INSR Armoring Protects CAR-NK and CAR-T Cells Against Tumor Stroma-Induced CAR Shedding by Maintaining Cholesterol Homeostasis.

While chimeric antigen receptor (CAR)-engineered T and NK cell therapies have revolutionized hematological malignancy treatment, translating this success to solid tumors remains a persistent challenge. Here, we demonstrated a stromal-associated metabolic mechanism in which cancer-associated fibroblasts (CAFs) and macrophage-associated populations in gastrointestinal cancers contribute to CAR loss through CYP27A1-derived 27-hydroxycholesterol (27HC). 27HC triggered membrane cholesterol depletion, destabilizing lipid rafts and promoting the release of full-length CAR in ARF6+CD81+ microvesicle-enriched fractions. NK92 cells chronically exposed to sublethal 27HC adapted to this stress via an AKT-FOXO1-insulin receptor (INSR) signaling cascade that restores cholesterol biosynthesis. Capitalizing on this mechanism, "metabolically armored" INSR-overexpressing CAR-NK92 and primary CAR-T cells were engineered, which stabilized lipid rafts, preserved CAR expression, and improved antitumor activity in patient-derived organoids, precision-cut tumor slices, and xenografts. Collectively, these findings identify a stromal-mediated metabolic immune evasion pathway in gastrointestinal tumors, uncovering a potential strategy to potentiate CAR-engineered therapies.