The advent of tissue engineering and biomedical techniques has significantly advanced the development of three-dimensional (3D) cell culture systems, particularly tumor organoids. These self-assembled 3D cell clusters closely replicate the histopathological, genetic, and phenotypic characteristics of primary tissues, making them invaluable tools in cancer research and drug screening.

This review addresses the challenges in developing in vitro models that accurately reflect tumor heterogeneity and explores the application of tumor organoids in cancer research, with a specific focus on the screening of natural products for antitumor therapies.

This review synthesizes information from major databases, including Chemical Abstracts, Medicinal and Aromatic Plants Abstracts, ScienceDirect, Google Scholar, Scopus, PubMed and Springer Link. Publications were selected without date restrictions, using terms such as 'organoid', 'natural product', 'pharmacological', 'extract', 'nanomaterial' and 'traditional uses'. Articles related to agriculture, ecology, synthetic work or published in languages other than English were excluded.

The review identifies key challenges related to the efficiency and variability of organoid generation and discusses ongoing efforts to enhance their predictive capabilities in drug screening and personalized medicine. Recent studies utilizing patient-derived organoid models for natural compound screening are highlighted, demonstrating the potential of these models in developing new classes of anticancer agents. The integration of natural products with patient-derived organoid models presents a promising approach for discovering novel anticancer compounds and elucidating their mechanisms of action.

Cholangiocytes are highly specialised cells participating in the pathobiology of various liver diseases and recognised to play a crucial role in response to liver injury. Cholangiocytes exhibit dramatic heterogeneity and plasticity, with distinct subtypes performing disparate functions during liver injury and regeneration. Acting as the liver progenitor cells, cholangiocytes can also convert to hepatocytes in the context of impaired hepatocyte proliferation. Harnessing the intrinsic regenerative ability of cholangiocytes is of great importance to alleviate liver injury and promote cholangiocyte-driven liver regeneration. Clinically, cholangiocytes and cholangiocyte organoids are expected to serve as favourable sources for cell therapy in cholangiopathies, which are known as a group of complex diseases involving the biliary system while lacking effective therapeutic options. A comprehensive understanding of the biological characteristics of cholangiocytes provides insights into developing cholangiocyte cell therapy for cholangiopathies. In this review, we discuss the critical role of cholangiocytes in liver injury and regeneration, reveal the underlying mechanism of cholangiocyte plasticity, and explore the prospects and challenges of using cholangiocytes as a source for cell therapy.

Malignant tumors notably decrease life expectancy. Despite advances in cancer diagnosis and treatment, the mechanisms underlying tumorigenesis, progression and drug resistance have not been fully elucidated. An emerging method to study tumors is tumor organoids, which are a three‑dimensional miniature structure. These retain the patient‑specific tumor heterogeneity while demonstrating the histological, genetic and molecular features of original tumors. Compared with conventional cancer cell lines and animal models, patient‑derived tumor organoids are more advanced at physiological and clinical levels. Their synergistic combination with other technologies, such as organ‑on‑a‑chip, 3D‑bioprinting, tissue‑engineered cell scaffolds and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‑associated protein 9, may overcome limitations of the conventional 3D organoid culture and result in the development of more appropriate model systems that preserve the complex tumor stroma, inter‑organ and intra‑organ communications. The present review summarizes the evolution of tumor organoids and their combination with advanced technologies, as well as the application of tumor organoids in basic and clinical research.

Cellular phenotypes are regulated by dynamic signalling processes that involve proteins, post-translational modifications, epigenetic events, and transcriptional responses. Functional perturbation studies are required to understand cell signalling mechanisms and organoids have recently emerged as scalable biomimetic models amenable to large-scale perturbation. Here, we review the recent advances in high-dimensional analysis of cell signalling in organoids. Single-cell technologies provide cell-type specific analysis of multiple biochemical modalities, enabling a deeper understanding of the signalling mechanisms driving cell-fate dynamics. Emerging multimodal techniques are further revealing coordination between signalling layers and are poised to increase our mechanistic understanding of cell signalling.

Patients with limbal dysfunction, which occurs when corneal epithelial stem cells are depleted, require the transplantation of donor corneal epithelial stem cells or donor-independent cell sources. This study aimed to establish organoids with limbal epithelial progenitor cell function from the central cornea, where stem cells do not reside in vivo. We confirmed the regenerative capacity of organoids in a rabbit limbal deficiency model.

After treatment with collagenase, central corneal epithelial cells were scraped from corneal tissue and seeded onto Matrigel. For comparison, cells were collected from the limbus. The cells were cultured in Limbal Phenotype Maintenance Medium (LPMM). After 1 month, the organoids were observed in terms of number and size, immunohistochemistry, cell cycle, and colony-forming efficiency. Organoids were also transplanted into a rabbit model of limbal deficiency.

Although we were able to form organoids from the central cornea, the number of organoids from the cornea was small (approximately one tenth compared to the limbus) after 1-month culture. Cornea-derived organoids were similar in shape and size to limbal-derived organoids, and expressed keratin 15 and p63, which are characteristics of the limbal epithelium, as well as collagen type IV, laminin, and tenascin-C, which are limbal basement membrane components. Cornea-derived organoids also showed colony forming efficiency, slow-cycling cells, and label-retaining cells. Transplanted corneal organoids were observed in the limbus of a rabbit limbal deficiency model, and the presence of organoid-derived cells extending into the host cornea was confirmed by immunohistochemistry using anti-human nuclei, -K12, -collagen type IV, and -laminin antibodies.

