Intrahepatic cholangiocarcinoma (ICC), an aggressive liver cancer, lacks simple and accurate clinical tests, which poses challenges to postoperative diagnosis and treatment. Recent studies have indicated that platelet levels might be relevant to the postoperative prognosis of ICC. However, their prognostic significance in ICC remains unclarified. This study included 218 ICC patients who underwent hepatic resection. Comprehensive analyses of patients' postoperative prognosis were conducted primarily focusing on their platelet levels associated with prognostic traits. To further investigate the underlying mechanism between platelet levels and patients' postoperative prognosis, we elucidated the association between platelets and tumor metastasis using HCCC-9810 and HUCC-T1 cells as well as mouse models. In the retrospective cohort study, elevated serum platelet levels (≥300 × 109/L) or tumoral platelet levels (≥0.23) individually indicated an unfavorable postoperative prognosis in individuals with ICC. Multivariate analysis showed that tumoral platelet levels can be an independent prognostic factor, while the loss of prognostic superiority of serum platelet levels in the analysis may be attributed to the influence of confounding inclusion variables. Epithelial/mesenchymal transition (EMT) marker expression changes in HCCC-9810 and HUCC-T1 cells with platelet treatment were analyzed to understand how platelets contribute to ICC malignant recurring progression. The significant role of the TGF-β/Smad2 pathway in ICC metastasis was identified. In addition, aspirin was found to have the potential to reduce ICC metastasis by inhibiting platelet function. In conclusion, this study indicated that ICC patients with postoperative serum platelet levels ≥300 × 109/L or tumoral platelet levels ≥0.23 have significantly higher risk of poor postoperative prognosis. This is due to platelet-derived TGFβ1 leading to EMT in ICC cells, thus promoting tumor metastasis.

Tumor dissemination is a key event in tumor progression. During this event, a main role is played by circulating tumor cells (CTCs), immune cells, and their interaction. How the immune system supports the survival and proliferation of CTCs is not fully elucidated. In this study we established an in-vitro co-culture system consisting of immune cells and CTCs from the same patient, which increased the success rate in the establishment of CTC-derived long-term cell cultures. In this system, we characterized the immune cells of successful co-cultures and the signals they exchange with cancer cells, including cytokines and extracellular vesicle (EV) content. Using this protocol, we stabilized four CTC-derived cell lines from patients with metastatic gastroesophageal cancer, which were cultured for over a year and characterized from a genetic and molecular point of view. The four cell lines harbor shared chromosomal aberrations including the amplification at 8q24.21 containing MYC and deletion 9p21.3 containing CDKN2A/B and the IFN type I cluster. The transcriptomic profile of CTC cell lines is distinct from primary tumors, and we detected the activation of E2F, G2M and MYC pathways and the downregulation of interferon response pathway. Each cell line shows a degree of invasiveness in zebrafish in-vivo, and the most invasive ones share the same mutation in RAB14 gene. In addition, the four cell lines secrete cell-line specific EVs containing microRNAs that target YAP, BRG1-AKT1, TCF8-HDAC pathways. Overall, we highlight how the immune system plays a key role in the proliferation of CTCs through EV signaling, and how CTC cell line genomic and transcriptomic alterations make these cells less visible from the immune system and likely responsible for the survival advantage in sites distant from the microenvironment of origin.

We aimed to evaluate the performance of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and their combination (combined NLR-PLR, CNP) in predicting overall survival (OS) and recurrence-free survival (RFS) in a large cohort of unselected hepatocellular carcinoma (HCC) patients.

Training and validation cohort data were retrieved from the Italian Liver Cancer (ITA.LI.CA) database. The optimal cut-offs of NLR and PLR were calculated according to the multivariable fractional polynomial and the minimum p value method. The continuous effect and best cut-off categories of NLR and PLR were analyzed using multivariable Cox regression analysis. A shrinkage procedure adjusted over-fitting hazard ratio (HR) estimates of best cut-off categories. C-statistic and integrated discrimination improvement (IDI) were calculated to evaluate the discrimination properties of the biomarkers when added to clinical survival models.

2,286 patients were split into training (n = 1,043) and validation (n = 1,243) cohorts. The optimal cut-offs for NLR and PLR were 1.45 and 188, respectively. NLR (HR 1.58, 95% CI 1.11-2.28, p = 0.014) and PLR (HR 1.79, 95% CI 1.11-2.90, p = 0.018) were independent predictors of OS. When incorporated into a clinical prognostic model that includes age, alpha-fetoprotein (AFP), the CHILD-Pugh score, and the Barcelona Clinic Liver Cancer (BCLC) staging system, CNP had a significant incremental value in predicting OS (IDI 1.3%, p = 0.04). Data were confirmed in the validation cohort. Neither NLR nor PLR significantly predicted RFS in the training cohort.

NLR, PLR, and CNP independently predicted shorter OS in HCC patients. The addition of CNP to the survival prediction model significantly improved the model's accuracy in predicting OS.

Despite the transformative impact of programmed cell death protein-1 (PD-1) blockade therapy on metastatic/advanced solid tumor treatment, its efficacy is hindered by a limited response rate. Platelets play a pivotal role in tumor metastasis by shielding circulating tumor cells and secreting immunosuppressive factors. We here demonstrate that selectively inducing apoptosis in tumor-associated platelets (TAPs) using ABT-737-loaded nanoparticles (cyclic arginine-glycine-aspartate containing peptide-modified ABT-737-loaded nanoparticles [cRGD-NP@A]) enhances the anti-metastatic efficacy of the anti-PD-1 antibody (aPD-1). cRGD-NP@A specifically binds to TAPs, disrupting platelet-tumor cell interactions and exposing tumor cells to immune surveillance in vivo. Combined with aPD-1, cRGD-NP@A substantially augments immune activation and reduces TAP-derived immunosuppressive factors, notably transforming growth factor β1 (TGF-β1), consequently improving anti-metastatic outcomes across multiple metastasis-bearing animal models without observable adverse effects. Our study underscores the importance of depleting TAPs to enhance PD-1 blockade therapy, presenting a promising strategy to improve response rates and clinical outcomes for patients with metastatic cancer.

