0 in overall DNA aneuploid samples, thus improving DNA ploidy assessment in FFPE carcinomas. Furthermore, the protocol is useful for studying molecular genetic alterations and intratumor heterogeneity in archival FFPE samples. Keratin-positive tumor cell fractions can be sorted for further molecular genetic analysis, while DNA from the sorted vimentin-positive stromal cells can serve as a reference when normal tissue of the patient is not available. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Multiparameter DNA content analysis of FFPE carcinomas Alternate Protocol 1: Immunocytochemistry for keratin and vimentin, and DNA labeling for blue and red excitation Alternate Protocol 2: Immunocytochemistry for keratin and vimentin, and DNA labeling for blue excitation Support Protocol: Sorting cell population from FFPE carcinomas.
Collapse
|
|
4
|
Thiosemicarbazones Can Act Synergistically with Anthracyclines to Downregulate CHEK1 Expression and Induce DNA Damage in Cell Lines Derived from Pediatric Solid Tumors. Int J Mol Sci 2022; 23:ijms23158549. [PMID: 35955683 PMCID: PMC9369312 DOI: 10.3390/ijms23158549] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Revised: 07/28/2022] [Accepted: 07/29/2022] [Indexed: 12/10/2022] Open
Abstract
Anticancer therapy by anthracyclines often leads to the development of multidrug resistance (MDR), with subsequent treatment failure. Thiosemicarbazones have been previously suggested as suitable anthracycline partners due to their ability to overcome drug resistance through dual Pgp-dependent cytotoxicity-inducing effects. Here, we focused on combining anthracyclines (doxorubicin, daunorubicin, and mitoxantrone) and two thiosemicarbazones (DpC and Dp44mT) for treating cell types derived from the most frequent pediatric solid tumors. Our results showed synergistic effects for all combinations of treatments in all tested cell types. Nevertheless, further experiments revealed that this synergism was independent of Pgp expression but rather resulted from impaired DNA repair control leading to cell death via mitotic catastrophe. The downregulation of checkpoint kinase 1 (CHEK1) expression by thiosemicarbazones and the ability of both types of agents to induce double-strand breaks in DNA may explain the Pgp-independent synergism between anthracyclines and thiosemicarbazones. Moreover, the concomitant application of these agents was found to be the most efficient approach, achieving the strongest synergistic effect with lower concentrations of these drugs. Overall, our study identified a new mechanism that offers an avenue for combining thiosemicarbazones with anthracyclines to treat tumors regardless the Pgp status.
Collapse
|
|
5
|
Bhavana S, Kusuma CG, Gubbiveeranna V, Sumachirayu CK, Ravikumar H, Nagaraju S. Green route synthesis of copper oxide nanoparticles using Vitex altissima [L] leaves extract and their potential anticancer activity against A549 cell lines and its apoptosis induction. INORG NANO-MET CHEM 2022. [DOI: 10.1080/24701556.2022.2081195] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Affiliation(s)
- S. Bhavana
- Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru, India
| | - C. G. Kusuma
- Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru, India
| | - Vinod Gubbiveeranna
- Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru, India
| | - C. K. Sumachirayu
- Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru, India
| | - H. Ravikumar
- Department of Life Science, Jnana Bharthi Campus, Bangalore University, Bangalore, India
| | - S. Nagaraju
- Department of Studies and Research in Biochemistry, Tumkur University, Tumakuru, India
| |
Collapse
|
|
6
|
The Effect of Clomiphene Citrate and Letrozole in Apoptotic Pathways and Cell Cycle in Human Primary Cumulus Cells and the Protective Effect of Estradiol. Reprod Sci 2022; 29:2272-2281. [PMID: 35513593 DOI: 10.1007/s43032-022-00961-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2021] [Accepted: 04/27/2022] [Indexed: 10/18/2022]
Abstract
Clomiphene citrate (CC) and letrozole are ovulatory stimulants that, despite high ovulation rates, achieve low pregnancy rates. This study aimed to investigate the in vitro effects of CC and letrozole, alone or in combination with estradiol, on apoptosis in human cumulus cells. We performed a controlled prospective study using primary cumulus cell cultures from patients undergoing in vitro fertilization (n=22). Alpha-inhibin immunocytochemistry was used to assess cell culture purity and morphology. Cell viability was evaluated by MTT assay, cell cycle status by flow cytometry, and Caspase-3, Bax and SOD-2, and S26 gene expression by qPCR. Cells were treated for 24 hours in 5 conditioned media: CC, CC + estradiol, letrozole, letrozole + estradiol and control. None of the treatments affected cell viability, but letrozole reduced the mean percentage of cells in the S phase compared to control (24.79 versus 21.70, p=0.0014). Clomiphene treatment increased mRNA expression of Bax (4 fold) and SOD-2 (2 fold), which was reversed by co-treatment with estradiol. SOD-2 expression increased in cells treated with letrozole compared to control (4 fold), which was also reversed by estradiol. These findings suggest that clomiphene citrate and letrozole do not significantly affect the viability of human cumulus cells. Still, the expression of genes involved in apoptosis was modulated by these drugs alone and in association with estradiol, suggesting that CC and letrozole may have direct effects on cumulus cells beyond their known mechanisms of action.
Collapse
|
|
7
|
Wyngaard GA, Skern-Mauritzen R, Malde K, Prendergast R, Peruzzi S. The salmon louse genome may be much larger than sequencing suggests. Sci Rep 2022; 12:6616. [PMID: 35459797 PMCID: PMC9033869 DOI: 10.1038/s41598-022-10585-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2021] [Accepted: 04/08/2022] [Indexed: 12/30/2022] Open
Abstract
The genome size of organisms impacts their evolution and biology and is often assumed to be characteristic of a species. Here we present the first published estimates of genome size of the ecologically and economically important ectoparasite, Lepeophtheirus salmonis (Copepoda, Caligidae). Four independent L. salmonis genome assemblies of the North Atlantic subspecies Lepeophtheirus salmonis salmonis, including two chromosome level assemblies, yield assemblies ranging from 665 to 790 Mbps. These genome assemblies are congruent in their findings, and appear very complete with Benchmarking Universal Single-Copy Orthologs analyses finding > 92% of expected genes and transcriptome datasets routinely mapping > 90% of reads. However, two cytometric techniques, flow cytometry and Feulgen image analysis densitometry, yield measurements of 1.3-1.6 Gb in the haploid genome. Interestingly, earlier cytometric measurements reported genome sizes of 939 and 567 Mbps in L. salmonis salmonis samples from Bay of Fundy and Norway, respectively. Available data thus suggest that the genome sizes of salmon lice are variable. Current understanding of eukaryotic genome dynamics suggests that the most likely explanation for such variability involves repetitive DNA, which for L. salmonis makes up ≈ 60% of the genome assemblies.
Collapse
Affiliation(s)
- Grace A Wyngaard
- Department of Biology, James Madison University, Harrisonburg, VA, USA
| | | | - Ketil Malde
- Institute of Marine Research, Bergen, Norway
- Department of Informatics, University of Bergen, Bergen, Norway
| | | | - Stefano Peruzzi
- Department of Arctic Marine Biology, UiT-the Arctic University of Norway, Tromsø, Norway.
| |
Collapse
|
|
8
|
Bioactive Low Molecular Weight Keratin Hydrolysates for Improving Skin Wound Healing. Polymers (Basel) 2022; 14:polym14061125. [PMID: 35335455 PMCID: PMC8955321 DOI: 10.3390/polym14061125] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2022] [Revised: 02/24/2022] [Accepted: 03/08/2022] [Indexed: 01/27/2023] Open
Abstract
Keratin biomaterials with high molecular weights were intensively investigated but few are marketed due to complex methods of extraction and preparation and limited understanding of their influence on cells behavior. In this context the aim of this research was to elucidate decisive molecular factors for skin homeostasis restoration induced by two low molecular weight keratin hydrolysates extracted and conditioned through a simple and green method. Two keratin hydrolysates with molecular weights of 3758 and 12,400 Da were physico-chemically characterized and their structure was assessed by circular dichroism (CD) and FTIR spectroscopy in view of bioactive potential identification. Other investigations were focused on several molecular factors: α1, α2 and β1 integrin mediated signals, cell cycle progression in pro-inflammatory conditions (TNFα/LPS stimulated keratinocytes and fibroblasts) and ICAM-1/VCAM-1 inhibition in human vascular endothelial cells. Flow cytometry techniques demonstrated a distinctive pattern of efficacy: keratin hydrolysates over-expressed α1 and α2 subunits, responsible for tight bounds between fibroblasts and collagen or laminin 1; both actives stimulated the epidermal turn-over and inhibited VCAM over-expression in pro-inflammatory conditions associated with bacterial infections. Our results offer mechanistic insights in wound healing signaling factors modulated by the two low molecular weight keratin hydrolysates which still preserve bioactive secondary structure.
Collapse
|
|
9
|
Buzgariu W, Aubry-Lachainaye JP, Galliot B. Studying Stem Cell Biology in Intact and Whole-Body Regenerating Hydra by Flow Cytometry. Methods Mol Biol 2022; 2450:373-398. [PMID: 35359319 PMCID: PMC9761490 DOI: 10.1007/978-1-0716-2172-1_20] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
The freshwater Hydra polyp is a versatile model to study whole-body regeneration from a developmental as well as a cellular point of view. The outstanding regenerative capacities of Hydra are based on its three populations of adult stem cells located in the central body column of the animal. There, these three populations, gastrodermal epithelial, epidermal epithelial, and interstitial, continuously cycle in homeostatic conditions, and their activity is locally regulated after mid-gastric bisection. Moreover, they present an unusual cycling behavior with a short G1 phase and a pausing in G2. This particular cell cycle has been studied for a long time with classical microscopic methods. We describe here two flow cytometry methods that provide accurate and reproducible quantitative data to monitor cell cycle regulation in homeostatic and regenerative contexts. We also present a cell sorting procedure based on flow cytometry, whereby stem cells expressing a fluorescent reporter protein in transgenic lines can be enriched for use in applications such as transcriptomic, proteomic, or cell cycle analysis.
Collapse
Affiliation(s)
- Wanda Buzgariu
- Department of Genetics and Evolution, iGE3, Faculty of Sciences, University of Geneva, Geneva, Switzerland.
| | | | - Brigitte Galliot
- Department of Genetics and Evolution, iGE3, Faculty of Sciences, University of Geneva, Geneva, Switzerland
| |
Collapse
|
|
10
|
Maarouf A, Boissard A, Henry C, Leman G, Coqueret O, Guette C, Lelièvre E. Anterior gradient protein 2 is a marker of tumor aggressiveness in breast cancer and favors chemotherapy‑induced senescence escape. Int J Oncol 2021; 60:5. [PMID: 34913074 PMCID: PMC8727137 DOI: 10.3892/ijo.2021.5295] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2021] [Accepted: 11/09/2021] [Indexed: 11/05/2022] Open
Abstract
Among the different chemotherapies available, genotoxic drugs are widely used. In response to these drugs, particularly doxorubicin, tumor cells can enter into senescence. Chemotherapy‑induced senescence (CIS) is a complex response. Long described as a definitive arrest of cell proliferation, the present authors and various groups have shown that this state may not be complete and could allow certain cells to reproliferate. The mechanism could be due to the activation of new signaling pathways. In the laboratory, the proteins involved in these pathways and triggering cell proliferation were studied. The present study determined a new role for anterior gradient protein 2 (AGR2) in vivo in patients and in vitro in a senescence escape model. AGR2's implication in breast cancer patients and proliferation of senescent cells was assessed based on a SWATH‑MS proteomic study of patients' samples and RNA interference technology on cell lines. First, AGR2 was identified and it was found that its concentration is higher in the serum of patients with breast cancer and that this high concentration is associated with metastasis occurrence. An inverse correlation between intratumoral AGR2 expression and the senescence marker p16 was also observed. This observation led to the study of the role of AGR2 in the CIS escape model. In this model, it was found that AGR2 is overexpressed in cells during senescence escape and that its loss considerably reduces this phenomenon. Furthermore, it was shown that the extracellular form of AGR2 stimulated the reproliferation of senescent cells. The power of proteomic analysis based on the SWATH‑MS approach allowed the present study to highlight the mammalian target of rapamycin (mTOR)/AKT signaling pathway in the senescence escape mechanism mediated by AGR2. Analysis of the two signaling pathways revealed that AGR2 modulated RICTOR and AKT phosphorylation. All these results showed that AGR2 expression in sera and tumors of breast cancer patients is a marker of tumor progression and metastasis occurrence. They also showed that its overexpression regulates CIS escape via activation of the mTOR/AKT signaling pathway.
Collapse
Affiliation(s)
- Amine Maarouf
- Paul Papin ICO Cancer Center, CRCINA, INSERM U1232, Université de Nantes, Université d'Angers, 49055 Angers, France
| | - Alice Boissard
- Paul Papin ICO Cancer Center, CRCINA, INSERM U1232, Université de Nantes, Université d'Angers, 49055 Angers, France
| | - Cécile Henry
- Paul Papin ICO Cancer Center, CRCINA, INSERM U1232, Université de Nantes, Université d'Angers, 49055 Angers, France
| | - Géraldine Leman
- Paul Papin ICO Cancer Center, CRCINA, INSERM U1232, Université de Nantes, Université d'Angers, 49055 Angers, France
| | - Olivier Coqueret
- Paul Papin ICO Cancer Center, CRCINA, INSERM U1232, Université de Nantes, Université d'Angers, 49055 Angers, France
| | - Catherine Guette
- Paul Papin ICO Cancer Center, CRCINA, INSERM U1232, Université de Nantes, Université d'Angers, 49055 Angers, France
| | - Eric Lelièvre
- Paul Papin ICO Cancer Center, CRCINA, INSERM U1232, Université de Nantes, Université d'Angers, 49055 Angers, France
| |
Collapse
|
|
11
|
Billaud A, Chevalier LM, Augereau P, Frenel JS, Passot C, Campone M, Morel A. Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes. Genome Med 2021; 13:174. [PMID: 34749799 PMCID: PMC8576946 DOI: 10.1186/s13073-021-00976-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Accepted: 09/23/2021] [Indexed: 11/25/2022] Open
Abstract
BACKGROUND Targeted therapies in oncology are promising but variants of uncertain significance (VUS) limit their use for clinical management and necessitate functional testing in vitro. Using BRCA1 and BRCA2 variants, which have consequences on PARP inhibitor sensitivity, and POLE variants, potential biomarkers of immunotherapy response, we developed a rapid functional assay based on CRISPR-Cas9 genome editing to determine the functional consequences of these variants having potentially direct implications on patients' access to targeted therapies. METHODS We first evaluated the functional impact of 26 BRCA1 and 7 BRCA2 variants by editing and comparing NGS results between the variant of interest and a silent control variant. Ten of these variants had already been classified as benign or pathogenic and were used as controls. Finally, we extended this method to the characterization of POLE VUS. RESULTS For the 23 variants that were unclassified or for which conflicting interpretations had been reported, 15 were classified as functionally normal and 6 as functionally abnormal. Another two variants were found to have intermediate consequences, both with potential impacts on splicing. We then compared these scores to the patients' responses to PARP inhibitors when possible. Finally, to prove the application of our method to the classification of variants from other tumor suppressor genes, we exemplified with three POLE VUS. Among them, two were classified with an intermediate functional impact and one was functionally abnormal. Eventually, four POLE variants previously classified in databases were also evaluated. However, we found evidence of a discordance with the classification, variant p.Leu424Val being found here functionally normal. CONCLUSIONS Our new rapid functional assay can be used to characterize the functional implication of BRCA1 and BRCA2 variants, giving patients whose variants were evaluated as functionally abnormal access to PARP inhibitor treatment. Retrospective analysis of patients' responses to PARP inhibitors, where accessible, was consistent with our functional score evaluation and confirmed the accuracy of our protocol. This method could potentially be extended to the classification of VUS from all essential tumor suppressor genes and can be performed within a timeframe compatible with clinical applications, thereby having a direct theranostic impact.
Collapse
Affiliation(s)
- Amandine Billaud
- Université d'Angers, Inserm, CRCINA, SFR ICAT, F-49000, Angers, France
- Institut de Cancérologie de l'Ouest Nantes-Angers, F-49000, Angers, France
| | - Louise-Marie Chevalier
- Université d'Angers, Inserm, CRCINA, SFR ICAT, F-49000, Angers, France
- Institut de Cancérologie de l'Ouest Nantes-Angers, F-49000, Angers, France
| | - Paule Augereau
- Institut de Cancérologie de l'Ouest Nantes-Angers, F-49000, Angers, France
| | - Jean-Sebastien Frenel
- Institut de Cancérologie de l'Ouest Nantes-Angers, F-49000, Angers, France
- Université de Nantes, Inserm, CRCINA, F-44000, Nantes, France
| | - Christophe Passot
- Institut de Cancérologie de l'Ouest Nantes-Angers, F-49000, Angers, France
| | - Mario Campone
- Institut de Cancérologie de l'Ouest Nantes-Angers, F-49000, Angers, France
- Université de Nantes, Inserm, CRCINA, F-44000, Nantes, France
| | - Alain Morel
- Université d'Angers, Inserm, CRCINA, SFR ICAT, F-49000, Angers, France.
- Institut de Cancérologie de l'Ouest Nantes-Angers, F-49000, Angers, France.
| |
Collapse
|
|
12
|
Patthey A, Boman K, Tavelin B, Lindquist D, Lundin E, Hultdin M. Combination of aneuploidy and high S-phase fraction indicates increased risk of relapse in stage I endometrioid endometrial carcinoma. Acta Oncol 2021; 60:1218-1224. [PMID: 34156893 DOI: 10.1080/0284186x.2021.1939146] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
INTRODUCTION Endometrioid endometrial carcinoma is a cancer type with generally excellent prognosis when diagnosed at an early stage, but there is a subset of patients with relapsing disease in spite of early diagnosis and surgical treatment. There is a need to find prognostic markers to identify these patients with increased risk of relapse. Depth of myometrial invasion, histological grade, and presence of lymphovascular invasion are known risk factors. DNA content (ploidy) and proliferation measured as S-phase fraction (SPF) have been discussed as prognostic markers but need additional evaluation. MATERIAL AND METHODS We evaluated relapse-free survival (RFS) with respect to ploidy and SPF, which was analyzed by flow cytometry on fresh tumor tissue, in a cohort of 1001 women treated for stage I endometrioid endometrial carcinoma in northern Sweden during the period of 1993-2010, with a median follow up time of 12.0 years. Data were obtained from historical records. RESULTS In simple analysis, both aneuploidy and high SPF were associated to increased risk of relapse with hazard ratios (HR) 2.37 (95% CI 1.52-3.70) and 1.94 (95% CI 1.24-3.02), respectively. Our data also confirmed stage, tumor grade, and ploidy as independent prognostic markers in an age adjusted cox regression multivariable analysis but we did not find SPF to contribute to prognosis. However, the combination of aneuploidy and high SPF identified a group of patients with increased risk of relapse, HR 2.02 (95% CI 1.19-3.44). CONCLUSION In this study, which is the largest study of ploidy and SPF in stage I endometrioid endometrial carcinoma using fresh frozen tissue, aneuploidy was shown to be an independent prognostic marker. Furthermore, the combination of aneuploidy and high SPF could be used to identify patients with increased risk of relapse.
Collapse
Affiliation(s)
- Annika Patthey
- Department of Medical Biosciences, Pathology, Umeå University, Umeå, Sweden
| | - Karin Boman
- Department of Radiation Sciences, Umeå University, Umeå, Sweden
| | - Björn Tavelin
- Department of Radiation Sciences, Umeå University, Umeå, Sweden
| | - David Lindquist
- Department of Clinical Sciences, Umeå University, Umeå, Sweden
| | - Eva Lundin
- Department of Medical Biosciences, Pathology, Umeå University, Umeå, Sweden
| | - Magnus Hultdin
- Department of Medical Biosciences, Pathology, Umeå University, Umeå, Sweden
| |
Collapse
|
|
13
|
Hubálek M, Flajšhans M. Simple Field Storage of Fish Samples for Measurement of DNA Content by Flow Cytometry. Cytometry A 2021; 99:743-752. [PMID: 33215865 PMCID: PMC8359303 DOI: 10.1002/cyto.a.24271] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2020] [Revised: 10/28/2020] [Accepted: 11/16/2020] [Indexed: 11/14/2022]
Abstract
Flow cytometry is an effective and widely used tool for determination of ploidy in fish, but it is not always possible to access the fresh samples for analysis. We investigated the potential for extended storage of fish tissue with sterlet and tench as representative species of Chondrostei and Teleostei, using blood and fin of subadult/adult specimens and tail of larvae. Thirteen procedures for extending storage, selected for rapidity and simplicity in both field and laboratory conditions, were tested for each tissue sample. Flow cytometry was applied to fresh tissue immediately after sampling and to tissue subjected to experimental protocols, always along with species-specific standard, after 1, 5, and 10 days storage at 0-4°C or freezing at -80°C. The fluorochrome 4',6-diamidine-2'-phenylindole dihydrochloride was used with excitation/emission maximum 358/461 nm. Based on the measurability of stored samples, evaluation of directly measured coefficients of variation of their DNA peaks and the changes in fluorescence intensity compared to fresh tissue, optimal procedures for extended storage of the selected tissue types of the model species are suggested. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.
Collapse
Affiliation(s)
- Martin Hubálek
- University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of HydrocenosesZátiší 728/II, 389 25VodňanyCzech Republic
| | - Martin Flajšhans
- University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of HydrocenosesZátiší 728/II, 389 25VodňanyCzech Republic
| |
Collapse
|
|
14
|
Fallah Y, Demas DM, Jin L, He W, Shajahan-Haq AN. Targeting WEE1 Inhibits Growth of Breast Cancer Cells That Are Resistant to Endocrine Therapy and CDK4/6 Inhibitors. Front Oncol 2021; 11:681530. [PMID: 34277427 PMCID: PMC8281892 DOI: 10.3389/fonc.2021.681530] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Accepted: 05/21/2021] [Indexed: 01/02/2023] Open
Abstract
Despite the success of antiestrogens in extending overall survival of patients with estrogen receptor positive (ER+) breast tumors, resistance to these therapies is prevalent. ER+ tumors that progress on antiestrogens are treated with antiestrogens and CDK4/6 inhibitors. However, 20% of these tumors never respond to CDK4/6 inhibitors due to intrinsic resistance. Here, we used endocrine sensitive ER+ MCF7 and T47D breast cancer cells to generate long-term estrogen deprived (LTED) endocrine resistant cells that are intrinsically resistant to CDK4/6 inhibitors. Since treatment with antiestrogens arrests cells in the G1 phase of the cell cycle, we hypothesized that a defective G1 checkpoint allows resistant cells to escape this arrest but increases their dependency on G2 checkpoint for DNA repair and growth, and hence, targeting the G2 checkpoint will induce cell death. Indeed, inhibition of WEE1, a crucial G2 checkpoint regulator, with AZD1775 (Adavosertib), significantly decreased cell proliferation and increased G2/M arrest, apoptosis and gamma-H2AX levels (a marker for DNA double stranded breaks) in resistant cells compared with sensitive cells. Thus, targeting WEE1 is a promising anti-cancer therapeutic strategy in standard therapy resistant ER+ breast cancer.
