RAMP3 is associated with macrophage-driven immune exclusion and promotes resistance to PD-1 blockade in breast cancer: integrated in vivo and single-cell transcriptomic analyses

Abstract Background Receptor activity-modifying protein 3 (RAMP3) has been implicated in stromal regulation in cancer. However, its role in tumor microenvironment (TME) remodeling and immune checkpoint resistance remains unclear. In this study, we aimed to define the role of RAMP3 in shaping the immunosuppressive TME and evaluate its potential as a therapeutic target to enhance immunotherapy responses. Methods We used syngeneic mouse models of breast cancer with genetic manipulation of RAMP3 to investigate tumor progression, immune remodeling, and response to immune checkpoint blockade in vivo. Tumor growth, metastasis, and survival were evaluated. Single-cell RNA sequencing was performed on murine tumor tissues, and a publicly available spatial transcriptomic dataset was analyzed to characterize immune cell states and spatial organization within the TME. Human breast cancer datasets from The Cancer Genome Atlas were analyzed to assess the clinical relevance of RAMP3-associated immune signatures. Results Our results suggest that RAMP3 contributed to the establishment of an immunosuppressive TME. In mouse models, RAMP3 deletion suppressed primary tumor growth, reduced lung metastasis, and improved survival. These effects were associated with a shift from M2-like tumor-associated macrophages to antigen-presenting macrophage states, increased CD8 + T cell infiltration, and reduced T cell exhaustion. Mechanistically, RAMP3 deficiency was associated with reduced IL-4/STAT6 signaling in macrophages and decreased expression of immunosuppressive genes, including the chemokine CCL8. Pharmacological inhibition of CCL synthesis phenocopied the effects of RAMP3 deficiency, and RAMP3 deficiency enhanced the efficacy of PD-1 blockade. Spatial transcriptomic analysis demonstrated co-localization of RAMP3 and CCL8 within the TME. In the TCGA-BRCA cohort, a higher weighted RAMP3/CD163/CD8A risk score was associated with poorer overall survival and remained significantly associated with overall survival in complete-case multivariable Cox models adjusted for clinicopathological variables. Conclusions RAMP3 was identified as a potential regulator of the immunosuppressive TME and its functional impact was demonstrated in vivo. Integration with human clinical datasets supports further investigation of RAMP3 as a potential therapeutic target for overcoming resistance to immune checkpoint therapy in breast cancer.