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While cell‐free liquid biopsy (cfLB) approaches provide simple and inexpensive disease monitoring, cell‐based liquid biopsy (cLB) may enable additional molecular genetic assessment of systemic disease heterogeneity and preclinical model development. We investigated 71 blood samples of 62 patients with various advanced cancer types and subjected enriched circulating tumor cells (CTCs) to organoid culture conditions. CTC‐derived tumoroid models were characterized by DNA/RNA sequencing and immunohistochemistry, as well as functional drug testing. Results were linked to molecular features of primary tumors, metastases, and CTCs; CTC enumeration was linked to disease progression. Of 52 samples with positive CTC counts (≥1) from eight different cancer types, only CTCs from two salivary gland cancer (SGC) patients formed tumoroid cultures (P = 0.0005). Longitudinal CTC enumeration of one SGC patient closely reflected disease progression during treatment and revealed metastatic relapse earlier than clinical imaging. Multiomics analysis and functional in vitro drug testing identified potential resistance mechanisms and drug vulnerabilities. We conclude that cLB might add a functional dimension (to the genetic approaches) in the personalized management of rare, difficult‐to‐treat cancers such as SGC. We quantified and cultured circulating tumor cells (CTCs) of 62 patients with various cancer types and generated CTC‐derived tumoroid models from two salivary gland cancer patients. Cellular liquid biopsy‐derived information enabled molecular genetic assessment of systemic disease heterogeneity and functional testing for therapy selection in both salivary gland cancer patients, which may provide a paradigm for other rare cancers.

Organoids are three‐dimensional (3D) miniaturized versions of organs or tissues that are derived from cells with stem potential and can self‐organize and differentiate into 3D cell masses, recapitulating the morphology and functions of their in vivo counterparts. Organoid culture is an emerging 3D culture technology, and organoids derived from various organs and tissues, such as the brain, lung, heart, liver, and kidney, have been generated. Compared with traditional bidimensional culture, organoid culture systems have the unique advantage of conserving parental gene expression and mutation characteristics, as well as long‐term maintenance of the function and biological characteristics of the parental cells in vitro. All these features of organoids open up new opportunities for drug discovery, large‐scale drug screening, and precision medicine. Another major application of organoids is disease modeling, and especially various hereditary diseases that are difficult to model in vitro have been modeled with organoids by combining genome editing technologies. Herein, we introduce the development and current advances in the organoid technology field. We focus on the applications of organoids in basic biology and clinical research, and also highlight their limitations and future perspectives. We hope that this review can provide a valuable reference for the developments and applications of organoids. Organoids can be generated by inducing and culturing pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), adult stem cells (ASCs), and tumor cells from healthy donors or patients. The potential biomedical applications for organoids include disease modeling, drug screening, toxicity assays, personalized medicine, biobank, biomarker discovery, and regenerative medicine.

The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200–1000 nm) and small EVs (50–200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

PurposeThe aim of this study was to set up reliable and reproducible culture conditions for 3D tumoroids derived from non-small cell lung cancer (NSCLC) cell lines to enable greater opportunity for successful cultivation of patient-derived samples.MethodsFour NSCLC cell lines, two adenocarcinomas (A549, NCI-H1975) and two squamous cell carcinomas (HCC-95, HCC-1588), were first cultured in traditional 2D settings. Their expected expression profiles concerning TTF-1, CK7, CK5, and p40 status were confirmed by immunohistochemistry (IHC) before the generation of 3D cultures. Tumoroids were established in the hydrogel GrowDex®-T, Nunclon™ Sphera™ flasks, BIOFLOAT™ plates, and Corning® Elplasia® plates. Western blot was used to verify antigen protein expression. Hematoxylin-eosin staining was used to evaluate the cell morphology in the 2D and 3D cultures. Mutational analysis of KRAS and EGFR by PCR on extracted DNA from 3D tumoroids generated from cells with known mutations (A549; KRAS G12S mutation, NCI-H1975; EGFR L858R/T790M mutations).ResultsWe successfully established 3D cultures from A549, NCI-H1975, HCC-95, and HCC-1588 with all four used cultivation methods. The adenocarcinomas (A549, NCI-H1975) maintained their original IHC features in the tumoroids, while the squamous cell carcinomas (HCC-95, HCC-1588) lost their unique markers in the cultures. PCR analysis confirmed persistent genetic changes where expected.ConclusionThe establishment of tumoroids from lung cancer cell lines is feasible with various methodologies, which is promising for future tumoroid growth from clinical lung cancer samples. However, analysis of relevant markers is a prerequisite and may need to be validated for each model and cell type.