Fc receptor γ subunit (FcRγ) activation plays a crucial role in cancer carcinogenesis. Here, we aimed to uncover the impact of FcRγ on circulating tumor cells (CTC) colonization and the underlying mechanism. FcRγ deficient (FcRγ-/-) mice were used to investigate the functional effects of FcRγ in cancer metastasis, and the results demonstrated that FcRγ deficiency significantly promotes metastasis. The tumor metastasis effect, antiplatelet, platelet or neutrophil infusion experiments were conducted with FcRγ deficient (FcRγ-/-) mice and wild type mice (WT), bearing B16F10 or LCC tumor cells. Blood routine test, flow cytometry, immunofluorescent staining and in vivo image were applied for analysis. Platelet adhesion and neutrophil chemotaxis were analyzed by flow cytometry and ELISA in vitro. Platelet adoptive model was used for mimicing early colonization stage. Our results indicated FcRγ deficiency significantly promoted tumor metastasis accompanied with increased number of platelet and neutrophil in the lung. Further investigation showed that FcRγ-/- platelet infusion, rather than FcRγ-/- neutrophils, promoted CTC colonization. While platelet inhibitor Aspirin abrogated the platelet-mediated infiltration of neutrophil in the lung. Mechanistically, platelet FcRγ deficiency facilitated the adhesion of platelets and cancer cells and increased secretion of CXCL5 and CXCL7 which triggered the platelet-induced neutrophil recruitment. In sum, our study indicates that FcRγ is a restrainer in controlling cancer metastasis through regulating the adhesion of platelets and cancer cells and recruiting more neutrophils, which provides a potential target for anti-metastatic therapies. The level of FcRγ expression in platelets could act as a novel biomarker for cancer metastasis.

Brain metastases are the most commonly diagnosed type of central nervous system tumor, yet the mechanisms of their occurrence are still widely unknown. Lung cancer, breast cancer, and melanoma are the most common etiologies, but renal and colorectal cancers have also been described as metastasizing to the brain. Regardless of their origin, there are common mechanisms for progression to all types of brain metastases, such as the creation of a suitable tumor microenvironment in the brain, priming of tumor cells, adaptations to survive spreading in lymphatic and blood vessels, and development of mechanisms to penetrate the blood-brain barrier. However, there are complex genetic and molecular interactions that are specific to every type of primary tumor, making the understanding of the metastatic progression of tumors to the brain a challenging field of study. In this review, we aim to summarize current knowledge on the pathophysiology of brain metastases, from specific genetic characteristics of commonly metastatic tumors to the molecular and cellular mechanisms involved in progression to the central nervous system. We also briefly discuss current challenges in targeted therapies for brain metastases and how there is still a gap in knowledge that needs to be overcome to improve patient outcomes.

Platelets have important roles in hemostasis but also actively participate in cancer metastasis and inflammatory processes. They are produced by large precursor cells, the megakaryocytes, residing mainly in the bone marrow. Clinically, elevated platelet counts and/or increased platelet-to-lymphocyte ratio are being explored as biomarkers of metastatic disease and to predict survival or response to therapy in certain cancers. Multiple mechanisms have been put forward on how platelets promote hematogenous metastasis stemming mainly from murine experimental models. Research is now beginning to explore the potential roles of megakaryocytes in solid cancer, myeloma, and lymphoma. Here, we review mechanisms on how platelets and megakaryocytes contribute to cancer progression and metastasis but also discuss potential cancer-suppressing functions mainly related to the regulation of vascular intratumor integrity. Recent developments in cancer immune checkpoint therapy are reviewed with a focus on the potential roles of platelets. Moreover, we review studies exploring platelets for targeted drug delivery systems in cancer therapy.

Circulating tumor cells (CTCs) are precursors of cancer metastasis. However, how CTCs evade immunosurveillance during hematogenous dissemination remains unclear.

We identified CTC-platelet adhesions by single-cell RNA sequencing and multiplex immunofluorescence of blood samples from multiple cancer types. Clinically, CTC-platelet aggregates were associated with significantly shorter progression-free survival and overall survival in patients with HCC. In vitro, ex vivo, and in vivo assays demonstrated direct platelet adhesions gifted cancer cells with an evasive ability from NK cell killing by upregulating inhibitory checkpoint CD155 (PVR cell adhesion molecule), therefore facilitating distant metastasis. Mechanistically, CD155 was transcriptionally regulated by the FAK/JNK/c-Jun cascade in a platelet contact-dependent manner. Further competition assays and cytotoxicity experiments revealed that CD155 on CTCs inhibited NK-cell cytotoxicity only by engaging with immune receptor TIGIT, but not CD96 and DNAM1, another 2 receptors for CD155. Interrupting the CD155-TIGIT interactions with a TIGIT antibody restored NK-cell immunosurveillance on CTCs and markedly attenuated tumor metastasis.

Our results demonstrated CTC evasion from NK-cell-mediated innate immunosurveillance mainly through immune checkpoint CD155-TIGIT, potentially offering an immunotherapeutic strategy for eradicating CTCs.

Brain metastases (BMs) are the most common intracranial tumors in adults and the major cause of cancer-related morbidity and mortality. The occurrence of BMs varies according to the type of primary tumors with most frequence in lung cancer, melanoma and breast cancer. Among of them, lung cancer has been reported to have a higher risk of BMs than other types of cancers with 40 ~ 50% of such patients will develop BMs during the course of disease. BMs lead to many neurological complications and result in a poor quality of life and short life span. Although the treatment strategies were improved for brain tumors in the past decades, the prognosis of BMs patients is grim. Poorly understanding of the molecular and cellular characteristics of BMs and the complicated interaction with brain microenvironment are the major reasons for the dismal prognosis of BM patients. Recent studies have enhanced understanding of the mechanisms of BMs. The newly identified potential therapeutic targets and the advanced therapeutic strategies have brought light for a better cure of BMs. In this review, we summarized the mechanisms of BMs during the metastatic course, the molecular and cellular landscapes of BMs, and the advances of novel drug delivery systems for overcoming the obstruction of blood-brain barrier (BBB). We further discussed the challenges of the emerging therapeutic strategies, such as synergistic approach of combining targeted therapy with immunotherapy, which will provide vital clues for realizing the precise and personalized medicine for BM patients in the future.

