For a long time, the immunosuppressive tumor microenvironment (TME) limits the efficacy of immunotherapies for solid tumors. In particular, tumor–stromal ECM alignment restricts T-cell infiltration, yet mechanistic in vitro models that reproduce this feature are scarce.

In this thesis, several novel in vitro platforms that recapitulate the structure and physiology of the tumor stroma were developed via microtissue self-assembly and used to systematically probe how stromal architecture modulates cytotoxic T-cell behavior. Using complementary 2D microfluidic and 3D spheroid-based models, key in vivo stromal features were closely reproduced, and it was demonstrated that condensed, aligned ECM fibers substantially impede T-cell infiltration and impair antitumor activity. In particular, circumferentially aligned fibers—tumor-associated collagen signature 2 (TACS-2)—significantly restricted T-cell entry by guiding cell movement, as antifibrotic treatment effectively restored T-cell penetration. Finally, tumor stromal ECM architecture was shown to be tunable by modulating growth-factor composition in the culture medium.

These results validate the in vitro platforms as robust, functional models of the tumor stroma, demonstrating that they reproduce ECM-alignment–mediated barriers to T-cell infiltration, and the platforms are established as experimental systems for mechanistic studies and for testing interventions to overcome stromal immunosuppression.