Ovarian cancer has the highest mortality of all of the gynecological malignancies. There are several distinct histotypes of this malignancy characterized by specific molecular events and clinical behavior. These histotypes have differing responses to platinum-based drugs that have been the mainstay of therapy for ovarian cancer for decades. For histotypes that initially respond to a chemotherapeutic regime of carboplatin and paclitaxel such as high-grade serous ovarian cancer, the development of chemoresistance is common and underpins incurable disease. Recent discoveries have led to the clinical use of PARP (poly ADP ribose polymerase) inhibitors for ovarian cancers defective in homologous recombination repair, as well as the anti-angiogenic bevacizumab. While predictive molecular testing involving identification of a genomic scar and/or the presence of germline or somatic BRCA1 or BRCA2 mutation are in clinical use to inform the likely success of a PARP inhibitor, no similar tests are available to identify women likely to respond to bevacizumab. Functional tests to predict patient response to any drug are, in fact, essentially absent from clinical care. New drugs are needed to treat ovarian cancer. In this review, we discuss applications to address the currently unmet need of developing physiologically relevant in vitro and ex vivo models of ovarian cancer for fundamental discovery science, and personalized medicine approaches. Traditional two-dimensional (2D) in vitro cell culture of ovarian cancer lacks critical cell-to-cell interactions afforded by culture in three-dimensions. Additionally, modelling interactions with the tumor microenvironment, including the surface of organs in the peritoneal cavity that support metastatic growth of ovarian cancer, will improve the power of these models. Being able to reliably grow primary tumoroid cultures of ovarian cancer will improve the ability to recapitulate tumor heterogeneity. Three-dimensional (3D) modelling systems, from cell lines to organoid or tumoroid cultures, represent enhanced starting points from which improved translational outcomes for women with ovarian cancer will emerge.

Background: Pancreatic neuroendocrine neoplasia (PanNEN) comprises a spectrum, from well-differentiated (i.e., G1, G2) pancreatic neuroendocrine tumors (PanNETs) to poorly differentiated carcinomas (PanNECs). Therapeutic progress is limited by the lack of representative preclinical models. Patient-derived organoids (PDOs) offer potential as translational models, but evidence remains scattered. Methods: We conducted a systematic review of PubMed (Jan 2009–Aug 2025) for original studies reporting on PDOs from PanNEN patients. Eligible studies were screened using the Rayyan software and data extracted from PDO take rates, validation methods, and clinical applications. Results: Twelve studies were included for qualitative and quantitative analyses. PDOs were successfully generated from both PanNETs (G1–G3; n = 26) and PanNECs (n = 6), primarily derived from primary tumors, but several studies also included metastatic sites. Take rates ranged from 33% to 100%, for a cumulative 33 PDOs from 44 attempts (overall take rate: 75%). Validation consistently employed histology, immunohistochemistry, and molecular profiling, with several studies incorporating xenotransplantation or omics approaches. PDOs demonstrated variable culture durations, from short-term (<3 weeks) to long-term (>20 passages). Drug screening studies (n = 7) revealed heterogenous responses to standard agents and pathways (everolimus, sunitinib, and temozolomide) and identified novel vulnerabilities, including EZH2 dependency, PI3K/CDK4/6 synergy, and Bcl-2-linked sensitivities in PanNECs. One study provided evidence of concordance between PDO drug sensitivity and patient responses. Conclusions: Research into PanNEN organoids remains limited. However, PDOs can preserve key histological and molecular features, enable pharmacotyping, and uncover candidate biomarkers for therapy. Despite feasibility across subtypes, progress is constrained by variability in culture success. Standardization and prospective validation are essential to advance PDOs as tools for personalized medicine in PanNENs.