The cellular programs that mediate therapy resistance are often important drivers of metastasis, a phenomenon that needs to be understood better to improve screening and treatment options for cancer patients. Although this issue has been studied extensively for chemotherapy, less is known about a causal link between resistance to radiation therapy and metastasis. We investigated this problem in triple-negative breast cancer (TNBC) and established that radiation resistant tumor cells have enhanced metastatic capacity, especially to bone. Resistance to radiation increases the expression of integrin β3 (ITGβ3), which promotes enhanced migration and invasion. Bioinformatic analysis and subsequent experimentation revealed an enrichment of RNA metabolism pathways that stabilize ITGβ3 transcripts. Specifically, the RNA binding protein heterogenous nuclear ribonucleoprotein L (HNRNPL), whose expression is regulated by Nrf2, mediates the formation of circular RNAs (circRNAs) that function as competing endogenous RNAs (ceRNAs) for the family of let-7 microRNAs that target ITGβ3. Collectively, our findings identify a novel mechanism of radiation-induced metastasis that is driven by alterations in RNA metabolism.

The progression of malignant tumors leads to the development of secondary tumors in various organs, including bones, the brain, liver, and lungs. This metastatic process severely impacts the prognosis of patients, significantly affecting their quality of life and survival rates. Research efforts have consistently focused on the intricate mechanisms underlying this process and the corresponding clinical management strategies. Consequently, a comprehensive understanding of the biological foundations of tumor metastasis, identification of pivotal signaling pathways, and systematic evaluation of existing and emerging therapeutic strategies are paramount to enhancing the overall diagnostic and treatment capabilities for metastatic tumors. However, current research is primarily focused on metastasis within specific cancer types, leaving significant gaps in our understanding of the complex metastatic cascade, organ-specific tropism mechanisms, and the development of targeted treatments. In this study, we examine the sequential processes of tumor metastasis, elucidate the underlying mechanisms driving organ-tropic metastasis, and systematically analyze therapeutic strategies for metastatic tumors, including those tailored to specific organ involvement. Subsequently, we synthesize the most recent advances in emerging therapeutic technologies for tumor metastasis and analyze the challenges and opportunities encountered in clinical research pertaining to bone metastasis. Our objective is to offer insights that can inform future research and clinical practice in this crucial field.

There are currently no relevant studies at home or abroad that combine inflammatory indicators and nomograms to predict the prognosis of gastrointestinal stromal tumor (GIST) patients after surgery. The purpose of this study was to investigate the predictive value of related inflammatory indicators [systemic immune-inflammation index (SII), neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR) and monocyte/Lymphocyte ratio (MLR)] in patients undergoing GIST surgery, incorporating relevant risk factors to establish a nomogram prediction model, with the aim of better predicting the prognosis of GIST patients.

To explore the relationships between the SII, NLR, PLR, and MLR and postoperative recurrence in patients with GIST.

This study retrospectively included patients who underwent GIST surgery from January 2014 to January 2017 and analyzed the potential relationships between the preoperative SII, NLR, PLR, and MLR and clinicopathological features. The independent risk factors influencing the prognosis of GIST patients were obtained via multivariate regression analysis, and a nomogram model based on the independent risk factors was established.

Among the 124 GIST patients included in the present study, 31 (25%) experienced recurrence within 5 years. Kaplan-Meier survival analysis revealed a correlation between the MLR and PLR and tumor size (P = 0.016 and P = 0.002, respectively). The preoperative SII, MLR, NLR, and PLR were significantly associated with recurrence-free survival (RFS) (P < 0.05). The multivariate analysis results identified the PLR, MLR, and targeted therapy as independent prognostic factors for patient outcomes.

Preoperative MLR and PLR, which are independent risk factors for GIST recurrence, were correlated with RFS. Nomograms based on the PLR, MLR and targeted therapy can be used for clinical treatment.

Tumor metastasis leads to circulating tumor cells (CTCs) that separate from primary malignant tumors and enter blood circulation. CTCs survive and engage with other cells to cope with obstacles, including shear stress, disease, immune attacks, and drugs. Platelets are the best partners for CTCs. Platelets provide a good protective layer for CTCs to ensure that are not monitored and cleared by the native immune system, and protected from shear stress and survive better. Here, we review current reports on platelet-CTC interaction and the clinical relevance of their combination and summarize new techniques for CTC capture and treatment based on platelet-CTC interaction. We discuss current data, identify its shortcomings, and suggest future developments.

Platelets, anucleate blood cells essential for hemostasis, are increasingly recognized for their role in cancer, challenging the traditional notion of their sole involvement in blood coagulation. It has been demonstrated that platelets establish bidirectional communication with tumor cells, contributing to tumor progression and metastasis through diverse molecular mechanisms such as modulation of proliferation, angiogenesis, epithelial-mesenchymal transition, resistance to anoikis, immune evasion, extravasation, chemoresistance, among other processes. Reciprocally, cancer significantly alters platelets in their count and composition, including mRNA, non-coding RNA, proteins, and lipids, product of both internal synthesis and the uptake of tumor-derived molecules. This phenomenon gives rise to tumor-educated platelets (TEPs), which are emerging as promising tools for the development of liquid biopsies. In this review, we provide a detailed overview of the dynamic roles of platelets in tumor development and progression as well as their use in diagnosis and prognosis. We also provide our view on current limitations, challenges and future research areas, including the need to design more efficient strategies for their isolation and analysis, as well as the validation of their sensitivity and specificity through large-scale and rigorous clinical trials. This research will not only enable the evaluation of their clinical viability but could also open new opportunities to enhance diagnostic accuracy and develop personalized treatments in oncology.

The prognostic value of the systemic immune-inflammation index (SII) for prostate cancer (PCa) patients receiving different treatments remains unclear. This research examined the relevance of SII in individuals undergoing radical prostatectomy (RP).

PubMed, Embase, Web of Science, Cochrane, Wanfang, and China National Knowledge Infrastructure (CNKI) dat3 abases were used to search literature up to May 2024. The quality was evaluated with Newcastle-Ottawa Scale. Outcomes examined were associations between SII and overall survival (OS), biochemical recurrence-free survival (BFS), and cancer-specific survival (CSS). Pooled analysis, Egger's test, and sensitivity analysis were conducted using Review Manager 5.4.1 and Stata 15.1. The GRADE system was employed to evaluate and grade the evidence for each outcome. Subgroup analyses were performed for outcomes with significant heterogeneity to evaluate the possible confounders, if data were sufficient.

