{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T18:31:14Z","timestamp":1789583474523,"version":"build-2803163510"},"reference-count":43,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T00:00:00Z","timestamp":1768521600000},"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":"publisher","award":["32471468"],"award-info":[{"award-number":["32471468"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in hematologic malignancies but has limited efficacy in solid tumors due to tumor microenvironment (TME) barriers that impede CAR T cell recognition, infiltration, and sustained function. Traditional 2D assays inadequately recapitulate these constraints, necessitating improved in vitro models. This study validated a 3D tumor spheroid platform using an agarose microwell system to generate uniform B7-H3-positive spheroids from multiple solid tumor cell lines, enabling the evaluation of CAR T cell activity. TME-relevant immune modulation under 3D conditions was analyzed by flow cytometry for B7-H3, MHC I\/II, and antigen processing machinery (APM), followed by co-culture with B7-H3 CAR T cells to assess cytotoxicity, spheroid integrity, tumor viability, and CAR T cell activation, exhaustion, and cytokine production. Two human cancer-cell-line-derived spheroids, DU 145 (prostate cancer) and SUM159 (breast cancer), retained B7-H3 expression, while MC38 (mouse colon cancer)-derived spheroids served as a B7-H3 negative control. Under 3D culture conditions, DU 145 and SUM159 spheroids acquire TME-like immune evasion characteristics and specifically downregulated MHC-I and APM (TAP1, TAP2, LMP7) with concurrent upregulation of MHC-II and calreticulin. Co-culture showed effective spheroid infiltration, cytotoxicity, and structural disruption, with infiltrating CAR T cells displaying higher CD4+ fraction, activation, exhaustion, effector\/terminal differentiation, and IFN-\u03b3\/TNF-\u03b1 production. This 3D platform recapitulates critical TME constraints and provides a cost-effective, feasible preclinical tool to assess CAR T therapies beyond conventional 2D assays.<\/jats:p>","DOI":"10.3390\/cells15020169","type":"journal-article","created":{"date-parts":[[2026,1,19]],"date-time":"2026-01-19T08:23:56Z","timestamp":1768811036000},"page":"169","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Three-Dimensional Tumor Spheroids Reveal B7-H3 CAR T Cell Infiltration Dynamics and Microenvironment-Induced Functional Reprogramming in Solid Tumors"],"prefix":"10.3390","volume":"15","author":[{"given":"Feng","family":"Chen","sequence":"first","affiliation":[{"name":"Key Laboratory of Luminescence Analysis and Molecular Sensing, Ministry of Education, School of Materials and Energy, Southwest University, Chongqing 400715, China"},{"name":"Division of Gastrointestinal and Oncologic Surgery, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-6116-6134","authenticated-orcid":false,"given":"Ke","family":"Ning","sequence":"additional","affiliation":[{"name":"Key Laboratory of Luminescence Analysis and Molecular Sensing, Ministry of Education, School of Materials and Energy, Southwest University, Chongqing 400715, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuanyuan","family":"Xie","sequence":"additional","affiliation":[{"name":"Key Laboratory of Luminescence Analysis and Molecular Sensing, Ministry of Education, School of Materials and Energy, Southwest University, Chongqing 400715, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoyan","family":"Yang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Luminescence Analysis and Molecular Sensing, Ministry of Education, School of Materials and Energy, Southwest University, Chongqing 400715, China"},{"name":"Division of Gastrointestinal and Oncologic Surgery, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6726-281X","authenticated-orcid":false,"given":"Ling","family":"Yu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Luminescence Analysis and Molecular Sensing, Ministry of Education, School of Materials and Energy, Southwest University, Chongqing 400715, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinhui","family":"Wang","sequence":"additional","affiliation":[{"name":"Division of Gastrointestinal and Oncologic Surgery, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"807","DOI":"10.1038\/s41423-020-0488-6","article-title":"The history and advances in cancer immunotherapy: Understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications","volume":"17","author":"Zhang","year":"2020","journal-title":"Cell. 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