Our data suggest that corneal organoids de-differentiated to gain a limbal phenotype and functionally supplied corneal epithelium in a rabbit limbal deficiency model for up to 1 month.

The biliary system is crucial for liver function, regulating bile production, secretion, and transport. Dysfunctions within this system can lead to various diseases, such as cholangiopathies and biliary fibrosis, which may progress from benign to malignant states like cholangiocarcinoma. While liver organoid research is well-established and technologically advanced, bile duct organoids (BDOs) offer significant potential. BDOs can accurately simulate the physiological structure and function of bile ducts, making them valuable tools for in-vitro biliary disease research. Here, we review the development of BDO models, focusing on stem cell-derived organoids and tissue-derived organoids. We also illustrate the role of cultivation strategies and extracellular scaffolds in supporting organoid growth and stability, including the influence of cellular components of the microenvironment and physicochemical factors. Furthermore, we discuss the applications of BDOs in biliary development, disease modeling, regenerative medicine, and drug screening. Additionally, we emphasize the transformative potential in BDO biobanks and personalized medicine, which helps to pave the way for innovative therapeutic strategies and personalized medicine. Finally, we summarize the current and prospective advancements in BDO technologies, highlighting the integration of emerging technologies such as artificial intelligence, 3D bioprinting, and organoid-on-chip systems. These technologies hold great promise for significantly enhancing both clinical and research applications in the field of biliary diseases.

Cholangiocarcinoma (CCA) is a rare but devastating liver cancer which is commonly diagnosed at a late stage and often resistant to chemotherapy. Bcl-2 family members, which control apoptotic cell death, are known to be involved in the chemoresistance of some cancer types. This study investigated the role of Bcl-2 family members in the chemoresistance of cholangiocarcinoma organoids (CCAOs) in both undifferentiated and matured branching phenotypes (BRCCAOs). Patient-derived CCAOs and BRCCAOs were cultured to assess chemoresistance to an FDA-approved anticancer drug panel by testing cell viability using ATP quantification and apoptotic cell death by cleaved caspase 3 staining. More specifically, sensitivity to the first-line drug gemcitabine was tested in combination with Bcl-2 family inhibitors or activators. We found that in gemcitabine-resistant CCAOs, inhibition of Bcl-xl could overcome gemcitabine resistance and induce apoptotic cell death. Although inhibition of Mcl-1 or activation of Bax induced spontaneous cell death, this could not overcome gemcitabine resistance. The BRCCAOs, which mimic tumor architecture better than CCAOs, show broader chemoresistance to anticancer drugs. Of note, in the resistant BRCCAOs, Bcl-xl inhibition could restore gemcitabine sensitivity. In conclusion, this study shows that targeting Bcl-xl can overcome chemoresistance to gemcitabine in CCA organoids. CCAOs and BRCCAOs provide good preclinical models for testing new drug combinations and assessing personalized therapeutic approaches.

Achieving high maturity and functionality in in vitro skeletal muscle models is essential for advancing our understanding of muscle biology, disease mechanisms, and drug discovery. However, current models struggle to fully recapitulate key features such as sarcomere structure, muscle fiber composition, and contractile function while also ensuring consistency and rapid production. Adult stem cells residing in muscle tissue are known for their powerful regenerative potential, yet tissue-derived skeletal muscle organoids have not been established. In this study, we introduce droplet-engineered skeletal muscle organoids derived from primary tissue using cascade-tubing microfluidics. These droplet-engineered organoids (DEOs) exhibit high maturity, including well-developed striated sarcomeres, spontaneous and stimulated contractions, and recapitulation of parental muscle fiber types. Notably, DEOs are produced in just 8 d without the need for primary cell culture-substantially accelerating the 50- to 60-d process required by classical organoid models. Additionally, the cascade-tubing microfluidics platform enables high-throughput production of hundreds of uniform DEO replicates from a small tissue sample, providing a scalable and reproducible solution for skeletal muscle research and drug screening.

Organoid models are powerful tools for evaluating cancer immunotherapy that provide a more accurate representation of the tumour microenvironment (TME) and immune responses than traditional models. This review focuses on the latest advancements in organoid technologies, including immune cell co-culture, 3D bioprinting, and microfluidic systems, which enhance the modelling of TME and facilitate the assessment of immune therapies such as immune checkpoint inhibitors (ICIs), CAR-T therapies, and oncolytic viruses. Although these models have great potential in personalised cancer treatment, challenges persist in immune cell diversity, long-term culture stability, and reproducibility. Future developments integrating artificial intelligence (AI), multi-omics, and high-throughput platforms are expected to improve the predictive power of organoid models and accelerate the clinical translation of immunotherapy.

Primary central nervous system lymphoma (PCNSL) exhibits substantial intratumoural and intertumoural heterogeneity, complicating the development of effective treatment methods. Existing in vitro models fail to simulate the cellular and mutational diversity of native tumours and require prolonged generation times. Therefore, we developed a culture method for patient-derived PCNSL organoids (CLOs) and evaluated the organoids through extensive molecular characterisation, histopathological analysis, single-nucleus RNA sequencing, bulk RNA sequencing and whole-exome sequencing. These CLOs accurately mimicked the histological attributes, gene expression landscapes and mutational profiles of their original tumours. Single-nucleus RNA sequencing also revealed that CLOs maintained cell-type heterogeneity and the molecular signatures of their original tumours. CLOs were generated within 2 weeks, demonstrating rapid development and reliability. Therapeutic profiling was performed on three selected CLOs treated with four standard drugs. The CLOs exhibited specific sensitivity to methotrexate, and resistance to dexamethasone, ibrutinib and rituximab, suggesting that CLOs may be valuable tools for reflecting drug sensitivities. Taken together, these results emphasise that CLOs effectively emulate the key characteristics of PCNSL, increasing the understanding of the genetic landscape of this complex disease. CLOs provide a rapid and reliable platform for exploring individualised treatment strategies, potentially accelerating the transition of research findings to clinical practice.

