Abstract

Regulation of Macrophage Polarization by Prostate Cancer-derived Extracellular Vesicles

NICOLE NARANJO, , THOMAS JEFFERSON UNIVRRSITY

Small extracellular vesicles (sEVs) mediate intercellular communication and carry cargoes that may alter the recipient cell phenotype and functionality. Our group has shown that   IFIT3 (Interferon induced protein with tetratricopeptide repeats) plays an intrinsic role as a regulator of STAT1 (Signal transducer and activator of transcription 1) expression in prostate cancer (PrCa) derived sEVs. STAT1 and IFIT3 are part of the Interferon Stimulated Genes protein family which are proteins that play key roles in macrophage polarization. To evaluate the role of PrCa sEVs in modulating the macrophage phenotype, we used CRISPR Cas9 methods to downregulate STAT1 or IFIT3 protein expression in PC3 PrCa cells (PC3-STAT1KO or PC3-IFIT3KO). Subsequently, we treated macrophages with sEVs derived from these cells and assessed surface expression of the macrophage M2 polarization markers CD163 or CD204. Our results show that CD163 and CD204 surface expression is dramatically reduced after treatment of macrophages with PC3 sEVs (mock or WT) as well as PC3 sEVs devoid of STAT1 or IFIT3. These results show that CD163 and CD204 reduction in macrophages is independent of STAT1 or IFIT3.  Furthermore, we demonstrate that the decrease of CD163 and CD204 protein levels in macrophages by PC3 sEV treatment is associated with decreased levels of their associated total RNA levels. In a parallel investigation, we performed proteomic analysis of macrophages after treatment with PC3, PC3-STAT1KO or PC3-IFIT3KO cell derived. Although polarization is independent of STAT1 or IFIT3, our Ingenuity Pathway analysis results show differential alterations of functional pathways in macrophages after treatment with PC3, PC3-STAT1KO or PC3-IFIT3KO cell derived sEVs. The functional pathways that are altered by STAT1 or IFIT3 are related to cellular movement, cellular proliferation, inflammatory response, and immune response. These results demonstrate that PrCa derived sEVs significantly alter the phenotype and protein content of macrophages, thus providing new avenues to explore the crucial contribution of PrCa derived sEVs in cancer progression.