Tumor-derived CEMIP drives macrophage reprogramming and EV-associated PD-L1 acquisition in colorectal cancer

Tumor-associated macrophages (TAMs) and tumor-derived extracellular vesicles (EVs) contribute to immune-checkpoint-blockade resistance in colorectal cancer (CRC), but the responsible intercellular signals remain incompletely defined. Syngeneic subcutaneous and orthotopic CRC models, human specimens, primary and cell-line-derived macrophages, differential-ultracentrifugation EV preparations, RNA sequencing, flow cytometry, immunoblotting, co-immunoprecipitation, donor-protein tracing, EV-dependence/add-back assays, and HSP90 rescue experiments were used. An exploratory immunotherapy-treated CRC cohort ( n = 71) was analyzed using prespecified CEMIP immunohistochemistry scoring and multivariable models. Endogenous CEMIP in CRC cells was associated with increased cytosolic calcium and increased EV output. EV-associated CEMIP was acquired by macrophages and competitively interfered with the HSP90-JAK2 complex, reducing JAK2/STAT1 phosphorylation and promoting a CD206-high/CD86-low macrophage state. HSP90 overexpression restored pathway activation, macrophage phenotype, and downstream CD8 + T-cell effector readouts. Donor-derived Flag-PD-L1 was detected in recipient macrophage membrane fractions, whereas donor-cell PD-L1 knockdown reduced recipient surface PD-L1, supporting EV-associated PD-L1 acquisition without defining the precise trafficking route. High CEMIP was associated with poorer disease control and shorter progression-free survival in the retrospective cohort. These findings define two spatially coordinated, functional axes of tumor-derived CEMIP in CRC: intrinsically, CEMIP drives cytosolic calcium-dependent EV output in tumor cells; extrinsically, EV-encapsulated CEMIP is acquired by TAMs to competitively disrupt the HSP90-JAK2 chaperone complex and suppress STAT1 signaling, operating in parallel with EV-mediated acquisition of tumor-derived PD-L1. This CEMIP-EV-TAM signaling axis represents a promising candidate target to overcome immunotherapy resistance in CRC.