Bladder cancer is one of the most common malignancies of the urinary system and there's a significant unmet need for new effective therapeutics for bladder cancer. The limited number of available models to study malignant bladder tumors is one of the obstructions in developing bladder cancer therapeutics. Patient-derived xenograft (PDX) and organoid (PDO) models are more representatives of human cancer than cell lines and cell line-derived xenograft (CDX) and are likely to be more promising and efficient in predicting drug response and finding new therapeutics.

Three pairs of patient-derived xenograft (PDX) models of bladder cancer and their corresponding PDX-derived organoids (PDXOs) were successfully established. These models were utilized to assess the efficacy of abemaciclib. The sensitivity of the drug was determined through the Cell Counting Kit-8 (CCK8) assay in PDXO cultures, corroborated by the EdU incorporation assay. Additionally, the in vivo tumor growth was monitored in the matched PDX models.

In vitro PDXO cultures and in vivo PDX tumor models consistently demonstrated that abemaciclib had varying degrees of inhibitory effects across different bladder cancer (BC) patients. Notably, our study further revealed that treatment with abemaciclib significantly modified the expression patterns of CyclinD1/CDK4. This was achieved by not only diminishing their expression levels but also by shifting their expression from a membrane-associated localization to the nucleus.

Our research provided compelling evidence attesting to the reliability and potential of PDX and PDXO models in the realm of precision medicine. These models are instrumental in identifying patients who are likely to respond favorably to a specific drug treatment.

Background/Objectives: Primary sclerosing cholangitis (PSC) is a rare, incurable liver disease characterized by chronic biliary inflammation and fibrosis. PSC is a significant risk factor for biliary tract cancer (BTC). This study aims to evaluate STAT3 expression in BTC and its prognostic significance as well as explore the potential of organoids derived from PSC and liver tumor patients as an in vitro model for testing novel therapeutic strategies in both PSC and BTC. Methods: Fresh tissue samples obtained from 10 PSC patients through targeted endoscopic retrograde cholangiography (ERC) and biopsy samples from liver tumor patients were used to establish organoid cultures. Organoids were treated with different agents and the therapeutic effect was measured by CellTiterGlo. Treatment with the JAK inhibitor baricitinib was followed by the measurement of cytokine concentrations in the supernatant. Archived formalin-fixed paraffin-embedded (FFPE) samples from 55 surgically resected BTC tumors were analyzed for STAT3 expression using immunohistochemistry. Results: We successfully established organoid cultures from all ERC samples. STAT3 protein expression was detected in 56% of tumor samples and 69% of the immune microenvironment. STAT3 positivity in the immune cell compartment was associated with longer disease-free survival, although the multivariate analysis could not confirm its value as an independent prognostic factor. Chemotherapy testing on liver tumor organoids showed various degrees of decreases in viability after treatment with gemcitabine, cisplatin, and cabozantinib. Baricitinib treatment significantly reduced IL-6 and MCP-1 secretion in cholangiocarcinoma Conclusions: The patient-derived organoid model of PSC and liver tumors is a valuable tool for testing novel and established therapeutic strategies, including JAK inhibitors and chemotherapy regimens. STAT3 expression in the immune microenvironment of BTC may serve as a prognostic marker. Further studies are needed to explore the integration of co-cultured organoid systems with stromal and immune components to improve physiological relevance.

With the rapid advancement of biomaterials and tissue engineering technologies, organoid research and its applications have made significant strides. Organoids are increasingly utilized in pharmacology, regenerative medicine, and precision clinical medicine. Current trends in organoid research are moving toward multifunctional composite three-dimensional cultivation and dynamic cultivation strategies. Key technologies driving this evolution, including 3D printing and microfluidics, continue to impact new areas of discovery and clinical relevance. This review provides a systematic overview of these emerging trends, discussing the strengths and limitations of these critical technologies and offering insight and research directions for professionals working in the organoid field.

Three-dimensional (3D) cell cultures have gained popularity in recent years due to their ability to represent complex tissues or organs more faithfully than conventional two-dimensional (2D) cell culture. This article reviews the application of both 2D and 3D microscopy approaches for monitoring and studying 3D cell cultures. We first summarize the most popular optical microscopy methods that have been used with 3D cell cultures. We then discuss the general advantages and disadvantages of various microscopy techniques for several broad categories of investigation involving 3D cell cultures. Finally, we provide perspectives on key areas of technical need in which there are clear opportunities for innovation. Our goal is to guide microscope engineers and biomedical end users toward optimal imaging methods for specific investigational scenarios and to identify use cases in which additional innovations in high-resolution imaging could be helpful.

