{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T14:42:12Z","timestamp":1787064132818,"version":"build-2736575974"},"reference-count":34,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T00:00:00Z","timestamp":1777334400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["82272920"],"award-info":[{"award-number":["82272920"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]},{"award":["82272920"],"award-info":[{"award-number":["82272920"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"State Key Laboratory of Systems Medicine for Cancer","award":["zz-RCPY-25-17"],"award-info":[{"award-number":["zz-RCPY-25-17"]}]},{"name":"State Key Laboratory of Systems Medicine for Cancer","award":["zz-RCPY-26-13"],"award-info":[{"award-number":["zz-RCPY-26-13"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Glypican-3 (GPC3)-targeted chimeric antigen receptor T (CAR-T) cell therapy is a promising approach for hepatocellular carcinoma (HCC), but marked interpatient variability and antigen heterogeneity limit its broader application. Here, we established a patient-derived organoid (PDO)-based platform to functionally evaluate autologous GPC3-targeted CAR-T cell activity in HCC. HCC PDOs preserved key histologic features and heterogeneous GPC3 expression patterns of the original tumors. In co-culture assays, CAR-T cell cytotoxicity was associated with GPC3 expression levels and was accompanied by IFN-\u03b3 and IL-2 release, supporting the feasibility of using PDOs for functional assessment of CAR-T cell sensitivity. We further found that matrix conditions strongly influenced organoid architecture, viral transduction, CAR-T cell infiltration, and killing efficiency, with lower Matrigel concentrations providing a more permissive setting for functional assessment. Importantly, in GPC3-low PDOs, pretreatment with the DNA methyltransferase inhibitor 5-azacytidine (5-AZA) reduced DNA methyltransferase 3 alpha (DNMT3A) expression, increased surface GPC3 expression, and significantly enhanced CAR-T-mediated cytotoxicity. Together, these findings provide proof-of-concept evidence supporting the use of HCC PDOs as a patient-derived platform for modeling selected determinants of GPC3-targeted CAR-T cell activity and for exploring combination strategies to improve therapeutic efficacy.<\/jats:p>","DOI":"10.3390\/cells15090799","type":"journal-article","created":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T13:32:22Z","timestamp":1777469542000},"page":"799","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Patient-Derived Organoid Modeling of Glypican-3 CAR-T Responses in Hepatocellular Carcinoma"],"prefix":"10.3390","volume":"15","author":[{"given":"Bohan","family":"Zhang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yun","family":"Deng","sequence":"additional","affiliation":[{"name":"Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Fudan University, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-5679-2037","authenticated-orcid":false,"given":"Mingshan","family":"Zhou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junfei","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China"},{"name":"Department of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiawen","family":"Wu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaofeng","family":"Lian","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miaoxin","family":"Zhu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min","family":"Zhou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Cao","sequence":"additional","affiliation":[{"name":"Department of Liver Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, 160 Pujian Road, Shanghai 200127, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1345","DOI":"10.1016\/S0140-6736(22)01200-4","article-title":"Hepatocellular carcinoma","volume":"400","author":"Vogel","year":"2022","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1038\/s41575-019-0186-y","article-title":"A global view of hepatocellular carcinoma: Trends, risk, prevention and management","volume":"16","author":"Yang","year":"2019","journal-title":"Nat. Rev. Gastroenterol. Hepatol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1830","DOI":"10.1200\/JCO.23.02745","article-title":"Systemic Therapy for Advanced Hepatocellular Carcinoma: ASCO Guideline Update","volume":"42","author":"Gordan","year":"2024","journal-title":"J. Clin. Oncol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"102175","DOI":"10.1016\/j.eclinm.2023.102175","article-title":"RUNX-3-expressing