Mechanistic insights into EOC-PMCs-Induced macrophage polarization via the Galnt15-PPARγ pathway

Ascites represents an extremely unique liquid immunosuppressive microenvironment of epithelial ovarian cancer (EOC), and it contains a substantial quantity of macrophages; however, these macrophages fail to exhibit effective anti-tumor activity. Peritoneal mesothelial cells (PMCs) constitute a critical component of ascites. Whether PMCs can modulate macrophages function through some mechanisms, thereby influencing the tumor immune microenvironment, remains to be elucidated.

Co-culture models of EOC-educated PMCs (EOC-PMCs) and macrophages in human and mouse were respectively constructed, and verified the differences of polarization markers, cytokines, and metabolic levels in macrophages after co-culture. Transcriptional sequencing experiments confirmed that polypeptide N-acetylgalactosaminyltransferase 15 (Galnt15) was highly expressed in macrophages after co-culture. Nuclear-cytoplasmic protein separation and immunofluorescence (IF) were used to confirm that the nuclear entry of peroxisome proliferator-activated receptor γ (PPARγ) increased and the polarization of macrophages was enhanced when Galnt15 was overexpressed. In vitro experiments, the overexpression and knockdown of Galnt15 were achieved using plasmids and siRNAs. Immunoprecipitation (IP) experiments verified that high expression of Galnt15 mediated the O-glycosylation of PPARγ, thereby affecting the nuclear entry of PPARγ. Site mutation and fusion protein detection were employed to explore the functional site of Galnt15-mediated O-glycosylation of the PPARγ protein. And an EOC model in situ was established on the ovary of C57BL/6J, and it was confirmed in vivo and in vitro that macrophages treated with EOC-PMCs played a pro-cancer role. Tissue microarrays confirmed that the expression of Galnt15 and PPARγ was related to patient prognosis.

EOC-PMCs led to high expression of Galnt15 in macrophages. Galnt15 promoted O-glycosylation and nuclear translocation of PPARγ, subsequently enhancing fatty acid oxidation (FAO) metabolism, and inducing macrophages polarization and secretion of pro-tumor cytokines. Animal experiments showed that EOC-PMCs increased the FAO metabolism level of macrophages to promote tumor development. Tissue microarray also confirmed that there was a positive correlation between the expression of Galnt15 and PPARγ, and patients with high expression of Galnt15/PPARγ had a poorer prognosis.

EOC-PMCs promote macrophages polarization through the Galnt15/PPARγ pathway, which is conducive to the formation of an immunosuppressive microenvironment in ascites and ultimately facilitates the progression of ovarian cancer.