The hepatitis B virus (HBV) only robustly infects primary human hepatocytes. This strict viral host and cell tropism has hampered the development of physiologically relevant in vitro culture models of HBV infection. Primary human hepatocytes (PHH) are robustly infected by HBV but are short-lived in tissue culture and rapidly lose their hepatocyte characteristics. Human tissue-derived liver organoids are a novel in vitro physiologically relevant model that supports infection by HBV and mitigates the limitations of PHH. Liver organoids are established by placing tissue fragments into a three-dimensional (3D) basement membrane-rich matrix dome bathed in medium containing supplements and growth factors to support organoid growth. The organoids can be expanded in vitro, cryopreserved, and are genetically stable. The expansion phase organoids, once differentiated to a hepatocyte phenotype, support HBV infection. We couple liver organoids with an adenoviral delivery system to achieve robust HBV infection. This robust model supports the full HBV virus replication cycle.

Hepatocellular carcinoma and cholangiocarcinoma, the two predominant histological subtypes of primary liver cancer, are characterized by a high global incidence and poor prognosis. Moreover, the therapeutic options are still limited, with surgical intervention being the predominant approach. Anchorage-Dependent Cell Death (Anoikis) is a form of regulated cell death triggered by the detachment of cells from their extracellular matrix, is crucial for maintaining tissue homeostasis. However, tumor cells often evade anoikis, a capability that is essential for their survival in the bloodstream and subsequent metastasis. Our study classified liver cancer into two distinct subtypes, C1 and C2, based on the differential expression of anoikis-related genes. Subtype C1 patients exhibited elevated expression of BRMS1, PTK2, and CASP8, which could serve as potential therapeutic targets for anoikis-based treatments. Conversely, subtype C2 patients showed higher expression of NTRK2, STAT3, SIK1, AKT1, and EGFR, suggesting these genes as promising therapeutic targets for C2 subtype liver cancer. Furthermore, employing Weighted Correlation Network analysis, machine learning models, and experimental validation, we identified NPY1R and HGF as potential biomarkers for the diagnosis and treatment of liver cancer.

Tumor heterogeneity is predicted to confer inferior clinical outcomes with precision-based strategies, however, modeling heterogeneity in a manner that still represents the tumor of origin remains a formidable challenge. Sequencing technologies are limited in their ability to identify rare subclonal populations and predict response to treatments for patients. Patient-derived organotypic cultures have significantly improved the modeling of cancer biology by faithfully representing the molecular features of primary malignant tissues. Patient-derived cancer organoid (PCO) cultures contain subclonal populations with the potential to recapitulate heterogeneity, although treatment response assessments commonly ignore diversity in the molecular profile or treatment response. Here, we demonstrate the advantage of evaluating individual PCO heterogeneity to enhance the sensitivity of these assays for predicting clinical response. Additionally, organoid subcultures identify subclonal populations with altered treatment response. Finally, dose escalation studies of PCOs to targeted anti-EGFR therapy are utilized which reveal divergent pathway expression when compared to pretreatment cultures. Overall, these studies demonstrate the importance of population-based organoid response assessments, the use of PCOs to identify molecular heterogeneity not observed with bulk tumor sequencing, and PCO heterogeneity for understanding therapeutic resistance mechanisms.

Organoids are a cutting-edge technology in the life sciences field, with applications in precision medicine, bionic organs, and toxicological evaluations of chemicals. Their 3D structure closely resembles that of real organs, allowing more accurate functional mimicry. The 3D organoid culture system can simulate the growth state of cells in vivo and establish a suspension culture system for organoid 3D culture by using scaffold-less or scaffold technology to avoid direct contact between cells and plastic culture vessels. Furthermore, organoids can simulate the pathophysiological state of tissues and organs in vitro. This paper primarily discusses the construction methodologies, as well as the advantages and disadvantages of 3D culture systems for both scaffold-free organoids and scaffolded organoids. This review also summarizes the application of organoid models in chemical toxicology evaluation, drug screening and functional evaluation, establishment of in vitro disease models, and research on disease occurrence and potential mechanisms. The aim is to provide a reference for the research and practical applications of organoid-related scientific fields.

Liver cancer poses a significant global health challenge owing to its increasing incidence and associated mortality rates. Traditional Chinese Medicine (TCM) has garnered attention for its potential in oncology, with formulations such as Hua Zheng San Ji Fang (HZSJF) exhibiting antineoplastic effects. HZSJF is clinically employed in China for cancer treatment; however, its molecular mechanisms in liver cancer remain elusive. TYRO3 plays a key role in tumor progression via the ERK signaling pathway, rendering it a potential therapeutic target. However, the effect of HZSJF on TYRO3 expression and its downstream signaling in liver cancer remains unexplored.

This study aimed to investigate the molecular mechanisms through which HZSJF alleviates liver cancer progression, focusing on its regulation of TYRO3 and the ERK signaling pathway.

TYRO3 expression in liver cancer and para-carcinoma tissues was analyzed using immunohistochemistry, reverse transcription-quantitative PCR, and western blotting. Liver cancer cells were used to investigate HZSJF-regulated pathways. Transcriptome sequencing was used to identify HZSJF-targeted genes. Cell proliferation, apoptosis, invasion, and migration were assessed using EdU, YO-PRO-1/PI staining, and transwell assays. ERK signaling involvement was examined using a specific inhibitor and validated in vivo using subcutaneous nude mouse tumor models.

HZSJF significantly inhibited TYRO3 expression and ERK pathway activation, reducing proliferation, invasion, and migration while promoting apoptosis. The ERK inhibitor corroborated the pathway's role in the antitumor effects of HZSJF. HZSJF suppressed tumor growth and TYRO3 expression in vivo.

HZSJF alleviated liver cancer progression through the ERK signaling pathway by inhibiting TYRO3 expression, presenting a potential therapeutic approach.

