CCR2/CXCR6 enhances tumor infiltration and antitumor efficacy of MSLN CAR-T cells.

Chimeric antigen receptor T (CAR-T) cell therapy has shown promise in treating solid tumors, but the clinical success is often limited by insufficient tumor infiltration. In this study, we sought to engineered two MSLN-targeted CAR-T cell variants, Intra2 and Intra6, expressing CCR2 and CXCR6, respectively, to improve their migration toward the tumor microenvironment. Flow cytometry confirmed stable receptor expression. In vitro assays demonstrated that both Intra2 and Intra6 CAR-T cells exhibited significantly improved functional phenotype, migration and invasion, as well as persistence in killing target cells compared to conventional MSLN CAR-T cells. Notably, in vivo, Intra6 CAR-T cells displayed superior antitumor effects, showing enhanced tumor suppression and reduced exhaustion. RNA sequencing analysis revealed that CXCR6 expression upregulated genes related to immune activation, migration, adhesion, and cytoskeletal remodeling, such as LFA-1, PAK1, and FSCN1, suggesting improved migration and transendothelial infiltration. RT-qPCR and flow cytometry confirmed higher LFA-1 expression and enhanced migratory capacity in Intra6 CAR-T cells. Importantly, LFA-1 was crucial for CXCR6-driven migration. These results suggest that chemokine receptor modification of MSLN-targeted CAR-T cells can significantly improve their tumor infiltration and therapeutic efficacy, offering a potential strategy to optimize CAR-T cell therapy for mesothelin-expressing cancers.  METHODS: We engineered two CAR-T cell variants, Intra2 and Intra6, by introducing chemokine receptors CCR2 and CXCR6, respectively. The migration and tumor infiltration capabilities of these modified cells were evaluated in vitro through migration assays and in vivo using tumor-bearing mouse models. The phenotypic characteristics of the CAR-T cells, including memory T cell subsets (stem cell memory and central memory), were analyzed by flow cytometry. Tumor growth inhibition was assessed, and markers of immune exhaustion and evasion were quantified.