RAB31 orchestrates CXCL2-CXCR4-mediated neutrophil recruitment and proangiogenic niche formation in colorectal cancer

BACKGROUND: The interaction between tumour-associated neutrophils (TANs) and angiogenesis plays a crucial role in tumour progression. However, the specific regulatory mechanisms by which different TANs populations influence angiogenesis in colorectal cancer (CRC) remain poorly understood. METHODS: The study integrates our own dataset with publicly available single-cell RNA sequencing (scRNA-seq) data to analyze the infiltration of various single-cell types in colorectal cancer (CRC). Using in vitro experiments, immunofluorescence, and animal models treated with anti-VEGFR antibodies, we investigated the role of tumor-associated neutrophils (TANs) in CRC and angiogenesis. We conducted RAB31 knockdown cell line, RNA sequencing, Western blotting, quantitative PCR, ELISA, tube formation assays, CCK8, and clonogenic assays, combined with in vivo experiments, to elucidate the impact of RAB31 in the tumor microenvironment (TME) and the mechanism by which tumor-derived RAB1 regulates TANs recruitment. RESULTS: Single-cell analysis revealed a significant infiltration of myeloid cells, particularly TANs, in CRC tumour tissue compared to normal tissue. RAB31 was found to be involved in malignant cell pathways, promoting CRC progression. Depletion of TANs substantially suppressed tumour growth and angiogenesis, while VEGFR blockade inhibited tumour growth and altered TANs infiltration in response to angiogenesis status. In vitro, knockdown of RAB31 expression inhibited tumour cell proliferation, and CXCL2 protein secretion was significantly reduced in shRab31-transfected tumour cells. In vivo, tumour tissue from shRab31 models showed decreased angiogenesis and reduced Neutrophil Extracellular Traps (NET) production, alongside reduced TANs infiltration. Moreover, CXCR4 expression was significantly elevated in TANs, and tumour cells influenced TANs through the CXCL2–CXCR4 axis. RAB31 regulated the PI3K–Akt signalling pathway, which is linked to neutrophil recruitment in CRC. CONCLUSIONS: Our findings suggest that tumour-derived RAB31 regulates the CXCL2–CXCR4 signalling pathway in CRC. RAB31 promotes CXCL2 secretion through the PI3K-Akt signalling pathway, facilitating TANs recruitment and angiogenesis, thereby driving tumour growth. This work enhances our understanding of the complex role of TANs and RAB31 in CRC progression and provides new insights into potential therapeutic strategies targeting the tumour microenvironment.