Out of 101 identified studies, eight studies involving 8,267 individuals were included. Patients with higher SII had shorter overall survival (HR: 1.89; 95% CI: 1.31-2.71; P = 0.0006), biochemical recurrence-free survival (HR: 1.55; 95% CI: 1.08-2.22; P = 0.02), and cancer-specific survival (HR: 3.63; 95% CI: 1.66-7.94; P = 0.001). The evidence for OS and CSS was rated very low-quality due to serious heterogeneity and/or imprecision. The prognostic value of SII for BFS was rated as low-quality evidence, given no serious risk observed. Subgroup analysis showed that, except for the subgroup aged >65 years (HR: 3.70; 95%CI: 0.91, 15.06, P=0.07), the prognostic value of SII for OS was not significant, but the prognostic value of SII for OS in other subgroups was still significant.

High SII was linked to shorter OS, BFS, and CSS in patients undergoing RP. However, the quality of the evidence provided by this study was low.

https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42024558431.

The interaction between platelets and tumor cells is a crucial step in the progression of tumor metastasis. Blocking platelet-tumor cell interaction is a potential target against metastasis. Ginsenoside Rb (G-Rb) exhibits potential anti-tumor pharmacological properties and may offer a therapeutic option for cancer.

This study aimed to investigate anti-metastatic effects of G-Rb through regulating the crosstalk of platelets with tumor cells.

In order to explore anti-metastatic effects of G-Rb in vitro, HepG2 cell and platelets were co-cultured to mimic the interaction of platelets with tumor cells. Wound healing and Transwell assays were used to assess the effect of G-Rb on cell migration and invasion. The expression of epithelial-mesenchymal transition (EMT)-related markers was determined by RT-qPCR and western blot assays. The aggregation and activation of platelets were detected by flow cytometry. Moreover, a lung metastasis model of mice was established to evaluate inhibitory effects of G-Rb in vivo. Metastatic nodules on the lung surface were counted and sections of lung tissues were stained by H&E.

G-Rb effectively suppressed tumor metastasis in the co-culture of platelets with HepG2 cell. First, G-Rb treatment significantly inhibited the migration and invasion of HepG2 cells induced by platelets. Second, the expressions of EMT-related markers, including N-cadherin, Snail, and MMP9, were decreased by the treatment of G-Rb in the presence of platelets. Meanwhile, G-Rb also suppressed platelet hyperactivity by regulating the adhesion to tumor cells, activation, TCIPA, and TGF-β1 secretion of platelets in vitro. In addition, the results of in vivo experiments proved G-Rb administration not only significantly decreased lung metastasis but also attenuated platelets aberrant aggregation and activation in vivo.

Our findings showed that G-Rb inhibited tumor metastasis and platelet activation through mediating platelet-tumor cell interaction, indicating the potential values of G-Rb in tumor metastasis therapy.

Circulating tumor cells (CTCs) are shed from primary tumors and travel through the body via circulation, eventually settling to form micrometastases under favorable conditions. Numerous studies have identified CTCs as a negative prognostic indicator for survival across various cancer types. CTCs mirror the current heterogeneity and genetic and biological state of tumors, making their study invaluable for understanding tumor progression, cell senescence, and cancer dormancy. However, their isolation and characterization still poses a major challenge that limits their clinical translation. A wide array of methods, each with different levels of specificity, utility, cost, and sensitivity, have been developed to isolate and characterize CTCs. Moreover, innovative techniques are emerging to address the limitations of existing methods. In this review, we provide insights into CTC biology addressing spectra of markers employed for molecular analysis and functional characterization. It also emphasizes current label-dependent and label-independent isolation procedures, addressing their strengths and limitations. SIGNIFICANCE: A comprehensive overview of CTC biology, their molecular and functional characterization, along with their current clinical utility will help in understanding the present-day extent to which the clinical potential of CTCs is getting tapped in personalized medicine.

Given the role of inflammation in cancer progression, the systemic immune-inflammation index (SII, defined as platelet × neutrophil/lymphocyte) has been suggested as an emerging prognostic marker in several solid malignant neoplasms. However, there are few studies on the prognostic value of SII in patients with limited-stage small cell lung cancer (LS-SCLC), and the optimal threshold of SII remains unclear in this population. This study calculated the optimal threshold of SII by a reasonable method and explored its association with survival outcomes.

This retrospective study reviewed clinical data of 572 patients with LS-SCLC. The threshold for SII was determined using an outcome-based method by maximizing the log-rank test statistic and the survival differences. Continuous time-dependent receiver operating characteristic curves (time-dependent ROC curves) were used to clarify the predictive ability of SII.

The thresholds of SII for overall survival (OS) and progression-free survival (PFS) were both 760.6, based on which patients were divided into low [292 cases (51.0%)] and high [280 cases (49.0%)] SII groups. The area under the time-dependent ROC curves of SII in 12-, 24-, and 36-months were 0.727, 0.708, and 0.680, respectively. The overall median OS and PFS were 26.0 months [95% confidence interval (CI): 23.8-28.2] and 13.0 months (95% CI: 11.3-14.7), respectively. Significantly improved OS [35.0 (95% CI: 30.0-40.0) vs. 19.0 months (95% CI: 17.1-20.9), P<0.001] and PFS [20.0 (95% CI: 17.3-22.7) vs. 11.0 months (95% CI: 9.9-12.1), P<0.001] was seen in the low SII group than that in the high SII group. In the multivariable survival analysis, SII remained an independent prognostic factor for OS [hazard ratio (HR): 1.699; 95% CI: 1.374-2.100; P=0.001] and PFS (HR: 1.482; 95% CI: 1.214-1.809; P<0.001).

Our study demonstrates that elevated SII is an independent adverse prognostic factor for LS-SCLC.

Chronic low-grade inflammation is recognized as a significant factor in various health outcomes, including the development and progression of breast cancer. The Systemic Immune-Inflammation Index (SII), a novel marker derived from routine blood counts, has been suggested as a predictor of all-cause mortality and cardiovascular mortality. However, its predictive value in a nationwide representative population, particularly for breast cancer incidence and mortality, is not well-established.

This study aimed to assess the association of SII and the risk of breast cancer incidence and all-cause mortality in breast cancer patients within the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2018. SII was calculated from complete blood count parameters. We used multifactor regression models to examine the associations between SII and the outcomes of interest.