Esophageal squamous cell carcinoma (ESCC) is a deadly cancer with a poor prognosis and a high recurrence rate after chemotherapy, posing a significant clinical challenge. To elucidate the molecular basis of chemotherapy (chemo)-resistance and to develop methods to effectively eliminate chemo-resistant tumor clones, we established an ESCC organoid (ESCCO) library from 24 ESCC patients of various stages, ages, and treatments. These ESCCOs faithfully recapitulate the oncogenic mutations observed in the original ESCC tissues and manifest tumorigenic properties when xenografted. The ESCCOs respond differently to cisplatin and 5-fluorouracil, chemotherapeutic agents commonly used to treat ESCC patients, with 7 ESCCOs exhibiting potent chemo-resistance. Notably, the chemo-resistant ESCCOs show higher genes involved in antioxidant stress response pathways and more accessible chromatin at their loci than the sensitive ESCCOs. These genes can serve as valuable biomarkers to stratify chemo-resistant ESCCs in histopathological specimens. Through drug screening using the ESCCO library, we reveal that fedratinib effectively induces cell death in chemo-resistant ESCCOs. Collectively, our human ESCCO model offers novel insights into the mechanism of chemo-resistance in ESCCs, which is critical for developing effective therapeutic approaches to eradicate the recurrence of ESCCs.

The cryopreservation of cancer tissues to generate frozen libraries is a common practice used worldwide for storing patient samples for later applications. However, frozen samples stored by existing methods cannot be used for initiating living cell cultures, such as patient-derived tumor organoids (PDOs), which offer great potential for personalized treatment. To overcome this challenge, we developed a novel procedure for culturing PDOs using frozen live tumor tissues. We show that tumor specimens stored using this technique maintain their viability and can be successfully used to generate organoids even after long-term freezing, with an impressive success rate of 95.2 %. Importantly, we found that the structural features, tumor marker expression, and drug responses of organoids derived from frozen tissues are similar to those derived from fresh tissues. Moreover, organoids derived from frozen tissues can be routinely passaged and frozen, making them ideal for high-throughput drug screening at any time. Notably, cryopreserved tumor tissues can also be utilized in air-liquid interface (ALI) culture. This method allows for preserving the original tumor microenvironment, making it an invaluable resource for conducting tests on antitumor drug responses, including immune checkpoint inhibitors (ICIs). This innovation has the potential to enable the identification of potentially effective drugs for patients and facilitate the development of novel therapeutic drugs. Thus, we have established protocols for the long-term cryopreservation of cancer tissues to maintain their viability and microenvironment, which are useful for personalized therapy.

Prediction of the potential for drug-induced liver injury (DILI) in the early stages of drug development is important. We developed an organoid-based and functional endpoint method for accurate prediction of DILI. To this end, hepatic organoids (HOs) derived from human pluripotent stem cells (hPSCs) were cocultured with hepatic stellate cells (HSCs) and THP-1 macrophages in Matrigel domes to mimic the cellular and physiological environment of the human liver. To validate our hepatotoxicity prediction model, we selected 12 hepatotoxic reference compounds. As indicators, we used factors related to mechanisms of hepatotoxicity and markers thereof, including factors related to oxidative stress and proinflammatory cytokines. We plotted radar graphs and calculated the relative areas of polygons to analyze the effects of drugs with different degrees of hepatotoxicity. The drugs in the severe DILI group significantly increased the levels of factors related to oxidative stress (ROS, GSSH, and catalase) compared to those in the no and mild DILI groups. The drugs in the severe group significantly increased the levels of inflammation-related factors (IL-1, IL-6, and IL-10). The drugs in the mild and severe groups highly significantly increased the activities of ALT and AST and the level of ALB compared to those in the no DILI group. In summary, the drugs in the severe DILI group had significantly greater effects on the factors analyzed than those in the no DILI group. Therefore, our hepatotoxicity evaluation method is suitable for predicting DILI in the early stages of drug development.

Fatty acid metabolism is closely associated with hepatocellular carcinoma (HCC). Elucidating the molecules that influence fatty acid metabolism in HCC is important for developing precision therapies. However, uncovering the precise molecular mechanisms underlying changes in fatty acid metabolism in tumour cells is challenging. In this study, we aimed to determine the characteristics of fatty acid metabolism in HCC.

We employed organoid models, single-cell RNA sequencing, and spatial transcriptomics to identify key genes involved in tumour fatty acid metabolism. Metabolomics, proteomics, metabolic flux analysis, and transmission electron microscopy were utilized to evaluate this metabolic process. Tumour malignancy was characterized using multi-species models. Changes in the immune microenvironment were analysed by time-of-flight mass cytometry and multiplexed immunohistochemistry. Gene knockdown targeting the liver was achieved using lipid nanoparticles.

Eukaryotic translation initiation factor 3 subunit f (eIF3f) is upregulated in HCC tissues and is associated with poor prognosis. eIF3f directly interacted with and stabilised long chain acyl CoA synthetase 4 (ACSL4) through K48-linked deubiquitination, promoting fatty acid biosynthesis and malignancy. The increased fatty acid levels in the tumour microenvironment indirectly reduced CD8+ T-cell infiltration. In addition, phosphorylated eIF3f enhanced the interaction between eIF3f and ACSL4.

Targeting the eIF3f-ACSL4-fatty acid biosynthesis axis could decelerate the progression of HCC and enhance anti-programmed cell death-1 efficacy, implicating eIF3f as a potential target for precision therapy in HCC.

