GTPase RAB31 supports ANXA1-driven macrophage education through clathrin-mediated endocytosis to suppress antitumor immunity.

backgroundTumor-associated macrophages (TAMs) play pivotal roles in shaping the tumor-microenvironment (TME) through functional plasticity, which is regulated by extrinsic and intrinsic signals. However, the role of vesicular trafficking in TAMs remains poorly understood. RAB31, a small GTPase enriched in myeloid cells, was proposed as a potential regulator of TAM polarization through clathrin-mediated endocytosis (CME). We hypothesized that RAB31 modulates TAM education by tumor-derived signals and thereby shapes antitumor immunity.

methodsWe profiled RAB31 expression in human cancers using public single-cell RNA sequencing (scRNA-seq) datasets and clinical sample immunofluorescence staining. Rab31 knockout mice were employed in subcutaneous tumor models. The TME was profiled by scRNA-seq, bulk RNA-seq, and flow cytometry. Bone marrow transplantation, adoptive cell transfer, and antibody-mediated depletion were performed to identify the effector cell populations. Co-immunoprecipitation coupled with mass spectrometry, receptor half-life assays, inhibitor intervention and lysosomal colocalization experiments dissected the molecular mechanism. Functional T-cell chemotaxis, activation, and anti-programmed death-ligand 1 (PD-L1) response assays were performed.

resultsRAB31 was highly expressed in TAMs across multiple cancers and correlated with poor prognosis and immunosuppressive TME. Rab31 deficiency reprogrammed TAMs to M1-like phenotype, enhanced CD8

conclusionsThese findings establish the CME/RAB31 pathway as an indispensable regulator of TAM polarization, underscoring the pivotal role of vesicular trafficking in the TME.