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Life sciences · Journal article
Frontiers in Cell and Developmental Biology · October 15, 2026
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Introduction Cancer immunotherapies require accurate preclinical models that recapitulate the complex cellular interactions within the human tumor microenvironment. While patient-derived organoids have emerged as powerful tools for cancer research, standard cultures lack the stromal and immune compartments necessary to evaluate immunotherapeutic mechanisms. This limitation hampers the development of next-generation targeted cytokine therapies. Methods We applied a 3D immune-containing colorectal cancer assembloid platform integrating patient-derived organoids, cancer-associated fibroblasts and allogeneic peripheral blood mononuclear cells within a collagen matrix. We characterized cellular responses to interferon-gamma (IFNg) and engineered IFNg receptor agonists targeted to fibroblast activation protein (FAP) through single-cell transcriptomics, immunohistochemistry, cytokine profiling, and functional T-cell migration assays. Results The assembloid model successfully recapitulated key features of IFNg signaling, including tissue growth suppression, enhanced chemokine secretion (CXCL10), and CD8 + T-cell recruitment. Single-cell transcriptomics revealed that fibroblasts were the most responsive cell population, undergoing a phenotypic transition from myofibroblasts toward antigen-presenting CAFs (apCAFs), characterized by robust upregulation of MHC class II genes. FAP-targeted IFNg molecules exhibited attenuated activity compared to recombinant IFNg, successfully avoiding upregulation of the severe immunosuppressive checkpoint molecule PD-L1. Discussion This 3D assembloid model provides a physiologically relevant platform for dissecting complex tumor-immune interactions and optimizing targeted immunotherapies. This approach enables the study of epithelial-stromal-immune crosstalk in cancer and offers a robust preclinical framework for screening and mechanistically validating next-generation targeted cytokine therapies.