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In the past decade, treatments targeting the immune system have revolutionized the cancer treatment field. Therapies such as immune checkpoint inhibitors have been approved as first-line treatment in a variety of solid tumors such as melanoma and non-small cell lung cancer while other therapies, for instance, chimeric antigen receptor (CAR) lymphocyte transfer therapies, are still in development. Although promising results are obtained in a small subset of patients, overall clinical efficacy of most immunotherapeutics is limited due to intertumoral heterogeneity and therapy resistance. Therefore, prediction of patient-specific responses would be of great value for efficient use of costly immunotherapeutic drugs as well as better outcomes. Because many immunotherapeutics operate by enhancing the interaction and/or recognition of malignant target cells by T cells, in vitro cultures using the combination of these cells derived from the same patient hold great promise to predict drug efficacy in a personalized fashion. The use of two-dimensional cancer cell lines for such cultures is unreliable due to altered phenotypical behavior of cells when compared with the in vivo situation. Three-dimensional tumor-derived organoids, better mimic in vivo tissue and are deemed a more realistic approach to study the complex tumor-immune interactions. In this review, we present an overview of the development of patient-specific tumor organoid-immune co-culture models to study the tumor-specific immune interactions and their possible therapeutic infringement. We also discuss applications of these models which advance personalized therapy efficacy and understanding the tumor microenvironment such as: (1) Screening for efficacy of immune checkpoint inhibition and CAR therapy screening in a personalized manner. (2) Generation of tumor reactive lymphocytes for adoptive cell transfer therapies. (3) Studying tumor-immune interactions to detect cell-specific roles in tumor progression and

Cholangiocarcinoma (CCA) is a type of liver cancer with an aggressive phenotype and dismal outcome in patients. The metastasis of CCA cancer cells to distant organs, commonly lung and lymph nodes, drastically reduces overall survival. However, mechanistic insight how CCA invades these metastatic sites is still lacking. This is partly because currently available models fail to mimic the complexity of tissue-specific environments for metastatic CCA. To create an in vitro model in which interactions between epithelial tumor cells and their surrounding extracellular matrix (ECM) can be studied in a metastatic setting, we combined patient-derived CCA organoids (CCAOs) (n=3) with decellularized human lung (n=3) and decellularized human lymph node (n=13). Decellularization resulted in removal of cells while preserving ECM structure and retaining important characteristics of the tissue origin. Proteomic analyses showed a tissue-specific ECM protein signature reflecting tissue functioning aspects. The macro and micro-scale mechanical properties, as determined by rheology and micro-indentation, revealed the local heterogeneity of the ECM. When growing CCAOs in decellularized lung and lymph nodes genes related to metastatic processes, including epithelial-to-mesenchymal transition and cancer stem cell plasticity, were significantly influenced by the ECM in an organ-specific manner. Furthermore, CCAOs exhibit significant differences in migration and proliferation dynamics dependent on the original patient tumor and donor of the target organ. In conclusion, CCA metastatic outgrowth is dictated both by the tumor itself as well as by the ECM of the target organ. Convergence of CCAOs with the ECM of its metastatic organs provide a new platform for mechanistic study of cancer metastasis.

Tumor initiation and progression are critically dependent on interaction of cancer cells with their cellular and extracellular microenvironment. Alterations in the composition, integrity, and mechanical properties of the extracellular matrix (ECM) dictate tumor processes including cell proliferation, migration, and invasion. Also in primary liver cancer, consisting of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), the dysregulation of the extracellular environment by liver fibrosis and tumor desmoplasia is pertinent. Yet, the exact changes occurring in liver cancer ECM remain uncharacterized and underlying tumor-promoting mechanisms remain largely unknown. Herein, an integrative molecular and mechanical approach is used to extensively characterize the ECM of HCC and CCA tumors by utilizing an optimized decellularization technique. We identified a myriad of proteins in both tumor and adjacent liver tissue, uncovering distinct malignancy-related ECM signatures. The resolution of this approach unveiled additional ECM-related proteins compared to large liver cancer transcriptomic datasets. The differences in ECM protein composition resulted in divergent mechanical properties on a macro- and micro-scale that are tumor-type specific. Furthermore, the decellularized tumor ECM was employed to create a tumor-specific hydrogel that supports patient-derived tumor organoids, which provides a new avenue for personalized medicine applications. Taken together, this study contributes to a better understanding of alterations to composition, stiffness, and collagen alignment of the tumor ECM that occur during liver cancer development.