Collapse
Affiliation(s)
- Yassi Fallah
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, United States
| | - Diane M Demas
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, United States
| | - Lu Jin
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, United States
| | - Wei He
- Program in Genetics, Bioinformatics, and Computational Biology, VT Biological Transport, Virginia Tech, Blacksburg, VA, United States
| | - Ayesha N Shajahan-Haq
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, United States
| |
Collapse
|
|
15
|
Costales P, Ríos-Lombardía N, Lorenzo-Herrero S, Morís F, González-Sabín J. Novel chiral naphthalimide-cycloalkanediamine conjugates: Design, synthesis and antitumor activity. Bioorg Chem 2021; 112:104859. [PMID: 33836453 DOI: 10.1016/j.bioorg.2021.104859] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2020] [Revised: 02/18/2021] [Accepted: 03/22/2021] [Indexed: 10/21/2022]
Abstract
A novel series of enantiopure naphthalimide-cycloalkanediamine conjugates were designed, synthetized and evaluated for in vitro cytotoxicity against human colon adenocarcinoma (LoVo), human lung adenocarcinoma (A549), human cervical carcinoma (Hela) and human promyelocytic leukemia cell lines (HL-60). The cytotoxicity of the compounds was highly dependent on size and relative stereochemistry of the cycloalkyl ring as well as length of the spacer. By contrast, any kind of enantioselection was observed for each pair of enantiomers. Flow cytometric analysis indicated that compounds 22 and 23 could effectively induce G2/M arrest in the four previous cell lines despite a mild apoptotic effect.
Collapse
Affiliation(s)
- Paula Costales
- EntreChem SL, Vivero Ciencias de la Salud, Santo Domingo de Guzmán, 33011 Oviedo, Spain
| | | | - Seila Lorenzo-Herrero
- EntreChem SL, Vivero Ciencias de la Salud, Santo Domingo de Guzmán, 33011 Oviedo, Spain
| | - Francisco Morís
- EntreChem SL, Vivero Ciencias de la Salud, Santo Domingo de Guzmán, 33011 Oviedo, Spain
| | - Javier González-Sabín
- EntreChem SL, Vivero Ciencias de la Salud, Santo Domingo de Guzmán, 33011 Oviedo, Spain.
| |
Collapse
|
|
16
|
Franceschini N, Verbruggen B, Tryfonidou MA, Kruisselbrink AB, Baelde H, de Visser KE, Szuhai K, Cleton-Jansen AM, Bovée JVMG. Transformed Canine and Murine Mesenchymal Stem Cells as a Model for Sarcoma with Complex Genomics. Cancers (Basel) 2021; 13:cancers13051126. [PMID: 33807947 PMCID: PMC7961539 DOI: 10.3390/cancers13051126] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 02/23/2021] [Accepted: 02/28/2021] [Indexed: 12/21/2022] Open
Abstract
Simple Summary Sarcomas are rare cancers of mesenchymal origin, the majority of which are characterized by many copy number alterations, amplifications, or deletions. Because of these complex genomics, it is notoriously difficult to identify driver events of malignant transformation. In this study, we show that murine and canine mesenchymal stem cells (MSCs) can be used to model spontaneous malignant transformation towards sarcomas with complex genomics. We show that these MSCs have an abnormal karyotype, many structural variants, and point mutations at whole genome sequencing analysis, and form sarcomas after injection into mice. Our cross-species analysis reveals that p53 loss is an early event in sarcomagenesis, and it was shown that MSCs with a knock-out in Trp53 transform earlier compared to wild-type MSCs. Our study points to the importance of p53 loss in the transformation process towards sarcomas with complex genomics. Abstract Sarcomas are rare mesenchymal tumors with a broad histological spectrum, but they can be divided into two groups based on molecular pathology: sarcomas with simple or complex genomics. Tumors with complex genomics can have aneuploidy and copy number gains and losses, which hampers the detection of early, initiating events in tumorigenesis. Often, no benign precursors are known, which is why good models are essential. The mesenchymal stem cell (MSC) is the presumed cell of origin of sarcoma. In this study, MSCs of murine and canine origin are used as a model to identify driver events for sarcomas with complex genomic alterations as they transform spontaneously after long-term culture. All transformed murine but not canine MSCs formed sarcomas after subcutaneous injection in mice. Using whole genome sequencing, spontaneously transformed murine and canine MSCs displayed a complex karyotype with aneuploidy, point mutations, structural variants, inter-chromosomal translocations, and copy number gains and losses. Cross-species analysis revealed that point mutations in Tp53/Trp53 are common in transformed murine and canine MSCs. Murine MSCs with a cre-recombinase induced deletion of exon 2–10 of Trp53 transformed earlier compared to wild-type murine MSCs, confirming the contribution of loss of p53 to spontaneous transformation. Our comparative approach using transformed murine and canine MSCs points to a crucial role for p53 loss in the formation of sarcomas with complex genomics.
Collapse
Affiliation(s)
- Natasja Franceschini
- Department of Pathology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; (N.F.); (B.V.); (A.B.K.); (H.B.); (A.-M.C.-J.)
| | - Bas Verbruggen
- Department of Pathology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; (N.F.); (B.V.); (A.B.K.); (H.B.); (A.-M.C.-J.)
| | - Marianna A. Tryfonidou
- Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands;
| | - Alwine B. Kruisselbrink
- Department of Pathology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; (N.F.); (B.V.); (A.B.K.); (H.B.); (A.-M.C.-J.)
| | - Hans Baelde
- Department of Pathology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; (N.F.); (B.V.); (A.B.K.); (H.B.); (A.-M.C.-J.)
| | - Karin E. de Visser
- Division of Tumour Biology & Immunology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands;
- Oncode Institute, Office Jaarbeurs Innovation Mile (JIM), Jaarbeursplein 6, 3521 AL Utrecht, The Netherlands
| | - Karoly Szuhai
- Department of Cell and Chemical Biology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands;
| | - Anne-Marie Cleton-Jansen
- Department of Pathology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; (N.F.); (B.V.); (A.B.K.); (H.B.); (A.-M.C.-J.)
| | - Judith V. M. G. Bovée
- Department of Pathology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; (N.F.); (B.V.); (A.B.K.); (H.B.); (A.-M.C.-J.)
- Correspondence: ; Tel.: +31-715266622
| |
Collapse
|
|
17
|
Inhibition of DNA Repair Pathways and Induction of ROS Are Potential Mechanisms of Action of the Small Molecule Inhibitor BOLD-100 in Breast Cancer. Cancers (Basel) 2020; 12:cancers12092647. [PMID: 32947941 PMCID: PMC7563761 DOI: 10.3390/cancers12092647] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2020] [Revised: 09/08/2020] [Accepted: 09/10/2020] [Indexed: 12/16/2022] Open
Abstract
BOLD-100, a ruthenium-based complex, sodium trans-[tetrachloridobis (1H-indazole) ruthenate (III)] (also known as IT-139, NKP1339 or KP1339), is a novel small molecule drug that demonstrated a manageable safety profile at the maximum tolerated dose and modest antitumor activity in a phase I clinical trial. BOLD-100 has been reported to inhibit the upregulation of the endoplasmic reticulum stress sensing protein GRP78. However, response to BOLD-100 varies in different cancer models and the precise mechanism of action in high-response versus low-response cancer cells remains unclear. In vitro studies have indicated that BOLD-100 induces cytostatic rather than cytotoxic effects as a monotherapy. To understand BOLD-100-mediated signaling mechanism in breast cancer cells, we used estrogen receptor positive (ER+) MCF7 breast cancer cells to obtain gene-metabolite integrated models. At 100 μM, BOLD-100 significantly reduced cell proliferation and expression of genes involved in the DNA repair pathway. BOLD-100 also induced reactive oxygen species (ROS) and phosphorylation of histone H2AX, gamma-H2AX (Ser139), suggesting disruption of proper DNA surveillance. In estrogen receptor negative (ER-) breast cancer cells, combination of BOLD-100 with a PARP inhibitor, olaparib, induced significant inhibition of cell growth and xenografts and increased gamma-H2AX. Thus, BOLD-100 is a novel DNA repair pathway targeting agent and can be used with other chemotherapies in ER- breast cancer.
Collapse
|
|
18
|
Brown PD, Walsh EJ. Genome size and lifestyle in gnesiotrochan rotifers. HYDROBIOLOGIA 2019; 844:105-115. [PMID: 31798186 PMCID: PMC6886742 DOI: 10.1007/s10750-018-3873-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/23/2018] [Revised: 11/30/2018] [Accepted: 12/20/2018] [Indexed: 06/10/2023]
Abstract
Gnesiotrochan rotifers display a variety of life styles ranging from taxa with free-swimming larval and sessile adult stages to those with motile adult stages and colonial habits. Several explanations for the C- value enigma posits that genome size is correlated with lifestyle. To investigate this, 13 gnesiotrochan species representing nine genera were measured by flow cytometry. Genome sizes (1C) within Gnesiotrocha ranged from 0.05 pg (Hexarthra mira and Hexarthra fennica) to 0.25 pg (Sinantherina ariprepes). Genome sizes varied within genera and species; e.g., H. fennica (El Huérfano, Mexico) was estimated to be 15% larger than that of H. mira and H. fennica (Keystone Wetland, TX, USA). Gnesiotrochan genome sizes are similar to those reported within Ploima, which range from 0.06 pg (Brachionus rotundiformis, B. dimidiatus) to 0.46 pg (B. asplanchnoidis). Within Gnesiotrocha, genome size was found to be significantly smaller in sessile versus motile species as well as in solitary versus colonial species. To account for phylogenetic background, Linear Mixed Models with hierarchical taxonomic ranks showed that there is a taxonomic component underlying genome size. This study provides the first estimates of genome size within the superorder, providing a baseline for genomic and evolutionary studies within the group.
Collapse
Affiliation(s)
- Patrick D Brown
- Department of Biological Sciences, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, USA 79968.
| | - Elizabeth J Walsh
- Department of Biological Sciences, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas, USA 79968.
| |
Collapse
|
|
19
|
Yunoki T, Kimura Y, Fujimori A. Maintenance Properties of Enzyme Molecule Stereostructure at High Temperature by Adsorption on Organo-Modified Magnetic Nanoparticle Layer Template. BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN 2019. [DOI: 10.1246/bcsj.20190102] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Affiliation(s)
- Takeru Yunoki
- Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
| | - Yusuke Kimura
- Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
| | - Atsuhiro Fujimori
- Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
| |
Collapse
|
|
20
|
Cossarizza A, Chang HD, Radbruch A, Acs A, Adam D, Adam-Klages S, Agace WW, Aghaeepour N, Akdis M, Allez M, Almeida LN, Alvisi G, Anderson G, Andrä I, Annunziato F, Anselmo A, Bacher P, Baldari CT, Bari S, Barnaba V, Barros-Martins J, Battistini L, Bauer W, Baumgart S, Baumgarth N, Baumjohann D, Baying B, Bebawy M, Becher B, Beisker W, Benes V, Beyaert R, Blanco A, Boardman DA, Bogdan C, Borger JG, Borsellino G, Boulais PE, Bradford JA, Brenner D, Brinkman RR, Brooks AES, Busch DH, Büscher M, Bushnell TP, Calzetti F, Cameron G, Cammarata I, Cao X, Cardell SL, Casola S, Cassatella MA, Cavani A, Celada A, Chatenoud L, Chattopadhyay PK, Chow S, Christakou E, Čičin-Šain L, Clerici M, Colombo FS, Cook L, Cooke A, Cooper AM, Corbett AJ, Cosma A, Cosmi L, Coulie PG, Cumano A, Cvetkovic L, Dang VD, Dang-Heine C, Davey MS, Davies D, De Biasi S, Del Zotto G, Cruz GVD, Delacher M, Bella SD, Dellabona P, Deniz G, Dessing M, Di Santo JP, Diefenbach A, Dieli F, Dolf A, Dörner T, Dress RJ, Dudziak D, Dustin M, Dutertre CA, Ebner F, Eckle SBG, Edinger M, Eede P, Ehrhardt GR, Eich M, Engel P, Engelhardt B, Erdei A, Esser C, Everts B, Evrard M, Falk CS, Fehniger TA, Felipo-Benavent M, Ferry H, Feuerer M, Filby A, Filkor K, Fillatreau S, Follo M, Förster I, Foster J, Foulds GA, Frehse B, Frenette PS, Frischbutter S, Fritzsche W, Galbraith DW, Gangaev A, Garbi N, Gaudilliere B, Gazzinelli RT, Geginat J, Gerner W, Gherardin NA, Ghoreschi K, Gibellini L, Ginhoux F, Goda K, Godfrey DI, Goettlinger C, González-Navajas JM, Goodyear CS, Gori A, Grogan JL, Grummitt D, Grützkau A, Haftmann C, Hahn J, Hammad H, Hämmerling G, Hansmann L, Hansson G, Harpur CM, Hartmann S, Hauser A, Hauser AE, Haviland DL, Hedley D, Hernández DC, Herrera G, Herrmann M, Hess C, Höfer T, Hoffmann P, Hogquist K, Holland T, Höllt T, Holmdahl R, Hombrink P, Houston JP, Hoyer BF, Huang B, Huang FP, Huber JE, Huehn J, Hundemer M, Hunter CA, Hwang WYK, Iannone A, Ingelfinger F, Ivison SM, Jäck HM, Jani PK, Jávega B, Jonjic S, Kaiser T, Kalina T, Kamradt T, Kaufmann SHE, Keller B, Ketelaars SLC, Khalilnezhad A, Khan S, Kisielow J, Klenerman P, Knopf J, Koay HF, Kobow K, Kolls JK, Kong WT, Kopf M, Korn T, Kriegsmann K, Kristyanto H, Kroneis T, Krueger A, Kühne J, Kukat C, Kunkel D, Kunze-Schumacher H, Kurosaki T, Kurts C, Kvistborg P, Kwok I, Landry J, Lantz O, Lanuti P, LaRosa F, Lehuen A, LeibundGut-Landmann S, Leipold MD, Leung LY, Levings MK, Lino AC, Liotta F, Litwin V, Liu Y, Ljunggren HG, Lohoff M, Lombardi G, Lopez L, López-Botet M, Lovett-Racke AE, Lubberts E, Luche H, Ludewig B, Lugli E, Lunemann S, Maecker HT, Maggi L, Maguire O, Mair F, Mair KH, Mantovani A, Manz RA, Marshall AJ, Martínez-Romero A, Martrus G, Marventano I, Maslinski W, Matarese G, Mattioli AV, Maueröder C, Mazzoni A, McCluskey J, McGrath M, McGuire HM, McInnes IB, Mei HE, Melchers F, Melzer S, Mielenz D, Miller SD, Mills KH, Minderman H, Mjösberg J, Moore J, Moran B, Moretta L, Mosmann TR, Müller S, Multhoff G, Muñoz LE, Münz C, Nakayama T, Nasi M, Neumann K, Ng LG, Niedobitek A, Nourshargh S, Núñez G, O’Connor JE, Ochel A, Oja A, Ordonez D, Orfao A, Orlowski-Oliver E, Ouyang W, Oxenius A, Palankar R, Panse I, Pattanapanyasat K, Paulsen M, Pavlinic D, Penter L, Peterson P, Peth C, Petriz J, Piancone F, Pickl WF, Piconese S, Pinti M, Pockley AG, Podolska MJ, Poon Z, Pracht K, Prinz I, Pucillo CEM, Quataert SA, Quatrini L, Quinn KM, Radbruch H, Radstake TRDJ, Rahmig S, Rahn HP, Rajwa B, Ravichandran G, Raz Y, Rebhahn JA, Recktenwald D, Reimer D, e Sousa CR, Remmerswaal EB, Richter L, Rico LG, Riddell A, Rieger AM, Robinson JP, Romagnani C, Rubartelli A, Ruland J, Saalmüller A, Saeys Y, Saito T, Sakaguchi S, de-Oyanguren FS, Samstag Y, Sanderson S, Sandrock I, Santoni A, Sanz RB, Saresella M, Sautes-Fridman C, Sawitzki B, Schadt L, Scheffold A, Scherer HU, Schiemann M, Schildberg FA, Schimisky E, Schlitzer A, Schlosser J, Schmid S, Schmitt S, Schober K, Schraivogel D, Schuh W, Schüler T, Schulte R, Schulz AR, Schulz SR, Scottá C, Scott-Algara D, Sester DP, Shankey TV, Silva-Santos B, Simon AK, Sitnik KM, Sozzani S, Speiser DE, Spidlen J, Stahlberg A, Stall AM, Stanley N, Stark R, Stehle C, Steinmetz T, Stockinger H, Takahama Y, Takeda K, Tan L, Tárnok A, Tiegs G, Toldi G, Tornack J, Traggiai E, Trebak M, Tree TI, Trotter J, Trowsdale J, Tsoumakidou M, Ulrich H, Urbanczyk S, van de Veen W, van den Broek M, van der Pol E, Van Gassen S, Van Isterdael G, van Lier RA, Veldhoen M, Vento-Asturias S, Vieira P, Voehringer D, Volk HD, von Borstel A, von Volkmann K, Waisman A, Walker RV, Wallace PK, Wang SA, Wang XM, Ward MD, Ward-Hartstonge KA, Warnatz K, Warnes G, Warth S, Waskow C, Watson JV, Watzl C, Wegener L, Weisenburger T, Wiedemann A, Wienands J, Wilharm A, Wilkinson RJ, Willimsky G, Wing JB, Winkelmann R, Winkler TH, Wirz OF, Wong A, Wurst P, Yang JHM, Yang J, Yazdanbakhsh M, Yu L, Yue A, Zhang H, Zhao Y, Ziegler SM, Zielinski C, Zimmermann J, Zychlinsky A. Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur J Immunol 2019; 49:1457-1973. [PMID: 31633216 PMCID: PMC7350392 DOI: 10.1002/eji.201970107] [Citation(s) in RCA: 723] [Impact Index Per Article: 120.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
Collapse
Affiliation(s)
- Andrea Cossarizza
- Department of Medical and Surgical Sciences for Children and Adults, Univ. of Modena and Reggio Emilia School of Medicine, Modena, Italy
| | - Hyun-Dong Chang
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Andreas Radbruch
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Andreas Acs
- Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany
| | - Dieter Adam
- Institut für Immunologie, Christian-Albrechts-Universität zu Kiel, Kiel, Germany
| | - Sabine Adam-Klages
- Institut für Transfusionsmedizin, Universitätsklinik Schleswig-Holstein, Kiel, Germany
| | - William W. Agace
- Mucosal Immunology group, Department of Health Technology, Technical University of Denmark, Kgs. Lyngby, Denmark
- Immunology Section, Lund University, Lund, Sweden
| | - Nima Aghaeepour
- Departments of Anesthesiology, Pain and Perioperative Medicine; Biomedical Data Sciences; and Pediatrics, Stanford University, Stanford, CA, USA
| | - Mübeccel Akdis
- Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland
| | - Matthieu Allez
- Université de Paris, Institut de Recherche Saint-Louis, INSERM U1160, and Gastroenterology Department, Hôpital Saint-Louis – APHP, Paris, France
| | | | - Giorgia Alvisi
- Laboratory of Translational Immunology, Humanitas Clinical and Research Center, Rozzano, Italy
| | | | - Immanuel Andrä
- Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany
| | - Francesco Annunziato
- Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
| | - Achille Anselmo
- Flow Cytometry Core, Humanitas Clinical and Research Center, Milan, Italy
| | - Petra Bacher
- Institut für Immunologie, Christian-Albrechts-Universität zu Kiel, Kiel, Germany
- Institut für Klinische Molekularbiologie, Christian-Albrechts Universität zu Kiel, Germany
| | | | - Sudipto Bari
- Division of Medical Sciences, National Cancer Centre Singapore, Singapore
- Cancer & Stem Cell Biology, Duke-NUS Medical School, Singapore
| | - Vincenzo Barnaba
- Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Rome, Italy
- Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy
- Istituto Pasteur - Fondazione Cenci Bolognetti, Rome, Italy
| | | | | | - Wolfgang Bauer
- Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria
| | - Sabine Baumgart
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Nicole Baumgarth
- Center for Comparative Medicine & Dept. Pathology, Microbiology & Immunology, University of California, Davis, CA, USA
| | - Dirk Baumjohann
- Institute for Immunology, Faculty of Medicine, Biomedical Center, LMU Munich, Planegg-Martinsried, Germany
| | - Bianka Baying
- Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
| | - Mary Bebawy
- Discipline of Pharmacy, Graduate School of Health, The University of Technology Sydney, Sydney, NSW, Australia
| | - Burkhard Becher
- Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland
- Comprehensive Cancer Center Zurich, Switzerland
| | - Wolfgang Beisker
- Flow Cytometry Laboratory, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, German Research Center for Environmental Health, München, Germany
| | - Vladimir Benes
- Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
| | - Rudi Beyaert
- Department of Biomedical Molecular Biology, Center for Inflammation Research, Ghent University - VIB, Ghent, Belgium
| | - Alfonso Blanco
- Flow Cytometry Core Technologies, UCD Conway Institute, University College Dublin, Dublin, Ireland
| | - Dominic A. Boardman
- Department of Surgery, The University of British Columbia, Vancouver, Canada
- BC Children’s Hospital Research Institute, Vancouver, Canada
| | - Christian Bogdan
- Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Erlangen, Germany
- Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg and Medical Immunology Campus Erlangen, Erlangen, Germany
| | - Jessica G. Borger
- Department of Immunology and Pathology, Monash University, Melbourne, Victoria, Australia
| | - Giovanna Borsellino
- Neuroimmunology and Flow Cytometry Units, Fondazione Santa Lucia IRCCS, Rome, Italy
| | - Philip E. Boulais
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA
- The Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Bronx, New York, USA
| | | | - Dirk Brenner
- Luxembourg Institute of Health, Department of Infection and Immunity, Experimental and Molecular Immunology, Esch-sur-Alzette, Luxembourg
- Odense University Hospital, Odense Research Center for Anaphylaxis, University of Southern Denmark, Department of Dermatology and Allergy Center, Odense, Denmark
- Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, Belvaux, Luxembourg
| | - Ryan R. Brinkman
- Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada
- Terry Fox Laboratory, BC Cancer, Vancouver, BC, Canada
| | - Anna E. S. Brooks
- University of Auckland, School of Biological Sciences, Maurice Wilkins Center, Auckland, New Zealand
| | - Dirk H. Busch
- Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany
- German Center for Infection Research (DZIF), Munich, Germany
- Focus Group “Clinical Cell Processing and Purification”, Institute for Advanced Study, Technische Universität München, Munich, Germany
| | - Martin Büscher
- Biophysics, R&D Engineering, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
| | - Timothy P. Bushnell
- Department of Pediatrics and Shared Resource Laboratories, University of Rochester Medical Center, Rochester, NY, USA
| | - Federica Calzetti
- University of Verona, Department of Medicine, Section of General Pathology, Verona, Italy
| | - Garth Cameron
- Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia
| | - Ilenia Cammarata
- Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Rome, Italy
| | - Xuetao Cao
- National Key Laboratory of Medical Immunology, Nankai University, Tianjin, China
| | - Susanna L. Cardell
- Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden
| | - Stefano Casola
- The FIRC Institute of Molecular Oncology (FOM), Milan, Italy
| | - Marco A. Cassatella
- University of Verona, Department of Medicine, Section of General Pathology, Verona, Italy
| | - Andrea Cavani
- National Institute for Health, Migration and Poverty (INMP), Rome, Italy
| | - Antonio Celada
- Macrophage Biology Group, School of Biology, University of Barcelona, Barcelona, Spain
| | - Lucienne Chatenoud
- Université Paris Descartes, Institut National de la Santé et de la Recherche Médicale, Paris, France
| | | | - Sue Chow
- Divsion of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto, Ontario, Canada
| | - Eleni Christakou
- Department of Immunobiology, School of Immunology and Microbial Sciences, King’s College London, UK
- National Institutes of Health Research Biomedical Research Centre at Guy’s and St. Thomas’ National Health Service, Foundation Trust and King’s College London, UK
| | - Luka Čičin-Šain
- Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany
| | - Mario Clerici
- IRCCS Fondazione Don Carlo Gnocchi, Milan, Italy
- Department of Physiopathology and Transplants, University of Milan, Milan, Italy
- Milan Center for Neuroscience, University of Milano-Bicocca, Milan, Italy
| | | | - Laura Cook
- BC Children’s Hospital Research Institute, Vancouver, Canada
- Department of Medicine, The University of British Columbia, Vancouver, Canada
| | - Anne Cooke
- Department of Pathology, University of Cambridge, Cambridge, UK
| | - Andrea M. Cooper
- Department of Respiratory Sciences, University of Leicester, Leicester, UK
| | - Alexandra J. Corbett
- Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia
| | - Antonio Cosma
- National Cytometry Platform, Luxembourg Institute of Health, Department of Infection and Immunity, Esch-sur-Alzette, Luxembourg
