ID #975 Rapid CAR T Testing in an Ex Vivo Platform Preserving Passage-Zero High Grade Pediatric Brain Tumor Tissues and their Native Immunosuppressive Microenvironment

Abstract

CAR T therapy has revolutionized treatment for pediatric hematologic malignancies; however, widespread adoption in pediatric brain tumors remains elusive, largely due to preclinical models failing to capture patient tumor heterogeneity and the immunosuppressive tumor microenvironment (TME). Conventional systems, including cell lines, xenografts, and organoids, often lack critical TME features, leading to overestimation of therapeutic potency. Progress in next-generation CAR T therapies requires more biologically representative models. We have therefore developed Screening Live Cancer Explants (SLiCE), a New Approach Methodology platform that engrafts living, passage-zero patient tumors onto substrates of living tissue. This design preserves tumor heterogeneity better than in vitro culture and enables investigation of how patient tumor-associated cells influence T cell activity. We tested B7-H3–targeted CAR T cells from three healthy donors against cell lines and passage-zero high-grade pediatric brain tumors (n = 2 medulloblastoma, 1 ATRT). B7-H3 expression was quantified by IHC (H-score) and flow cytometry. CAR T or control T cells were added to SLiCE-engrafted tumors at effector-to-target ratios from 1:3 to 3:1. Tumor kill was measured by live tumor cell bioluminescence within four days of seeding. Killing of B7H3+ tumors occurred only in the presence of CAR+ T cells and not non-transduced T cells; killing among multiple tumor specimens collected from the same patient positively correlated with B7H3 expression (assessed by flow cytometry). Notably, the ATRT tumor showed minimal killing despite high B7-H3 expression (H-score = 152). Real-time cytokine secretion profiling and Flow/CyTOF analyses revealed active, inhibitory human myeloid cells, regulatory T cells and PD-L1+ tumor cells, features likely suppressing CAR T activity. These findings indicate SLiCE maintains key immunosuppressive TME components in pediatric brain tumors and can dissect mechanisms of immune evasion. Ongoing studies aim to overcome these barriers. These findings underscore SLiCE’s potential to accelerate development of CAR T therapies tailored for pediatric brain tumors.