Fatty acid metabolism is closely associated with hepatocellular carcinoma (HCC), yet the underlying mechanisms involved remain unclear. Here, we found that eIF3f is upregulated in HCC and is associated with poor prognosis. eIF3f interacts with and stabilizes ACSL4, thereby promoting fatty acid biosynthesis. Additionally, increased fatty acid levels reduce CD8+ T-cell infiltration and activation. These findings are of significant importance for clinicians and researchers in the field of HCC treatment, as eIF3f inhibition combined with anti-PD-1 therapy significantly improved anti-tumour efficacy in a mouse model and could offer therapeutic benefits for patients. These findings have practical implications, as eIF3f could serve as a novel therapeutic target in HCC. However, further clinical studies are needed to confirm the efficacy of eIF3f targeting in human patients.

The incidence of intracranial aneurysms (IAs) is markedly elevated in postmenopausal women compared to men and premenopausal women, a disparity historically linked to declining estrogen levels. Emerging evidence, however, suggests that the expression and functional roles of estrogen receptors (ERs), including ERα, ERβ, and GPER1, in vascular tissues may implicate estrogen-independent pathways in vascular aging and related pathologies. An integrative bioinformatics approach, combining three IA datasets (GSE75436, GSE122897, GSE54083) and two vascular endothelial cell senescence (VECS) datasets (GSE214476, GSE102397) from the Gene Expression Omnibus (GEO) database, was employed to investigate this hypothesis and define shared molecular mechanisms. This cross-disease differential expression analysis identified 452 significantly downregulated genes, suggesting conserved pathogenic pathways in IA and VECS. Among ERs, GPER1 was uniquely downregulated in both conditions. Subsequent weighted gene co-expression network analysis and subsequent module clustering revealed ACACB as a hub gene co-expressed with GPER1 and inversely correlated with IA and VECS progression. In vitro validation confirmed that GPER1 expression was reduced during VECS and that GPER1 silencing decreased ACACB expression and accelerated endothelial senescence, supporting its estrogen-independent role in vascular homeostasis. Computational pharmacological screening further identified PD0325901, SCH772984, and selumetinib as potential therapeutic agents targeting both GPER1 and ACACB, offering a dual-pathway therapeutic strategy. The identification of GPER1 and ACACB as potential target genes associated with IA and VECS provides a framework for developing therapies that circumvent hormone dependency, addressing an unmet need in the treatment of IA and age-related vascular pathologies.

Precision medicine is a personalized medical model based on the individual's genome, phenotype, and lifestyle that provides tailored treatment plans for patients. In this context, tumor organoids, a 3-dimensional preclinical model based on patient-derived tumor cell self-organization, combined with digital analysis methods, such as high-throughput sequencing and image processing technology, can be used to analyze the genome, transcriptome, and cellular heterogeneity of tumors, so as to accurately track and assess the growth process, genetic characteristics, and drug responsiveness of tumor organoids, thereby facilitating the implementation of precision medicine. This interdisciplinary approach is expected to promote the innovation of cancer diagnosis and enhance personalized treatment. In this review, the characteristics and culture methods of tumor organoids are summarized, and the application of multi-omics, such as bioinformatics and artificial intelligence, and the digital methods of organoids in precision medicine research are discussed. Finally, this review explores the main causes and potential solutions for the bottleneck in the clinical translation of digital tumor organoids, proposes the prospects of multidisciplinary cooperation and clinical transformation to narrow the gap between laboratory and clinical settings, and provides references for research and development in this field.

Tumor organoid-based drug sensitivity prediction is a new approach for precision medicine, which has wide applications in cancer treatment and attracts increasing attention. In the field of breast cancer, conventional organoid culture methods often require more than three weeks of culture period. The culture time greatly limits the further extension of the application scenarios of breast cancer organoids. We developed a fluid system that builds on the conventional organoid "dome" culture method, which continuously and stably supplies the nutrients for the growth of breast cancer organoids. We demonstrated that this is an effective optimization method, which can shorten the culture period of breast cancer organoids without significant changes in histological characteristics and drug sensitivity features.

In-depth exploration into the dysregulation of lipid metabolism in hepatocellular carcinoma (HCC) has contributed to the development of advanced antitumor strategies. CRSP8 is a critical component of mediator multiprotein complex involved in transcriptional recruiting. However, the regulatory mechanisms of CRSP8 on fatty acid metabolism reprogramming and HCC progression remain unclear.

In-silico/house dataset analysis, lipid droplets (LDs) formation, HCC mouse models and targeted lipidomic analysis were performed to determine the function of CRSP8 on regulating lipid metabolism in HCC. The subcellular colocalization and live cell imaging of LDs, transmission electron microscopy, co-immunoprecipitation and luciferase reporter assay were employed to investigate their potential mechanism.

CRSP8 was identified as a highly expressed oncogene essential for the proliferation and aggressiveness of HCC in vitro and in vivo. The tumor promotion of CRSP8 was accompanied by LDs accumulation and increased de novo fatty acids (FAs) synthesis. Moreover, CRSP8 diminished the colocalization between LC3 and LDs to impair lipophagy in a nuclear-localized PPARα-dependent manner, which decreased the mobilization of FAs from LDs degradation and hindered mitochondrial fatty acid oxidation. Mechanistically, the small ras family GTPase RAN was transcriptionally activated by CRSP8, leading to the reinforcement of RAN/CRM1-mediated nuclear export. CRSP8-induced enhanced formation of RAN/CRM1/PPARα nucleus-cytoplasm shuttling heterotrimer orchestrated cytoplasmic translocation of PPARα, attenuated nPPARα-mediated lipophagy and fatty acid catabolism, subsequently exacerbated HCC progression. In CRSP8-enriched HCC, lipid synthesis inhibitor Orlistat effectively reshaped the immunosuppressive tumor microenvironment (TME) and improved the efficacy of anti-PD-L1 therapy in vivo.