| | - Lorenzo Cosmi
- Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
| | - Pierre G. Coulie
- de Duve Institute, Université catholique de Louvain, Brussels, Belgium
| | - Ana Cumano
- Unit Lymphopoiesis, Department of Immunology, Institut Pasteur, Paris, France
| | - Ljiljana Cvetkovic
- Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Van Duc Dang
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Chantip Dang-Heine
- Clinical Research Unit, Berlin Institute of Health (BIH), Charite Universitätsmedizin Berlin, Berlin, Germany
| | - Martin S. Davey
- Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
- Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia
| | - Derek Davies
- Flow Cytometry Scientific Technology Platform, The Francis Crick Institute, London, UK
| | - Sara De Biasi
- Department of Surgery, Medicine, Dentistry and Morphological Sciences, Univ. of Modena and Reggio Emilia, Modena, Italy
| | | | - Gelo Victoriano Dela Cruz
- Novo Nordisk Foundation Center for Stem Cell Biology – DanStem, University of Copenhagen, Copenhagen, Denmark
| | - Michael Delacher
- Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany
- Chair for Immunology, University Regensburg, Germany
| | - Silvia Della Bella
- Department of Medical Biotechnologies and Translational Medicine, University of Milan, Milan, Italy
| | - Paolo Dellabona
- Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy
| | - Günnur Deniz
- Istanbul University, Aziz Sancar Institute of Experimental Medicine, Department of Immunology, Istanbul, Turkey
| | | | - James P. Di Santo
- Innate Immunty Unit, Department of Immunology, Institut Pasteur, Paris, France
- Institut Pasteur, Inserm U1223, Paris, France
| | - Andreas Diefenbach
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Charité - Universitätsmedizin Berlin, Laboratory of Innate Immunity, Department of Microbiology, Infectious Diseases and Immunology, Berlin, Germany
- Berlin Institute of Health (BIH), Berlin, Germany
| | - Francesco Dieli
- University of Palermo, Central Laboratory of Advanced Diagnosis and Biomedical Research, Department of Biomedicine, Neurosciences and Advanced Diagnostics, Palermo, Italy
| | - Andreas Dolf
- Flow Cytometry Core Facility, Institute of Experimental Immunology, University of Bonn, Bonn, Germany
| | - Thomas Dörner
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Dept. Medicine/Rheumatology and Clinical Immunology, Charité Universitätsmedizin Berlin, Germany
| | - Regine J. Dress
- Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
| | - Diana Dudziak
- Department of Dermatology, Laboratory of Dendritic Cell Biology, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), University Hospital Erlangen, Erlangen, Germany
| | - Michael Dustin
- Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK
| | - Charles-Antoine Dutertre
- Program in Emerging Infectious Disease, Duke-NUS Medical School, Singapore
- Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
| | - Friederike Ebner
- Institute of Immunology, Centre for Infection Medicine, Department of Veterinary Medicine, Freie Universität Berlin, Germany
| | - Sidonia B. G. Eckle
- Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia
| | - Matthias Edinger
- Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany
- Department of Internal Medicine III, University Hospital Regensburg, Germany
| | - Pascale Eede
- Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Neuropathology, Germany
| | | | - Marcus Eich
- Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany
| | - Pablo Engel
- University of Barcelona, Faculty of Medicine and Health Sciences, Department of Biomedical Sciences, Barcelona, Spain
| | | | - Anna Erdei
- Department of Immunology, University L. Eotvos, Budapest, Hungary
| | - Charlotte Esser
- Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany
| | - Bart Everts
- Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands
| | - Maximilien Evrard
- Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
| | - Christine S. Falk
- Institute of Transplant Immunology, Hannover Medical School, MHH, Hannover, Germany
| | - Todd A. Fehniger
- Division of Oncology, Washington University School of Medicine, St. Louis, MO, USA
| | - Mar Felipo-Benavent
- Laboratory of Cytomics, Joint Research Unit CIPF-UVEG, Principe Felipe Research Center, Valencia, Spain
| | - Helen Ferry
- Experimental Medicine Division, Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Markus Feuerer
- Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany
- Chair for Immunology, University Regensburg, Germany
| | - Andrew Filby
- The Flow Cytometry Core Facility, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
| | | | - Simon Fillatreau
- Institut Necker-Enfants Malades, Université Paris Descartes Sorbonne Paris Cité, Faculté de Médecine, AP-HP, Hôpital Necker Enfants Malades, INSERM U1151-CNRS UMR 8253, Paris, France
| | - Marie Follo
- Department of Medicine I, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Universitaetsklinikum FreiburgLighthouse Core Facility, Zentrum für Translationale Zellforschung, Klinik für Innere Medizin I, Freiburg, Germany
| | - Irmgard Förster
- Immunology and Environment, LIMES Institute, University of Bonn, Bonn, Germany
| | | | - Gemma A. Foulds
- John van Geest Cancer Research Centre, Nottingham Trent University, Nottingham, UK
| | - Britta Frehse
- Institute for Systemic Inflammation Research, University of Luebeck, Luebeck, Germany
| | - Paul S. Frenette
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA
- The Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Bronx, New York, USA
- Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Stefan Frischbutter
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Dermatology, Venereology and Allergology
| | - Wolfgang Fritzsche
- Nanobiophotonics Department, Leibniz Institute of Photonic Technology (IPHT), Jena, Germany
| | - David W. Galbraith
- School of Plant Sciences and Bio5 Institute, University of Arizona, Tucson, USA
- Honorary Dean of Life Sciences, Henan University, Kaifeng, China
| | - Anastasia Gangaev
- Division of Molecular Oncology and Immunology, the Netherlands Cancer Institute, Amsterdam, The Netherlands
| | - Natalio Garbi
- Institute of Experimental Immunology, University of Bonn, Germany
| | - Brice Gaudilliere
- Stanford Department of Anesthesiology, Perioperative and Pain Medicine, Stanford University School of Medicine, CA, USA
| | - Ricardo T. Gazzinelli
- Fundação Oswaldo Cruz - Minas, Laboratory of Immunopatology, Belo Horizonte, MG, Brazil
- Department of Mecicine, University of Massachusetts Medical School, Worcester, MA, USA
| | - Jens Geginat
- INGM - Fondazione Istituto Nazionale di Genetica Molecolare “Ronmeo ed Enrica Invernizzi”, Milan, Italy
| | - Wilhelm Gerner
- Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Austria
- Christian Doppler Laboratory for Optimized Prediction of Vaccination Success in Pigs, Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Austria
| | - Nicholas A. Gherardin
- Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia
| | - Kamran Ghoreschi
- Department of Dermatology, Venereology and Allergology, Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Lara Gibellini
- Department of Surgery, Medicine, Dentistry and Morphological Sciences, Univ. of Modena and Reggio Emilia, Modena, Italy
| | - Florent Ginhoux
- Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
- Translational Immunology Institute, SingHealth Duke-NUS Academic Medical Centre, Singapore
- Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Keisuke Goda
- Department of Bioengineering, University of California, Los Angeles, California, USA
- Department of Chemistry, University of Tokyo, Tokyo, Japan
- Institute of Technological Sciences, Wuhan University, Wuhan, China
| | - Dale I. Godfrey
- Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia
| | | | - Jose M. González-Navajas
- Alicante Institute for Health and Biomedical Research (ISABIAL), Alicante, Spain
- Networked Biomedical Research Center for Hepatic and Digestive Diseases (CIBERehd), Madrid, Spain
| | - Carl S. Goodyear
- Institute of Infection Immunity and Inflammation, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow Biomedical Research Centre, Glasgow, UK
| | - Andrea Gori
- Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, University of Milan
| | - Jane L. Grogan
- Cancer Immunology Research, Genentech, South San Francisco, CA, USA
| | | | - Andreas Grützkau
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Claudia Haftmann
- Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland
| | - Jonas Hahn
- Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Department of Medicine 3, Rheumatology and Immunology, Universitätsklinikum Erlangen, Erlangen
| | - Hamida Hammad
- Department of Internal Medicine and Pediatrics, Faculty of Medicine and Health Sciences, Zwijnaarde, Belgium
| | | | - Leo Hansmann
- Berlin Institute of Health (BIH), Berlin, Germany
- German Cancer Consortium (DKTK), partner site Berlin, Berlin, Germany
- Department of Hematology, Oncology, and Tumor Immunology, Charité - Universitätsmedizin Berlin, Campus Virchow Klinikum, Berlin, Germany
| | - Goran Hansson
- Department of Medicine and Center for Molecular Medicine at Karolinska University Hospital, Solna, Sweden
| | | | - Susanne Hartmann
- Institute of Immunology, Centre for Infection Medicine, Department of Veterinary Medicine, Freie Universität Berlin, Germany
| | - Andrea Hauser
- Department of Internal Medicine III, University Hospital Regensburg, Germany
| | - Anja E. Hauser
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin
- Department of Rheumatology and Clinical Immunology, Berlin Institute of Health, Berlin, Germany
| | - David L. Haviland
- Flow Cytometry, Houston Methodist Hospital Research Institute, Houston, TX, USA
| | - David Hedley
- Divsion of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto, Ontario, Canada
| | - Daniela C. Hernández
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Charité - Universitätsmedizin Berlin, Medical Department I, Division of Gastroenterology, Infectiology and Rheumatology, Berlin, Germany
| | - Guadalupe Herrera
- Cytometry Service, Incliva Foundation. Clinic Hospital and Faculty of Medicine, University of Valencia, Valencia, Spain
| | - Martin Herrmann
- Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Department of Medicine 3, Rheumatology and Immunology, Universitätsklinikum Erlangen, Erlangen
| | - Christoph Hess
- Immunobiology Laboratory, Department of Biomedicine, University and University Hospital Basel, Basel, Switzerland
- Cambridge Institute of Therapeutic Immunology & Infectious Disease, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, UK
| | - Thomas Höfer
- German Cancer Research Center (DKFZ), Division of Theoretical Systems Biology, Heidelberg, Germany
| | - Petra Hoffmann
- Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany
- Department of Internal Medicine III, University Hospital Regensburg, Germany
| | - Kristin Hogquist
- Center for Immunology, University of Minnesota, Minneapolis, MN, USA
| | - Tristan Holland
- Institute of Experimental Immunology, University of Bonn, Germany
| | - Thomas Höllt
- Leiden Computational Biology Center, Leiden University Medical Center, Leiden, The Netherlands
- Computer Graphics and Visualization, Department of Intelligent Systems, TU Delft, Delft, The Netherlands
| | | | - Pleun Hombrink
- Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
- Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
| | - Jessica P. Houston
- Department of Chemical & Materials Engineering, New Mexico State University, Las Cruces, NM, USA
| | - Bimba F. Hoyer
- Rheumatologie/Klinische Immunologie, Klinik für Innere Medizin I und Exzellenzzentrum Entzündungsmedizin, Universitätsklinikum Schleswig-Holstein, Kiel, Germany
| | - Bo Huang
- Department of Immunology & National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medical College, Beijing, China
| | - Fang-Ping Huang
- Institute for Advanced Study (IAS), Shenzhen University, Shenzhen, China
| | - Johanna E. Huber
- Institute for Immunology, Faculty of Medicine, Biomedical Center, LMU Munich, Planegg-Martinsried, Germany
| | - Jochen Huehn
- Experimental Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany
| | - Michael Hundemer
- Department of Hematology, Oncology and Rheumatology, University Heidelberg, Heidelberg, Germany
| | - Christopher A. Hunter
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - William Y. K. Hwang
- Department of Hematology, Singapore General Hospital, Singapore
- Cancer & Stem Cell Biology, Duke-NUS Medical School, Singapore
- Executive Offices, National Cancer Centre Singapore, Singapore
| | - Anna Iannone
- Department of Diagnostic Medicine, Clinical and Public Health, Univ. of Modena and Reggio Emilia, Modena, Italy
| | - Florian Ingelfinger
- Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland
| | - Sabine M Ivison
- Department of Surgery, The University of British Columbia, Vancouver, Canada
- BC Children’s Hospital Research Institute, Vancouver, Canada
| | - Hans-Martin Jäck
- Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Peter K. Jani
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Max Planck Institute for Infection Biology, Berlin, Germany
| | - Beatriz Jávega
- Laboratory of Cytomics, Joint Research Unit CIPF-UVEG, Department of Biochemistry and Molecular Biology, University of Valencia, Valencia, Spain
| | - Stipan Jonjic
- Department of Histology and Embryology/Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
| | - Toralf Kaiser
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Tomas Kalina
- Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University, Prague, Czech Republic
| | - Thomas Kamradt
- Jena University Hospital, Institute of Immunology, Jena, Germany
| | | | - Baerbel Keller
- Department of Rheumatology and Clinical Immunology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Steven L. C. Ketelaars
- Division of Molecular Oncology and Immunology, the Netherlands Cancer Institute, Amsterdam, The Netherlands
| | - Ahad Khalilnezhad
- Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
- Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Srijit Khan
- Department of Immunology, University of Toronto, Toronto, ON, Canada
| | - Jan Kisielow
- Institute of Molecular Health Sciences, ETH Zurich, Zürich, Switzerland
| | - Paul Klenerman
- Experimental Medicine Division, Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Jasmin Knopf
- Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Department of Medicine 3, Rheumatology and Immunology, Universitätsklinikum Erlangen, Erlangen
| | - Hui-Fern Koay
- Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia
| | - Katja Kobow
- Department of Neuropathology, Universitätsklinikum Erlangen, Germany
| | - Jay K. Kolls
- John W Deming Endowed Chair in Internal Medicine, Center for Translational Research in Infection and Inflammation Tulane School of Medicine, New Orleans, LA, USA
| | - Wan Ting Kong
- Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
| | - Manfred Kopf
- Institute of Molecular Health Sciences, ETH Zurich, Zürich, Switzerland
| | - Thomas Korn
- Department of Neurology, Technical University of Munich, Munich, Germany
| | - Katharina Kriegsmann
- Department of Hematology, Oncology and Rheumatology, University Heidelberg, Heidelberg, Germany
| | - Hendy Kristyanto
- Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands
| | - Thomas Kroneis
- Division of Cell Biology, Histology & Embryology, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria
| | - Andreas Krueger
- Institute for Molecular Medicine, Goethe University Frankfurt, Frankfurt am Main, Germany
| | - Jenny Kühne
- Institute of Transplant Immunology, Hannover Medical School, MHH, Hannover, Germany
| | - Christian Kukat
- FACS & Imaging Core Facility, Max Planck Institute for Biology of Ageing, Cologne, Germany
| | - Désirée Kunkel
- Flow & Mass Cytometry Core Facility, Charité - Universitätsmedizin Berlin and Berlin Institute of Health, Berlin, Germany
- BCRT Flow Cytometry Lab, Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin
| | - Heike Kunze-Schumacher
- Institute for Molecular Medicine, Goethe University Frankfurt, Frankfurt am Main, Germany
| | - Tomohiro Kurosaki
- WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan
| | - Christian Kurts
- Institute of Experimental Immunology, University of Bonn, Germany
| | - Pia Kvistborg
- Division of Molecular Oncology and Immunology, the Netherlands Cancer Institute, Amsterdam, The Netherlands
| | - Immanuel Kwok
- Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
- School of Biological Sciences, Nanyang Technological University, Singapore
| | - Jonathan Landry
- Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
| | - Olivier Lantz
- INSERM U932, PSL University, Institut Curie, Paris, France
| | - Paola Lanuti
- Department of Medicine and Aging Sciences, Centre on Aging Sciences and Translational Medicine (Ce.S.I.-Me.T.), University “G. d’Annunzio” of Chieti-Pescara, Chieti, Italy
| | - Francesca LaRosa
- IRCCS Fondazione Don Carlo Gnocchi, Milan, Italy
- Milan Center for Neuroscience, University of Milano-Bicocca, Milan, Italy
| | - Agnès Lehuen
- Institut Cochin, CNRS8104, INSERM1016, Department of Endocrinology, Metabolism and Diabetes, Université de Paris, Paris, France
| | | | - Michael D. Leipold
- The Human Immune Monitoring Center (HIMC), Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, CA, USA
| | - Leslie Y.T. Leung
- Department of Immunology, University of Toronto, Toronto, ON, Canada
| | - Megan K. Levings
- Department of Surgery, The University of British Columbia, Vancouver, Canada
- BC Children’s Hospital Research Institute, Vancouver, Canada
- School of Biomedical Engineering, The University of British Columbia, Vancouver, Canada
| | - Andreia C. Lino
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Dept. Medicine/Rheumatology and Clinical Immunology, Charité Universitätsmedizin Berlin, Germany
| | - Francesco Liotta
- Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
| | | | - Yanling Liu
- Department of Immunology, University of Toronto, Toronto, ON, Canada
| | - Hans-Gustaf Ljunggren
- Center for Infectious Medicine, Department of Medicine Huddinge, ANA Futura, Karolinska Institutet, Stockholm, Sweden
| | - Michael Lohoff
- Inst. f. Med. Mikrobiology and Hospital Hygiene, University of Marburg, Germany
| | - Giovanna Lombardi
- King’s College London, “Peter Gorer” Department of Immunobiology, London, UK
| | | | - Miguel López-Botet
- IMIM(Hospital de Mar Medical Research Institute), University Pompeu Fabra, Barcelona, Spain
| | - Amy E. Lovett-Racke
- Department of Microbial Infection and Immunity, Ohio State University, Columbus, OH, USA
| | - Erik Lubberts
- Department of Rheumatology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
| | - Herve Luche
- Centre d’Immunophénomique - CIPHE (PHENOMIN), Aix Marseille Université (UMS3367), Inserm (US012), CNRS (UMS3367), Marseille, France
| | - Burkhard Ludewig
- Institute of Immunobiology, Kantonsspital St.Gallen, St. Gallen, Switzerland
| | - Enrico Lugli
- Laboratory of Translational Immunology, Humanitas Clinical and Research Center, Rozzano, Italy
- Flow Cytometry Core, Humanitas Clinical and Research Center, Milan, Italy
| | - Sebastian Lunemann
- Department of Virus Immunology, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
| | - Holden T. Maecker
- Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, CA, USA
| | - Laura Maggi
- Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
| | - Orla Maguire
- Flow and Image Cytometry Shared Resource, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Florian Mair
- Fred Hutchinson Cancer Research Center, Vaccine and Infectious Disease Division, Seattle, WA, USA
| | - Kerstin H. Mair
- Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Austria
- Christian Doppler Laboratory for Optimized Prediction of Vaccination Success in Pigs, Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Austria
| | - Alberto Mantovani
- Istituto Clinico Humanitas IRCCS and Humanitas University, Pieve Emanuele, Milan, Italy
- William Harvey Research Institute, Queen Mary University, London, United Kingdom
| | - Rudolf A. Manz
- Institute for Systemic Inflammation Research, University of Luebeck, Luebeck, Germany
| | - Aaron J. Marshall
- Department of Immunology, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada
| | | | - Glòria Martrus
- Department of Virus Immunology, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
| | - Ivana Marventano
- IRCCS Fondazione Don Carlo Gnocchi, Milan, Italy
- Milan Center for Neuroscience, University of Milano-Bicocca, Milan, Italy
| | - Wlodzimierz Maslinski
- National Institute of Geriatrics, Rheumatology and Rehabilitation, Department of Pathophysiology and Immunology, Warsaw, Poland
| | - Giuseppe Matarese
- Treg Cell Lab, Dipartimento di Medicina Molecolare e Biotecologie Mediche, Università di Napoli Federico II and Istituto per l’Endocrinologia e l’Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (IEOS-CNR), Napoli, Italy
| | - Anna Vittoria Mattioli
- Department of Surgery, Medicine, Dentistry and Morphological Sciences, Univ. of Modena and Reggio Emilia, Modena, Italy
- Lab of Clinical and Experimental Immunology, Humanitas Clinical and Research Center, Rozzano, Milan, Italy
| | - Christian Maueröder
- Cell Clearance in Health and Disease Lab, VIB Center for Inflammation Research, Ghent, Belgium
- Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
| | - Alessio Mazzoni
- Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
| | - James McCluskey
- Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia
| | - Mairi McGrath
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Helen M. McGuire
- Ramaciotti Facility for Human Systems Biology, and Discipline of Pathology, The University of Sydney, Camperdown, Australia
| | - Iain B. McInnes
- Institute of Infection Immunity and Inflammation, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow Biomedical Research Centre, Glasgow, UK
| | - Henrik E. Mei
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Fritz Melchers
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Max Planck Institute for Infection Biology, Berlin, Germany
| | - Susanne Melzer
- Clinical Trial Center Leipzig, University Leipzig, Leipzig, Germany
| | - Dirk Mielenz
- Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Stephen D. Miller
- Interdepartmental Immunobiology Center, Dept. of Microbiology-Immunology, Northwestern Univ. Medical School, Chicago, IL, USA
| | - Kingston H.G. Mills
- Trinity College Dublin, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Dublin, Ireland
| | - Hans Minderman
- Flow and Image Cytometry Shared Resource, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Jenny Mjösberg
- Center for Infectious Medicine, Department of Medicine Huddinge, ANA Futura, Karolinska Institutet, Stockholm, Sweden
- Department of Clinical and Experimental Medine, Linköping University, Linköping, Sweden
| | - Jonni Moore
- Abramson Cancer Center Flow Cytometry and Cell Sorting Shared Resource, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
| | - Barry Moran
- Trinity College Dublin, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Dublin, Ireland
| | - Lorenzo Moretta
- Department of Immunology, IRCCS Bambino Gesu Children’s Hospital, Rome, Italy
| | - Tim R. Mosmann
- David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY, USA
| | - Susann Müller
- Centre for Environmental Research - UFZ, Department Environmental Microbiology, Leipzig, Germany
| | - Gabriele Multhoff
- Institute for Innovative Radiotherapy (iRT), Experimental Immune Biology, Helmholtz Zentrum München, Neuherberg, Germany
- Radiation Immuno-Oncology Group, Center for Translational Cancer Research Technische Universität München (TranslaTUM), Klinikum rechts der Isar, Munich, Germany
| | - Luis Enrique Muñoz
- Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Department of Medicine 3, Rheumatology and Immunology, Universitätsklinikum Erlangen, Erlangen
| | - Christian Münz
- Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland
- Comprehensive Cancer Center Zurich, Switzerland
| | - Toshinori Nakayama
- Department of Immunology, Graduate School of Medicine, Chiba University, Chiba city, Chiba, Japan
| | - Milena Nasi
- Department of Surgery, Medicine, Dentistry and Morphological Sciences, Univ. of Modena and Reggio Emilia, Modena, Italy
| | - Katrin Neumann
- Institute of Experimental Immunology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Lai Guan Ng
- Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
- Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
- School of Biological Sciences, Nanyang Technological University, Singapore
- Discipline of Dermatology, University of Sydney, Sydney, New South Wales, Australia
- State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China
| | - Antonia Niedobitek
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Sussan Nourshargh
- Barts and The London School of Medicine and Dentistry, Queen Mary University of London, UK
| | - Gabriel Núñez
- Department of Pathology and Rogel Cancer Center, the University of Michigan, Ann Arbor, Michigan, USA
| | - José-Enrique O’Connor
- Laboratory of Cytomics, Joint Research Unit CIPF-UVEG, Department of Biochemistry and Molecular Biology, University of Valencia, Valencia, Spain
| | - Aaron Ochel
- Institute of Experimental Immunology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Anna Oja
- Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
| | - Diana Ordonez
- Flow Cytometry Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
| | - Alberto Orfao
- Department of Medicine, Cancer Research Centre (IBMCC-CSIC/USAL), Cytometry Service, University of Salamanca, CIBERONC and Institute for Biomedical Research of Salamanca (IBSAL), Salamanca, Spain
| | - Eva Orlowski-Oliver
- Burnet Institute, AMREP Flow Cytometry Core Facility, Melbourne, Victoria, Australia
| | - Wenjun Ouyang
- Inflammation and Oncology, Research, Amgen Inc, South San Francisco, USA
| | | | - Raghavendra Palankar
- Department of Transfusion Medicine, Institute of Immunology and Transfusion Medicine, University Medicine Greifswald, Greifswald, Germany
| | - Isabel Panse
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Kovit Pattanapanyasat
- Center of Excellence for Flow Cytometry, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Malte Paulsen
- Flow Cytometry Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
| | - Dinko Pavlinic
- Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
| | - Livius Penter
- Department of Hematology, Oncology, and Tumor Immunology, Charité - Universitätsmedizin Berlin, Campus Virchow Klinikum, Berlin, Germany
| | - Pärt Peterson
- Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia
| | - Christian Peth
- Biophysics, R&D Engineering, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
| | - Jordi Petriz
- Functional Cytomics Group, Josep Carreras Leukaemia Research Institute, Campus ICO-Germans Trias i Pujol, Universitat Autònoma de Barcelona, UAB, Badalona, Spain
| | - Federica Piancone
- IRCCS Fondazione Don Carlo Gnocchi, Milan, Italy
- Milan Center for Neuroscience, University of Milano-Bicocca, Milan, Italy
| | - Winfried F. Pickl
- Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria
| | - Silvia Piconese
- Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Rome, Italy
- Istituto Pasteur - Fondazione Cenci Bolognetti, Rome, Italy
| | - Marcello Pinti
- Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - A. Graham Pockley
- John van Geest Cancer Research Centre, Nottingham Trent University, Nottingham, UK
- Chromocyte Limited, Electric Works, Sheffield, UK
| | - Malgorzata Justyna Podolska
- Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Department of Medicine 3, Rheumatology and Immunology, Universitätsklinikum Erlangen, Erlangen
- Department for Internal Medicine 3, Institute for Rheumatology and Immunology, AG Munoz, Universitätsklinikum Erlangen, Erlangen, Germany
| | - Zhiyong Poon
- Department of Hematology, Singapore General Hospital, Singapore
| | - Katharina Pracht
- Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Immo Prinz
- Institute of Immunology, Hannover Medical School, Hannover, Germany
| | | | - Sally A. Quataert
- David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY, USA
| | - Linda Quatrini
- Department of Immunology, IRCCS Bambino Gesu Children’s Hospital, Rome, Italy
| | - Kylie M. Quinn
- School of Biomedical and Health Sciences, RMIT University, Bundoora, Victoria, Australia
- Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
| | - Helena Radbruch
- Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Neuropathology, Germany
| | - Tim R. D. J. Radstake
- Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
| | - Susann Rahmig
- Regeneration in Hematopoiesis, Leibniz-Institute on Aging, Fritz-Lipmann-Institute (FLI), Jena, Germany
| | - Hans-Peter Rahn
- Preparative Flow Cytometry, Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany
| | - Bartek Rajwa
- Bindley Biosciences Center, Purdue University, West Lafayette, IN, USA
| | - Gevitha Ravichandran
- Institute of Experimental Immunology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Yotam Raz
- Department of Internal Medicine, Groene Hart Hospital, Gouda, The Netherlands
| | - Jonathan A. Rebhahn
- David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY, USA
| | | | - Dorothea Reimer
- Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany
| | | | - Ester B.M. Remmerswaal
- Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
- Renal Transplant Unit, Division of Internal Medicine, Academic Medical Centre, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
| | - Lisa Richter
- Core Facility Flow Cytometry, Biomedical Center, Ludwig-Maximilians-University Munich, Germany
| | - Laura G. Rico
- Functional Cytomics Group, Josep Carreras Leukaemia Research Institute, Campus ICO-Germans Trias i Pujol, Universitat Autònoma de Barcelona, UAB, Badalona, Spain
| | - Andy Riddell
- Flow Cytometry Scientific Technology Platform, The Francis Crick Institute, London, UK
| | - Aja M. Rieger
- Department of Medical Microbiology and Immunology, University of Alberta, Alberta, Canada
| | - J. Paul Robinson
- Purdue University Cytometry Laboratories, Purdue University, West Lafayette, IN, USA
| | - Chiara Romagnani
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Charité - Universitätsmedizin Berlin, Medical Department I, Division of Gastroenterology, Infectiology and Rheumatology, Berlin, Germany
| | - Anna Rubartelli
- Cell Biology Unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy
| | - Jürgen Ruland
- Institut für Klinische Chemie und Pathobiochemie, Fakultät für Medizin, Technische Universität München, München, Germany
| | - Armin Saalmüller
- Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine Vienna, Austria
| | - Yvan Saeys
- Data Mining and Modeling for Biomedicine, VIB-UGent Center for Inflammation Research, Ghent, Belgium
- Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Ghent, Belgium
| | - Takashi Saito
- RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
| | - Shimon Sakaguchi
- WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan
| | - Francisco Sala de-Oyanguren
- Flow Cytometry Facility, Ludwig Cancer Institute, Faculty of Medicine and Biology, University of Lausanne, Epalinges, Switzerland
| | - Yvonne Samstag
- Heidelberg University, Institute of Immunology, Section of Molecular Immunology, Heidelberg, Germany
| | - Sharon Sanderson
- Translational Immunology Laboratory, NIHR BRC, University of Oxford, Kennedy Institute of Rheumatology, Oxford, UK
| | - Inga Sandrock
- Institute of Immunology, Hannover Medical School, Hannover, Germany
| | - Angela Santoni
- Department of Molecular Medicine, Sapienza University of Rome, IRCCS, Neuromed, Pozzilli, Italy
| | - Ramon Bellmàs Sanz
- Institute of Transplant Immunology, Hannover Medical School, MHH, Hannover, Germany
| | - Marina Saresella
- IRCCS Fondazione Don Carlo Gnocchi, Milan, Italy
- Milan Center for Neuroscience, University of Milano-Bicocca, Milan, Italy
| | | | - Birgit Sawitzki
- Charité – Universitätsmedizin Berlin, and Berlin Institute of Health, Institute of Medical Immunology, Berlin, Germany
| | - Linda Schadt
- Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland
- Comprehensive Cancer Center Zurich, Switzerland
| | - Alexander Scheffold
- Institut für Immunologie, Christian-Albrechts-Universität zu Kiel, Kiel, Germany
| | - Hans U. Scherer
- Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands
| | - Matthias Schiemann
- Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany
| | - Frank A. Schildberg
- Clinic for Orthopedics and Trauma Surgery, University Hospital Bonn, Bonn, Germany
| | | | - Andreas Schlitzer
- Quantitative Systems Biology, Life & Medical Sciences Institute, University of Bonn, Bonn, Germany
| | - Josephine Schlosser
- Institute of Immunology, Centre for Infection Medicine, Department of Veterinary Medicine, Freie Universität Berlin, Germany
| | - Stephan Schmid
- Internal Medicine I, University Hospital Regensburg, Germany
| | - Steffen Schmitt
- Flow Cytometry Core Facility, German Cancer Research Centre (DKFZ), Heidelberg, Germany
| | - Kilian Schober
- Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany
| | - Daniel Schraivogel
- Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
| | - Wolfgang Schuh
- Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Thomas Schüler
- Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Magdeburg, Germany
| | - Reiner Schulte
- University of Cambridge, Cambridge Institute for Medical Research, Cambridge, UK
| | - Axel Ronald Schulz
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Sebastian R. Schulz
- Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Cristiano Scottá
- King’s College London, “Peter Gorer” Department of Immunobiology, London, UK
| | - Daniel Scott-Algara
- Institut Pasteur, Cellular Lymphocytes Biology, Immunology Departement, Paris, France
| | - David P. Sester
- TRI Flow Cytometry Suite (TRI.fcs), Translational Research Institute, Wooloongabba, QLD, Australia
| | | | - Bruno Silva-Santos
- Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Portugal
| | | | - Katarzyna M. Sitnik
- Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany
| | - Silvano Sozzani
- Dept. Molecular Translational Medicine, University of Brescia, Brescia, Italy
| | - Daniel E. Speiser
- Department of Oncology, University of Lausanne and CHUV, Epalinges, Switzerland
| | | | - Anders Stahlberg
- Lundberg Laboratory for Cancer, Department of Pathology, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
| | | | - Natalie Stanley
- Departments of Anesthesiology, Pain and Perioperative Medicine; Biomedical Data Sciences; and Pediatrics, Stanford University, Stanford, CA, USA
| | - Regina Stark
- Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
- Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
| | - Christina Stehle
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Charité - Universitätsmedizin Berlin, Medical Department I, Division of Gastroenterology, Infectiology and Rheumatology, Berlin, Germany
| | - Tobit Steinmetz
- Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Hannes Stockinger
- Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria
| | | | - Kiyoshi Takeda
- WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan
| | - Leonard Tan
- Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
- Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Attila Tárnok
- Departement for Therapy Validation, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany
- Institute for Medical Informatics, Statistics and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany
- Department of Precision Instruments, Tsinghua University, Beijing, China
| | - Gisa Tiegs
- Institute of Experimental Immunology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | | | - Julia Tornack
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- BioGenes GmbH, Berlin, Germany
| | - Elisabetta Traggiai
- Novartis Biologics Center, Mechanistic Immunology Unit, Novartis Institute for Biomedical Research, NIBR, Basel, Switzerland
| | - Mohamed Trebak
- Department of Cellular and Molecular Physiology, Penn State University College of Medicine, PA, United States
| | - Timothy I.M. Tree
- Department of Immunobiology, School of Immunology and Microbial Sciences, King’s College London, UK
- National Institutes of Health Research Biomedical Research Centre at Guy’s and St. Thomas’ National Health Service, Foundation Trust and King’s College London, UK
| | | | - John Trowsdale
- Department of Pathology, University of Cambridge, Cambridge, UK
| | | | - Henning Ulrich
- Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP, Brazil
| | - Sophia Urbanczyk
- Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Willem van de Veen
- Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland
- Christine Kühne Center for Allergy Research and Education (CK-CARE), Davos, Switzerland
| | - Maries van den Broek
- Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland
- Comprehensive Cancer Center Zurich, Switzerland
| | - Edwin van der Pol
- Vesicle Observation Center; Biomedical Engineering & Physics; Laboratory Experimental Clinical Chemistry; Amsterdam University Medical Centers, Location AMC, The Netherlands
| | - Sofie Van Gassen
- Data Mining and Modeling for Biomedicine, VIB-UGent Center for Inflammation Research, Ghent, Belgium
- Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Ghent, Belgium
| | | | - René A.W. van Lier
- Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
| | - Marc Veldhoen
- Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Portugal
| | | | - Paulo Vieira
- Unit Lymphopoiesis, Department of Immunology, Institut Pasteur, Paris, France
| | - David Voehringer
- Department of Infection Biology, University Hospital Erlangen, Friedrich-Alexander University Erlangen-Nuremberg (FAU), Erlangen, Germany
| | - Hans-Dieter Volk
- BIH Center for Regenerative Therapies (BCRT) Charité Universitätsmedizin Berlin and Berlin Institute of Health, Core Unit ImmunoCheck
| | - Anouk von Borstel
- Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
- Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia
| | | | - Ari Waisman
- Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg University of Mainz, Mainz, Germany
| | | | - Paul K. Wallace
- Roswell Park Comprehensive Cancer Center, Elm and Carlton Streets, Buffalo, NY, USA
| | - Sa A. Wang
- Dept of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Xin M. Wang
- The Scientific Platforms, the Westmead Institute for Medical Research, the Westmead Research Hub, Westmead, New South Wales, Australia
| | | | | | - Klaus Warnatz
- Department of Rheumatology and Clinical Immunology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Gary Warnes
- Flow Cytometry Core Facility, Blizard Institute, Queen Mary London University, London, UK
| | - Sarah Warth
- BCRT Flow Cytometry Lab, Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin
| | - Claudia Waskow
- Regeneration in Hematopoiesis, Leibniz-Institute on Aging, Fritz-Lipmann-Institute (FLI), Jena, Germany
- Faculty of Biological Sciences, Friedrich Schiller University Jena, Jena, Germany
| | | | - Carsten Watzl
- Department for Immunology, Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund (IfADo), Dortmund, Germany
| | - Leonie Wegener
- Biophysics, R&D Engineering, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
| | - Thomas Weisenburger
- Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany
| | - Annika Wiedemann
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Dept. Medicine/Rheumatology and Clinical Immunology, Charité Universitätsmedizin Berlin, Germany
| | - Jürgen Wienands
- Institute for Cellular & Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Anneke Wilharm
- Institute of Immunology, Hannover Medical School, Hannover, Germany
| | - Robert John Wilkinson
- Department of Infectious Disease, Imperial College London, UK
- Wellcome Centre for Infectious Diseases Research in Africa and Department of Medicine, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Republic of South Africa
- Tuberculosis Laboratory, The Francis Crick Institute, London, UK
| | - Gerald Willimsky
- Cooperation Unit for Experimental and Translational Cancer Immunology, Institute of Immunology (Charité - Universitätsmedizin Berlin) and German Cancer Research Center (DKFZ), Berlin, Germany
| | - James B. Wing
- WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan
| | - Rieke Winkelmann
- Institut für Immunologie, Christian-Albrechts-Universität zu Kiel, Kiel, Germany
| | - Thomas H. Winkler
- Department of Biology, Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany
| | - Oliver F. Wirz
- Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland
| | - Alicia Wong
- Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
| | - Peter Wurst
- University Bonn, Medical Faculty, Bonn, Germany
| | - Jennie H. M. Yang
- Department of Immunobiology, School of Immunology and Microbial Sciences, King’s College London, UK
- National Institutes of Health Research Biomedical Research Centre at Guy’s and St. Thomas’ National Health Service, Foundation Trust and King’s College London, UK
| | - Juhao Yang
- Experimental Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany
| | - Maria Yazdanbakhsh
- Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands
| | | | - Alice Yue
- School of Computing Science, Simon Fraser University, Burnaby, Canada
| | - Hanlin Zhang
- Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK
| | - Yi Zhao
- Department of Rheumatology and Immunology, West China Hospital, Sichuan University, Chengdu, Sichuan, China
| | - Susanne Maria Ziegler
- Department of Virus Immunology, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
| | - Christina Zielinski
- German Center for Infection Research (DZIF), Munich, Germany
- Institute of Virology, Technical University of Munich, Munich, Germany
- TranslaTUM, Technical University of Munich, Munich, Germany
| | - Jakob Zimmermann
- Maurice Müller Laboratories (Department of Biomedical Research), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Bern, Switzerland
| | | |
Collapse
|
|
21
|
Bechsgaard J, Schou MF, Vanthournout B, Hendrickx F, Knudsen B, Settepani V, Schierup MH, Bilde T. Evidence for Faster X Chromosome Evolution in Spiders. Mol Biol Evol 2019; 36:1281-1293. [PMID: 30912801 PMCID: PMC6526907 DOI: 10.1093/molbev/msz074] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
In species with chromosomal sex determination, X chromosomes are predicted to evolve faster than autosomes because of positive selection on recessive alleles or weak purifying selection. We investigated X chromosome evolution in Stegodyphus spiders that differ in mating system, sex ratio, and population dynamics. We assigned scaffolds to X chromosomes and autosomes using a novel method based on flow cytometry of sperm cells and reduced representation sequencing. We estimated coding substitution patterns (dN/dS) in a subsocial outcrossing species (S. africanus) and its social inbreeding and female-biased sister species (S. mimosarum), and found evidence for faster-X evolution in both species. X chromosome-to-autosome diversity (piX/piA) ratios were estimated in multiple populations. The average piX/piA estimates of S. africanus (0.57 [95% CI: 0.55-0.60]) was lower than the neutral expectation of 0.75, consistent with more hitchhiking events on X-linked loci and/or a lower X chromosome mutation rate, and we provide evidence in support of both. The social species S. mimosarum has a significantly higher piX/piA ratio (0.72 [95% CI: 0.65-0.79]) in agreement with its female-biased sex ratio. Stegodyphus mimosarum also have different piX/piA estimates among populations, which we interpret as evidence for recurrent founder events. Simulations show that recurrent founder events are expected to decrease the piX/piA estimates in S. mimosarum, thus underestimating the true effect of female-biased sex ratios. Finally, we found lower synonymous divergence on X chromosomes in both species, and the male-to-female substitution ratio to be higher than 1, indicating a higher mutation rate in males.
Collapse
Affiliation(s)
| | - Mads Fristrup Schou
- Department of Bioscience, Aarhus University, Aarhus C, Denmark.,Department of Biology, Lund University, SE-223 62 Lund, Sweden
| | - Bram Vanthournout
- Department of Bioscience, Aarhus University, Aarhus C, Denmark.,Evolution and Optics of Nanostructure Group (EON), Biology Department, Ghent University, Ghent, Belgium
| | - Frederik Hendrickx
- Royal Belgian Institute of Natural Sciences, Brussels, Belgium.,Terrestrial Ecology Unit (TEREC), Biology Department, Ghent University, Ghent, Belgium
| | | | | | - Mikkel Heide Schierup
- Department of Bioscience, Aarhus University, Aarhus C, Denmark.,Bioinformatics Research Centre (BiRC), Aarhus University, Aarhus C, Denmark
| | - Trine Bilde
- Department of Bioscience, Aarhus University, Aarhus C, Denmark
| |
Collapse
|
|
22
|
Vanthournout B, Busck MM, Bechsgaard J, Hendrickx F, Schramm A, Bilde T. Male spiders control offspring sex ratio through greater production of female-determining sperm. Proc Biol Sci 2019; 285:rspb.2017.2887. [PMID: 29563266 DOI: 10.1098/rspb.2017.2887] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2017] [Accepted: 02/26/2018] [Indexed: 01/07/2023] Open
Abstract
Sex allocation theory predicts that when sons and daughters have different reproductive values, parents should adjust offspring sex ratio towards the sex with the higher fitness return. Haplo-diploid species directly control offspring sex ratio, but species with chromosomal sex determination (CSD) were presumed to be constrained by Mendelian segregation. There is now increasing evidence that CSD species can adjust sex ratio strategically, but the underlying mechanism is not well understood. One hypothesis states that adaptive control is more likely to evolve in the heterogametic sex through a bias in gamete production. We investigated this hypothesis in males as the heterogametic sex in two social spider species that consistently show adaptive female-biased sex ratio and in one subsocial species that is characterized by equal sex ratio. We quantified the production of male (0) and female (X) determining sperm cells using flow cytometry, and show that males of social species produce significantly more X-carrying sperm than 0-sperm, on average 70%. This is consistent with the production of more daughters. Males of the subsocial species produced a significantly lower bias of 54% X-carrying sperm. We also investigated whether inter-genomic conflict between hosts and their endosymbionts may explain female bias. Next generation sequencing showed that five common genera of bacterial endosymbionts known to affect sex ratio are largely absent, ruling out that endosymbiont bacteria bias sex ratio in social spiders. Our study provides evidence for paternal control over sex allocation through biased gamete production as a mechanism by which the heterogametic sex in CSD species adaptively adjust offspring sex ratio.