A total of 21,058 female participants were included in the study, of which 557 (2.7%) were identified as having breast cancer. After adjusting for multiple potential confounders, the relationship between SII and the incidence of breast cancer revealed an inverse L-shaped association. The optimal inflection point for SII/100 was determined to be 5.09. Below this threshold, there was a significant increase in the risk of breast cancer (OR=1.05, 95% CI: 1.02-1.09). Within the breast cancer population, SII exhibited a J-shaped relationship with all-cause mortality. The optimal inflection point for SII/100 in this context was 5.22, and above this threshold, there was a marked escalation in all-cause mortality (HR=1.09, 95% CI: 1.04-1.14).

The SII, as a novel inflammatory composite index, is significantly associated with the risk of breast cancer incidence and all-cause mortality in breast cancer patients. These findings highlight the importance of monitoring systemic inflammation and suggest that SII could serve as a valuable prognostic tool.

Metastasis is the leading cause of cancer-related deaths and poses a treatment challenge. Although studies have shown the importance of epithelial-mesenchymal transition (EMT) and metabolic reprogramming during cancer metastasis, the link between EMT and metabolic reprogramming, as well as the underlying molecular mechanisms by which both mediate cancer cell invasion and metastasis have not been elucidated. Here, we observed that interactions between platelets and cancer cells promote the secretion of TGF-β, thereby initiating EMT, promoting the invasion, and altering the metastatic and metabolic potential of colon cancer cells. TGF-β activates the AKT signaling pathway to enhance HK1 and HK2 expression in cancer cells, leading to increased glucose consumption, ATP production, and precise modulation of cell cycle distribution. In an energy-deficient model induced by oxidative phosphorylation (OXPHOS) inhibition with oligomycin A, TGF-β-induced highly metastatic HCT116 (H-HCT116) cells adapt by upregulating HK expression and glycolytic metabolism, while concurrently decreasing cell proliferation to conserve energy for survival. Mechanistically, H-HCT116 cells regulate cell division rates by downregulating CDK2, CDK4, and Cyclin D1 protein expression and upregulating p21 expression. Furthermore, H-HCT116 cells display enhanced motility, which is linked to increased mitochondrial metabolic activity. These findings indicated that cancer cells-platelets interaction secreted TGF-β activates cancer metastasis potential by inducing metabolic reprogramming and bioenergetic adaptation. The present study provides new insights into the adaptive strategies of highly metastatic cancer cells under adverse conditions and indicates that targeting glycolysis and metabolic reprogramming could serve as a viable approach to prevent cancer metastasis.

Despite being discovered decades ago, metastasis remains a formidable challenge in cancer treatment. During the intermediate phase of metastasis, tumor cells detach from primary tumor or metastatic sites and travel through the bloodstream and lymphatic system to distant tissues. These tumor cells in the circulation are known as circulating tumor cells (CTCs), and a higher number of CTCs has been linked to poor prognoses in various cancers. The blood is an inhospitable environment for any foreign cells, including CTCs, as they face numerous challenges, such as the shear stress within blood vessels and their interactions with blood and immune cells. However, the exact mechanisms by which CTCs survive the hostile conditions of the bloodstream remain enigmatic. Platelets have been studied for their interactions with tumor cells, promoting their survival, growth, and metastasis. This review explores the latest clinical methods for enumerating CTCs, recent findings on platelet-CTC crosstalk, and current research on antiplatelet therapy as a potential strategy to inhibit metastasis, offering new therapeutic insights.

Laboratory and clinical data have provided insights into the role of platelets in promoting CTC survival, while clinical advancements in CTC enumeration offer improved prognostic tools.

CTCs play a critical role in metastasis, and their interactions with platelets aid their survival in the hostile environment of the bloodstream. Understanding this crosstalk offers insights into potential therapeutic strategies, including antiplatelet therapy, to inhibit metastasis and improve cancer treatment outcomes.

Tumor immunity has garnered increasing attention in cancer treatment and progression. However, there is still a challenge in understanding the mechanisms of specific molecules affecting the clinical prognosis and tumor microenvironment (TME).

Here, we applied the ESTIMATE algorithm to calculate the immune and stromal scores in 504 HNSC cases from TCGA. Patients were grouped according to the median value of the immune and stromal. Clinicopathological characteristics and differentially expressed genes (DEG) were analyzed. Subsequently, LASSO, COX regression, survival analysis, and clinicopathological characteristics were conducted. Subsequently, SLC2A3 was determined as a predictive factor that high expression of SLC2A3 at the mRNA and protein levels predicted a worse clinical prognosis. GSEA25099 was utilized for external validation of immune infiltration, while tissue PCR, IHC, and Western Blot were used to confirm the expression levels of SLC2A3.

A series of immune-infiltration analyses showed that SLC2A3 expression was negatively correlated with CD8+ T cells, significantly affecting the survival prognosis of HNSC. In the GSEA analysis, the high expression of SLC2A3 was mainly enriched for immune-related biological processes. Meanwhile, high expression of SLC2A3 possessed higher TIDE scores and was also strongly positively correlated with a series of immune checkpoints affecting survival prognosis, thus causing greater susceptibility to immune escape.

Conclusively, SLC2A3 is a potential oncogene and factor of HNSC development, notably by an altered state of the immune microenvironment, immune-suppressive regulation, and immune escape.

The association between aspirin and hepatocellular carcinoma (HCC) has been reported to prevent carcinogenesis caused by hepatitis B or C virus infection. The objective of this study was to investigate the prognostic impact of aspirin in patients who underwent liver resection for HCC.

Data for 1032 patients who underwent primary resection for HCC between 2000 and 2019 were reviewed. There were 87 patients (8.4%) who took aspirin (aspirin group) and 945 (91.6%) who did not (non-aspirin group). Short-term outcomes, recurrence-free survival (RFS), and overall survival (OS) were compared between two groups in the matched cohort using propensity-score matching.

The median patient follow-up was 42.6 months (95% confidence interval 3.12-136.8 months). There was no significant difference in short-term outcomes, including bleeding events. RFS and OS after liver resection in the aspirin group were significantly better than those in the non-aspirin group in the unmatched cohort [5-year RFS rate: 50.3% vs 31.4%, hazard ratio (HR) 0.55, P = 0.0002; 5-year OS rate: 82.9% vs 70.2%, HR 0.46, P = 0.002]. In the matched cohort, RFS and OS after liver resection in the aspirin group were also significantly better than those in the non-aspirin group (5-year RFS rate: 50.3% vs 32.0%, HR 0.60, P = 0.003; 5-year OS rate: 82.9% vs 74.6%, HR 0.56, P = 0.03).

