SPINK4 affected M2 macrophage polarization to promote colorectal cancer malignant phenotype by PI3K/AKT pathway.

Abstract

This study aimed to elucidate the oncogenic role of SPINK4 in colorectal cancer (CRC) and its underlying mechanism regulating macrophage polarization in the tumor microenvironment. In vitro, we constructed SPINK4-overexpressing and SPINK4-knockdown CRC cell lines (HT29 and HCT116) to evaluate their effects on cell proliferation, migration, and invasion. We then established a macrophage-CRC cell co-culture system to explore whether SPINK4 promotes macrophage recruitment and M2 polarization. Mechanistically, we not only regulated the PI3K/AKT pathway using inhibitor LY294002 and activator 740Y-P but also specifically knocked down PI3K via small interfering RNA (siRNA) to confirm if SPINK4's function depends on this pathway. In vivo, we established HCT116 xenograft models in nude mice, monitored tumor volume and weight, evaluated tumor proliferation by Ki67 immunohistochemistry, quantified tumor-infiltrating macrophages by flow cytometry, and detected M1/M2 macrophage marker expression by RT-qPCR. Experimental results showed SPINK4 overexpression significantly enhanced CRC cell proliferation, migration, and invasion, induced macrophage recruitment and M2 polarization, and upregulated IL-33, IL-4, IL-10, CSF1, CCL2, and VEGF-C secretion. Inhibiting PI3K/AKT reversed these effects. In vivo, SPINK4 overexpression activated PI3K/AKT, promoting tumor growth and M2 macrophage infiltration. Collectively, SPINK4 acts as an oncogene to promote macrophage recruitment and M2 polarization via PI3K/AKT, driving CRC malignant progression.