Collapse
Affiliation(s)
- Bram Vanthournout
- Department of Bioscience, Section for Genetics, Ecology and Evolution, Aarhus University, Ny Munkegade 114, Building 1540, 8000 Aarhus C, Denmark.,Biology Department, Evolution and Optics of Nanostructures Group, Ghent University, K.L. Ledeganckstraat 35, 9000 Gent, Belgium
| | - Mette Marie Busck
- Department of Bioscience, Section for Microbiology, Aarhus University, Ny Munkegade 114, Building 1540, 8000 Aarhus C, Denmark
| | - Jesper Bechsgaard
- Department of Bioscience, Section for Genetics, Ecology and Evolution, Aarhus University, Ny Munkegade 114, Building 1540, 8000 Aarhus C, Denmark
| | - Frederik Hendrickx
- Biology Department, Terrestrial Ecology Unit, Ghent University, K.L. Ledeganckstraat 35, 9000 Gent, Belgium.,Entomology Department, Royal Belgian Institute of Natural Sciences, Vautierstraat 29, 1000 Brussels, Belgium
| | - Andreas Schramm
- Department of Bioscience, Section for Microbiology, Aarhus University, Ny Munkegade 114, Building 1540, 8000 Aarhus C, Denmark
| | - Trine Bilde
- Department of Bioscience, Section for Genetics, Ecology and Evolution, Aarhus University, Ny Munkegade 114, Building 1540, 8000 Aarhus C, Denmark
| |
Collapse
|
|
23
|
Growth-inhibition of cell lines derived from B cell lymphomas through antagonism of serotonin receptor signaling. Sci Rep 2019; 9:4276. [PMID: 30862884 PMCID: PMC6414675 DOI: 10.1038/s41598-019-40825-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2018] [Accepted: 02/25/2019] [Indexed: 11/21/2022] Open
Abstract
A majority of lymphomas are derived from B cells and novel treatments are required to treat refractory disease. Neurotransmitters such as serotonin and dopamine influence activation of B cells and the effects of a selective serotonin 1A receptor (5HT1A) antagonist on growth of a number of B cell-derived lymphoma cell lines were investigated. We confirmed the expression of 5HT1A in human lymphoma tissue and in several well-defined experimental cell lines. We discovered that the pharmacological inhibition of 5HT1A led to the reduced proliferation of B cell-derived lymphoma cell lines together with DNA damage, ROS-independent caspase activation and apoptosis in a large fraction of cells. Residual live cells were found ‘locked’ in a non-proliferative state in which a selective transcriptional and translational shutdown of genes important for cell proliferation and metabolism occurred (e.g., AKT, GSK-3β, cMYC and p53). Strikingly, inhibition of 5HT1A regulated mitochondrial activity through a rapid reduction of mitochondrial membrane potential and reducing dehydrogenase activity. Collectively, our data suggest 5HT1A antagonism as a novel adjuvant to established cancer treatment regimens to further inhibit lymphoma growth.
Collapse
|
|
24
|
Vezain M, Lecuyer M, Rubio M, Dupé V, Ratié L, David V, Pasquier L, Odent S, Coutant S, Tournier I, Trestard L, Adle-Biassette H, Vivien D, Frébourg T, Gonzalez BJ, Laquerrière A, Saugier-Veber P. A de novo variant in ADGRL2 suggests a novel mechanism underlying the previously undescribed association of extreme microcephaly with severely reduced sulcation and rhombencephalosynapsis. Acta Neuropathol Commun 2018; 6:109. [PMID: 30340542 PMCID: PMC6195752 DOI: 10.1186/s40478-018-0610-5] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2018] [Accepted: 09/29/2018] [Indexed: 12/13/2022] Open
Abstract
Extreme microcephaly and rhombencephalosynapsis represent unusual pathological conditions, each of which occurs in isolation or in association with various other cerebral and or extracerebral anomalies. Unlike microcephaly for which several disease-causing genes have been identified with different modes of inheritance, the molecular bases of rhombencephalosynapsis remain unknown and rhombencephalosynapsis presents mainly as a sporadic condition consistent with de novo dominant variations. We report for the first time the association of extreme microcephaly with almost no sulcation and rhombencephalosynapsis in a fœtus for which comparative patient-parent exome sequencing strategy revealed a heterozygous de novo missense variant in the ADGRL2 gene. ADGRL2 encodes latrophilin 2, an adhesion G-protein-coupled receptor whose exogenous ligand is α-latrotoxin. Adgrl2 immunohistochemistry and in situ hybridization revealed expression in the telencephalon, mesencephalon and rhombencephalon of mouse and chicken embryos. In human brain embryos and fœtuses, Adgrl2 immunoreactivity was observed in the hemispheric and cerebellar germinal zones, the cortical plate, basal ganglia, pons and cerebellar cortex. Microfluorimetry experiments evaluating intracellular calcium release in response to α-latrotoxin binding showed significantly reduced cytosolic calcium release in the fœtus amniocytes vs amniocytes from age-matched control fœtuses and in HeLa cells transfected with mutant ADGRL2 cDNA vs wild-type construct. Embryonic lethality was also observed in constitutive Adgrl2−/− mice. In Adgrl2+/− mice, MRI studies revealed microcephaly and vermis hypoplasia. Cell adhesion and wound healing assays demonstrated that the variation increased cell adhesion properties and reduced cell motility. Furthermore, HeLa cells overexpressing mutant ADGRL2 displayed a highly developed cytoplasmic F-actin network related to cytoskeletal dynamic modulation. ADGRL2 is the first gene identified as being responsible for extreme microcephaly with rhombencephalosynapsis. Increased cell adhesion, reduced cell motility and cytoskeletal dynamic alterations induced by the variant therefore represent a new mechanism responsible for microcephaly.
Collapse
|
|
25
|
Mikalsen SG, Mikalsen LTG, Sandvik JA, Aarnes EK, Fenne S, Flatmark K, Lyng H, Edin NFJ, Pettersen EO. Low dose-rate irradiation with [ 3H]-labelled valine to selectively target hypoxic cells in a human colorectal cancer xenograft model. Acta Oncol 2018; 57:1216-1224. [PMID: 29630428 DOI: 10.1080/0284186x.2018.1457223] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Abstract
BACKGROUND Earlier in vitro studies show that irradiation with an ultra-low dose-rate of 15 mGy/h delivered with [3H]-valine leads to loss of clonogenicity in hypoxic T-47D cells. Here, the aim was to determine if [3H]-valine could be used to deliver low dose-rate irradiation in a colorectal cancer model. METHODS Clonogenicity was measured in cultured cancer cell line HT29 irradiated with 15 mGy/h combined with intermittent hypoxia. Mice with HT29 xenografts were irradiated by repeated injections of [3H]-valine intravenously. The activity in the tumor tissue was measured by scintillation counting and tumor growth, hypoxic fraction and tritium distribution within tumors were assessed by pimonidazole staining and autoradiography. RESULTS Ultra-low dose-rate irradiation decreased clonogenicity in hypoxic colorectal cancer cells. In vivo, the tumor growth, hypoxic fraction and weight of the mice were similar between the treated and untreated group. Autoradiography showed no [3H]-valine uptake in hypoxic tumor regions in contrast to aerobic tissue. CONCLUSION Continuous low-dose-rate irradiation was well tolerated by aerobic tissue. This indicates a potential use of low dose-rate irradiation to target hypoxic tumor cells in combination with high dose-rate irradiation to eradicate the well oxygenated tumor regions. However, [3H]-valine is not the appropriate method to deliver ultra-low dose-rate irradiation in vivo.
Collapse
Affiliation(s)
- Stine Gyland Mikalsen
- Department of Physics, University of Oslo, Oslo, Norway
- Department of Medical Physics, Oslo University Hospital, Oslo, Norway
| | | | | | | | - Siri Fenne
- Department of Physics, University of Oslo, Oslo, Norway
| | - Kjersti Flatmark
- Department of Tumour Biology, Oslo University Hospital, Oslo, Norway
| | - Heidi Lyng
- Department of Radiation Biology, Oslo University Hospital, Oslo, Norway
| | | | | |
Collapse
|
|
26
|
Najid A, Ratinaud MH. Comparative Studies of Steroidogenesis Inhibitors (Econazole, Ketoconazole) on Human Breast Cancer MCF-7 Cell Proliferation by Growth Experiments, Thymidine Incorporation and Flow Cytometric DNA Analysis. TUMORI JOURNAL 2018; 77:385-90. [PMID: 1781035 DOI: 10.1177/030089169107700504] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The effects of aminoglutethimide, econazole and ketoconazole on human breast cancer cells in culture were compared with those of tamoxifen using four methods (growth experiments, thymidine incorporation, monoparameter and bivariate DNA content flow cytometry analysis). Aminoglutethimide (1 nM-10 microM) had no effect on cell proliferation after 8 days of treatment and did not decrease thymidine tritiated incorporation during logarithmic phase. Even at 20 microM, similar results were obtained with flow cytometry. Econazole and ketoconazole (1 nM-1 microM) decreased MCF-7 cell proliferation in a time- and dose-dependent fashion. They also decreased tritiated thymidine incorporation. By using flow cytometry and a monoclonal antibody against bromodeoxyuridine, we showed an accumulation of MCF-7 cells treated by imidazoles derivatives in G0-G1 phase of the cell cycle.
Collapse
Affiliation(s)
- A Najid
- Laboratoire de Biochimie, Faculté de Pharmacie, Université de Limoges, France
| | | |
Collapse
|
|
27
|
Darzynkiewicz Z, Huang X, Zhao H. Analysis of Cellular DNA Content by Flow Cytometry. ACTA ACUST UNITED AC 2018; 82:7.5.1-7.5.20. [DOI: 10.1002/cpcy.28] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Affiliation(s)
- Zbigniew Darzynkiewicz
- Department of Pathology and Brander Cancer Research Institute, New York Medical College Valhalla New York
| | - Xuan Huang
- Hematology/Oncology, Case Western Reserve University Cleveland Ohio
| | - Hong Zhao
- Department of Pathology and Brander Cancer Research Institute, New York Medical College Valhalla New York
| |
Collapse
|
|
28
|
Darzynkiewicz Z, Huang X, Zhao H. Analysis of Cellular DNA Content by Flow Cytometry. ACTA ACUST UNITED AC 2018; 119:5.7.1-5.7.20. [DOI: 10.1002/cpim.36] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Affiliation(s)
- Zbigniew Darzynkiewicz
- Department of Pathology and Brander Cancer Research Institute, New York Medical College Valhalla New York
| | - Xuan Huang
- Hematology/Oncology, Case Western Reserve University Cleveland Ohio
| | - Hong Zhao
- Department of Pathology and Brander Cancer Research Institute, New York Medical College Valhalla New York
| |
Collapse
|
|
29
|
Favero GM, Paz JL, Otake AH, Maria DA, Caldini EG, de Medeiros RSS, Deus DF, Chammas R, Maranhão RC, Bydlowski SP. Cell internalization of 7-ketocholesterol-containing nanoemulsion through LDL receptor reduces melanoma growth in vitro and in vivo: a preliminary report. Oncotarget 2018; 9:14160-14174. [PMID: 29581835 PMCID: PMC5865661 DOI: 10.18632/oncotarget.24389] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2017] [Accepted: 01/25/2018] [Indexed: 01/01/2023] Open
Abstract
Oxysterols are cholesterol oxygenated derivatives which possess several biological actions. Among oxysterols, 7-ketocholesterol (7KC) is known to induce cell death. Here, we hypothesized that 7KC cytotoxicity could be applied in cancer therapeutics. 7KC was incorporated into a lipid core nanoemulsion. As a cellular model the murine melanoma cell line B16F10 was used. The nanoparticle (7KCLDE) uptake into tumor cells was displaced by increasing amounts of low-density-lipoproteins (LDL) suggesting a LDL-receptor-mediated cell internalization. 7KCLDE was mainly cytostatic, which led to an accumulation of polyploid cells. Nevertheless, a single dose of 7KCLDE killed roughly 10% of melanoma cells. In addition, it was observed dissipation of the transmembrane potential, evidenced with flow cytometry; presence of autophagic vacuoles, visualized and quantified with flow cytometry and acridine orange; and presence of myelin figures, observed with ultrastructural microscopy. 7KCLDE impaired cytokenesis was accompanied by changes in cellular morphology into a fibroblastoid shape which is supported by cytoskeletal rearrangements, as shown by the increased actin polymerization. 7KCLDE was injected into B16 melanoma tumor-bearing mice. 7KCLDE accumulated in the liver and tumor. In melanoma tumor 7KCLDE promoted a >50% size reduction, enlarged the necrotic area, and reduced intratumoral vasculature. 7KCLDE increased the survival rates of animals, without hematologic or liver toxicity. Although more pre-clinical studies should be performed, our preliminary results suggested that 7KCLDE is a promising novel preparation for cancer chemotherapy.
Collapse
Affiliation(s)
- Giovani M Favero
- Laboratory of Genetics and Molecular Hematology (LIM31), Department of Hematology, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.,Department of General Biology, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil
| | - Jessica L Paz
- Laboratory of Genetics and Molecular Hematology (LIM31), Department of Hematology, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil
| | - Andréia H Otake
- Centro de Investigação Translacional em Oncologia (LIM24), Departamento de Radiologia e Oncologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.,Instituto do Cancer do Estado de Sao Paulo (ICESP), SP, Brazil
| | - Durvanei A Maria
- Biochemistry and Biophysics Laboratories, Instituto Butantan, Sao Paulo, SP, Brazil
| | - Elia G Caldini
- Laboratory for Cell Biology, Department of Pathology, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, Brazil
| | - Raphael S S de Medeiros
- Centro de Investigação Translacional em Oncologia (LIM24), Departamento de Radiologia e Oncologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.,Instituto do Cancer do Estado de Sao Paulo (ICESP), SP, Brazil
| | - Debora F Deus
- Laboratory of Metabolism and Lipids, Heart Institute (InCor), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil
| | - Roger Chammas
- Centro de Investigação Translacional em Oncologia (LIM24), Departamento de Radiologia e Oncologia, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.,Instituto do Cancer do Estado de Sao Paulo (ICESP), SP, Brazil
| | - Raul C Maranhão
- Laboratory of Metabolism and Lipids, Heart Institute (InCor), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.,Faculdade de Ciencias Farmaceuticas, Universidade de Sao Paulo, Sao Paulo, SP, Brazil
| | - Sergio P Bydlowski
- Laboratory of Genetics and Molecular Hematology (LIM31), Department of Hematology, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, Brazil
| |
Collapse
|
|
30
|
Yang Y, Zhou W, Wu J, Yao L, Xue L, Zhang Q, Wang Z, Wang X, Dong S, Zhao J, Yin D. Antitumor activity of nimotuzumab in combination with cisplatin in lung cancer cell line A549 in vitro. Oncol Lett 2018; 15:5280-5284. [PMID: 29552167 DOI: 10.3892/ol.2018.7923] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2016] [Accepted: 01/03/2018] [Indexed: 12/17/2022] Open
Abstract
Nimotuzumab, a humanized IgG1 monoclonal antibody against epidermal growth factor receptor (EGFR), increases radiosensitivity in lung cancer. Cisplatin is an effective antitumor agent in lung cancer. In the present study, the antitumor activity of nimotuzumab combined with cisplatin was investigated in A549 lung cancer cells. Viability, cell cycle distribution and cyclin D1 expression were assessed following treatment with nimotuzumab alone, cisplatin alone, nimotuzumab in combination with cisplatin, and nimotuzumab followed sequentially by cisplatin. The inhibitory effect on cell viability of nimotuzumab sequentially followed by cisplatin was higher compared with cisplatin alone (82.17±1.62 vs. 56.97±1.42%). Compared with treatment by cisplatin alone, cell cycle analysis by flow cytometry demonstrated that the percentage of cells in the G0/G1 phase was increased when A549 cells were treated with nimotuzumab followed sequentially by cisplatin (P<0.01). However, the proportion of cells in G0/G1 phase was decreased when A549 cells were treated with nimotuzumab and cisplatin simultaneously compared with cisplatin alone (P<0.05). Cyclin D1 expression was decreased in all chemotherapy treatment groups; the most significant decrease was in A549 cells treated with nimotuzumab followed sequentially by cisplatin. Nimotuzumab may enhance the antitumor activity of cisplatin on A549 cells. The cell cycle arrest at G0/G1 observed may have been due to decreased cyclin D1 levels. Potential antagonism was identified when A549 cells were treated with nimotuzumab and cisplatin simultaneously, indicating that targeted therapy may be more effective when administered prior to conventional chemotherapy.
Collapse
Affiliation(s)
- Yanhong Yang
- Department of Oncology, Qinhuangdao No. 1 People's Hospital, Qinhuangdao, Hebei 066000, P.R. China
| | - Wenwen Zhou
- Department of Oncology, Qinhuangdao No. 1 People's Hospital, Qinhuangdao, Hebei 066000, P.R. China
| | - Jiandong Wu
- Department of Oncology, Qinhuangdao No. 1 People's Hospital, Qinhuangdao, Hebei 066000, P.R. China
| | - Lixin Yao
- Department of Oncology, Qinhuangdao No. 1 People's Hospital, Qinhuangdao, Hebei 066000, P.R. China
| | - Lei Xue
- Department of Oncology, Qinhuangdao No. 1 People's Hospital, Qinhuangdao, Hebei 066000, P.R. China
| | - Qianyi Zhang
- College of Pharmacy, University of Tasmania, Hobart, TAS 7001, Australia
| | - Zhenzhen Wang
- Department of Oncology, Qinhuangdao No. 1 People's Hospital, Qinhuangdao, Hebei 066000, P.R. China.,Postgraduate College of Chengde Medical University, Chengde, Hebei 067000, P.R. China
| | - Xiaoyu Wang
- Postgraduate College of Chengde Medical University, Chengde, Hebei 067000, P.R. China
| | - Shu Dong
- Biotecan Medical Diagnostics Co., Ltd., Shanghai 201203, P.R. China.,Department of Medicine, Zhangjiang Center for Translational Medicine, Shanghai 201203, P.R. China
| | - Jiangman Zhao
- Biotecan Medical Diagnostics Co., Ltd., Shanghai 201203, P.R. China.,Department of Medicine, Zhangjiang Center for Translational Medicine, Shanghai 201203, P.R. China
| | - Duanduan Yin
- Department of Oncology, Qinhuangdao No. 1 People's Hospital, Qinhuangdao, Hebei 066000, P.R. China
| |
Collapse
|
|
31
|
Abstract
The aim of this study was to reexamine the prognostic role of tumor cell kinetics measured by S-phase fraction (SPF) and to establish its clinically relevant threshold values. SPF was determined by flow cytometry in a group of 920 consecutive breast cancer patients, all followed at our institute for 10 years (1988 to 1998). Mean age was 60.5 years (27–89 years). Median follow-up was 63 months (3–150 months). All patients had initial surgical treatment. SPF quartiles were: Q1=3.08%, median value = 5.98%, Q3=10.22%. A significant difference in overall specific survival was obtained between two populations divided by a cutoff at Q1 (p<0.0001). A multifactorial analysis including SPF and known prognostic factors such as tumor size, node status, histological grade, ER and PR status was performed using the Cox model in a population of 719 patients: univariate analysis showed that each of these factors had significant influence on overall survival. Multivariate analysis selected three of them, ranked by decreasing order of hazard ratio (HR) value: SPF (HR: 3.88, p<0.001), tumor size (HR: 2.49, p<0.001) and nodal status (HR: 2.28, p<0.001). In addition, when tumors were stratified according to SPF quartile values, there were statistically different overall survival curves in patients with small tumors (<2 cm) and in axillary node-negative patients.
Collapse
|
|
32
|
Corver WE, Demmers J, Oosting J, Sahraeian S, Boot A, Ruano D, Wezel TV, Morreau H. ROS-induced near-homozygous genomes in thyroid cancer. Endocr Relat Cancer 2018; 25:83-97. [PMID: 29066502 DOI: 10.1530/erc-17-0288] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/29/2017] [Accepted: 10/24/2017] [Indexed: 12/17/2022]
Abstract
A near-homozygous genome (NHG) is especially seen in a subset of follicular thyroid cancer of the oncocytic type (FTC-OV). An NHG was also observed in the metabolically relatively quiescent cell lines XTC.UC1, a model for FTC-OV, and in FTC-133, -236 and -238, the latter three derived from one single patient with follicular thyroid cancer. FTC-236 subclones showed subtle whole-chromosome differences indicative of sustained reciprocal mitotic missegregations. Reactive oxygen species (ROS) scavenger experiments reduced the number of chromosomal missegregations in XTC.UC1 and FTC-236, while pCHK2 was downregulated in these cells. Treatment with antimycin A increased ROS indicated by enhanced MitoSOX Red and pCHK2 fluorescence in metaphase cells. In a selected set of oncocytic follicular thyroid tumors, increasing numbers of whole-chromosome losses were observed toward an aggressive phenotype, but with retention of chromosome 7. Together, ROS activates CHK2 and links to the stepwise loss of whole chromosomes during tumor progression in these lesions. We postulate that sequential loss of whole chromosomes is a dominant driver of the oncogenesis of a subset of follicular thyroid tumors.
