Abstract

Immune evasion limits the efficacy of cancer immunotherapy, yet the metabolic basis underlying differential immune sensitivity remains poorly explored. Here, we established an integrated platform that couples mass spectrometry imaging (MSI) with a patient-derived tumor organoid/immune cell coculture system to delineate metabolic adaptations driving tumor immune resistance. By optimizing a pretreatment workflow that preserves morphological integrity, we achieved in situ MSI mapping of phospholipids, fatty acids, taurine, glutathione, and other metabolites within tumor organoids. Colorectal cancer (CRC) organoids segregate into immune-sensitive and immune-resistant subtypes according to their responses to cocultured peripheral blood mononuclear cells (PBMCs), and the resistant fraction exhibits significantly elevated B7-H3 expression. MSI of these organoids further revealed that phospholipid metabolism is markedly reprogrammed in immune-resistant organoids. This metabolic signature was corroborated by MSI of human CRC tissues, and dynamic MSI of organoids exposed to PBMCs showed that immune-resistant variants preserved a significantly larger phospholipid pool than did their sensitive counterparts, indicating superior membrane-lipid conservation under immune pressure. This integrated MSI-organoid immunity platform offers a broadly applicable framework for dissecting immune-resistance mechanisms and highlights phospholipid metabolism as a potential targetable axis to restore antitumor immunity in cancer treatment.