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  3. Engineering a multilayered 3D stromal barrier model for quantitative analysis of T cell infiltration and cytotoxicity

Engineering a multilayered 3D stromal barrier model for quantitative analysis of T cell infiltration and cytotoxicity

  • Acta Biomater. 2025 Sep 10:S1742-7061(25)00677-4. doi: 10.1016/j.actbio.2025.09.012.
Rii Morimura 1,  Isana Nada 1,  Yuka Mizue 2,  Eiji Shinozaki 3,  Naoya Fujita 4,  Ryohei Katayama 5,  Michiya Matsusaki 6,  Yoshihiko Hirohashi 7,  Shiro Kitano 8,  Toshihiko Torigoe 2
Affiliations

Affiliations

  • 1 Business Development Division, Technical Research Institute, TOPPAN Holdings Inc., Saitama 345-8508, Japan; Division of Clinical Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan.
  • 2 Department of Pathology, Sapporo Medical University, Sapporo 060-8556, Japan.
  • 3 Department of Gastroenterological Chemotherapy, The Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan.
  • 4 Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan.
  • 5 Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan.
  • 6 Department of Applied Chemistry Graduate School of Engineering Osaka University, Osaka 565-0871, Japan.
  • 7 Department of Pathology, Sapporo Medical University, Sapporo 060-8556, Japan. Electronic address: hirohash@sapmed.ac.jp.
  • 8 Business Development Division, Technical Research Institute, TOPPAN Holdings Inc., Saitama 345-8508, Japan. Electronic address: shiro.kitano@toppan.co.jp.
PMID: 40939760 DOI: 10.1016/j.actbio.2025.09.012
Abstract

The development of immunocompetent three-dimensional (3D) culture systems is critical for advancing in vitro models that enable precise analysis of immune-tumor interactions. Here, we report a biomaterial-based method for engineering a multilayered 3D stromal construct using the cell-assembled viscous tissues (CAViTs) approach. This system enables spatial compartmentalization of Cancer cells and stromal components, including fibroblasts and endothelial cells, thereby mimicking the tumor microenvironment (TME). When co-cultured with tumor-specific cytotoxic T lymphocytes (CTLs), the system permits quantitative analysis of T cell infiltration and cytotoxicity. Moreover, we constructed a cancer-associated fibroblast (CAF)-rich stroma to model immune exclusion. Drug screening using this model identified histone deacetylase (HDAC) inhibitors as agents capable of reducing stromal barrier function by downregulating ECM components, thereby enhancing T cell penetration. This platform provides a robust, tunable, and reproducible in vitro model for investigating immune-stroma dynamics and accelerating immunotherapeutic discovery. STATEMENT OF SIGNIFICANCE: We present a biomaterial-based method for engineering a multilayered 3D stromal construct using the cell-assembled viscous tissues approach. This system enables spatial compartmentalization of Cancer cells and stromal components, closely mimicking the tumor microenvironment. Within this model, tumor cell killing by CTLs was successfully observed, resembling a "hot tumor" phenotype. Furthermore, we established a CAF-rich stroma to recapitulate immune exclusion. Drug screening using this platform revealed that HDAC inhibitors enhanced CTL-mediated cytotoxicity. Overall, this platform provides a robust, tunable, and reproducible in vitro model for investigating immune-stroma interactions and accelerating the discovery of novel immunotherapeutic strategies.

Keywords

3D culture; Cancer associated fibroblast; Cell-assembled viscous tissues; HDAC inhibitor; Tumor microenvironment.

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