Our study establishes that CRSP8-driven fatty acid metabolism reprogramming facilitates HCC progression via the RAN/CRM1/PPARα nucleus-cytoplasm shuttling heterotrimer and impaired lipophagy-derived catabolism. Targeting the energy supply sourced from lipids could represent a promising therapeutic strategy for treating CRSP8-sufficient HCC.

Developing and providing the right therapy for the right patient (or personalized targeted treatments) is key to reducing side-effects and improving survival in childhood cancers. Most efforts aiming to personalize childhood cancer treatment use genomic analysis of malignancies to identify potentially targetable genetic events. But it is becoming clear that not all patients will have an actionable change, and in those that do there is no additional way to determine if treatments will be effective. Ex vivo drug screening is a laboratory technique used to test the effects of various drugs or compounds, on biological tissues or cells that have been removed from an organism. This information is then used to predict which cancer treatments will be most effective based on the therapeutic response in the tissue or cells removed from that individual. Its utility in personalizing treatments in childhood cancer is increasingly recognized. In this review we describe the different methods for ex vivo drug screening and the advantages and disadvantages of each technique. We also present recent evidence that ex vivo screening may have utility in a variety of childhood malignancies including an overview of current clinical trials appraising its use. Finally, we discuss the research questions and hurdles that must be overcome before ex vivo screening can be widely used in pediatric oncology.

Liver cancer poses a global health challenge with limited therapeutic options. Notably, the limited success of current therapies in patients with primary liver cancers (PLCs) may be attributed to the high heterogeneity of both hepatocellular carcinoma (HCCs) and intrahepatic cholangiocarcinoma (iCCAs). This heterogeneity evolves over time as tumor-initiating stem cells, or cancer stem cells (CSCs), undergo (epi)genetic alterations or encounter microenvironmental changes within the tumor microenvironment. These modifications enable CSCs to exhibit plasticity, differentiating into various resistant tumor cell types. Addressing this challenge requires urgent efforts to develop personalized treatments guided by biomarkers, with a specific focus on targeting CSCs. The lack of effective precision treatments for PLCs is partly due to the scarcity of ex vivo preclinical models that accurately capture the complexity of CSC-related tumors and can predict therapeutic responses. Fortunately, recent advancements in the establishment of patient-derived liver cancer cell lines and organoids have opened new avenues for precision medicine research. Notably, patient-derived organoid (PDO) cultures have demonstrated self-assembly and self-renewal capabilities, retaining essential characteristics of their respective in vivo tissues, including both inter- and intratumoral heterogeneities. The emergence of PDOs derived from PLCs serves as patient avatars, enabling preclinical investigations for patient stratification, screening of anticancer drugs, efficacy testing, and thereby advancing the field of precision medicine. This review offers a comprehensive summary of the advancements in constructing PLC-derived PDO models. Emphasis is placed on the role of CSCs, which not only contribute significantly to the establishment of PDO cultures but also faithfully capture tumor heterogeneity and the ensuing development of therapy resistance. The exploration of PDOs' benefits in personalized medicine research is undertaken, including a discussion of their limitations, particularly in terms of culture conditions, reproducibility, and scalability.

Prostate cancer (PC) is the most frequently diagnosed malignancy among men and contributes significantly to cancer-related mortality. While recent advances in in vitro PC modeling systems have been made, there remains a lack of robust preclinical models that faithfully recapitulate the genetic and phenotypic characteristics across various PC subtypes-from localized PC (LPC) to castration-resistant PC (CRPC)-along with associated stromal cells. Here, we established human PC assembloids from LPC and CRPC tissues by reconstituting tumor organoids with corresponding cancer-associated fibroblasts (CAFs), thereby incorporating aspects of the tumor microenvironment (TME). Established PC organoids exhibited high concordance in genomic landscape with parental tumors, and the tumor assembloids showed a higher degree of phenotypic similarity to parental tumors compared to tumor organoids without CAFs. PC assembloids displayed increased proliferation and reduced sensitivity to anti-cancer treatments, indicating that PC assembloids are potent tools for understanding PC biology, investigating the interaction between tumor and CAFs, and identifying personalized therapeutic targets.

Chemoresistance remains a major hurdle in ovarian cancer (OC) treatment, as many patients eventually develop resistance to platinum-based chemotherapy and/or PARP inhibitors (PARPi).

We performed transcriptome-wide analysis by RNA sequencing (RNA-seq) data of platinum-resistant and -sensitive OC tissues. We demonstrated the role of LINC02776 in platinum resistance in OC cells, mice models and patient-derived organoid (PDO) models.

We identify the long noncoding RNA LINC02776 as a critical factor of platinum resistance. Elevated expression of LINC02776 is observed in platinum-resistant OC and serves as an independent prognostic factor for OC patients. Functionally, silencing LINC02776 reduces proliferation and DNA damage repair in OC cells, thereby enhancing sensitivity to platinum and PARPi in both xenograft mouse models and patient-derived organoid (PDO) models with acquired chemoresistance. Mechanistically, LINC02776 binds to the catalytic domain of poly (ADP-ribose) polymerase 1 (PARP1), promoting PARP1-dependent polyADP-ribosylation (PARylation) and facilitating homologous recombination (HR) restoration. Additionally, high HIF-1α expression in platinum-resistant tissues further stimulates LINC02776 transcription.

