The RNA binding protein IGF2BP2/IMP2 alters the cargo of cancer cell-derived extracellular vesicles supporting tumor-associated macrophages

Abstract

Background Tumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 afects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization. Methods EVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and fow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte® system and macrophage matrix degradation potential by zymography. Changes in the meta‑ bolic activity of macrophages were quantifed using a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafsh larvae, and macrophages were isolated by fuorescence-activated cell sorting. Results EVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle mark‑ ers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafsh larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a signif‑ cant diference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed diferences in protein cargo in KO vs. WT EVs, with the diferentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidifcation rate than KO EV-treated cells. Conclusion Our results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs’ actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype.