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.

The ability to establish organoids composed exclusively of tumour rather than healthy cells is essential for their implementation into clinical practice. Organoids have recently emerged as a powerful tool to expand patient material in culture and generate modifiable 3D models derived from humans or animal models. For translational research, they enable the creation of model systems for an ever-increasing number of cell types and diseases. And in personalised medicine, they potentially allow for functional drug testing with high predictive power in certain settings. We found that using biopsy material from untreated, early-stage primary breast cancer patients poses significant challenges for consistently culturing tumour cells as organoids. Specifically, we observed frequent outgrowth of genetically normal, non-cancerous epithelial cells. We analysed >100 biopsy samples from early-stage breast cancer and present our large collection of >70 organoid lines. We also show methods of assessing successful tumour cell culture in a time, and cost-efficient manner, proving a high rate (>85%) of normal cell overgrowth in early-stage breast cancer organoids. Finally, we show a number of successful attempts to culture cancer organoids from mastectomy-derived tissue of advanced, metastatic breast cancer. We conclude that the usefulness of organoids from early breast cancer for translational research and personalised medicine, especially guidance of adjuvant or post-surgical maintenance therapy, is strongly limited by the low success rate of culturing cancerous cells under organoid conditions.

Temozolomide (TMZ) is the first-line chemotherapeutic agent for glioblastoma (GBM) therapy; however, resistance to TMZ remains a major obstacle in GBM treatment. The aim of this study is to elucidate the mechanisms underlying TMZ resistance and explore how to enhance the sensitivity of GBM to TMZ.

GBM organoids were generated from patient samples, and organoid-based TMZ sensitivity testing was performed. Transcriptome sequencing was conducted on GBM organoids, which identified Centromere protein U (CENPU) as a novel key gene mediating TMZ resistance. Histopathological assessments were carried out using immunohistochemistry (IHC) and Hematoxylin and Eosin (HE) staining. Single-cell sequencing data were utilized to determine the functional states of CENPU in GBM cells. Intracranial and subcutaneous glioma mouse models were constructed to evaluate the effect of CENPU on TMZ sensitivity. The underlying mechanisms were further investigated using immunofluorescence, lentivirus transduction, co-immunoprecipitation, mass spectrometry, alkaline comet assay et al. RESULTS: CENPU was found to be highly expressed in TMZ-resistant GBM organoids and enhanced the TMZ resistance of GBM cells by promoting DNA damage repair. Its abnormal expression correlates with poor clinical outcomes in glioma patients. In vivo studies demonstrated that downregulation of CENPU enhances the sensitivity of GBM to TMZ. Correspondingly, rescue of CENPU expression reversed this effect on TMZ sensitivity in GBM cells. Mechanistically, CENPU cooperates with TRIM5α to promote the ubiquitination and degradation of RPS3 by inducing its polyubiquitination at the K214 residue. This process subsequently activates the ERK1/2 pathway and promotes the expression of E2F1 and RAD51. Consequently, the degradation of RPS3 and upregulation of RAD51 in GBM cells enhance DNA damage repair, thereby contributing to TMZ resistance.

Our study identified CENPU as a novel key gene mediating TMZ resistance and elucidated its molecular mechanisms, providing a new target to overcome TMZ resistance in GBM.

Ruthenium-based metallodrugs have garnered attention as a promising alternative for anticancer therapy, aiming to overcome chemoresistance and severe side effects linked to platinum-based drugs. However, ruthenium complexes tested in clinical trials to date have yielded unsatisfactory results. This study synthesized a positively charged ruthenium complex (Ru-2) that effectively penetrated cancer cells and exhibited superior cytotoxicity to cisplatin in vitro against cancer cell lines and organoids. Ru-2 selectively targeted mitochondria, disrupting their function by depolarizing mitochondrial membrane potential, elevating reactive oxygen species production, and impairing both oxidative phosphorylation and the tricarboxylic acid cycle. Furthermore, Ru-2 triggered endoplasmic reticulum (ER) stress and apoptosis. Integrative transcriptomic and proteomic analyses, performed using RNA sequencing and mass spectrometry, identified key molecular changes in cancer cells treated with Ru-2. For enhanced in vivo application, we developed a transferrin-based nanomedicine formulation, TF/Ru-2, incorporating Ru-2 into transferrin. In vivo studies demonstrated that both Ru-2 and TF/Ru-2 exhibited superior antitumor efficacy and improved biosafety compared to cisplatin. This study presents a novel ruthenium complex and a transferrin-based drug delivery platform with significant potential for future cancer therapies.

Oral cancer poses significant health risks with an increasing incidence annually. Despite advancements in treatment methods, their efficacy is frequently constrained by cancer heterogeneity and drug resistance, leading to minimal improvement in the 5-year survival rate. Therefore, there is a critical need for new treatment methods leaded by representative preclinical research models. Compared to other models, organoids can more precisely simulate the tissue structure, genetic characteristics, and tumor microenvironment (TME) of in vivo tumors, exhibiting high tumor specificity. This makes organoid technology a valuable tool in investigating tumor development, mechanisms of metastasis, drug screening, prediction of clinical responses, and personalized patient treatment. Moreover, integrating organoid technology with other biotechnologies could expand its applications in tissue regeneration. Although organoid technology is increasingly utilized in oral cancer research, a systematic review in this field is absent. This paper is to bridge the gap by reviewing the development and current status of organoid research, highlighting its applications, future prospects, and challenges in oral cancer. It aims to provide novel insights into the role of organoids in precision treatment and regenerative medicine for oral cancer.

Tumours of the appendix - a vestigial digestive organ attached to the colon - are rare. Although we estimate that around 3,000 new appendiceal cancer cases are diagnosed annually in the USA, the challenges of accurately diagnosing and identifying this tumour type suggest that this number may underestimate true population incidence. In the current absence of disease-specific screening and diagnostic imaging modalities, or well-established risk factors, the incidental discovery of appendix tumours is often prompted by acute presentations mimicking appendicitis or when the tumour has already spread into the abdominal cavity - wherein the potential misclassification of appendiceal tumours as malignancies of the colon and ovaries also increases. Notwithstanding these diagnostic difficulties, our understanding of appendix carcinogenesis has advanced in recent years. However, there persist considerable challenges to accelerating the pace of research discoveries towards the path to improved treatments and cures for patients with this group of orphan malignancies. The premise of this Expert Recommendation article is to discuss the current state of the field, to delineate unique challenges for the study of appendiceal tumours, and to propose key priority research areas that will deliver a more complete picture of appendix carcinogenesis and metastasis. The Appendix Cancer Pseudomyxoma Peritonei (ACPMP) Research Foundation Scientific Think Tank delivered a consensus of core research priorities for appendiceal tumours that are poised to be ground-breaking and transformative for scientific discovery and innovation. On the basis of these six research areas, here, we define the first 'cells to society' research framework for appendix tumours.

Identifying therapeutic targets for signet ring cell carcinoma (SRCC) of the colon and rectum is a clinical challenge because of the lack of patient-derived organoids (PDO) or patient-derived xenografts (PDX). To address this unmet need, we present a robust method for establishing PDO and PDX models. We demonstrate that these models identify novel therapeutic strategies targeting therapy resistance and peritoneal metastasis.

We derived nine PDO and PDX models from patients with colorectal SRCC. Detailed histopathologic characterization confirmed the fidelity of these models to the original tumors. Drug sensitivity assays were conducted in vitro and in vivo to assess the therapeutic efficacy and impact on peritoneal metastasis. An RNA sequencing analysis was performed to identify critical pathways contributing to therapy resistance and metastatic progression.

We successfully developed and characterized PDO and PDX models from nine patients with SRCC. The SRCC PDO and PDX models exhibited histopathologic features consistent with those of the original tumors, including high mucin content and eccentric nuclei. They demonstrated increased sensitivity to FOLFIRI combined with paclitaxel or vincristine, reducing peritoneal metastasis. RNA sequencing analysis revealed the upregulation of autophagy genes in SRCC. Treatment with chloroquine alone resulted in decreased tumor growth and peritoneal metastasis.

Our study establishes PDO and PDX models as robust platforms for studying SRCC and identifying potential therapeutic strategies. Combining FOLFIRI with paclitaxel/vincristine or chloroquine alone inhibits tumor growth and prevents peritoneal metastasis, showing promise for clinical translation. These findings suggest that combining FOLFIRI with intraperitoneal paclitaxel warrants further investigation in phase I clinical trials for patients with SRCC.

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.

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 tissue 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 interaction between tumor and CAFs, and identifying personalized therapeutic targets.

Cancer-associated fibroblasts (CAFs) are key players in cancer development and therapy, and they exhibit multifaceted roles in the tumor microenvironment (TME). From their diverse cellular origins, CAFs undergo phenotypic and functional transformation upon interacting with tumor cells and their presence can adversely influence treatment outcomes and the severity of the cancer. Emerging evidence from single-cell RNA sequencing (scRNA-seq) studies have highlighted the heterogeneity and plasticity of CAFs, with subtypes identifiable through distinct gene expression profiles and functional properties. CAFs influence cancer development through multiple mechanisms, including regulation of extracellular matrix (ECM) remodeling, direct promotion of tumor growth through provision of metabolic support, promoting epithelial-mesenchymal transition (EMT) to enhance cancer invasiveness and growth, as well as stimulating cancer stem cell properties within the tumor. Moreover, CAFs can induce an immunosuppressive TME and contribute to therapeutic resistance. In this review, we summarize the fundamental knowledge and recent advances regarding CAFs, focusing on their sophisticated roles in cancer development and potential as therapeutic targets. We discuss various strategies to target CAFs, including ECM modulation, direct elimination, interruption of CAF-TME crosstalk, and CAF normalization, as approaches to developing more effective treatments. An improved understanding of the complex interplay between CAFs and TME is crucial for developing new and effective targeted therapies for cancer.

Statins, commonly used to lower cholesterol, are associated with improved prognosis in colorectal cancer (CRC), though their effectiveness varies. This study investigates the anti-cancer effects of atorvastatin in CRC using patient-derived organoids (PDOs) and PDO-derived xenograft (PDOX) models. Our findings reveal that atorvastatin induces mitochondrial dysfunction, leading to apoptosis in cancer cells. In response, cancer cells induce mitophagy to clear damaged mitochondria, enhancing survival and reducing statin efficacy. Analysis of a clinical cohort confirms mitophagy's role in diminishing statin effectiveness. Importantly, inhibiting mitophagy significantly enhances the anti-cancer effects of atorvastatin in CRC PDOs, xenograft models, and azoxymethane (AOM)-dextran sulfate sodium (DSS) mouse models. These findings identify mitophagy as a critical pro-survival mechanism in CRC during statin treatment, providing insights into the variable responses observed in epidemiological studies. Targeting this vulnerability through combination therapy can elicit potent therapeutic responses.

Docetaxel resistance in gastric cancer poses a major therapeutic challenge. In this study, we established docetaxel-sensitive and -resistant gastric cancer organoids and performed single-cell RNA sequencing to identify cellular and molecular alterations. We observed significant shifts in cell populations, with increased secretory, immune-chemotactic, and transitional gastric cancer cells in the resistant group. Key resistance-related genes, including FOS, IFI27, and PTTG1IP, were upregulated in resistant organoids and gastric cancer patients. A pseudo-time trajectory analysis revealed that resistant cells predominantly occupied terminal differentiation stages. Knocking down FOS, IFI27, and PTTG1IP enhanced docetaxel sensitivity in both cell lines and organoids, regulating ROS production, autophagy, and apoptosis. In vivo, silencing these genes reduced tumor growth in response to docetaxel. These findings suggest that targeting FOS, IFI27, and PTTG1IP could overcome resistance and improve treatment outcomes for gastric cancer patients.

Several patient-derived tumor models have emerged recently. However, soft tissue sarcomas (STSs) present a challenge in developing preclinical drug-testing models due to their non-epithelial and complex nature. Here, we report a model termed patient-derived tumor-like cell clusters (PTCs) derived from STS patients. PTCs result from the self-assembly and proliferation of mesenchymal stem cells (MSCs), epithelial cells, and immune cells, faithfully recapitulating the morphology and function of the original tumors. Through standardized culture and drug-response assessment protocols, PTCs facilitate personalized drug testing, evaluating hundreds of therapies within two weeks. Notably, PTCs exhibit 100% accuracy in distinguishing between complete or partial response and disease progression. We demonstrate the utility of PTCs in guiding chemotherapy selection for a patient with relapse and metastases following conventional therapy, who exhibited a positive response after non-conventional therapy identified through PTC. These findings underscore the potential of PTCs for prospective use in clinical decision-making regarding therapy selection.

Malignant phyllodes tumours (MPT) of the breast are rare fibroepithelial neoplasms. It exhibits rapid growth, large size, and a high local recurrence rate.

In this study, we established novel patient-derived organoid (PDO) models from two primary MPT samples and conducted comprehensive genetic profiling and drug screening.

The PDO models faithfully recapped the histopathological and molecular features of the primary tumours, including stromal overgrowth, leaf-like projections, and the expression of key diagnostic markers. Drug testing revealed significant heterogeneity in response profiles to chemotherapeutic reagents between the two MPT-derived organoids, implying the importance of personalised drug testing. Next-generation sequencing analysis identified recurrent mutations in TP53, RB1, EGFR, ATM, and RECQL4, which correlated with the drug sensitivity profiles observed in the organoid models. Targeted therapeutic drugs, such as Abemaciclib (targeting the RB1 pathway) with an IC50 value of 1.744 µM, and Alflutinib Mesylate (targeting the EGFR pathway) with an IC50 value of 0.9150 µM, exhibited significant cytotoxic effects in the MPT2 organoid models.

This study highlights the novel application of PDOs for studying the molecular landscape of MPTs and identifying effective therapeutic targets, offering a promising platform for guiding personalised treatment strategies for this rare and challenging cancer.

Long-term treatment options for conventional chemo-endocrine therapy and molecular-pathway-based targeted therapy are associated with acquired therapy resistance and the emergence of drug-resistant cancer-initiating stem cell populations, leading to the progression of metastatic disease. These treatment options are based on the expression status of estrogen receptor-α (ER-α), progesterone receptor (PR) hormone receptors, and/or of human epidermal growth factor receptor-2 (HER-2). The breast cancer subtypes Luminal A, Luminal B, and HER-2-enriched express hormone/growth factor receptors and exhibit a favorable response to hormone receptor modulators and growth factor receptor antagonists. The triple-negative breast cancer subtype lacks the expression of hormone/growth factor receptors and responds only to cytotoxic conventional chemotherapy. The clinical limitations, due to the modest therapeutic responses of chemo-resistant cancer-initiating stem cells, emphasize the need for the identification of stem cells targeting testable alternatives for therapy-resistant breast cancer. Developed drug-resistant stem cell models exhibit upregulated expression of select cellular biomarker tumor spheroid (TS) formations and cluster of differentiation44 (CD44), DNA-binding protein (NANOG), and octamer-binding protein-4 (OCT-4) molecular biomarkers that represent novel experimentally modifiable quantitative endpoints. Naturally occurring dietary phytochemicals and nutritional herbs containing polyphenols, flavones, terpenes, saponins, lignans, and tannins have documented human consumption, lack systemic toxicity, lack phenotypic drug resistance, and exhibit preclinical efficacy. Constituent bioactive agents may provide testable stem cell-targeting alternatives. The present report provides an overview of (i) clinically relevant cellular models and drug-resistant cancer stem cell models for breast cancer subtypes, (ii) evidence for preclinical efficacy and mechanistic leads for natural phytochemicals and nutritional herbs, and (iii) the potential for the stem cell-targeting efficacy of natural bioactive agents as testable drug candidates for therapy-resistant breast cancer.

Head and neck cancers (HNC) represent an extremely heterogeneous group of diseases with a poorly predictable therapy outcome. Patient-derived tumor organoids (PDTO) offer enormous potential for individualized therapy testing and a better mechanistic understanding of the main HNC drivers.

Here, we have established a comprehensive molecularly and functionally characterized head and neck organoid biobank (HNOB) recapitulating the clinically relevant subtypes of TP53 mutant and human papillomavirus type 16 (HPV 16) infection-driven HNC. Organoids were exposed to radiotherapy, and responses were correlated with clinical data. Genetically engineered normal and tumor organoids were used for testing the direct functional consequences of TP53-loss and HPV infection.

The HNOB consisting of 18 organoid models, including 15 tumor models, was generated. We identified subtype-associated transcriptomic signatures and pathological features, including sensitivity to TP53 stabilization by the MDM2 inhibitor Nutlin-3. Furthermore, we describe an in vitro radio response assay revealing phenotypic heterogeneity linked to the individual patient's treatment outcome, including relapse probability. Using genetically engineered organoids, the possibility of co-existence of both cancer drivers was confirmed. TP53 loss, as well as HPV, increased growth in normal and tumor organoids. TP53 loss-of-function alone was insufficient to promote radiation resistance, whereas HPV 16 oncogenes E6/E7 mediated radiosensitivity via induction of cell cycle arrest.

Our results highlight the translational value of the head and neck organoid models not only for patient stratification but also for mechanistic validation of therapy responsiveness of specific cancer drivers.

Radiotherapy (RT) is a central pillar of a multimodal cancer treatment approach. The ongoing advances in the fields of RT, imaging technologies, cancer biology, and others yield the potential to refine the use of RT. The European Organisation for Research and Treatment of Cancer (EORTC) hosted a dedicated workshop to identify and prioritize key research questions and to define future RT-based treatment strategies to improve the survival and quality of life of cancer patients.

An initial call for relevant RT research topics led to the formation of workgroups to develop these into new clinical research proposals and projects. The EORTC Radiation Oncology Scientific Council (ROSC) State of Science workshop was held in Brussels, Belgium, in February 2024, bringing together EORTC members and international stakeholders to connect and work on the proposals.

Four topics of interest were identified: I) De-escalation of RT, minimizing toxicity while maintaining patients' quality of life, II) Technology-driven RT utilizing advances in treatment techniques, such as spatially fractionated RT to improve outcomes in patients with bulky disease and localized high tumor burden, III) Biology-driven RT, integrating the rapid advances in cancer biology and functional imaging to guide and personalize RT, and IV) New indications adding value and expanding the use of RT.

The EORTC ROSC State of Science workshop prioritized clinical questions to be addressed in prospective clinical research projects to advance RT care and improve patient outcomes.

Consensus regarding the hyperthermic intraperitoneal chemotherapy (HIPEC) for colorectal cancer (CRC) regimen remains elusive. In this study, patient-derived tumor organoids from CRC were utilized as a preclinical model for in vitro drug testing of HIPEC regimens commonly used in clinical practice. This approach was used to facilitate the clinical formulation of HIPEC.

Tumor tissues and corresponding clinical data were obtained from patients diagnosed with CRC at the Sixth Affiliated Hospital of Sun Yat-Sen University. Qualified samples were cultured and passaged. We aimed to assess the sensitivity of in vitro hyperthermic perfusion using five different regimens, i.e. mitomycin C, mitomycin C combined with cisplatin, mitomycin C combined with 5-fluorouracil, oxaliplatin, and oxaliplatin combined with 5-fluorouracil.

Tumor organoids obtained from 46 patients with CRC were cultured, and in vitro hyperthermic perfusion experiments were conducted on 42 organoids using five different regimens. The average inhibition rate of mitomycin C was 85.2% (95% confidence interval [CI] 80.4-89.9%), mitomycin C combined with cisplatin was 85.5% (95% CI 80.2-90.7%), mitomycin C combined with 5-fluorouracil was 65.6% (95% CI 59.6-71.6%), oxaliplatin was 37.9% (95% CI 31.5-44.3%), and oxaliplatin combined with 5-fluorouracil was 40.7% (95% CI 33.9-47.5%).

In vitro hyperthermic perfusion demonstrates that the inhibition rate of mitomycin C, both alone and in combination with cisplatin, surpasses that of the combination of mitomycin C with 5-fluorouracil and oxaliplatin. In clinical practice, the combination of mitomycin C and cisplatin can be regarded as the optimal choice for HIPEC in CRC.

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.

Glioblastoma (GBM) has a fatal prognosis because of its aggressive and invasive characteristics. Understanding the mechanism of invasion necessitates an elucidation of the relationship between tumor cells and the tumor microenvironment. However, there has been a scarcity of suitable models to investigate this. In this study, we established a glioblastoma-cerebral organoid assembloid (GCOA) model by co-culturing patient-derived GBM tumoroids and human cerebral organoids. Tumor cells from the tumoroids infiltrated the cerebral organoids, mimicking the invasive nature of the parental tumors. Using time-lapse imaging, various invasion patterns of cancer cells within cerebral organoids resembling a normal tissue milieu were monitored. Both single- and collective-cell invasion was captured in real-time. We also confirmed the formation of an intercellular tumor network and tumor-normal-cell interactions. Furthermore, the transcriptomic characterization of GCOAs revealed distinct features of invasive tumor cells. Overall, this study established the GCOA as a three-dimensional (3D) in vitro assembloid model to investigate invasion mechanisms and interactions between tumor cells and their microenvironment.

Studying the human immune system in vivo is challenging and often not possible. Therefore, most human immunology studies have been predominantly confined to peripheral blood analyses, which by themselves have inherent limitations, as many immune reactions take place within tissues. For example, potent antibody responses that contribute to fighting infections and provide protection following vaccination require cellular interactions between B cells and T cells in specialized micro-anatomical structures called germinal centers, which are found in secondary lymphoid organs such as spleen, lymph nodes, and tonsils. Thus, there is a clear demand for novel enhanced experimental systems that faithfully recapitulate the intricate dynamics of the human immune system as much as possible. In this review, we discuss recent advances in versatile human tonsil/adenoid tissue-based ex vivo immune organoid cultures as well as related cancer and autoimmunity-focused experimental setups. These systems have been implemented as translational immunology platforms for in-depth analyses of human B and T cell-mediated immune responses, thereby facilitating mechanistic studies as well as drug and vaccine testing in a human-first approach.

Approximately 70% of breast cancer (BC) cases are luminal-type (estrogen receptor-positive, ER+), suitable for endocrine therapy with tamoxifen as the most commonly used drug. However, about 30% of these patients develop tamoxifen resistance due to various mechanisms, primarily involving PI3K pathway activation through mutations or unknown pathways. Here, we discover, via bioinformatics analysis and clinical samples, that N6 adenine-specific DNA methyltransferase 1 (N6AMT1) is highly expressed in luminal breast cancer but downregulated in tamoxifen-resistant (TamR) BC cells. ChIP-qPCR and luciferase reporter assays showed that FOXA1 binds to the N6AMT1 promoter and enhances its transcription. In TamR models, FOXA1 and N6AMT1 are downregulated, increasing p110α protein levels (but not mRNA), phospho-AKT levels, and tamoxifen resistance. In vivo, N6AMT1 overexpression enhanced tamoxifen sensitivity, while knockdown reduced it; this sensitivity could be restored with the p110α inhibitor A66. Clinically, decreased N6AMT1 expression correlates with poor prognosis in luminal BC patients. In TamR BC organoids, combining tamoxifen with A66 further reduced growth compared to either treatment alone. Mechanistically, increased p110α levels result from inhibited degradation by E3 ubiquitin ligase NEDD4L. These findings suggest N6AMT1 as a potential luminal breast cancer biomarker and highlight the N6AMT1-p110α pathway as a therapeutic target to sensitize cells to tamoxifen.

Hepatic organoid-based modelling, through the elucidation of a range of in vivo biological processes and the recreation of the intricate liver microenvironment, is yielding groundbreaking insights into the pathophysiology and personalized medicine approaches for liver diseases.

This study was designed to analyse the global scientific output of hepatic organoid research and assess current achievements and future trends through bibliometric analysis.

Articles were retrieved from the Web of Science Core Collection, and CiteSpace 6.3.R1 was employed to analyse the literature, including outputs, journals, and countries, among others.

Between 2010 and 2024, a total of 991 articles pertaining to hepatic organoid research were published. The journal Hepatology published the greatest number of papers, and journals with an impact factor greater than 10 constituted 60% of the top 10 journals. The United States and Utrecht University were identified as the most prolific country and institution, respectively. Clevers H emerged as the most prolific author, whereas Huch M had the highest number of cocitations, suggesting that both are ideal candidates for academic collaboration. Research on hepatic organoids has exhibited a progressive shift in focus, evolving from initial investigations into model building, differentiation research in stem cells, bile ducts, and progenitor cells, to a broader spectrum encompassing lipid metabolism, single-cell RNA sequencing, and therapeutic applications. The phrases exhibiting citation bursts from 2022 to 2024 include "drug resistance", "disease model", and "patient-derived tumor organoids".

Research on hepatic organoids has increased over the past decade and is expected to continue to grow. Key research areas include applications for liver diseases and drug development. Future trends likely to gain focus include patient-derived tumour organoids, disease modelling, and personalized medicine.

Cell cultures, organs-on-chip and microphysiological systems become increasingly relevant as in vitro models, e.g., in drug development, disease modelling, toxicology or cancer research. It has been underlined repeatedly that culture conditions and metabolic cues have a strong or even essential influence on the reproducibility and validity of such experiments but are often not appropriately measured or controlled. Here we review microsensor systems for cell metabolism for the continuous measurement of culture conditions in microfluidic and lab-on-chip platforms. We identify building blocks, features and essential advantages to underline the relevance of these systems and to derive appropriate requirements for development and practical use. We discuss different formats and geometries of cell culture, microfluidics and the resulting consequences for sensor placement, as the prerequisite for understanding the various approaches and classification of the systems. The major chemical and biosensors based on electrochemical and optical principles are discussed for general understanding and to contextualize current developments. We then review selected recent sensor systems with real-world implementations of sensing in cell cultures and organs-on-chip, employing a helpful characterization. That includes formats and cell models, microfluidic systems and sensor types applied in static and dynamic monitoring of 2D and 3D cell cultures, as well as single spheroids. We discuss notable advances, particularly with respect to sensor performance and the demonstration of long-term continuous measurements. We outline current approaches to system fabrication technologies, material choice, and interfacing, and comment on recent trends. Finally, we conclude with critical remarks on the current state of sensors in cell culture monitoring and identify avenues for future improvements for both developers and users of such systems, which will lead to better and more predictive in vitro models.

Despite missense mutation accounts for over 60% of p53 alterations while homozygous deletion (HOM) for only 5% or less in advanced bladder cancer cases, most of the previously reported mouse models are deficient of p53. Accordingly, few studies have addressed the mechanisms of missense mutation occurrence and its functional advantage over HOM in bladder cancer development. Organoids derived from Krt5-expressing mouse urothelium (K5-mUrorganoid) demonstrated the crucial role of Pten loss in driving loss of wild-type allele of Trp53 (Trp53R172H/LOH), which conferred tumorigenic ability to K5-mUrorganoid in athymic mice. These tumors recapitulated the histological and genetic characteristics of the human basal-squamous subtype bladder cancer. Both Trp53R172H/Δ; PtenΔ/Δ and Trp53Δ/Δ; PtenΔ/Δ K5-mUrorganoids formed tumors in athymic mice, whereas only Trp53R172H/Δ; PtenΔ/Δ K5-mUrorganoid formed tumors even when directly inoculated in immunocompetent syngeneic mice. The absence of wild-type Trp53 was associated with upregulation of proliferative signaling, and the presence of a mutant Trp53 allele was associated with immune-excluded microenvironment. This study highlights the functional significance of p53 mutant LOH in bladder carcinogenesis conferring several hallmarks of cancer such as sustaining proliferative signaling and avoiding immune destruction, thus provides a novel immunocompetent mouse model of urothelial carcinoma harboring p53 mutations as a novel tool for cancer immunology research.

Canine apocrine gland anal sac adenocarcinoma (AGASACA) is a rare, malignant tumor in dogs. To date, few cell lines are available and used to establish the current treatment protocols. Organoids are three-dimensional cell cultures derived mainly from stem cells and can reproduce tissue's epithelial structure, function, and genetics, and thus, of great promise in precision medicine. In the current investigation, 10 AGASACA organoid lines were developed from surgically removed tissues of AGASACA-affected dogs and analyzed for comparison with the original tissues. AGASACA organoids were successfully generated from all cases and were positive for CK7, HER2, p53, p63, VEGF, and Ki67, and negative for CK20, consistent with previous reports in dogs and humans. Electron microscopic imaging of AGASACA organoids showed organelles, including numerous granules and fat droplets that characterize apocrine gland cells. AGASACA organoids were tumorigenic in vivo in immunodeficient mice. In addition, treatment of the AGASACA organoids with carboplatin, mitoxantrone, toceranib, and lapatinib revealed different sensitivity profiles among lineages, with carboplatin and lapatinib, in particular, being divided into sensitive and resistant ones. In contrast, mitoxantrone and toceranib showed generally high efficacy in all organoids. In conclusion, our established AGASACA organoids have the potential to be an experimental tool for the development of novel therapies for canine and human apocrine gland adenocarcinoma.

This study aimed to explore the mechanisms through which microRNAs (miRNAs) regulate 5-fluorouracil (5-FU) sensitivity in colorectal cancer (CRC) using organoid models. Fresh tissue samples from CRC tumors were collected, and CRC organoids were isolated and cultured. The consistency between CRC organoids and their derived tissues was validated. CRC organoids were treated with 5-FU, and ATP activity was measured. High-throughput sequencing of CRC organoids, combined with Gene Expression Omnibus (GEO) data analysis, was performed to examine miRNA expression following 5-FU treatment. Next, we investigated the cellular function of miR-526b-5p in CRC organoids and cells. Dual-luciferase reporter assays validated the binding of miR-526b-5p to the 3' UTR of TP53 mRNA. We successfully established CRC organoids that exhibited characteristics consistent with their source tissues. 5-FU treatment suppressed the proliferation and ATP activity of CRC organoids. High-throughput sequencing of CRC organoids, combined with GEO data analysis and quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation, revealed that hsa-miR-526b-5p levels were elevated following 5-FU treatment in CRC organoids and cells. Furthermore, hsa-miR-526b-5p was upregulated in CRC tissues compared to adjacent normal tissues, correlating with poor survival in CRC patients. Overexpression of hsa-miR-526b-5p mitigated the inhibitory effects of 5-FU on CRC organoid proliferation, migration, invasion, and ferroptosis. In contrast, silencing of hsa-miR-526b-5p impaired cell function and ferroptosis. Additionally, overexpression of hsa-miR-526b-5p decreased TP53 mRNA and protein levels while increasing the expression of SLC7A11 mRNA and protein. Silencing of hsa-miR-526b-5p resulted in the opposite effect. hsa-miR-526b-5p directly targeted and inhibited TP53 expression. Overexpression of TP53 diminished the promotive effect of hsa-miR-526b-5p on ferroptosis-related proteins GPX4 and SLC7A11, whereas inhibition of TP53 reversed the impact of hsa-miR-526b-5p silencing. Our study demonstrates that hsa-miR-526b-5p targets TP53 to regulate 5-FU sensitivity in CRC through the ferroptosis pathway based on CRC organoid models.

A drug to be successfully launched in the market requires a significant amount of capital, resources and time, where the unsuccessful results in the last stages lead to catastrophic failure for discovering drugs. This is the very reason which calls for the invention of innovative models that can closely mimic the human in vivo model for producing reliable results. Throughout the innovation line, there has been improvement in the rationale in silico designing but yet there is requirement for in vitro-in vivo correlations. During the evolving of the drug testing models, the 3D models produced by different methods have been proven to produce better results than the traditional 2D models. However, the in vitro fabrications of live tissues are still bottleneck in realizing their complete potential. There is an urgent need for the development of single, standard and simplified in vitro 3D tissue models that can be reliable for investigating the biological and pathological aspects of drug discovery, which is yet to be achieved. The existing pre-clinical models have considerable drawbacks despite being the gold standard in pre-clinical research. The major drawback being the interspecies differences and low reliability on the generated results. This gap could be overcome by the fabrication of bioengineered human disease models for drug screening. The advancement in the fabrication of 3D models will provide a valuable tool in screening drugs at different stages as they are one step closer to bio-mimic human tissues. In this review, we have discussed on the evolution of preclinical studies, and different models, including mini tissues, spheroids, organoids, bioengineered three dimensional models and organs on chips. Furthermore, we provide details of different disease models fabricated across various organs and their applications. In addition to this, the review also focuses on the limitations and the current prospects of the role of three dimensionally bioprinted models in drug screening and development.

Tumor treatment has undergone revolutionary changes with the development of immunotherapy, especially immune checkpoint inhibitors. Because not all patients respond positively to immune therapeutic agents, and severe immune-related adverse events (irAEs) are frequently observed, the development of the biomarkers evaluating the response of a patient is key for the application of immunotherapy in a wider range. Recently, various multi-omics features measured by high-throughput technologies, such as tumor mutation burden (TMB), gene expression profiles, and DNA methylation profiles, have been proved to be sensitive and accurate predictors of the response to immunotherapy. A large number of predictive models based on these features, utilizing traditional machine learning or deep learning frameworks, have also been proposed. In this review, we aim to cover recent advances in predicting tumor immunotherapy response using multi-omics features. These include new measurements, research cohorts, data sources, and predictive models. Key findings emphasize the importance of TMB, neoantigens, MSI, and mutational signatures in predicting ICI responses. The integration of bulk and single-cell RNA sequencing has enhanced our understanding of the tumor immune microenvironment and enabled the identification of predictive biomarkers like PD-L1 and IFN-γ signatures. Public datasets and machine learning models have also improved predictive tools. However, challenges remain, such as the need for large and diverse clinical datasets, standardization of multi-omics data, and model interpretability. Future research will require collaboration among researchers, clinicians, and data scientists to address these issues and enhance cancer immunotherapy precision.

Organoid culture has emerged as a forefront technology in the life sciences field. As "in vitro micro-organs", organoids can faithfully recapitulate the organogenesis process, and conserve the key structure, physiological function and pathological state of the original tissue or organ. Consequently, it is widely used in basic and clinical studies, becoming important preclinical models for studying diseases and developing therapies. Here, we introduced the definition and advantages of organoids and described the development and advances in hepatobiliary organoids research. We focus on applying hepatobiliary organoids in benign and malignant diseases of the liver and biliary tract, drug research, and regenerative medicine to provide valuable reference information for the application of hepatobiliary organoids. Despite advances in research and treatment, hepatobiliary diseases including carcinoma, viral hepatitis, fatty liver and bile duct defects have still been conundrums of the hepatobiliary field. It is necessary and crucial to study disease mechanisms, establish efficient and accurate research models and find effective treatment strategies. The organoid culture technology shed new light on solving these issues. However, the technology is not yet mature, and many hurdles still exist that need to be overcome. The combination with new technologies such as CRISPR-HOT, organ-on-a-chip may inject new vitality into future development.

Iron oxide-based nanoparticles are promising materials for cancer thermal therapy and immunotherapy. However, several proofs of concept reported data with murine tumor models that might have limitations for clinical translation. Magnetite is nowadays the most popular nanomaterial, but doping with distinct ions can enhance thermal therapy, namely, magnetic nanoparticle hyperthermia (MNH) and photothermal therapy (PTT). In this study, we used a 3D alveolar reconstructed A549 lung cancer tissue model and investigated the thermal properties, toxicity, and impact of the thermal dose on tissue viability and inflammatory response using magnetite codoped with 40% Zn and 2% Co divalent ions. The ZnCo-doped magnetite nanoparticles are not toxic up to an NP concentration of 30 mg/mL. PTT showed a better heat generation response than MNH under the evaluated conditions, while NP showed a high external photothermal conversion efficiency of ∼1.3 g·L-1·cm-1 at 808 nm. PTT study is carried out at different temperatures, 43 and 47 °C, for 15 min. Tissue viability decreased with increasing thermal dose, while intracelullar ROS levels increased, mitochondrial activity decreased, and active caspase-3 increased, suggesting cell death via apoptosis. Nanoparticles and PTT did not influence the cytokine TNF, IL-10, IL-1B, and IL-12p70. In contrast, IL-6 and IL-8 were triggered by NP and PTT. Increased expression of IL-6 and IL-8 with higher thermal doses is correlated with tissue injury results, suggesting the potential role in activating and attracting immune cells to the site of thermal-mediated tissue injury.

Over recent decades, in vitro and in vivo models have significantly advanced brain cancer research; however, each presents distinct challenges for accurately mimicking in situ conditions. In response, organotypic slice cultures have emerged as a promising model recapitulating precisely specific in vivo phenotypes through an ex vivo approach. Ex vivo organotypic brain slice models can integrate biological relevance and patient-specific variability early in drug discovery, thereby aiming for more precise treatment stratification. However, the challenges of obtaining representative fresh brain tissue, ensuring reproducibility, and maintaining essential central nervous system (CNS)-specific conditions reflecting the in situ situation over time have limited the direct application of ex vivo organotypic slice cultures in robust clinical trials. In this review, we explore the benefits and possible limitations of ex vivo organotypic brain slice cultures in neuro-oncological research. Additionally, we share insights from clinical experts in neuro-oncology on how to overcome these current limitations and improve the practical application of organotypic brain slice cultures beyond academic research.

Patient-derived tumor organoids have been leveraged for disease modeling and preclinical studies but rarely applied in real time to aid with interpretation of patient treatment responses in clinics. We recently demonstrated early efficacy signals in a first-in-human, phase 1 study of dual-targeting chimeric antigen receptor (CAR)-T cells (EGFR-IL13Rα2 CAR-T cells) in patients with recurrent glioblastoma. Here, we analyzed six sets of patient-derived glioblastoma organoids (GBOs) treated concurrently with the same autologous CAR-T cell products as patients in our phase 1 study. We found that CAR-T cell treatment led to target antigen reduction and cytolysis of tumor cells in GBOs, the degree of which correlated with CAR-T cell engraftment detected in patients' cerebrospinal fluid (CSF). Furthermore, cytokine release patterns in GBOs mirrored those in patient CSF samples over time. Our findings highlight a unique trial design and GBOs as a valuable platform for real-time assessment of CAR-T cell bioactivity and insights into immunotherapy efficacy.

In this study, we present an oncolytic virus (OV) evaluation system established using microfluidic organ-on-a-chip (OOC) systems and patient-derived organoids (PDOs), which was used in the development of a novel oncolytic virus, AD4-GHPE. An OV offers advantages such as good targeting ability and minimal side effects, and it has achieved significant breakthroughs when combined with immunotherapy in recent clinical trials. The development of OVs has become an emerging research focus. PDOs can preserve the heterogeneity of in situ tumor tissues, whereas microfluidic OOC systems can automate and standardize various experimental procedures. These systems have been applied in cutting-edge drug screening and cell therapy experiments; however, their use in functionally complex oncolytic viruses remains to be explored. In this study, we constructed a novel recombinant oncolytic adenovirus, AD4-GHPE, and evaluated OOC systems and PDOs through various functional validations in hypopharyngeal and breast cancer organoids. The results confirmed that AD4-GHPE exhibits three antitumor mechanisms, namely, tumor-specific cytotoxicity, a reduction in programmed death ligand 1 (PD-L1) expression in tumor cells to increase CD8+ T-cell activity, and granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion. The evaluation system combining OOC systems and PDOs was efficient and reliable, providing personalized OV treatment recommendations for patients and offering industrialized and standardized research ideas for the development of OVs.