Collapse
Affiliation(s)
- Willem E Corver
- Department of Pathology Leiden University Medical CenterLeiden, Netherlands
| | - Joris Demmers
- Department of Pathology Leiden University Medical CenterLeiden, Netherlands
| | - Jan Oosting
- Department of Pathology Leiden University Medical CenterLeiden, Netherlands
| | - Shima Sahraeian
- Department of Pathology Leiden University Medical CenterLeiden, Netherlands
| | - Arnoud Boot
- Department of Pathology Leiden University Medical CenterLeiden, Netherlands
| | - Dina Ruano
- Department of Pathology Leiden University Medical CenterLeiden, Netherlands
| | - Tom van Wezel
- Department of Pathology Leiden University Medical CenterLeiden, Netherlands
| | - Hans Morreau
- Department of Pathology Leiden University Medical CenterLeiden, Netherlands
| |
Collapse
|
|
33
|
Mikalsen SG, Jeppesen Edin N, Sandvik JA, Pettersen EO. Separation of two sub-groups with different DNA content after treatment of T-47D breast cancer cells with low dose-rate irradiation and intermittent hypoxia. Acta Radiol 2018; 59:26-33. [PMID: 28350256 DOI: 10.1177/0284185117699999] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Background Previous studies have shown that combined treatment with internal ultra-low dose-rate irradiation selectively inactivated hypoxic T-47D breast cancer cells after three to five weeks of treatment. However, 2-3% of the hypoxic cells were found to survive and restart proliferation upon re-oxygenation. Purpose To investigate the metastatic potential and characteristics of radiosensitivity of these surviving cells, named T - 47 DS. Material and Methods The T - 47 DS cells were grown in ambient air without irradiation. A cloning experiment identified two sub-groups with different DNA content ([Formula: see text] and [Formula: see text]). Furthermore, radiosensitivity and presence of hyper-radiosensitivity (HRS) was measured by Co-60 challenge irradiation and relative migration was determined by scratch assays. Results The two subpopulations of T - 47 DS had different DNA content; one had abnormally high DNA content ([Formula: see text]) and one had DNA content similar to wild-type T-47D cells ([Formula: see text]). HRS was surprisingly present in cells of the cloned population [Formula: see text], but was absent in cells of both [Formula: see text] and T - 47 DS. The radio response of T - 47 DS, [Formula: see text] at higher radiation doses were similar to that of T-47D cells, and neither subpopulation showed increased migration compared with wild-type T-47D. Conclusion No increase in the risk of metastasis was found and only slight changes in radiosensitivity in response to conventional clinical doses was observed. Thus, the data suggest that if ultra-low dose-rate irradiation is used for targeting the hypoxic tumor fraction, conventional high dose-rate irradiation can be used to eradicate eventual surviving cells as well as cells in the well oxygenated areas of the tumor.
Collapse
Affiliation(s)
- Stine Gyland Mikalsen
- Department of Physics, University of Oslo, Oslo, Norway
- Department of Medical Physics, Oslo University Hospital, Oslo, Norway
| | | | | | | |
Collapse
|
|
34
|
Noronha S, Alt LAC, Scimeca TE, Zarou O, Obrzut J, Zanotti B, Hayward EA, Pillai A, Mathur S, Rojas J, Salamah R, Chandar N, Fay MJ. Preclinical evaluation of the Aurora kinase inhibitors AMG 900, AZD1152-HQPA, and MK-5108 on SW-872 and 93T449 human liposarcoma cells. In Vitro Cell Dev Biol Anim 2017; 54:71-84. [PMID: 29197031 DOI: 10.1007/s11626-017-0208-4] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2017] [Accepted: 10/10/2017] [Indexed: 11/26/2022]
Abstract
Liposarcoma is a malignant soft tissue tumor that originates from adipose tissue and is one of the most frequently diagnosed soft tissue sarcomas in humans. There is great interest in identifying novel chemotherapeutic options for treating liposarcoma based upon molecular alterations in the cancer cells. The Aurora kinases have been identified as promising chemotherapeutic targets based on their altered expression in many human cancers and cellular roles in mitosis and cytokinesis. In this study, we investigated the effects of an Aurora kinase A inhibitor (MK-5108), an Aurora kinase B inhibitor (AZD1152-HQPA), and a pan-Aurora kinase inhibitor (AMG 900) on undifferentiated SW-872 and well-differentiated 93T449 human liposarcoma cells. Treatment of the SW-872 and 93T449 cells with MK-5108 (0-1000 nM), AZD1152-HQPA (0-1000 nM), and AMG 900 (0-1000 nM) for 72 h resulted in a dose-dependent decrease in the total viable cell number. Based upon the EC50 values, the potency of the three Aurora kinase inhibitors in the SW-872 cells was as follows: AMG 900 (EC50 = 3.7 nM) > AZD1152-HQPA (EC50 = 43.4 nM) > MK-5108 (EC50 = 309.0 nM), while the potency in the 93T449 cells was as follows: AMG 900 (EC50 = 6.5 nM) > AZD1152-HQPA (EC50 = 74.5 nM) > MK-5108 (EC50 = 283.6 nM). The percentage of polyploidy after 72 h of drug treatment (0-1000 nM) was determined by propidium iodide staining and flow cytometric analysis. AMG 900 caused a significant increase in polyploidy starting at 25 nM in the SW-872 and 93T449 cells, and AZD1152-HQPA caused a significant increase starting at 100 nM in the SW-872 cells and 250 nM in the 93T449 cells. The Aurora kinase A inhibitor MK-5108 did not significantly increase the percentage of polyploid cells at any of the doses tested in either cell line. The expression of Aurora kinase A and B was evaluated in the SW-872 cells versus differentiated adipocytes and human mesenchymal stem cells by real-time RT-PCR and Western blot analysis. Aurora kinase A and B mRNA expression was significantly increased in the SW-872 cells versus the differentiated adipocytes and human mesenchymal stem cells. Western blot analysis revealed a ~ 48 kDa immunoreactive band for Aurora kinase A that was not present in the differentiated adipocytes or the human mesenchymal stem cells. A ~ 39 kDa immunoreactive band for Aurora kinase B was detected in the SW-872 cells, differentiated adipocytes, and human mesenchymal stem cells. A smaller immunoreactive band for Aurora kinase B was detected in the SW-872 cells but not in the differentiated adipocytes and human mesenchymal stem cells, and this may reflect the expression of a truncated splice variant of Aurora kinase B that has been associated with poor patient prognosis. The 93T449 cells demonstrated decreased expression of Aurora kinase A and B mRNA and protein compared to the SW-872 cells, and also expressed the truncated form of Aurora kinase B. The results of these in vitro studies indicate that Aurora kinase inhibitors should be further investigated as possible chemotherapeutic agents for human liposarcoma.
Collapse
Affiliation(s)
- Sandhya Noronha
- Physician Assistant Program, College of Health Sciences, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA
| | - Lauren A C Alt
- Department of Biomedical Sciences, College of Health Sciences, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA
| | - Taylor E Scimeca
- Department of Biomedical Sciences, College of Health Sciences, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA
| | - Omran Zarou
- Department of Biomedical Sciences, College of Health Sciences, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA
| | - Justyna Obrzut
- Department of Biomedical Sciences, College of Health Sciences, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA
| | - Brian Zanotti
- Department of Microbiology and Immunology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA
| | - Elizabeth A Hayward
- Department of Biomedical Sciences, College of Health Sciences, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA
| | - Akhil Pillai
- Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA
| | - Shubha Mathur
- Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA
| | - Joseph Rojas
- Department of Biomedical Sciences, College of Health Sciences, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA
| | - Ribhi Salamah
- Department of Biomedical Sciences, College of Health Sciences, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA
| | - Nalini Chandar
- Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA
| | - Michael J Fay
- Department of Biomedical Sciences, College of Health Sciences, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA.
- Department of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st Street, Downers Grove, IL, 60515, USA.
| |
Collapse
|
|
35
|
Cytotoxic Effect on Human Myeloma Cells and Leukemic Cells by the Agaricus blazei Murill Based Mushroom Extract, Andosan™. BIOMED RESEARCH INTERNATIONAL 2017; 2017:2059825. [PMID: 29238712 PMCID: PMC5697368 DOI: 10.1155/2017/2059825] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/13/2017] [Revised: 10/03/2017] [Accepted: 10/10/2017] [Indexed: 11/29/2022]
Abstract
Agaricus blazei Murill is an edible mushroom of the Basidiomycetes family, which has been found to contain a number of compounds with antitumor properties, such as proteoglycans and ergosterol. In the present investigation, we show that the commercial mushroom product Andosan, which contains 82.4% Agaricus blazei Murill, together with medicinal mushrooms Hericium erinaceus (14.7%) and Grifola frondosa (2.9%), has a cytotoxic effect on primary myeloma cells, other myeloma cell lines, and leukemia cell lines in vitro. Although the exact content and hence the mechanisms of action of the Andosan extract are unknown, we have found in this investigation indications of cell cycle arrest when myeloma cell lines are cultivated with Andosan. This may be one of the possible explanations for the cytotoxic effects of Andosan.
Collapse
|
|
36
|
Cossarizza A, Chang HD, Radbruch A, Akdis M, Andrä I, Annunziato F, Bacher P, Barnaba V, Battistini L, Bauer WM, Baumgart S, Becher B, Beisker W, Berek C, Blanco A, Borsellino G, Boulais PE, Brinkman RR, Büscher M, Busch DH, Bushnell TP, Cao X, Cavani A, Chattopadhyay PK, Cheng Q, Chow S, Clerici M, Cooke A, Cosma A, Cosmi L, Cumano A, Dang VD, Davies D, De Biasi S, Del Zotto G, Della Bella S, Dellabona P, Deniz G, Dessing M, Diefenbach A, Di Santo J, Dieli F, Dolf A, Donnenberg VS, Dörner T, Ehrhardt GRA, Endl E, Engel P, Engelhardt B, Esser C, Everts B, Dreher A, Falk CS, Fehniger TA, Filby A, Fillatreau S, Follo M, Förster I, Foster J, Foulds GA, Frenette PS, Galbraith D, Garbi N, García-Godoy MD, Geginat J, Ghoreschi K, Gibellini L, Goettlinger C, Goodyear CS, Gori A, Grogan J, Gross M, Grützkau A, Grummitt D, Hahn J, Hammer Q, Hauser AE, Haviland DL, Hedley D, Herrera G, Herrmann M, Hiepe F, Holland T, Hombrink P, Houston JP, Hoyer BF, Huang B, Hunter CA, Iannone A, Jäck HM, Jávega B, Jonjic S, Juelke K, Jung S, Kaiser T, Kalina T, Keller B, Khan S, Kienhöfer D, Kroneis T, Kunkel D, Kurts C, Kvistborg P, Lannigan J, Lantz O, Larbi A, LeibundGut-Landmann S, Leipold MD, Levings MK, Litwin V, Liu Y, Lohoff M, Lombardi G, Lopez L, Lovett-Racke A, Lubberts E, Ludewig B, Lugli E, Maecker HT, Martrus G, Matarese G, Maueröder C, McGrath M, McInnes I, Mei HE, Melchers F, Melzer S, Mielenz D, Mills K, Mirrer D, Mjösberg J, Moore J, Moran B, Moretta A, Moretta L, Mosmann TR, Müller S, Müller W, Münz C, Multhoff G, Munoz LE, Murphy KM, Nakayama T, Nasi M, Neudörfl C, Nolan J, Nourshargh S, O'Connor JE, Ouyang W, Oxenius A, Palankar R, Panse I, Peterson P, Peth C, Petriz J, Philips D, Pickl W, Piconese S, Pinti M, Pockley AG, Podolska MJ, Pucillo C, Quataert SA, Radstake TRDJ, Rajwa B, Rebhahn JA, Recktenwald D, Remmerswaal EBM, Rezvani K, Rico LG, Robinson JP, Romagnani C, Rubartelli A, Ruckert B, Ruland J, Sakaguchi S, Sala-de-Oyanguren F, Samstag Y, Sanderson S, Sawitzki B, Scheffold A, Schiemann M, Schildberg F, Schimisky E, Schmid SA, Schmitt S, Schober K, Schüler T, Schulz AR, Schumacher T, Scotta C, Shankey TV, Shemer A, Simon AK, Spidlen J, Stall AM, Stark R, Stehle C, Stein M, Steinmetz T, Stockinger H, Takahama Y, Tarnok A, Tian Z, Toldi G, Tornack J, Traggiai E, Trotter J, Ulrich H, van der Braber M, van Lier RAW, Veldhoen M, Vento-Asturias S, Vieira P, Voehringer D, Volk HD, von Volkmann K, Waisman A, Walker R, Ward MD, Warnatz K, Warth S, Watson JV, Watzl C, Wegener L, Wiedemann A, Wienands J, Willimsky G, Wing J, Wurst P, Yu L, Yue A, Zhang Q, Zhao Y, Ziegler S, Zimmermann J. Guidelines for the use of flow cytometry and cell sorting in immunological studies. Eur J Immunol 2017; 47:1584-1797. [PMID: 29023707 PMCID: PMC9165548 DOI: 10.1002/eji.201646632] [Citation(s) in RCA: 399] [Impact Index Per Article: 49.9] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Affiliation(s)
- Andrea Cossarizza
- Department of Medical and Surgical Sciences for Children and Adults, Univ. of Modena and Reggio Emilia School of Medicine, Modena, Italy
| | - Hyun-Dong Chang
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Andreas Radbruch
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Mübeccel Akdis
- Swiss Institute of Allergy and Asthma Research (SIAF), University Zurich, Davos, Switzerland
| | - Immanuel Andrä
- Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany
| | | | | | - Vincenzo Barnaba
- Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Via Regina Elena 324, 00161 Rome, Italy
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Rome, Italy
| | - Luca Battistini
- Neuroimmunology and Flow Cytometry Units, Santa Lucia Foundation, Rome, Italy
| | - Wolfgang M Bauer
- Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria
| | - Sabine Baumgart
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Burkhard Becher
- University of Zurich, Institute of Experimental Immunology, Zürich, Switzerland
| | - Wolfgang Beisker
- Flow Cytometry Laboratory, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, German Research Center for Environmental Health
| | - Claudia Berek
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Alfonso Blanco
- Flow Cytometry Core Technologies, UCD Conway Institute, University College Dublin, Dublin, Ireland
| | - Giovanna Borsellino
- Neuroimmunology and Flow Cytometry Units, Santa Lucia Foundation, Rome, Italy
| | - Philip E Boulais
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, USA
- The Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Bronx, New York, USA
| | - Ryan R Brinkman
- Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada
- Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada
| | - Martin Büscher
- Biopyhsics, R&D Engineering, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
| | - Dirk H Busch
- Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany
- DZIF - National Centre for Infection Research, Munich, Germany
- Focus Group ''Clinical Cell Processing and Purification", Institute for Advanced Study, Technische Universität München, Munich, Germany
| | - Timothy P Bushnell
- Department of Pediatrics and Shared Resource Laboratories, University of Rochester Medical Center, Rochester NY, United States of America
| | - Xuetao Cao
- Institute of Immunology, Zhejiang University School of Medicine, Hangzhou 310058, China
- National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai 200433, China
- Department of Immunology & Center for Immunotherapy, Institute of Basic Medical Sciences, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100005, China
| | | | | | - Qingyu Cheng
- Medizinische Klinik mit Schwerpunkt Rheumatologie und Medizinische Immunolologie Charité Universitätsmedizin Berlin, Berlin, Germany
| | - Sue Chow
- Divsion of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto, Ontario, Canada
| | - Mario Clerici
- University of Milano and Don C Gnocchi Foundation IRCCS, Milano, Italy
| | - Anne Cooke
- Department of Pathology, University of Cambridge, Cambridge, United Kingdom
| | - Antonio Cosma
- CEA - Université Paris Sud - INSERM U, Immunology of viral infections and autoimmune diseases, France
| | - Lorenzo Cosmi
- Department of Experimental and Clinical Medicine, University of Firenze, Firenze, Italia
| | - Ana Cumano
- Lymphopoiesis Unit, Immunology Department Pasteur Institute, Paris, France
| | - Van Duc Dang
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Derek Davies
- Flow Cytometry Facility, The Francis Crick Institute, London, United Kingdom
| | - Sara De Biasi
- Department of Surgery, Medicine, Dentistry and Morphological Sciences, Univ. of Modena and Reggio Emilia, Modena, Italy
| | | | - Silvia Della Bella
- University of Milan, Department of Medical Biotechnologies and Translational Medicine
- Humanitas Clinical and Research Center, Lab of Clinical and Experimental Immunology, Rozzano, Milan, Italy
| | - Paolo Dellabona
- Experimental Immunology Unit, Head, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milano, Italy
| | - Günnur Deniz
- Istanbul University, Aziz Sancar Institute of Experimental Medicine, Department of Immunology, Istanbul, Turkey
| | | | | | | | - Francesco Dieli
- University of Palermo, Department of Biopathology, Palermo, Italy
| | - Andreas Dolf
- Institute of Experimental Immunology, University Bonn, Bonn, Germany
| | - Vera S Donnenberg
- Department of Cardiothoracic Surgery, School of Medicine, University of Pittsburgh, PA
| | - Thomas Dörner
- Department of Medicine/Rheumatology and Clinical Immunology, Charite Universitätsmedizin Berlin, Germany
| | | | - Elmar Endl
- Department of Molecular Medicine and Experimental Immunology, (Core Facility Flow Cytometry) University of Bonn, Germany
| | - Pablo Engel
- Department of Biomedical Sciences, University of Barcelona, Barcelona, Spain
| | - Britta Engelhardt
- Professor for Immunobiology, Director, Theodor Kocher Institute, University of Bern, Bern, Switzerland
| | - Charlotte Esser
- IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany
| | - Bart Everts
- Leiden University Medical Center, Department of Parasitology, Leiden, The Netherlands
| | - Anita Dreher
- Swiss Institute of Allergy and Asthma Research (SIAF), University Zurich, Davos, Switzerland
| | - Christine S Falk
- Institute of Transplant Immunology, IFB-Tx, MHH Hannover Medical School, Hannover, Germany
- German Center for Infectious diseases (DZIF), TTU-IICH, Hannover, Germany
| | - Todd A Fehniger
- Divisions of Hematology & Oncology, Department of Medicine, Washington University School of Medicine, St Louis, MO
| | - Andrew Filby
- The Flow Cytometry Core Facility, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
| | - Simon Fillatreau
- Institut Necker-Enfants Malades (INEM), INSERM U-CNRS UMR, Paris, France
- Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France
- Assistance Publique - Hôpitaux de Paris (AP-HP), Hôpital Necker Enfants Malades, Paris, France
| | - Marie Follo
- Department of Medicine I, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Irmgard Förster
- Immunology and Environment, Life & Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany
| | | | - Gemma A Foulds
- John van Geest Cancer Research Centre, Nottingham Trent University, Nottingham, UK
| | - Paul S Frenette
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, USA
- Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA
| | - David Galbraith
- University of Arizona, Bio Institute, School of Plant Sciences and Arizona Cancer Center, Tucson, Arizona, USA
| | - Natalio Garbi
- Institute of Experimental Immunology, University Bonn, Bonn, Germany
- Department of Molecular Immunology, Institute of Experimental Immunology, Bonn, Germany
| | | | - Jens Geginat
- INGM, Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy
| | - Kamran Ghoreschi
- Flow Cytometry Core Facility, Department of Dermatology, University Medical Center, Eberhard Karls University Tübingen, Germany
| | - Lara Gibellini
- Department of Surgery, Medicine, Dentistry and Morphological Sciences, Univ. of Modena and Reggio Emilia, Modena, Italy
| | | | - Carl S Goodyear
- Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow
| | - Andrea Gori
- Clinic of Infectious Diseases, "San Gerardo" Hospital - ASST Monza, University Milano-Bicocca, Monza, Italy
| | - Jane Grogan
- Genentech, Department of Cancer Immunology, South San Francisco, California, USA
| | - Mor Gross
- Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
| | - Andreas Grützkau
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | | | - Jonas Hahn
- Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Department of Internal Medicine, Rheumatology and Immunology, Universitätsklinikum Erlangen, Erlangen
| | - Quirin Hammer
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Anja E Hauser
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Immundynamics, Charité - Universitätsmedizin Berlin, Berlin, Germany
| | | | - David Hedley
- Divsion of Medical Oncology and Hematology, Princess Margaret Hospital, Toronto, Ontario, Canada
| | - Guadalupe Herrera
- Cytometry Service, Incliva Foundation. Clinic Hospital and Faculty of Medicine, The University of Valencia. Av. Blasco Ibáñez, Valencia, Spain
| | - Martin Herrmann
- Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Department of Internal Medicine, Rheumatology and Immunology, Universitätsklinikum Erlangen, Erlangen
| | - Falk Hiepe
- Medizinische Klinik mit Schwerpunkt Rheumatologie und Medizinische Immunolologie Charité Universitätsmedizin Berlin, Berlin, Germany
| | - Tristan Holland
- Department of Molecular Immunology, Institute of Experimental Immunology, Bonn, Germany
| | - Pleun Hombrink
- Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands
| | - Jessica P Houston
- Chemical and Materials Engineering, New Mexico State University, Las Cruces, NM, 88003, USA
| | - Bimba F Hoyer
- Medizinische Klinik mit Schwerpunkt Rheumatologie und Medizinische Immunolologie Charité Universitätsmedizin Berlin, Berlin, Germany
| | - Bo Huang
- Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Department of Immunology, Institute of Basic Medical Sciences & State Key Laboratory of Medical Molecular Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
- Clinical Immunology Center, Chinese Academy of Medical Sciences, Beijing, China
| | - Christopher A Hunter
- Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Anna Iannone
- Department of Diagnostic Medicine, Clinical and Public Health, Univ. of Modena and Reggio Emilia, Modena, Italy
| | - Hans-Martin Jäck
- Division of Molecular Immunology, Internal Medicine III, Nikolaus-Fiebiger-Center of MolecularMedicine, University Hospital Erlangen, Erlangen, Germany
| | - Beatriz Jávega
- Laboratory of Cytomics, Joint Research Unit CIPF-UVEG, Department of Biochemistry and Molecular Biology, The University of Valencia. Av. Blasco Ibáñez, Valencia, Spain
| | - Stipan Jonjic
- Faculty of Medicine, Center for Proteomics, University of Rijeka, Rijeka, Croatia
- Department for Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
| | - Kerstin Juelke
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Steffen Jung
- Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
| | - Toralf Kaiser
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Tomas Kalina
- Department of Paediatric Haematology and Oncology, Second Faculty of Medicine, Charles University and University Hospital Motol, Prague, Czech Republic
| | - Baerbel Keller
- Center for Chronic Immunodeficiency (CCI), Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Srijit Khan
- Department of Immunology, University of Toronto, Toronto, Canada
| | - Deborah Kienhöfer
- Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Department of Internal Medicine, Rheumatology and Immunology, Universitätsklinikum Erlangen, Erlangen
| | - Thomas Kroneis
- Medical University of Graz, Institute of Cell Biology, Histology & Embryology, Graz, Austria
| | - Désirée Kunkel
- BCRT Flow Cytometry Lab, Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin
| | - Christian Kurts
- Institute of Experimental Immunology, University Bonn, Bonn, Germany
| | - Pia Kvistborg
- Division of immunology, the Netherlands Cancer Institute, Amsterdam
| | - Joanne Lannigan
- University of Virginia School of Medicine, Flow Cytometry Shared Resource, Charlottesville, VA, USA
| | - Olivier Lantz
- INSERM U932, Institut Curie, Paris 75005, France
- Laboratoire d'immunologie clinique, Institut Curie, Paris 75005, France
- Centre d'investigation Clinique en Biothérapie Gustave-Roussy Institut Curie (CIC-BT1428), Institut Curie, Paris 75005, France
| | - Anis Larbi
- Singapore Immunology Network (SIgN), Principal Investigator, Biology of Aging Program
- Director Flow Cytomerty Platform, Immunomonitoring Platform, Agency for Science Technology and Research (A*STAR), Singapore
- Department of Medicine, University of Sherbrooke, Qc, Canada
- Faculty of Sciences, ElManar University, Tunis, Tunisia
| | | | - Michael D Leipold
- The Human Immune Monitoring Center (HIMC), Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, CA, USA
| | - Megan K Levings
- Department of Surgery, University of British Columbia & British Columbia Children's Hospital Research Institute, Vancouver, BC, Canada
| | | | - Yanling Liu
- Department of Immunology, University of Toronto, Toronto, Canada
| | - Michael Lohoff
- Institute for Medical Microbiology and Hospital Hygiene, University of Marburg, Marburg 35043, Germany
| | - Giovanna Lombardi
- MRC Centre for Transplantation, King's College London, Guy's Hospital, SE1 9RT London, UK
| | | | - Amy Lovett-Racke
- Department of Microbial Infection and Immunity, Ohio State University, Columbus, OH, USA
| | - Erik Lubberts
- Erasmus MC, University Medical Center, Department of Rheumatology, Rotterdam, The Netherlands
