Rapid and dynamic reprogramming within the tumor microenvironment drives EDA-CAR-T dysfunction and compromised therapeutic efficacy in solid tumors

Abstract

BackgroundChimeric antigen receptor (CAR)-T cell therapies efficacy in solid tumors remains limited, largely due to the profoundly immunosuppressive tumor microenvironment (TME) which drives CAR-T cells to dysfunction and poor persistence. A comprehensive understanding of the dynamic interplay between CAR-T cells and the TME is therefore critical for the rational design of more effective CAR-T strategies for solid cancers. MethodsHere, we performed single-cell RNA sequencing of tumor samples from immunocompetent mice treated with stroma-targeting EDA-CAR-T cells, profiling CAR-T cell states and TME programs at the peak of antitumor response and during subsequent tumor progression. ResultsOur analysis revealed a marked temporal remodeling of EDA-CAR-T cells within the TME, where early antitumor efficacy is associated with concurrent expansion of cytotoxic effector CD8 CAR-T cells and activation of memory CD4 CAR-T subsets. Moreover, EDA-CAR-T cells effectively engaged the myeloid compartment, resulting in strengthened communication networks involving T cell activation. However, by tumor progression, EDA-CAR-T cells suffered a widespread transcriptional reprogramming towards dysfunction, characterized by loss of effector programs alongside induction of exhaustion and immunoregulatory pathways within the TME, including PD-L1/PD-L2 and TGF{beta} signaling, which impairs sustained immune responses. Notably, early CAR-T cell activation led to increased susceptibility to TME-mediated immunosuppression, revealing EDA-CAR-T-specific soluble galectin-mediated cell-to-cell interaction networks. ConclusionsTogether, this works offers a high-resolution view of CAR-T cell dynamics within the solid TME, uncovering cellular and molecular mechanisms of rapid functional decline and identifying regulatory pathways within the TME that can be exploited to improve CAR-T cell therapy efficacy in solid tumors. KEY MESSAGES OF THE ARTICLEO_ST_ABSWhat is already known on this topicC_ST_ABSThe determinants of CAR-T cell therapeutic efficacy in solid tumors remain poorly defined, largely due to the complexity of the immunosuppressive tumor microenvironment. In this effort, it is necessary to perform comprehensive and detailed mechanistic studies that capture CAR-T cell dynamics within the solid tumor microenvironment to understand treatment failure. What this study addsWe performed single-cell profiling of stroma-targeting EDA-CAR-T cells, revealing their dynamic reprogramming toward dysfunction within the solid tumor microenvironment. We dissected CAR-T cell states and their cell-to-cell interactions with the tumor microenvironment across response and tumor progression and identified mechanisms linking CAR-T cell functionality and therapeutic failure. How this study might affect research, practice or policyThis study provides comprehensive mechanistic insights from an immunocompetent model that can be leveraged to identify shared determinants of CAR-T cell functionality in solid tumors and potentially guide the rational development of improved CAR-T cell therapies.