Novel Roles of Splice-Derived Tumor Mirtrons in the Regulation of Macrophage Dichotomy 2310054

Abstract

Background: Tumor-derived exosomes play key roles in cell-cell communication within the tumor microenvironment and have been shown to drive macrophage polarization towards an M2-like phenotype.We recently identified a novel class of splicing generated small RNAs, known as mirtrons, are highly packed in exosomes and potentially modulate macrophage plasticity. However, molecular mechanisms for this exosome-driven phenomenon are not completely clear. Human macrophages (U937) were co-cultured with exosomes derived from human lung (H23), prostate (PC3), and a variety subtypes of breast cancer cell lines (T47D, SKBR3, and MDA-MB-468). Expression of mirtrons including miR-7110, -cv002, and -6756 in cancer cells and in exosomes was assessed by qPCR. Immunofluorescent imaging was used to assess exosomal small RNA uptake in macrophages and their intracellular localization. Genomic characterization of M2-associated mirtrons in association with exosome exposure was performed using ChIP-seq. All three mirtrons–particularly cv002–were consistently enriched in tumor-derived exosomes relative to their parent tumor cells across all cancer cell lines examined. However, mirtron expression levels in exosomes or the cytoplasm did not correlate with tumor aggressiveness in breast cancer. Immunofluorescence imaging demonstrated that mirtrons carried by tumor-derived exosomes are taken up by human monocyte U937 cells and are predominantly localized to the cytoplasm, consistent with the behavior of conventional miRNAs. Notably, a subset of mirtrons was also observed to translocate into the nucleus and interact with monocyte genomic DNA, as supported by ChIP-seq analyses. Importantly, these mirtron—DNA interactions were enriched at M2-associated gene loci, suggesting a novel regulatory mechanism underlying macrophage polarization. Our results showed a novel mechanism through exosomal delivery of tumor derived mirtrons to manipulate M2-like macrophage polarization. NIGMS (R16GM153648) Immune Response Regulation: Molecular Mechanisms (IRM)