Cytoskeletal engineering through Formin-like 1 overexpression enhances T cell infiltration and antitumor potency in solid tumors

Abstract

Solid tumors are often surrounded by abnormal vasculature and a dense collagen-rich extracellular matrix that severely restrict the infiltration of T cells, including tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR)-T cells. These physical barriers represent a major obstacle to the efficacy of adoptive T cell therapies in solid tumors. We previously identified Formin-like 1 (FMNL1) as a cytoskeletal regulator critical for T cell extravasation and migration through restrictive environments, making it a promising target to improve T cell infiltration into tumors. Here, we developed a bioengineering platform to enhance T cell cytoskeletal dynamics by overexpressing FMNL1 in TILs and CAR-T cells. FMNL1 overexpression significantly increased T cell migration through restrictive pores in transwell assays, supporting enhanced migratory capacity of T cells under mechanically constraining conditions. Importantly, FMNL1 overexpression did not impair T cell reactivation or cytotoxic function in vitro. In murine models of melanoma and lung carcinoma characterized by limited effector T cell infiltration, FMNL1-overexpressing TILs and CAR-T cells had significantly increased accumulation at tumor sites compared to controls. Importantly, enhanced tumor accumulation resulted in improved therapeutic activity, as adoptive transfer of FMNL1-overexpressing CAR-T cells limited tumor growth and prolonged the survival of tumor-bearing mice in multiple melanoma models. Together, our findings identify FMNL1 as a broadly applicable cytoskeletal engineering target to enhance T cell accumulation and persistence in restrictive tumor microenvironments, thereby overcoming a fundamental limitation of adoptive cellular immunotherapy in solid tumors.