| | - Burkhard Ludewig
- Institute of Immunobiology, Kantonsspital St. Gallen, St. Gallen, Switzerland
| | - Enrico Lugli
- Laboratory of Translational Immunology, Humanitas Clinical and Research Center, Rozzano, Milan, Italy
- Humanitas Flow Cytometry Core, Humanitas Clinical and Research Center, Rozzano, Milan, Italy
| | - Holden T Maecker
- The Human Immune Monitoring Center (HIMC), Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, CA, USA
| | - Glòria Martrus
- Department of Virus Immunology, Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
| | - Giuseppe Matarese
- Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, Napoli, Italy and Istituto per l'Endocrinologia e l'Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (IEOS-CNR), Napoli, Italy
| | - Christian Maueröder
- Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Department of Internal Medicine, Rheumatology and Immunology, Universitätsklinikum Erlangen, Erlangen
| | - Mairi McGrath
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Iain McInnes
- Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow
| | - Henrik E Mei
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Fritz Melchers
- Senior Group on Lymphocyte Development, Max Planck Institute for Infection Biology, Berlin, Germany
| | - Susanne Melzer
- Clinical Trial Center Leipzig, University Leipzig, Leipzig, Germany
| | - Dirk Mielenz
- Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Kingston Mills
- Trinity Biomedical Sciences Institute, Trinity College Dublin, the University of Dublin, Dublin, Ireland
| | - David Mirrer
- Swiss Institute of Allergy and Asthma Research (SIAF), University Zurich, Davos, Switzerland
| | - Jenny Mjösberg
- Center for Infectious Medicine, Department of Medicine, Karolinska Institute Stockholm, Sweden
- Department of Clinical and Experimental Medicine, Linköping University, Sweden
| | - Jonni Moore
- Department of Pathology and Laboratory Medicine, Perelman School of Medicine of the University of Pennsylvania, Philadelphia, Pennsylvania
| | - Barry Moran
- Trinity Biomedical Sciences Institute, Trinity College Dublin, the University of Dublin, Dublin, Ireland
| | - Alessandro Moretta
- Department of Experimental Medicine, University of Genova, Genova, Italy
- Centro di Eccellenza per la Ricerca Biomedica-CEBR, Genova, Italy
| | - Lorenzo Moretta
- Department of Immunology, IRCCS Bambino Gesu Children's Hospital, Rome, Italy
| | - Tim R Mosmann
- David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY, USA
| | - Susann Müller
- Centre for Environmental Research - UFZ, Department Environemntal Microbiology, Leipzig, Germany
| | - Werner Müller
- Bill Ford Chair in Cellular Immunology, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom
| | - Christian Münz
- University of Zurich, Institute of Experimental Immunology, Zürich, Switzerland
| | - Gabriele Multhoff
- Department of Radiation Oncology, Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany
- Institute for Innovative Radiotherapy (iRT), Experimental Immune Biology, Helmholtz Zentrum München, Neuherberg, Germany
| | - Luis Enrique Munoz
- Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Department of Internal Medicine, Rheumatology and Immunology, Universitätsklinikum Erlangen, Erlangen
| | - Kenneth M Murphy
- Department of Pathology and Immunology, School of Medicine, Washington University in St. Louis, St. Louis, MO, USA
- Howard Hughes Medical Institute, School of Medicine, Washington University in St. Louis, St. Louis, MO, USA
| | - Toshinori Nakayama
- Department of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan
| | - Milena Nasi
- Department of Surgery, Medicine, Dentistry and Morphological Sciences, Univ. of Modena and Reggio Emilia, Modena, Italy
| | - Christine Neudörfl
- Institute of Transplant Immunology, IFB-Tx, MHH Hannover Medical School, Hannover, Germany
| | - John Nolan
- The Scintillon Institute, Nancy Ridge Drive, San Diego, CA, USA
| | - Sussan Nourshargh
- Centre for Microvascular Research, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom
| | - José-Enrique O'Connor
- Laboratory of Cytomics, Joint Research Unit CIPF-UVEG, Department of Biochemistry and Molecular Biology, The University of Valencia. Av. Blasco Ibáñez, Valencia, Spain
| | - Wenjun Ouyang
- Department of Inflammation and Oncology, Amgen Inc., South San Francisco, CA, USA
| | | | - Raghav Palankar
- Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Ferdinand-Sauerbruch-Straße, 17489, Greifswald, Germany
| | - Isabel Panse
- Kennedy Institute of Rheumatology, University of Oxford, Oxford, United Kingdom
| | - Pärt Peterson
- Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia
| | - Christian Peth
- Biopyhsics, R&D Engineering, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
| | - Jordi Petriz
- Josep Carreras Leukemia Research Institute, Barcelona, Spain
| | - Daisy Philips
- Division of immunology, the Netherlands Cancer Institute, Amsterdam
| | - Winfried Pickl
- Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria
| | - Silvia Piconese
- Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Via Regina Elena 324, 00161 Rome, Italy
- Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Rome, Italy
| | - Marcello Pinti
- Department of Life Sciences, Univ. of Modena and Reggio Emilia, Modena, Italy
| | - A Graham Pockley
- John van Geest Cancer Research Centre, Nottingham Trent University, Nottingham, UK
- Chromocyte Limited, Electric Works, Sheffield, UK
| | - Malgorzata Justyna Podolska
- Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Department of Internal Medicine, Rheumatology and Immunology, Universitätsklinikum Erlangen, Erlangen
| | - Carlo Pucillo
- Univeristy of Udine - Department of Medicine, Lab of Immunology, Udine, Italy
| | - Sally A Quataert
- David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY, USA
| | - Timothy R D J Radstake
- Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Bartek Rajwa
- Bindley Biosciences Center, Purdue University, West Lafayette, In, USA
| | - Jonathan A Rebhahn
- David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY, USA
| | | | - Ester B M Remmerswaal
- Department of Experimental Immunology and Renal Transplant Unit, Division of Internal Medicine, Academic Medical Centre, The Netherlands
| | - Katy Rezvani
- Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA
| | - Laura G Rico
- Josep Carreras Leukemia Research Institute, Barcelona, Spain
| | - J Paul Robinson
- The SVM Professor of Cytomics & Professor of Biomedical Engineering, Purdue University Cytometry Laboratories, Purdue University, West Lafayette, IN, USA
| | - Chiara Romagnani
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | | | - Beate Ruckert
- Swiss Institute of Allergy and Asthma Research (SIAF), University Zurich, Davos, Switzerland
| | - Jürgen Ruland
- Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technische Universität München, Munich, Germany
- German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany
- German Center for Infection Research (DZIF), partner site Munich, Munich, Germany
| | - Shimon Sakaguchi
- Laboratory of Experimental Immunology, WPI Immunology Frontier Research Center (IFReC), Osaka University, Suita 565-0871, Japan
- Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
| | - Francisco Sala-de-Oyanguren
- Laboratory of Cytomics, Joint Research Unit CIPF-UVEG, Department of Biochemistry and Molecular Biology, The University of Valencia. Av. Blasco Ibáñez, Valencia, Spain
| | - Yvonne Samstag
- Institute of Immunology, Section Molecular Immunology, Ruprecht-Karls-University, D-69120, Heidelberg, Germany
| | - Sharon Sanderson
- Translational Immunology Laboratory, NIHR BRC, University of Oxford, Kennedy Institute of Rheumatology,Oxford, United Kingdom
| | - Birgit Sawitzki
- Charité-Universitaetsmedizin Berlin, Corporate Member of Freie Universitaet Berlin, Humboldt-Universitaet zu Berlin
- Berlin Institute of Health, Institute of Medical Immunology, Augustenburger Platz 1, 13353 Berlin, Germany
| | - Alexander Scheffold
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
- Charité - Universitätsmedizin Berlin, Germany
| | - Matthias Schiemann
- Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany
| | - Frank Schildberg
- Harvard Medical School, Department of Microbiology and Immunobiology, Boston, MA, USA
| | | | - Stephan A Schmid
- Klinik und Poliklinik für Innere Medizin I, Universitätsklinikum Regensburg, Regensburg, Germany
| | - Steffen Schmitt
- Imaging and Cytometry Core Facility, Flow Cytometry Unit, German Cancer Research Centre (DKFZ), Heidelberg, Germany
| | - Kilian Schober
- Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany
| | - Thomas Schüler
- Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Magdeburg, Germany
| | - Axel Ronald Schulz
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Ton Schumacher
- Division of immunology, the Netherlands Cancer Institute, Amsterdam
| | - Cristiano Scotta
- MRC Centre for Transplantation, King's College London, Guy's Hospital, SE1 9RT London, UK
| | | | - Anat Shemer
- Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
| | | | - Josef Spidlen
- Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada
| | | | - Regina Stark
- Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands
| | - Christina Stehle
- Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany
| | - Merle Stein
- Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Tobit Steinmetz
- Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Dept. of Internal Medicine III, University of Erlangen-Nuremberg, Erlangen, Germany
| | - Hannes Stockinger
- Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria
| | - Yousuke Takahama
- Division of Experimental Immunology, Institute of Advanced Medical Sciences, University of Tokushima, Tokushima, Japan
| | - Attila Tarnok
- Departement for Therapy Validation, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany
- Institute for Medical Informatics, IMISE, Leipzig, Germany
| | - ZhiGang Tian
- School of Life Sciences and Medical Center, Institute of Immunology, Key Laboratory of Innate Immunity and Chronic Disease of Chinese Academy of Science, University of Science and Technology of China, Hefei, China
- Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
| | - Gergely Toldi
- University of Birmingham, Institute of Immunology and Immunotherapy, Birmingham, UK
| | - Julia Tornack
- Senior Group on Lymphocyte Development, Max Planck Institute for Infection Biology, Berlin, Germany
| | | | | | - Henning Ulrich
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo
| | | | - René A W van Lier
- Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands
| | | | | | - Paulo Vieira
- Unité Lymphopoiese, Institut Pasteur, Paris, France
| | - David Voehringer
- Department of Infection Biology, University Hospital Erlangen, Wasserturmstr. 3/5, 91054 Erlangen, Germany
| | | | | | - Ari Waisman
- Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg University of Mainz, Mainz, Germany
| | | | | | - Klaus Warnatz
- Center for Chronic Immunodeficiency (CCI), Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Sarah Warth
- BCRT Flow Cytometry Lab, Berlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin Berlin
| | | | - Carsten Watzl
- Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund, IfADo, Department of Immunology, Dortmund, Germany
| | - Leonie Wegener
- Biopyhsics, R&D Engineering, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
| | - Annika Wiedemann
- Department of Medicine/Rheumatology and Clinical Immunology, Charite Universitätsmedizin Berlin, Germany
| | - Jürgen Wienands
- Universitätsmedizin Göttingen, Georg-August-Universität, Abt. Zelluläre und Molekulare Immunologie, Humboldtallee 34, 37073 Göttingen, Germany
| | - Gerald Willimsky
- Cooperation Unit for Experimental and Translational Cancer Immunology, Institute of Immunology (Charité - Universitätsmedizin Berlin) and German Cancer Research Center (DKFZ), Berlin, Germany
| | - James Wing
- Laboratory of Experimental Immunology, WPI Immunology Frontier Research Center (IFReC), Osaka University, Suita 565-0871, Japan
- Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
| | - Peter Wurst
- Institute of Experimental Immunology, University Bonn, Bonn, Germany
| | | | - Alice Yue
- School of Computing Science, Simon Fraser University, Burnaby, Canada
| | | | - Yi Zhao
- Department of Rheumatology & Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - Susanne Ziegler
- Department of Virus Immunology, Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
| | - Jakob Zimmermann
- Maurice Müller Laboratories (DKF), Universitätsklinik für Viszerale Chirurgie und Medizin Inselspital, University of Bern, Murtenstrasse, Bern
| |
Collapse
|
|
37
|
Carloni S, Gallerani G, Tesei A, Scarpi E, Verdecchia GM, Virzì S, Fabbri F, Arienti C. DNA ploidy and S-phase fraction analysis in peritoneal carcinomatosis from ovarian cancer: correlation with clinical pathological factors and response to chemotherapy. Onco Targets Ther 2017; 10:4657-4664. [PMID: 29033584 PMCID: PMC5614767 DOI: 10.2147/ott.s141117] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Objective We investigated the correlation between ploidy or S-phase fraction (SPF) and the clinical pathological characteristics of patients with peritoneal carcinomatosis from ovarian cancer. We also assessed their relation with the in vivo and in vitro response to several chemotherapeutic agents. Patients and methods Fifty-three patients with peritoneal carcinomatosis from ovarian cancer were enrolled. Frozen tumor tissue was dissociated by a detergent–trypsin method, and the resulting cell suspension was stained with RNase A and propidium iodide. Samples were then analyzed for ploidy and SPF by flow cytometry. Fresh tumor tissue was dissociated by enzymatic digestion, and cells were exposed to different concentrations of cisplatin, adriamycin, carboplatin, gemcitabine and taxol for 72 hours. In vitro drug sensitivity was then measured using the sulforhodamine B assay. Results No significant correlation was found between ploidy or SPF and patient characteristics, even though primary carcinomas were mainly hyperdiploid and more proliferative than recurrent tumors. SPF differed significantly among ploidy categories (P=0.01), and high SPF was associated with short-term survival (P=0.48). Patients with multiploid tumors were the most resistant to platinum-based chemotherapy, whereas those with hyperdiploid tumors were the most responsive. In vitro multiploid tumors were the least sensitive, while hypodiploid samples showed the highest sensitivity to the tested drugs. Sensitivity to adriamycin was significantly correlated with ploidy (P=0.03), whereas sensitivity to taxol was correlated with SPF (P=0.04). Conclusion Our results indicate that ploidy and SPF could facilitate the choice of therapy for patients with peritoneal carcinomatosis.
Collapse
Affiliation(s)
- Silvia Carloni
- Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola
| | - Giulia Gallerani
- Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola
| | - Anna Tesei
- Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola
| | - Emanuela Scarpi
- Unit of Biostatistics and Clinical Trials, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola
| | | | | | - Francesco Fabbri
- Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola
| | - Chiara Arienti
- Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola
| |
Collapse
|
|
38
|
Chemosensitivity of BRCA1-Mutated Ovarian Cancer Cells and Established Cytotoxic Agents. Int J Gynecol Cancer 2017; 27:1571-1578. [PMID: 28604461 DOI: 10.1097/igc.0000000000001052] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
Abstract
OBJECTIVE Serous adenocarcinomas that arise in patients with inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 are initially well treatable with platinum/paclitaxel. For recurrent disease in patients with BRCA1 or BRCA2 mutations, olaparib treatment is available. To study additional therapeutic regimens, a better understanding of the cellular and molecular mechanisms of the tumors in in vitro models is important. METHODS/MATERIALS From a high-grade serous ovarian tumor of a BRCA1 mutation carrier, we established 3 distinct cell line subclones, OVCA-TR3.1, -2, and -3. Immunohistochemical characterization, flow cytometric analyses, chemosensitivity, and somatic mutation profiling were performed. RESULTS The cell lines expressed AE1/AE3, Pax8, WT-1, OC125, estrogen receptor (ER), and p53, comparable to the primary tumor. Synergism could be shown in the combination treatment eremophila-1-(10)-11(13)-dien-12,8β-olide (EPD), with cisplatin, whereas combination with olaparib did not show synergism. Eremophila-1-(10)-11(13)-dien-12,8β-olide, a sesquiterpene lactone, is a novel chemotherapeutic agent. The inherited BRCA1 c.2989_2990dupAA mutation was confirmed in the cell lines. Loss of heterozygosity of BRCA1 was detected in each cell line, as well as a homozygous TP53 c.722C>A mutation. Flow cytometry showed that all cell lines had a distinct DNA index. CONCLUSIONS Three new isogenic ovarian cancer cell lines were developed from a patient with a germ line BRCA1 mutation. Chemosensitivity profiling of the cell lines showed high tolerance for olaparib. Treatment with EPD proved synergistic with cisplatin. The effects of EPD will be further investigated for future clinical efficacy.
Collapse
|
|
39
|
Orfanidis K, Wäster P, Lundmark K, Rosdahl I, Öllinger K. Evaluation of tubulin β-3 as a novel senescence-associated gene in melanocytic malignant transformation. Pigment Cell Melanoma Res 2017; 30:243-254. [PMID: 28024114 DOI: 10.1111/pcmr.12572] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2016] [Accepted: 12/17/2016] [Indexed: 12/22/2022]
Abstract
Malignant melanoma might develop from melanocytic nevi in which the growth-arrested state has been broken. We analyzed the gene expression of young and senescent human melanocytes in culture and compared the gene expression data with a dataset from nevi and melanomas. A concordant altered gene expression was identified in 84 genes when comparing the growth-arrested samples with proliferating samples. TUBB3, which encodes the microtubule protein tubulin β-3, showed a decreased expression in senescent melanocytes and nevi and was selected for further studies. Depletion of tubulin β-3 caused accumulation of cells in the G2/M phase and decreased proliferation and migration. Immunohistochemical assessment of tubulin β-3 in benign lesions revealed strong staining in the superficial part of the intradermal components, which faded with depth. In contrast, primary melanomas exhibited staining without gradient in a disordered pattern and strong staining of the invasive front. Our results describe an approach to find clinically useful diagnostic biomarkers to more precisely identify cutaneous malignant melanoma and present tubulin β-3 as a candidate marker.
Collapse
Affiliation(s)
- Kyriakos Orfanidis
- Department of Dermatology and Venereology, Linköping University, Linköping, Sweden.,Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
| | - Petra Wäster
- Experimental Pathology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
| | - Katarzyna Lundmark
- Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden.,Department of Clinical Pathology and Clinical Genetics, Linköping University, Linköping, Sweden
| | - Inger Rosdahl
- Department of Dermatology and Venereology, Linköping University, Linköping, Sweden.,Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
| | - Karin Öllinger
- Experimental Pathology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden
| |
Collapse
|
|
40
|
Jonsson M, Ragnum HB, Julin CH, Yeramian A, Clancy T, Frikstad KAM, Seierstad T, Stokke T, Matias-Guiu X, Ree AH, Flatmark K, Lyng H. Hypoxia-independent gene expression signature associated with radiosensitisation of prostate cancer cell lines by histone deacetylase inhibition. Br J Cancer 2016; 115:929-939. [PMID: 27599042 PMCID: PMC5061908 DOI: 10.1038/bjc.2016.278] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Revised: 07/22/2016] [Accepted: 08/11/2016] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND Histone deacetylase inhibitors (HDACis) like vorinostat are promising radiosensitisers in prostate cancer, but their effect under hypoxia is not known. We investigated gene expression associated with radiosensitisation of normoxic and hypoxic prostate cancer cells by vorinostat. METHODS Cells were exposed to vorinostat under normoxia or hypoxia and subjected to gene expression profiling before irradiation and clonogenic survival analysis. RESULTS Pretreatment with vorinostat led to radiosensitisation of the intrinsically radioresistant DU 145 cells, but not the radiosensitive PC-3 and 22Rv1 cells, and was independent of hypoxia status. Knockdown experiments showed that the sensitisation was not caused by repression of hypoxia-inducible factor HIF1 or tumour protein TP53. Global deregulation of DNA repair and chromatin organisation genes was associated with radiosensitisation under both normoxia and hypoxia. A radiosensitisation signature with expression changes of 56 genes was generated and valid for both conditions. For eight signature genes, baseline expression also correlated with sensitisation, showing potential as pretreatment biomarker. The hypoxia independence of the signature was confirmed in a clinical data set. CONCLUSIONS Pretreatment with HDACi may overcome radioresistance of hypoxic prostate tumours by similar mechanisms as under normoxia. We propose a gene signature to predict radiosensitising effects independent of hypoxia status.
Collapse
Affiliation(s)
- Marte Jonsson
- Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Pb 4950, Nydalen, 0424 Oslo, Norway
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
| | - Harald Bull Ragnum
- Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Pb 4950, Nydalen, 0424 Oslo, Norway
| | - Cathinka Halle Julin
- Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Pb 4950, Nydalen, 0424 Oslo, Norway
| | - Andree Yeramian
- Department of Pathology and Molecular Genetics HUAV, University of Lleida, Lleida, Spain
| | - Trevor Clancy
- Department of Tumor Biology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Kari-Anne Myrum Frikstad
- Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Pb 4950, Nydalen, 0424 Oslo, Norway
| | - Therese Seierstad
- Department of Radiology and Nuclear Medicine, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Trond Stokke
- Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Pb 4950, Nydalen, 0424 Oslo, Norway
| | - Xavier Matias-Guiu
- Department of Pathology and Molecular Genetics HUAV, University of Lleida, Lleida, Spain
| | - Anne Hansen Ree
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
- Department of Tumor Biology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
- Department of Oncology, Akershus University Hospital, Lørenskog, Norway
| | - Kjersti Flatmark
- Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
- Department of Tumor Biology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
- Department of Gastroenterological Surgery, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
| | - Heidi Lyng
- Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Pb 4950, Nydalen, 0424 Oslo, Norway
| |
Collapse
|
|
41
|
Magalhaes LG, Marques FB, da Fonseca MB, Rogério KR, Graebin CS, Andricopulo AD. Discovery of a Series of Acridinones as Mechanism-Based Tubulin Assembly Inhibitors with Anticancer Activity. PLoS One 2016; 11:e0160842. [PMID: 27508497 PMCID: PMC4980028 DOI: 10.1371/journal.pone.0160842] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2016] [Accepted: 07/26/2016] [Indexed: 12/16/2022] Open
Abstract
Microtubules play critical roles in vital cell processes, including cell growth, division, and migration. Microtubule-targeting small molecules are chemotherapeutic agents that are widely used in the treatment of cancer. Many of these compounds are structurally complex natural products (e.g., paclitaxel, vinblastine, and vincristine) with multiple stereogenic centers. Because of the scarcity of their natural sources and the difficulty of their partial or total synthesis, as well as problems related to their bioavailability, toxicity, and resistance, there is an urgent need for novel microtubule binding agents that are effective for treating cancer but do not have these disadvantages. In the present work, our lead discovery effort toward less structurally complex synthetic compounds led to the discovery of a series of acridinones inspired by the structure of podophyllotoxin, a natural product with important microtubule assembly inhibitory activity, as novel mechanism-based tubulin assembly inhibitors with potent anticancer properties and low toxicity. The compounds were evaluated in vitro by wound healing assays employing the metastatic and triple negative breast cancer cell line MDA-MB-231. Four compounds with IC50 values between 0.294 and 1.7 μM were identified. These compounds showed selective cytotoxicity against MDA-MB-231 and DU-145 cancer cell lines and promoted cell cycle arrest in G2/M phase and apoptosis. Consistent with molecular modeling results, the acridinones inhibited tubulin assembly in in vitro polymerization assays with IC50 values between 0.9 and 13 μM. Their binding to the colchicine-binding site of tubulin was confirmed through competitive assays.