Use of aspirin was associated with better prognosis for patients who underwent primary resection for HCC.

Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal hepatobiliary malignancy with an increasing incidence annually. Extensive research has elucidated the existence of a reciprocal interaction between platelets and cancer cells, which promotes tumor proliferation and metastasis. This study aims to investigate the function and mechanism underlying iCCA progression driven by the interplay between platelets and tumor cells, aiming to provide novel therapeutic strategies for iCCA.

The associations between platelets and cancer development were investigated by analyzing the peripheral blood platelet count, degree of platelet activation and infiltration in the microenvironment of patients with iCCA. By co-culturing tumor cells with platelets, the influence of platelet stimulation on the epithelial-mesenchymal transition (EMT), proliferation, and metastasis of iCCA cells was assessed through in vitro and in vivo experiments. Quantitative proteomic profiling was conducted to identify key downstream targets that were altered in tumor cells following platelet stimulation. The RNA interference technique was utilized to investigate the impacts of gene silencing on the malignant biological behaviors of tumor cells.

Compared with healthy adults, patients with iCCA presented significantly higher levels of peripheral blood platelet counts, platelet activation and infiltration degrees, which were also found to be correlated with patient prognosis. Platelet stimulation greatly facilitated the EMT of iCCA cells, leading to enhanced proliferative and metastatic capabilities. Mechanistically, proteomic profiling identified a total of 67 up-regulated and 40 down-regulated proteins in iCCA cells co-cultured with platelets. Among these proteins, two elevated targets ILK and ITGB3, were further demonstrated to be partially responsible for platelet-induced iCCA progression, which might depend on their regulatory effects on FAK/PI3K/AKT signaling transduction.

Our data revealed that platelet-related indices were abnormally ascendant in iCCA patients compared to healthy adults. Co-culturing with platelets enhanced the progression of EMT, and the motility and viability of iCCA cells in vitro and in vivo. Proteomic profiling discovered that platelets promoted the development of iCCA through FAK/PI3K/AKT pathway by means of elevating the expression of ILK and ITGB3, indicating that both proteins are promising therapeutic targets for iCCA with the guidance of platelet-related indices.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related death worldwide, with no precise method for early detection. Circulating tumor cells (CTCs) expressing the dynamic polarity of the cytoskeletal membrane protein, ezrin, have been proposed to play a crucial role in tumor progression and metastasis. This study investigated the diagnostic and prognostic potential of polarized circulating tumor cells (p-CTCs) in HCC patients. CTCs were isolated from the peripheral blood of 20 HCC patients and 18 patients with nonmalignant liver disease (NMLD) via an OncoQuick® kit and immunostained with Ezrin-Alexa Fluor 488®, CD146-PE, and CD45-APC. A fluorescence microscopy was then performed for analysis. The HCC group exhibited significantly higher levels of p-CTCs, with median values of 0.56 p-CTCs/mL, compared to 0.02 p-CTCs/mL (p = 0.03) in the NMLD group. CTCs were detected in 95% of the HCC patients, with a sensitivity of 95% and specificity of 89%. p-CTCs were present in 75% of the HCC patients, with a sensitivity of 75% and a specificity of 94%. Higher p-CTC counts were associated with the significantly longer overall survival in HCC patients (p = 0.05). These findings suggest that p-CTCs could serve as valuable diagnostic and prognostic markers for HCC. The incorporation of p-CTCs into diagnostic strategies could enhance therapeutic decision-making and improve patient outcomes.

Efforts to deconvolve the complex interactions of cancer cells with other components of the tumor micro- and macro-environment have exposed a common tendency for cancers to subvert systems physiology and exploit endogenous programs involved in homeostatic control of metabolism, immunity, regeneration, and repair. Many such programs are engaged in the healing response to surgery which, together with other abrupt biochemical changes in the perioperative period, provide an opportunity for the macroevolution of residual disease. This review relates contemporary perspectives of cancer as a systemic disease with the overlapping biology of host responses to surgery and events within the perioperative period. With a particular focus on examples of cancer cell plasticity and changes within the host, we explore how perioperative inflammation and acute metabolic, neuroendocrine, and immune dyshomeostasis might contribute to cancer evolution within this contextually short, yet crucially influential timeframe, and highlight potential therapeutic opportunities within to further optimize surgical cancer care and its long-term oncological outcomes.

There is currently no research on the correlation between novel inflammatory indexes systemic immune-inflammation index (SII), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and the risk of anemia in chronic kidney disease (CKD) population, as well as survival analysis in CKD with anemia.

This investigation encompassed 4444 adult subjects out of the National Health and Nutrition Examination Survey (NHANES) between 2005 and 2018. The study utilized multi-variable logistic regression to assess the relationship between SII, NLR, PLR, and anemia risk occurrence in CKD population. Survival differences in CKD patients with anemia, based on varying levels of SII, NLR, and PLR were evaluated employing Kaplan-Meier and Cox proportional hazards models.

The adjusted logistic regression model demonstrates that SII, NLR, and PLR are associated with the risk of anemia occurrence in CKD population. Kaplan-Meier's analysis reveals significant differences in survival rates among CKD patients with anemia stratified by NLR levels. The adjusted Cox proportional hazards model shows that the higher NLR group has a 30% elevated risk of all-cause mortality contrasted with lower group (hazard ratio, HR: 1.30, confidence interval (CI) [1.01, 1.66], p value <.04). Restricted cubic spline (RCS) demonstrates no nonlinear relationship between NLR and all-cause mortality. Lastly, sub-cohort analysis indicates that in populations with diabetes, hypertension, and hyperuricemia, NLR levels have a greater impact on all-cause mortality.

Controlling inflammation may reduce the occurrence of anemia in CKD populations, with NLR serving to be a potential prognostic indicator for survival results within CKD patients suffering from co-morbid anemia.