Cancer is a heterogeneous disease. Each individual tumor is unique and characterized by structural, cellular, genetic and molecular features. Therefore, patient-derived cancer models are indispensable tools in cancer research and have been actively introduced into the healthcare system. For instance, patient-derived models provide a good reproducibility of susceptibility and resistance of cancer cells against drugs, allowing personalized therapy for patients. In this article, we review the advantages and disadvantages of the following patient-derived models of cancer: (1) PDC—patient-derived cell culture, (2) PDS—patient-derived spheroids and PDO—patient-derived organoids, (3) PDTSC—patient-derived tissue slice cultures, (4) PDX—patient-derived xenografts, humanized PDX, as well as PDXC—PDX-derived cell cultures and PDXO—PDX-derived organoids. We also provide an overview of current clinical investigations and new developments in the area of patient-derived cancer models. Moreover, attention is paid to databases of patient-derived cancer models, which are collected in specialized repositories. We believe that the widespread use of patient-derived cancer models will improve our knowledge in cancer cell biology and contribute to the development of more effective personalized cancer treatment strategies.

Current pre-clinical models of cancer fail to recapitulate the cancer cell behavior in primary tumors primarily because of the lack of a deeper understanding of the effects that the microenvironment has on cancer cell phenotype. Transcriptomic profiling of 4T1 murine mammary carcinoma cells from 2D and 3D cultures, subcutaneous or orthotopic allografts (from immunocompetent or immunodeficient mice), as well as ex vivo tumoroids, revealed differences in molecular signatures including altered expression of genes involved in cell cycle progression, cell signaling and extracellular matrix remodeling. The 3D culture platforms had more in vivo-like transcriptional profiles than 2D cultures. In vivo tumors had more cells undergoing epithelial-to-mesenchymal transition (EMT) while in vitro cultures had cells residing primarily in an epithelial or mesenchymal state. Ex vivo tumoroids incorporated aspects of in vivo and in vitro culturing, retaining higher abundance of cells undergoing EMT while shifting cancer cell fate towards a more mesenchymal state. Cellular heterogeneity surveyed by scRNA-seq revealed that ex vivo tumoroids, while rapidly expanding cancer and fibroblast populations, lose a significant proportion of immune components. This study emphasizes the need to improve in vitro culture systems and preserve syngeneic-like tumor composition by maintaining similar EMT heterogeneity as well as inclusion of stromal subpopulations.

Background Hepatocellular carcinoma (HCC) is characterised by a remarkable molecular heterogeneity and resistance to current therapies. The use of established HCC cell lines has been the gold standard for fundamental and drug screening studies. However, each cell line is a single clone lacking cell heterogeneity, thus limiting the evaluation of anticancer efficacy, with a consequent drop of translatability regarding their clinical effectiveness, notably in relation to HCC complexity. Additionally, 2D monolayer cultures do not reproduce cell-cell and cell-matrix interactions, known to influence biological, molecular, and signalling features of cancer cells and their response to treatments. Methods A panel of primary HCC cells and tumoroids was generated from primary tumours of the Alb-R26 Met mice. Morphological, molecular, and signalling features were evaluated by histology, imaging, RT-qPCR, and western blots. Proliferation capability and drug efficiency were evaluated through cell viability assays. Results We report the establishment of eight primary cells from distinct spontaneous HCC of the Alb-R26 Met mouse model. By evaluating their morphological, molecular, and signalling pathway characteristics, we illustrate their inter-/intra- tumour heterogeneity. We show their biological features by reporting their distinct proliferation rate and resistance to RTK inhibitors used in the clinic for HCC treatments. Moreover, we document their ability to generate tumoroids, optimizing a protocol for HCC 3D cultures. The robustness of the methodology we established is illustrated by the maintenance of morphological, molecular, and growth features along several passages. Finally, we exemplify the value of this tumoroid panel for anticancer treatment evaluation by assessing the effectiveness of a new combinatorial therapy for HCC that we recently identified. Conclusions Outcomes provide a robust methodology for the generation of HCC tumoroids and designate such heterogeneous tumoroid panel as a valuable setting for disease modelling and drug discovery.

We describe a method for quantifying the contractile forces that tumor spheroids collectively exert on highly nonlinear three-dimensional collagen networks. While three-dimensional traction force microscopy for single cells in a nonlinear matrix is computationally complex due to the variable cell shape, here we exploit the spherical symmetry of tumor spheroids to derive a scale-invariant relationship between spheroid contractility and the surrounding matrix deformations. This relationship allows us to directly translate the magnitude of matrix deformations to the total contractility of arbitrarily sized spheroids. We show that our method is accurate up to strains of 50% and remains valid even for irregularly shaped tissue samples when considering only the deformations in the far field. Finally, we demonstrate that collective forces of tumor spheroids reflect the contractility of individual cells for up to 1 hr after seeding, while collective forces on longer timescales are guided by mechanical feedback from the extracellular matrix.