Collapse
Affiliation(s)
- Luma G. Magalhaes
- Laboratório de Química Medicinal e Computacional, Centro de Pesquisa e Inovação em Biodiversidade e Fármacos, Instituto de Física de São Carlos, Universidade de São Paulo, 13563–120, São Carlos-SP, Brazil
| | - Fernando B. Marques
- Laboratório de Diversidade Molecular e Química Medicinal, Departamento de Química, Universidade Federal Rural do Rio de Janeiro, 23897–000, Seropédica-RJ, Brazil
| | - Marina B. da Fonseca
- Laboratório de Diversidade Molecular e Química Medicinal, Departamento de Química, Universidade Federal Rural do Rio de Janeiro, 23897–000, Seropédica-RJ, Brazil
| | - Kamilla R. Rogério
- Laboratório de Diversidade Molecular e Química Medicinal, Departamento de Química, Universidade Federal Rural do Rio de Janeiro, 23897–000, Seropédica-RJ, Brazil
| | - Cedric S. Graebin
- Laboratório de Diversidade Molecular e Química Medicinal, Departamento de Química, Universidade Federal Rural do Rio de Janeiro, 23897–000, Seropédica-RJ, Brazil
| | - Adriano D. Andricopulo
- Laboratório de Química Medicinal e Computacional, Centro de Pesquisa e Inovação em Biodiversidade e Fármacos, Instituto de Física de São Carlos, Universidade de São Paulo, 13563–120, São Carlos-SP, Brazil
- * E-mail:
| |
Collapse
|
|
42
|
Mayorga M, García-Valtuille A, Fernández F, Val-Bernal JF, Cabrera E. Adenocarcinoma of the Uterine Cervix with Massive Signet-Ring Cell Differentiation. Int J Surg Pathol 2016. [DOI: 10.1177/106689699700500304] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Primary signetring cell adenocarcinoma of the cervix is very rare and less common than metastatic signet-ring cell adenocarcinoma. To the best of the authors' knowldge only one genuine case has been reported to date. Tvvo cases of primary signeting cell adenocarcinoma of the uterine cervix in patients aged 68 and 74 years are presented. The tumors were in stage IB. The light microscopic findings were conirmed by histochemical and immunohistochemical study. DNA nuclear analysis by flow cytometry of the neoplasms revealed an aneuploid (tetraploid) pattern. The paients had no evidence of recurrent or metastatic disease 35 and 25 months after a radical hysterectomy with bilateral salpingo-oophorectomy and lymph node dissecion respectively. Primary signet-ring cell adenocarcinoma of the cervix should be diferentiated from metastatic adenocarcinoma, endocervical involvement by signeting cell carcinoma of the endometrium, benign mucinfilled signet-ring cell aggregates that may accumulate in mucosal folds, microglandular hyperplasia, muciarminophilic histiocytosis, and other malignant neoplasms that may have signet ringlike cells and deserve consideration such as squamous cell carcinoma, maligant lymphoma, myeloma, and malignant melanoma. Although very rare, signeting cell adenocarcinoma of the cervix can exist as a primary tumor. Distinction beween a primary neoplasm and a metastasis to the cervix is decisive for treatment and prognosis.
Collapse
Affiliation(s)
| | | | | | | | - Ernesto Cabrera
- Department of Anatomical Pathology, Marques de Valdecilla University Hospital, University of Cantabria Medical School, Santander, Spain
| |
Collapse
|
|
43
|
Cabrera E, Fernández F, Gómez-Román J, Val-Bernal JF. Basaloid Squamous Cell Carcinoma of the Esophagus Immunohistochemistry and Flow Cytometric DNA Analysis in Two Cases. Int J Surg Pathol 2016. [DOI: 10.1177/106689699604030407] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Basaloid squamous cell carcinoma is a well-established anatomopathologic entity of the upper aerodigestive tract and is more aggressive than the conventional squamous cell carcinoma. Only seven cases of this variant have been reported in the esophagus. Two cases of basaloid squamous carcinoma in the middle and lower third of the esophagus in two men aged 59 and 60 years are presented. Tumor stages were IIA and IV at the time of total esophagectomy, and survival times were 34 and 4 months, respectively. Histologically, the two tumors were composed of nests of cells with a basaloid appearance, the first with a predominantly cribriform pattern and the second with a solid lobular pattern and intense comedonecrosis; in the adjacent squamous epithelium of both tumors, carcinoma in situ was observed focally. Immunoreactivity was poor for high-molecular-weight cytokeratins and absent for those of low molecular weight. Staining for epithelial membrane antigen was weakly positive, enolase was moderately positive, and no reactivity for carcinoembryonic antigen, S100 protein, and chromogranin was observed. The flow cytometric analysis showed both tumors to be hyperdiploid with an index of cell proliferation of over 50%.
Collapse
Affiliation(s)
- Ernesto Cabrera
- From the Department of Anatomical Pathology, “Marqués de Valdecilla” University Hospital, Faculty of Medicine, University of Cantabria, Santander, Spain
| | - Fidel Fernández
- From the Department of Anatomical Pathology, “Marqués de Valdecilla” University Hospital, Faculty of Medicine, University of Cantabria, Santander, Spain
| | - Javier Gómez-Román
- From the Department of Anatomical Pathology, “Marqués de Valdecilla” University Hospital, Faculty of Medicine, University of Cantabria, Santander, Spain
| | - J. Fernado Val-Bernal
- From the Department of Anatomical Pathology, “Marqués de Valdecilla” University Hospital, Faculty of Medicine, University of Cantabria, Santander, Spain
| |
Collapse
|
|
44
|
Hoekstra AS, Addie RD, Ras C, Seifar RM, Ruivenkamp CA, Briaire-de Bruijn IH, Hes FJ, Jansen JC, Corssmit EPM, Corver WE, Morreau H, Bovée JVMG, Bayley JP, Devilee P. Parent-of-origin tumourigenesis is mediated by an essential imprinted modifier in SDHD-linked paragangliomas: SLC22A18 and CDKN1C are candidate tumour modifiers. Hum Mol Genet 2016; 25:3715-3728. [PMID: 27402879 DOI: 10.1093/hmg/ddw218] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2016] [Revised: 06/28/2016] [Accepted: 06/30/2016] [Indexed: 12/11/2022] Open
Abstract
Mutations in SDHD and SDHAF2 (both located on chromosome 11) give rise to hereditary paraganglioma almost exclusively after paternal transmission of the mutation, and tumours often show loss of the entire maternal copy of chromosome 11. The 'Hensen' model postulates that a tumour modifier gene located on chromosome 11p15, a region known to harbour a cluster of imprinted genes, is essential to tumour formation. We observed decreased protein expression of the 11p15 candidate genes CDKN1C, SLC22A18 and ZNF215 evaluated in 60 SDHD-mutated tumours compared to normal carotid body tissue and non-SDH mutant tumours.We then created stable knockdown in vitro models, reasoning that the simultaneous knockdown of SDHD and a maternally expressed 11p15 modifier gene would enhance paraganglioma-related cellular characteristics compared to SDHD knockdown alone. Knockdown of SDHD in SNB19 and SHSY5Y cells resulted in the accumulation of succinate, the stabilization of HIF1 protein and a reduction in cell proliferation.Compared to single knockdown of SDHD, knockdown of SDHD together with SLC22A18 or with CDKN1C led to small but significant increases in cell proliferation and resistance to apoptosis, and to a gene expression profile closely related to the known transcriptional profile of SDH-deficient tumours. Of the 60 SDHD tumours investigated, four tumours showing retention of chromosome 11 showed SLC22A18 and CDKN1C expression levels comparable to levels in tumours showing loss of chromosome 11, suggesting loss of protein expression despite chromosomal retention.Our data strongly suggest that SLC22A18 and/or CDKN1C are tumour modifier genes involved in the tumourigenesis of SDHD-linked paraganglioma.
Collapse
Affiliation(s)
| | - Ruben D Addie
- Center for Proteomics and Metabolomics
- Department of Pathology
| | - Cor Ras
- Department of Biotechnology, Delft University of Technology, Delft, The Netherlands
| | - Reza M Seifar
- Department of Biotechnology, Delft University of Technology, Delft, The Netherlands
| | | | | | | | | | - Eleonora P M Corssmit
- Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands
| | | | | | | | | | | |
Collapse
|
|
45
|
Carbonari M. New use for an old reagent: Cell cycle analysis of DNA content using flow cytometry in formamide treated cells. Cytometry A 2016; 89:498-503. [PMID: 26866418 DOI: 10.1002/cyto.a.22823] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2015] [Revised: 11/10/2015] [Accepted: 01/08/2016] [Indexed: 11/12/2022]
Abstract
Formamide has long been one of the most widely used reagents in the study of nucleic acids. However, the use of formamide for treating cells to be analyzed by flow cytometry is a recent development and is restricted to measuring telomere lengths by flow-FISH. In this field, we have published several papers in order to observe the effects of formamide treatment on cells at room temperature. We therefore discovered that, with suitable modifications, a short and simple incubation in this ionizing solvent facilitates cell cycle analysis by flow cytometry, equivalent or superior to that obtained with treatments in alcohol, acetone or detergent in hypotonic solution. Even using a bulky and problematic stain (low quantum efficiency and G-C base preference), such as 7-aminoactinomycin D (7-AAD) which, on the other hand, has the advantage of being excited at 488 nm and does not bind to the RNA, it is possible to obtain excellent coefficients of variation and (G2-M) mode/(G0-G1) mode ratios. These parameters, especially if stained cells are washed before acquisition, arrive at optimal values. It is noteworthy that the ability to wash the cells stained for DNA content analysis without affecting the stoichiometry of the staining has not been described elsewhere in the literature. With formamide treatment the doublets are practically absent, sample recovery is efficient, as well as the preservation of physical parameters, and the stained cells can be stored for at least 10 days at room temperature before acquisition. © 2016 International Society for Advancement of Cytometry.
Collapse
Affiliation(s)
- Maurizio Carbonari
- Immunology Laboratory, Department of Clinical Medicine, University of Rome "Sapienza,", Rome, Italy
| |
Collapse
|
|
46
|
Trojan PJJ, Bohatch-Junior MS, Otuki MF, Souza-Fonseca-Guimarães F, Svidnicki PV, Nogaroto V, Fernandes D, Krum EA, Favero GM. Pravastatin induces cell cycle arrest and decreased production of VEGF and bFGF in multiple myeloma cell line. BRAZ J BIOL 2016; 76:59-65. [PMID: 26909624 DOI: 10.1590/1519-6984.11914] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2014] [Accepted: 10/17/2014] [Indexed: 01/19/2023] Open
Abstract
Multiple myeloma (MM) is a B cell bone marrow neoplasia characterized by inflammation with an intense secretion of growth factors that promote tumor growth, cell survival, migration and invasion. The aim of this study was to evaluate the effects of pravastatin, a drug used to reduce cholesterol, in a MM cell line.Cell cycle and viability were determinate by Trypan Blue and Propidium Iodide. IL6, VEGF, bFGF and TGFβ were quantified by ELISA and qRT-PCR including here de HMG CoA reductase. It was observed reduction of cell viability, increase of cells in G0/G1 phase of the cell cycle and reducing the factors VEGF and bFGF without influence on 3-Methyl-Glutaryl Coenzyme A reductase expression.The results demonstrated that pravastatin induces cell cycle arrest in G0/G1 and decreased production of growth factors in Multiple Myeloma cell line.
Collapse
Affiliation(s)
- P J J Trojan
- Laboratório Multidisciplinar de Ciências Biológicas e da Saúde, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil
| | - M S Bohatch-Junior
- Laboratório Multidisciplinar de Ciências Biológicas e da Saúde, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil
| | - M F Otuki
- Departamento de Ciências Farmacêuticas, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil
| | - F Souza-Fonseca-Guimarães
- Unit Cytokines and Inflammation, Department Infection and Epidemiology Institut Pasteur, Paris, France
| | - P V Svidnicki
- Departamento de Biologia Molecular, Estrutural e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil
| | - V Nogaroto
- Departamento de Biologia Molecular, Estrutural e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil
| | - D Fernandes
- Departamento de Ciências Farmacêuticas, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil
| | - E A Krum
- Departamento de Ciências Farmacêuticas, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil
| | - G M Favero
- Laboratório Multidisciplinar de Ciências Biológicas e da Saúde, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil
| |
Collapse
|
|
47
|
Sunde L, Lund H, J Sebire N, Grove A, Fisher RA, Niemann I, Kjeldsen E, Andreasen L, Hansen ES, Bojesen A, Bolund L, Nyegaard M. Paternal Hemizygosity in 11p15 in Mole-like Conceptuses: Two Case Reports. Medicine (Baltimore) 2015; 94:e1776. [PMID: 26554776 PMCID: PMC4915877 DOI: 10.1097/md.0000000000001776] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
Abstract
Hydatidiform mole is an abnormal human pregnancy characterized by the fetus being absent or nonviable, and the chorionic villi being vesicular and with trophoblastic hyperplasia. Most often, the mole phenotype is seen in conceptuses with an excess of paternally inherited genome set(s) relative to maternally inherited genome set(s), suggesting that the phenotype is caused by an excess of genome with a paternal imprinting pattern. However, it is unknown if correct parental origin of every imprinted gene is crucial for normal early differentiation or if abnormal parental imprinting of only one, or some, gene(s) can cause the mole phenotype.Two conceptuses included in the Danish Mole Project stood out since they presented with vesicular chorionic villi and without signs of fetal differentiation, and had apparently biparental diploid genomes, and no mutations in NLRP7 or KHDC3L were detected in the mothers. These conceptuses were subjected to a centralized histopathological revision and their genetic complements were scrutinized using fluorescence in situ hybridization, and DNA-marker and array comparative genomic hybridization analyses. Both conceptuses showed dysmorphic chorionic villi with some similarities to hydatidiform moles; however, no definite florid trophoblast hyperplasia was observed. Both conceptuses showed paternal hemizygosity of 11pter-11p15.4, most likely in nonmosaic state.Our findings suggest that the product of one (or a few) maternally expressed gene(s) on the tip of chromosome 11 is necessary for normal early embryonic differentiation. However, since the present two cases did not exhibit all features of hydatidiform moles, it is likely that abnormal parental imprinting of genes in other regions contribute to the phenotype of a hydatidiform mole.
Collapse
Affiliation(s)
- Lone Sunde
- From the Department of Clinical Genetics, Aarhus University Hospital, Aarhus N, Denmark (LS); Institute of Pathology, Aalborg University Hospital, Aalborg, Denmark (HL, AG); Trophoblastic Tumour Screening and Treatment Centre, Department of Oncology, Imperial College Healthcare NHS (NJS, RF); Institute of Child Health, University College London (NJS); Institute of Reproductive and Developmental Biology, Department of Surgery and Cancer, Imperial College London, London, UK (RF); Department of Gynaecology and Obstetrics, Aarhus University Hospital, Aarhus N (IN); Hemodiagnostic Laboratory, CancercytogeneticSection, Aarhus University Hospital, Aarhus C, Denmark (EK); Department of Immunology and Biochemistry, Vejle Sygehus, Vejle, Denmark (LA); Department of Pathology, Aarhus University Hospital, Aarhus C, Denmark (EH); Department of Clinical Genetics, Vejle Sygehus, Vejle, Denmark (AB); Department of Biomedicine, Aarhus University, Aarhus C, Denmark (LS, LB, MN); and Beijing Genomics Institute/HuaDa-Shenzhen, Shenzhen, China (LB)
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
48
|
Asano T, Sato A, Isono M, Okubo K, Ito K, Asano T. Bortezomib and belinostat inhibit renal cancer growth synergistically by causing ubiquitinated protein accumulation and endoplasmic reticulum stress. Biomed Rep 2015; 3:797-801. [PMID: 26623018 DOI: 10.3892/br.2015.523] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2015] [Accepted: 09/15/2015] [Indexed: 01/07/2023] Open
Abstract
There is no curative treatment for advanced renal cancer, and a novel treatment approach is urgently required. Inducing ubiquitinated protein accumulation and endoplasmic reticulum (ER) stress has recently emerged as a new approach in the treatment of malignancies. In the present study, we hypothesized that the histone deacetylase inhibitor belinostat would increase the amount of unfolded proteins in cells by inhibiting heat-shock protein (HSP) 90, and that the proteasome inhibitor bortezomib would inhibit their degradation by inhibiting the proteasome, thus causing ubiquitinated protein accumulation and ER stress synergistically. The combination of bortezomib and belinostat induced significant increases in apoptosis and inhibited renal cancer growth synergistically (combination indexes <1). The combination also suppressed colony formation significantly (P<0.05). As co-treatment with the pan-caspase inhibitor Z-VAD-FMK changed the number of Annexin V-positive cells, this combination-induced apoptosis was considered caspase dependent. Mechanistically, the combination synergistically caused ubiquitinated proteins to accumulate and induced ER stress, as evidenced by the increased expression of glucose-regulated protein 78 and HSP70. To the best of our knowledge, this is the first study demonstrating the beneficial combined effect of bortezomib and belinostat in renal cancer cells. The study provides a basis for clinical studies with the combination in patients with advanced renal cancer.
Collapse
Affiliation(s)
- Takako Asano
- Department of Urology, National Defense Medical College, Tokorozawa, Saitama 359-8513, Japan
| | - Akinori Sato
- Department of Urology, National Defense Medical College, Tokorozawa, Saitama 359-8513, Japan
| | - Makoto Isono
- Department of Urology, National Defense Medical College, Tokorozawa, Saitama 359-8513, Japan
| | - Kazuki Okubo
- Department of Urology, National Defense Medical College, Tokorozawa, Saitama 359-8513, Japan
| | - Keiichi Ito
- Department of Urology, National Defense Medical College, Tokorozawa, Saitama 359-8513, Japan
| | - Tomohiko Asano
- Department of Urology, National Defense Medical College, Tokorozawa, Saitama 359-8513, Japan
| |
Collapse
|
|
49
|
Aizen D, Sarfstein R, Bruchim I, Weinstein D, Laron Z, Werner H. Proliferative and signaling activities of insulin analogues in endometrial cancer cells. Mol Cell Endocrinol 2015; 406:27-39. [PMID: 25697343 DOI: 10.1016/j.mce.2015.02.011] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/17/2013] [Revised: 01/15/2015] [Accepted: 02/12/2015] [Indexed: 12/29/2022]
Abstract
Insulin analogues have been developed to achieve further improvement in the therapy of diabetes. However, modifications introduced into the insulin molecule may enhance their affinity for the insulin-like growth factor-1 receptor (IGF1R). Hyperinsulinemia has been identified as a risk factor for endometrial cancer. We hypothesized that insulin analogues may elicit atypical proliferative and signaling activities in endometrial cancer cells. Our results demonstrate that glargine, but not detemir, stimulated cell proliferation, displayed an anti-apoptotic effect, and had a positive effect on cell cycle progression in endometrial cancer cell lines ECC-1 and USPC-1. In addition, we showed that glargine and detemir induced dual activation of the insulin receptor (INSR) and IGF1R in both cell types. Furthermore, we showed that glargine elicited signaling events that are markedly different from those induced by insulin. In conclusion, our data support the concept that, although insulin analogues were designed to display insulin-like metabolic effects, glargine and, possibly, additional analogues exhibit IGF1-like activities and, accordingly, may function as IGF1 analogues.
Collapse
Affiliation(s)
- Daniel Aizen
- Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Rive Sarfstein
- Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Ilan Bruchim
- Gynecologic Oncology Unit, Department of Obstetrics and Gynecology, Hillel Yaffe Medical Center, Hadera, Israel
| | - Doron Weinstein
- Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Zvi Laron
- Endocrine and Diabetes Research Unit, Schneider Children's Medical Center, Petah Tikva 49292, Israel
| | - Haim Werner
- Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
| |
Collapse
|
|
50
|
Anbumani S, Mohankumar MN. Gamma radiation induced cell cycle perturbations and DNA damage in Catla Catla as measured by flow cytometry. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 2015; 113:18-22. [PMID: 25483367 DOI: 10.1016/j.ecoenv.2014.11.011] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/05/2014] [Revised: 11/08/2014] [Accepted: 11/11/2014] [Indexed: 06/04/2023]
Abstract
Gamma radiation induced cell cycle perturbations and DNA damage in Catla catla were analyzed in erythrocytes at different time points using flow cytometry (FCM). Protracted exposure to radiation induced damage between days 12 and 45. Disturbances in cell cycle machinery, i.e., proportional increase and decrease in Gap0 or quiescent/Gap1 (G0/G1), Synthesis (S) and Gap2/Mitotic (G2/M) phases were observed at both acute and protracted treatments. Both acute and protracted exposures induced apoptosis with a notable significance between days 3 and 6 at protracted and on day 45 at acute doses. Fish exposed protractedly avail some DNA repair mechanisms than acutely exposed. This is the first study to analyze radiation induced DNA damage under laboratory conditions and suggests that flow cytometry can also be an alternate tool to screen genotoxicity induced by ionizing radiation in fish.
Collapse
Affiliation(s)
- S Anbumani
- Radiological Safety Division, Indira Gandhi Centre for Atomic Research, Kalpakkam, Tamilnadu 603 102, India.
| | - Mary N Mohankumar
- Radiological Safety Division, Indira Gandhi Centre for Atomic Research, Kalpakkam, Tamilnadu 603 102, India.
| |
Collapse
|