Cervical cancer ranks as the fourth most common and fatal cancer among women worldwide. Studies have demonstrated a strong association between purinergic platelet signaling and tumor progression in this type of cancer. The literature shows that neoplastic cells, when in the bloodstream, secrete adenosine triphosphate (ATP) and adenosine nucleotide diphosphate (ADP) that act on their corresponding platelet P2Y and P2X receptors. The interaction of these nucleotides with their receptors results in platelet activation and degranulation, ensuing several consequences, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor, matrix metalloproteinases, ADP, and ATP. These molecules play essential roles in angiogenesis and tumor metastasis in cervical cancer. Several purinergic receptors are found in endothelial cells. Their activation, especially P2Y2, by the nucleotides released by platelets can induce relaxation of the endothelial barrier and consequent extravasation of tumor cells, promoting the development of metastases. Cancer cells that enter the bloodstream during the metastatic process are also subject to high shear stress and immune surveillance. In this context, activated platelets bind to circulating tumor cells and protect them against shear stress and the host's immune system, especially against natural killer cells, facilitating their spread throughout the body. Furthermore, activation of the P2Y12 receptor present on the platelet surface promotes the release of VEGF, the main inducer of angiogenesis in cervical cancer, in addition to increasing the concentration of several other pro-angiogenic molecules. Therefore, this review will address the role of platelet purinergic signaling in tumor progression of cervical cancer and propose possible therapeutic targets.

The current constraints associated with cancer diagnosis and molecular profiling, which rely on invasive tissue biopsies or clinical imaging, have spurred the emergence of the liquid biopsy field. Liquid biopsy involves the extraction of circulating tumor cells (CTCs), circulating free or circulating tumor DNA (cfDNA or ctDNA), circulating cell-free RNA (cfRNA), extracellular vesicles (EVs), and tumor-educated platelets (TEPs) from bodily fluid samples. Subsequently, these components undergo molecular characterization to identify biomarkers that are critical for early cancer detection, prognosis, therapeutic assessment, and post-treatment monitoring. These innovative biosources exhibit characteristics analogous to those of the primary tumor from which they originate or interact. This review comprehensively explores the diverse technologies and methodologies employed for processing these biosources, along with their principal clinical applications.

Epithelial ovarian carcinoma (EOC) is the eighth most common cancer in women and the leading cause of gynaecological cancer death, predominantly due to the absence of effective screening tools, advanced stage at diagnosis, and high rates of recurrence. Circulating tumour cells (CTCs), a rare subset of tumour cells that disseminate from a tumour and migrate into the circulation, play a pivotal role in the metastatic cascade, and therefore hold promise as biomarkers for disease monitoring and prognostication. Exploring CTCs from liquid biopsies is an appealing approach for research and clinical practice, given it is minimally invasive, facilitates serial sampling and enables the capture of the entire spectrum of cancer cells circulating in the blood. The prognostic utility of CTC enumeration has been FDA-approved for clinical use in metastatic breast, prostate, and colorectal cancers. However, the unique biology of EOC, discussed herein, compounds the detection and characterisation complexities already inherent in CTC research, consequently hindering progress towards clinical applications. The aim of this review is to provide an overview of both the biological and clinical challenges encountered in harnessing the power of CTCs in EOC.

Recent studies have identified SLC2A3 as being abnormally upregulated in multiple tumor types, correlating with poor survival and disrupted microenvironments. However, its prognostic significance in head and neck squamous cell carcinoma (HNSC) remains underexplored. In this study, SLC2A3 was screened as a potential risk gene influencing both immune and tumor components within the tumor microenvironment (TME) of 504 HNSC patients from the TCGA database. Immune infiltration analyses and clinical significance on SLC2A3 were conducted using ESTIMATE, CIBERSORT, ssGSEA, TIMER and clinical prognosis parameters. Additionally, the single-cell dataset is used to analyze the expression of SLC2A3 in various subpopulations. The magnetic activated cell sorting (MACS) is used to isolate CD8+ T cells from PBMCs or tumor tissues. Flow cytometry is used to identify purified and activated CD8+ T cells. GSEA and WB were used to investigate the molecular mechanism of SLC2A3 in CD8+ T cells. The co-culture system of CD8+ T cells and TU686 was used to investigate the effects of SLC2A3 on immune cells and tumor development. In this study, SLC2A3 was identified as a potential risk gene affecting both immune cells and tumor components within the TME of 504 HNSC patients derived from the TCGA database. We conducted immune infiltration analyses and assessed the clinical significance of SLC2A3 using various bioinformatics tools, including ESTIMATE, CIBERSORT, ssGSEA, and TIMER, along with clinical prognosis parameters. The single-cell RNA sequencing dataset was utilized to examine SLC2A3 expression across different cellular subpopulations. Magnetic activated cell sorting (MACS) was employed to isolate CD8+ T cells from peripheral blood mononuclear cells (PBMCs) or tumor tissues. Flow cytometry was implemented to confirm the purity and activation state of the isolated CD8+ T cells. GSEA and Western blot were applied to explore the molecular mechanisms underlying SLC2A3's role in CD8+ T cells. Lastly, a co-culture system involving CD8+ T cells and TU686 tumor cells was established to study the impact of SLC2A3 on immune cell function and tumor progression. SLC2A3 emerges as an actively variable gene within the immune and stromal components of the TME, linked to aggravated immune infiltration and poor clinical outcomes. The upregulated expression of SLC2A3 is predominantly enriched in immune-related biological processes and linked to the suppression of CD8+ T cells, which are crucial for the survival of HNSC patients. Furthermore, SLC2A3 exhibits specific overexpression in CD8+ T cells and may potentially trigger ferroptosis. Knockdown of SLC2A3 led to a significant increase in the proliferation of CD8+ T cells compared to those without knockdown. In co-culture systems, CD8+ T cells with SLC2A3 knockdown demonstrated an enhanced ability to eliminate tumor cells compared to those without the knockdown. SLC2A3 is associated with changes in the TME and prognostic indicators. Moreover, high SLC2A3 expression in CD8+ T cells may drive cell death through ferroptosis, fostering tumor progression.

Pain is a prevalent discomfort symptom associated with cancer, yet the correlations and potential mechanisms between pain and the efficacy of cancer immunotherapy remain uncertain.

