Abstract 2177: Spatial transcriptomic and functional analysis of HLA/PD-1/CTLA4 multi-gene edited CD19 CAR-iNK cells targeting pericytes in the glioblastoma tumor microenvironment to inhibit tumor migration and growth

Abstract Targeting glioblastoma (GBM) stem cell-derived pericytes in the perivascular niche offers a promising strategy to overcome intratumoral heterogeneity and inhibit tumor migration. By targeting CD19-positive pericytes, CD19 chimeric antigen receptor (CAR)-expressing natural killer (NK) cells can mitigate off-tumor toxicity; however, their interactions within the GBM tumor microenvironment (TME) require further investigation. In this study, we developed a GBM TME xenograft model by transplanting GBM-blood vessel assembloids (GBVA) into immunodeficient mice to study the effects of CD19 CAR-expressing iPSC-derived NK (CAR-iNK) cells on pericytes and their interactions within the TME. Additionally, we enhanced the efficacy of CD19 CAR-iNK cells by knocking out PD1 and CTLA4, enabling these cells to better target GBM within an immunosuppressive TME. As a result, GBVA xenografts recapitulated the GBM TME including human pericytes and macrophages, providing a robust in vivo platform for validating CD19 CAR-iNK cell efficacy against GBM. Optimization of CAR-iNK cell dosing and efficacy resulted in significant antitumor effects. Spatial transcriptome analysis using SLACS (Spatially Linked Antibody and cDNA Sequencing) revealed that CD19 CAR-iNK cell infiltration altered gene expression in the TME, elucidating mechanisms of GBM lysis, migration inhibition, and evasion of immunosuppressive influences. These findings underscore the therapeutic potential of CD19 CAR-iNK cells for GBM treatment through precise targeting of pericytes and modulation of the TME. Citation Format: Kyung-Sun Kang. Spatial transcriptomic and functional analysis of HLA/PD-1/CTLA4 multi-gene edited CD19 CAR-iNK cells targeting pericytes in the glioblastoma tumor microenvironment to inhibit tumor migration and growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2177.