A 3D Ex Vivo Tumor‐Immune Coculture System Mimicking In Vivo Tumor Environmental Stress on CD8+ T Cells Exhaustion

Abstract Dissection of exhaustion trajectories of immune cells under tumor selection pressure in the tumor microenvironment (TME) elucidates the underlying machinery in anti‐tumor immunity, which still lacks easy‐to‐use models to decipher. Herein, gelatin methacryloyl (GelMA)–poly (ethylene oxide) (PEO) based 3D hydrogel microspheroids are constructed with non‐immunogenicity and controllable macroporous structure to establish a tumor‐immune cell coculture (3D‐HyGTIC) system. In 3D‐HyGTIC system, when immune cells embarked, stepwise up‐regulation of main immune checkpoints (ICs) molecules is observed with compromised cytokine production in CD8 + T cells, the trajectory of which is in lineage correlation with in vivo grafted tumors. Reinvigoration of CD8 + T cells is more obvious with the addition of an anti‐PD‐1 regimen at the early time point, which is recapitulated during the coculture of patient‐derived tumor fragments (PDTF) and autologous T cells. Moreover, the upregulation of LAG‐3 on CD8 + T cells after anti‐PD‐1 treatment is uncovered. Sequential addition of anti‐LAG‐3 successfully rescues the otherwise failed reactivation of CD8 + T cells. Therefore, the 3D‐HyGTIC system is not only inclined to mimic the early differentiation trajectories of tumor‐infiltrating CD8 + T cells but also may facilitate an evaluation of the efficacy of IC blockades and guide the designing of combination immunotherapy.