Allogeneic Immune Cell Perfusion Inhibits the Growth of Vascularized 3D In Vitro Tumor Models, Induces Vascular Regression and Desmoplasia, but Promotes Tumor Cell Invasion

ABSTRACT Advanced in vitro platforms incorporating vascularized tumors offer a promising approach to dissect biological interactions between cancer, stromal, and immune components, as well as for biological drug testing. Here, we employed a vascularized 3D bioreactor system to evaluate the impact of allogeneic peripheral blood mononuclear cell (PBMC) perfusion on breast cancer spheroids embedded within self‐organizing endothelial and stromal matrices. PBMC introduction results in rapid vascular regression, with reduced vessel density and interconnectivity of the self‐assembled networks. Tumor spheroids exposed to PBMC show increased apoptosis and pyroptosis, resulting in spheroid size reduction. Interestingly, this is accompanied by enhanced peripheral tumor cell proliferation and invasive dissemination into the surrounding matrix. While tumor spheroids alone stabilize vascular networks and activate stromal components, PBMC perfusion triggers further stromal activation and desmoplasia, indicating inflammation and immune‐mediated cytotoxicity. This approach demonstrates the multifaceted impact of allogeneic immune cell perfusion, including tumor suppression, vascular regression, stromal activation, and invasive tumor behavior, collectively reshaping the tumor microenvironment through innate immune‐driven inflammation. These findings emphasize the importance of accounting for donor mismatch and innate immune activation in designing translationally relevant vascularized tumor models, and they support the development of autologous systems.