Non-small cell lung cancer (NSCLC) patients who received immune checkpoint inhibitors (ICIs) in the inpatient department of Guangdong Provincial Hospital of Chinese Medicine from January 1, 2018, to December 31, 2021, were retrospectively enrolled. Through cox regression analysis, prognostic factors and independent prognostic factors affecting the efficacy of ICIs were identified, and a nomogram model was constructed. Hub cancer-related pain genes (CRPGs) were identified through bioinformatic analysis. Finally, the expression levels of hub CRPGs were detected using an enzyme-linked immunosorbent assay (ELISA).

Before PSM, a total of 222 patients were enrolled in this study. Univariate and multivariate cox analysis indicated that bone metastasis and NRS scores were independent prognostic factors for the efficacy of ICIs. After PSM, a total of 94 people were enrolled in this study. Univariate cox analysis and multivariate cox analysis indicated that age, platelets, Dnlr, liver metastasis, bone metastasis, and NRS scores were independent prognostic factors for the efficacy of ICIs. A nomogram was constructed based on 6 independent prognostic factors with AUC values of 0.80 for 1-year, 0.73 for 2-year, and 0.80 for 3-year survival. ELISA assay results indicated that the level of CXCL12 significantly decreased compared to baseline after pain was relieved.

Baseline pain is an independent prognostic factor affecting the efficacy of ICIs in lung cancer, potentially through CXCL12-mediated inflammation promotion and immunosuppression.

Metastatic disease and cancer recurrence are the primary causes of cancer-related deaths. Circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) are the driving forces behind the spread of cancer cells. The emergence and development of liquid biopsy using rare CTCs as a minimally invasive strategy for early-stage tumor detection and improved tumor management is a promising advancement in recent years. However, before blood sample analysis and clinical translation, precise isolation of CTCs from patients' blood based on their biophysical properties, followed by molecular identification of CTCs using single-cell multi-omics technologies is necessary to understand tumor heterogeneity and provide effective diagnosis and monitoring of cancer progression. Additionally, understanding the origin, morphological variation, and interaction between CTCs and the primary and metastatic tumor niche, as well as and regulatory immune cells, will offer new insights into the development of CTC-based advanced tumor targeting in the future clinical trials.

Platelets can dynamically regulate tumor development and progression. Nevertheless, research on the predictive value and specific roles of platelets in gastric cancer (GC) is limited. This research aims to establish a predictive platelets-related gene signature in GC with prognostic and therapeutic implications.

We downloaded the transcriptome data and clinical materials of GC patients (n=378) from The Cancer Genome Atlas (TCGA) database. Prognostic platelets-related genes screened by univariate Cox regression were included in Least Absolute Shrinkage and Selection Operator (LASSO) analysis to construct a risk model. Kaplan-Meier curves and receiver operating characteristic curves (ROCs) were performed in the TCGA cohort and three independent validation cohorts. A nomogram integrating the risk score and clinicopathological features was constructed. Functional enrichment and tumor microenvironment (TME) analyses were performed. Drug sensitivity prediction was conducted through The Cancer Therapeutics Response Portal (CTRP) database. Finally, the expression of ten signature genes was validated by quantitative real-time PCR (qRT-PCR).

A ten-gene (SERPINE1, ANXA5, DGKQ, PTPN6, F5, DGKB, PCDH7, GNG11, APOA1, and TF) predictive risk model was finally constructed. Patients were categorized as high- or low-risk using median risk score as the threshold. The area under the ROC curve (AUC) values for the 1-, 2-, and 3-year overall survival (OS) in the training cohort were 0.670, 0.695, and 0.707, respectively. Survival analysis showed a better OS in low-risk patients in the training and validation cohorts. The AUCs of the nomogram for predicting 1-, 2-, and 3-year OS were 0.708, 0.763, and 0.742, respectively. TME analyses revealed a higher M2 macrophage infiltration and an immunosuppressive TME in the high-risk group. Furthermore, High-risk patients tended to be more sensitive to thalidomide, MK-0752, and BRD-K17060750.

The novel platelets-related genes signature we identified could be used for prognosis and treatment prediction in GC.

Cancer metastasis remains a major challenge in the clinical management of triple-negative breast cancer (TNBC). The NF-κB signaling pathway has been implicated as a crucial factor in the development of metastases, but the underlying molecular mechanisms remain largely unclear.

PTPN20 expression was evaluated using data from the Sweden Cancerome Analysis Network-Breast and The Cancer Genome Atlas database, as well as by western blotting and immunohistochemistry in 88 TNBC patients. The ability of PTPN20 to activate NF-κB was assessed by luciferase reporter assays. The effects of PTPN20 overexpression and knockdown via short hairpin RNA were examined in TNBC cell lines by wound healing and transwell matrix penetration assays. Additionally, we analyzed the growth and metastasis abilitiy of 4T1 xenograft tumors in nude mice.

PTPN20 levels were elevated in TNBC cell lines and patient samples compared to controls, and higher protein levels correlated with metastasis-free survival. Overexpression of PTPN20 enhanced migration and invasion in vitro, and promoted lung metastasis in vivo. Our finding revealed that PTPN20 activates NF-κB signaling by dephosphorylating p65 at Ser468, preventing its binding to COMMD1, thereby protecting p65 from degradation. Downregulation of PTPN20 effectively inhibit, while p65 S468A mutant restored the migratory and invasive abilities of TNBC cells.

Collectively, our results demonstrate that PTPN20 plays a critical role in TNBC metastasis through the activation of NF-κB signaling. We propose that PTPN20 may serve as a novel prognostic marker and potential therapeutic target for the treatment of TNBC.

Circulating tumor cells (CTCs) are cancer cells that detach from the primary tumor and enter the bloodstream, where they can seed new metastatic lesions in distant organs. CTCs are often associated with white blood cells (WBCs), especially neutrophils, the most abundant and versatile immune cells in the blood. Neutrophils can interact with CTCs through various mechanisms, such as cell-cell adhesion, cytokine secretion, protease release, and neutrophil extracellular traps (NETs) formation. These interactions can promote the survival, proliferation, invasion, and extravasation of CTCs, as well as modulate the pre-metastatic niche and the tumor microenvironment. Therefore, inhibiting CTC-neutrophils interaction could be a potential strategy to reduce tumor metastasis and improve the prognosis of cancer patients. In this review, we summarize the current literature on CTC-neutrophils interaction' role in tumor metastasis and discuss the possible therapeutic approaches to target this interaction.