Our findings suggest that targeting LINC02776 represents a promising therapeutic strategy for OC patients who have developed resistance to platinum or PARPi.

LINC02776 promotes OC cell proliferation by regulating DNA damage and apoptosis signaling pathways. LINC02776 binds PARP1 to promote DNA damage-triggered PARylation in OC cells. LINC02776 mediates cisplatin and olaparib resistance in OC cells by enhancing PARP1-mediated PARylation activity and regulating the PARP1-mediated HR pathway. The high expression of LINC02776 is induced by HIF-1α in platinum-resistant OC cells and tissues.

Many species, including fruit flies (Drosophila melanogaster), are sexually dimorphic. Phenotypic variation in morphology, physiology, and behavior can affect development, reproduction, health, and aging. Therefore, designating sex as a variable and sex-blocking should be considered when designing experiments. The brain regulates phenotypes throughout the lifespan by balancing survival and reproduction, and sex-specific development at each life stage is likely. Changes in morphology and physiology are governed by differential gene expression, a quantifiable molecular marker for age- and sex-specific variations. We assessed the fruit fly brain transcriptome at three adult ages for gene expression signatures of sex, age, and sex-by-age: 6698 genes were differentially expressed between sexes, with the most divergence at 3 days. Between ages, 31.1% of 6084 differentially expressed genes (1890 genes) share similar expression patterns from 3 to 7 days in females, and from 7 to 14 days in males. Most of these genes (90.5%, 1712) were upregulated and enriched for chemical stimulus detection and/or cilium regulation. Our data highlight an important delay in male brain gene regulation compared to females. Because significant delays in expression could confound comparisons between sexes, studies of sexual dimorphism at phenotypically comparable life stages rather than chronological age should be more biologically relevant.

The engineering construction of the liver has attracted enormous attention. Organoids, as emerging miniature three-dimensional cultivation units, hold significant potential in the biomimetic simulation of liver structure and function. Despite notable successes, organoids still face limitations such as high variability and low maturity. To overcome these challenges, engineering strategies have been established to maintain organoid stability and enhance their efficacy, laying the groundwork for the development of advanced liver organoids. The present review comprehensively summarizes the construction of engineered liver organoids and their prospective applications in biomedicine. Initially, we briefly present the latest research progress on matrix materials that maintain the three-dimensional morphology of organoids. Next, we discuss the manipulative role of engineering technologies in organoid assembly. Additionally, we outline the impact of gene-level regulation on organoid growth and development. Further, we introduce the applications of liver organoids in disease modeling, drug screening and regenerative medicine. Lastly, we overview the current obstacles and forward-looking perspectives on the future of engineered liver organoids. We anticipate that ongoing innovations in engineered liver organoids will lead to significant advancements in medical applications.

As a highly heterogeneous cancer, hepatocellular carcinoma (HCC) shows different response rates to the multi-kinase inhibitor lenvatinib. Thus, it is important to explore genetic biomarkers for precision lenvatinib therapy in HCC.

The effect and mechanism of AXIN1 mutation on HCC were revealed by cell proliferation assay, long-term clone formation assay, sphere formation assay and small molecule inhibitor library screening. A new therapeutic strategy targeting HCC with AXIN1 mutation was evaluated in humanized models (patient-derived xenograft [PDX] and patient-derived organoid [PDO]).

Based on The Cancer Genome Atlas (TCGA) data, we screened 6 most frequently lost tumour suppressor genes in HCC (TP53, ARID1A, AXIN1, CDKN2A, ARID2 and PTEN) and identified AXIN1 as the most crucial gene for lenvatinib sensitivity. Further study showed that AXIN1-knockout HCC cells had a more malignant phenotype and lower sensitivity to lenvatinib in vitro and in vivo. Mechanistically, the WNT pathway and its target gene c-Myc were activated when AXIN1 was missing, and the expression of tumour suppressor p15 was inhibited by transcription co-repressors c-Myc and Miz-1, resulting in the exacerbation of the resistant phenotype. Screening of a library of epigenetic-related enzyme inhibitors showed that a KDM5B inhibitor up-regulated p15 expression, leading to increased sensitivity to lenvatinib in vitro and in vivo.

AXIN1-deficient patients have a lower response to lenvatinib, which may be associated with suppression of p15 mediated by WNT pathway activation. KDM5B inhibitors can restore p15 levels, resulting in efficient killing of resistant cells in HCC.

Genome-wide functional genetic screening has been widely used in the biomedicine field, which makes it possible to find a needle in a haystack at the genetic level. In cancer research, gene mutations are closely related to tumor development, metastasis, and recurrence, and the use of state-of-the-art powerful screening technologies, such as clustered regularly interspaced short palindromic repeat (CRISPR), to search for the most critical genes or coding products provides us with a new possibility to further refine the cancer mapping and provide new possibilities for the treatment of cancer patients. The use of CRISPR screening for the most critical genes or coding products has further refined the cancer atlas and provided new possibilities for the treatment of cancer patients. Immunotherapy, as a highly promising cancer treatment method, has been widely validated in the clinic, but it could only meet the needs of a small proportion of cancer patients. Finding new immunotherapy targets is the key to the future of tumor immunotherapy. Here, we revisit the application of functional screening in cancer immunology from different perspectives, from the selection of diverse in vitro and in vivo screening models to the screening of potential immune checkpoints and potentiating genes for CAR-T cells. The data will offer fresh therapeutic clues for cancer patients.