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process.

Monitoring tumor growth dynamics is crucial for understanding cancer. To establish an in vitro method for the continuous assessment of patient-specific tumor growth, tumor organoids were generated from patients with intrahepatic CCA (iCCA). Organoid growth was monitored for 48 h by label-free live brightfield imaging. Growth kinetics were calculated and validated by MTS assay as well as immunohistochemistry of Ki67 to determine proliferation rates. We exposed iCCA organoids (iCCAOs) and non-tumor intrahepatic cholangiocyte organoids (ICOs) to sub-therapeutic concentrations of sorafenib. Monitoring the expansion rate of iCCAOs and ICOs revealed that iCCAO growth was inhibited by sorafenib in a time- and dose-dependent fashion, while ICOs were unaffected. Quantification of the proliferation marker Ki67 confirmed inhibition of iCCAO growth by roughly 50% after 48 h of treatment with 4 µM sorafenib. We established a robust analysis pipeline combining brightfield microscopy and a straightforward image processing approach for the label-free growth monitoring of patient-derived iCCAOs. Combined with bioanalytical validation, this approach is suitable for a fast and efficient high-throughput drug screening in tumor organoids to develop patient-specific systemic treatment options.

Hepatic, pancreatic, and biliary (HPB) organoids are powerful tools for studying development, disease, and regeneration. As organoid research expands, the need for clear definitions and nomenclature describing these systems also grows. To facilitate scientific communication and consistent interpretation, we revisit the concept of an organoid and introduce an intuitive classification system and nomenclature for describing these 3D structures through the consensus of experts in the field. To promote the standardization and validation of HPB organoids, we propose guidelines for establishing, characterizing, and benchmarking future systems. Finally, we address some of the major challenges to the clinical application of organoids.

The well-established 3D organoid culture method enabled efficient expansion of cholangiocyte-like cells from intrahepatic (IHBD) and extrahepatic bile duct (EHBD) tissue biopsies. The extensive expansion capacity of these organoids enables various applications, from cholangiocyte disease modelling to bile duct tissue engineering. Recent research demonstrated the feasibility of culturing cholangiocyte organoids from bile, which was minimal-invasive collected via endoscopic retrograde pancreaticography (ERCP). However, a detailed analysis of these bile cholangiocyte organoids (BCOs) and the cellular region of origin was not yet demonstrated. In this study, we characterize BCOs and mirror them to the already established organoids initiated from IHBD- and EHBD-tissue. We demonstrate successful organoid-initiation from extrahepatic bile collected from gallbladder after resection and by ERCP or percutaneous transhepatic cholangiopathy from a variety of patients. BCOs initiated from these three sources of bile all show features similar to in vivo cholangiocytes. The regional-specific characteristics of the BCOs are reflected by the exclusive expression of regional common bile duct genes (HOXB2 and HOXB3) by ERCP-derived BCOs and gallbladder-derived BCOs expressing gallbladder-specific genes. Moreover, BCOs have limited hepatocyte-fate differentiation potential compared to intrahepatic cholangiocyte organoids. These results indicate that organoid-initiating cells in bile are likely of local (extrahepatic) origin and are not of intrahepatic origin. Regarding the functionality of organoid initiating cells in bile, we demonstrate that BCOs efficiently repopulate decellularized EHBD scaffolds and restore the monolayer of cholangiocyte-like cells in vitro. Bile samples obtained through minimally invasive procedures provide a safe and effective alternative source of cholangiocyte organoids. The shedding of (organoid-initiating) cholangiocytes in bile provides a convenient sour