CAR T cells targeting glypican-3 in patients with heavily pretreated advanced hepatocellular carcinoma: A phase I trial","volume":"63","author":"Fu","year":"2023","journal-title":"eClinicalMedicine"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1186\/s13045-024-01625-7","article-title":"Optimizing CAR-T cell therapy for solid tumors: Current challenges and potential strategies","volume":"17","author":"Ai","year":"2024","journal-title":"J. Hematol. Oncol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3979","DOI":"10.1158\/1078-0432.CCR-19-3259","article-title":"Chimeric Antigen Receptor-Glypican-3 T-Cell Therapy for Advanced Hepatocellular Carcinoma: Results of Phase I Trials","volume":"26","author":"Shi","year":"2020","journal-title":"Clin. Cancer Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1016\/j.cpt.2025.12.002","article-title":"Safety and efficacy of JWATM204, a novel Glypican-3 (GPC3)-targeted CAR T cell therapy for advanced hepatocellular carcinoma: A phase I dose-escalation study","volume":"4","author":"Yang","year":"2026","journal-title":"Cancer Pathog. Ther."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1186\/s13045-022-01278-4","article-title":"Tumor organoids: Applications in cancer modeling and potentials in precision medicine","volume":"15","author":"Xu","year":"2022","journal-title":"J. Hematol. Oncol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"920","DOI":"10.1126\/science.aao2774","article-title":"Patient-derived organoids model treatment response of metastatic gastrointestinal cancers","volume":"359","author":"Vlachogiannis","year":"2018","journal-title":"Science"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Su, C., Olsen, K.A., Bond, C.E., and Whitehall, V.L.J. (2023). The Efficacy of Using Patient-Derived Organoids to Predict Treatment Response in Colorectal Cancer. Cancers, 15.","DOI":"10.3390\/cancers15030805"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"5015","DOI":"10.1158\/1078-0432.CCR-25-1564","article-title":"Patient-Derived Organoids Predict Treatment Response in Metastatic Colorectal Cancer","volume":"31","author":"Smabers","year":"2025","journal-title":"Clin. Cancer Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1424","DOI":"10.1038\/nm.4438","article-title":"Human primary liver cancer-derived organoid cultures for disease modeling and drug screening","volume":"23","author":"Broutier","year":"2017","journal-title":"Nat. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1363","DOI":"10.1016\/j.celrep.2018.07.001","article-title":"Organoid Models of Human Liver Cancers Derived from Tumor Needle Biopsies","volume":"24","author":"Nuciforo","year":"2018","journal-title":"Cell Rep."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Henke, E., Nandigama, R., and Ergun, S. (2019). Extracellular Matrix in the Tumor Microenvironment and Its Impact on Cancer Therapy. Front. Mol. Biosci., 6.","DOI":"10.3389\/fmolb.2019.00160"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Nguyen, D.T., Ogando-Rivas, E., Liu, R., Wang, T., Rubin, J., Jin, L., Tao, H., Sawyer, W.W., Mendez-Gomez, H.R., and Cascio, M. (2022). CAR T Cell Locomotion in Solid Tumor Microenvironment. Cells, 11.","DOI":"10.3390\/cells11121974"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1186\/s12943-023-01744-8","article-title":"Extracellular matrix remodeling in tumor progression and immune escape: From mechanisms to treatments","volume":"22","author":"Yuan","year":"2023","journal-title":"Mol. Cancer"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"123","DOI":"10.2147\/ITT.S296161","article-title":"Priming Leukemia with 5-Azacytidine Enhances CAR T Cell Therapy","volume":"10","author":"Xu","year":"2021","journal-title":"Immunotargets Ther."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Kim, D.J. (2025). The Role of the DNA Methyltransferase Family and the Therapeutic Potential of DNMT Inhibitors in Tumor Treatment. Curr. Oncol., 32.","DOI":"10.3390\/curroncol32020088"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1792","DOI":"10.1038\/s42003-025-09154-w","article-title":"Multi-omic analysis of hepatocellular carcinoma reveals aberrant cis-regulatory changes and dysregulated retrotransposons with prognostic potential","volume":"8","author":"Cheng","year":"2025","journal-title":"Commun. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Harjunpaa, H., Llort Asens, M., Guenther, C., and Fagerholm, S.C. (2019). Cell Adhesion Molecules and Their Roles and Regulation in the Immune and Tumor Microenvironment. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.01078"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.stem.2019.10.010","article-title":"Patient-Derived Organoids Predict Chemoradiation Responses of Locally Advanced Rectal Cancer","volume":"26","author":"Yao","year":"2020","journal-title":"Cell Stem Cell"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Klein, E., Hau, A.C., Oudin, A., Golebiewska, A., and Niclou, S.P. (2020). Glioblastoma Organoids: Pre-Clinical Applications and Challenges in the Context of Immunotherapy. Front. Oncol., 10.","DOI":"10.3389\/fonc.2020.604121"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1016\/j.cell.2019.08.052","article-title":"Integrated Proteogenomic Characterization of HBV-Related Hepatocellular Carcinoma","volume":"179","author":"Gao","year":"2019","journal-title":"Cell"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e121490","DOI":"10.1172\/jci.insight.121490","article-title":"Human primary liver cancer organoids reveal intratumor and interpatient drug response heterogeneity","volume":"4","author":"Li","year":"2019","journal-title":"JCI Insight"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1046","DOI":"10.1007\/s11596-025-00109-0","article-title":"Novel Prognostic Scoring Systems for Severe CRS after Anti-CD19 CAR-T-Cells in Acute B-Lymphoblastic Leukemia","volume":"45","author":"Ke","year":"2025","journal-title":"Curr. Med. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1038\/s41591-018-0036-4","article-title":"Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells","volume":"24","author":"Norelli","year":"2018","journal-title":"Nat. Med."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Nishida, T., and Kataoka, H. (2019). Glypican 3-Targeted Therapy in Hepatocellular Carcinoma. Cancers, 11.","DOI":"10.3390\/cancers11091339"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e011698","DOI":"10.1136\/jitc-2025-011698","article-title":"Predictive value of preclinical models for CAR-T cell therapy clinical trials: A systematic review and meta-analysis","volume":"13","author":"Gregor","year":"2025","journal-title":"J. Immunother. Cancer"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1016\/j.stem.2024.11.010","article-title":"Patient-derived glioblastoma organoids as real-time avatars for assessing responses to clinical CAR-T cell therapy","volume":"32","author":"Logun","year":"2025","journal-title":"Cell Stem Cell"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1491","DOI":"10.1038\/s41423-024-01228-9","article-title":"Specific ECM degradation potentiates the antitumor activity of CAR-T cells in solid tumors","volume":"21","author":"Zheng","year":"2024","journal-title":"Cell. Mol. Immunol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"101077","DOI":"10.1016\/j.mtbio.2024.101077","article-title":"Heat-inducible CAR-T overcomes adverse mechanical tumor microenvironment in a 3D bioprinted glioblastoma model","volume":"26","author":"Tang","year":"2024","journal-title":"Mater. Today Bio"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.cell.2019.11.036","article-title":"A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity","volume":"180","author":"Jacob","year":"2020","journal-title":"Cell"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1038\/s43856-022-00150-3","article-title":"Patient-derived tumor organoids for personalized medicine in a patient with rare hepatocellular carcinoma with neuroendocrine differentiation: A case report","volume":"2","author":"Meier","year":"2022","journal-title":"Commun. Med."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Malerba, S., Vladimirov, M., Goyal, A., Dulskas, A., Bausys, A., Cwalinski, T., Girnyi, S., Skokowski, J., Duka, R., and Molchanov, R. (2026). Artificial Intelligence Applications in Gastric Cancer Surgery: Bridging Early Diagnosis and Responsible Precision Medicine. J. Clin. Med., 15.","DOI":"10.3390\/jcm15062208"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/9\/799\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,30]],"date-time":"2026-04-30T08:21:02Z","timestamp":1777537262000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/9\/799"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,28]]},"references-count":34,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["cells15090799"],"URL":"https:\/\/doi.org\/10.3390\/cells15090799","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,28]]}}}