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Characterization of zebrafish melanoma-derived interstitial EVs and their ncRNA content

ABSTRACT: Extracellular vesicles (EVs) are membranous particles released by all cell types. Their role as functional carriers of bioactive molecules is boosted in cancer where they can be either secreted in biological fluids or found in the intercellular space (interstitial EVs, iEVs). Here we have opti-mised a method for the isolation and characterization of zebrafish iEVs from whole melanoma tissues. Zebrafish melanoma iEVs are in the range of ~ 140 nm by nanoparticle tracking analysis (NTA) and TEM analysis. Western blot revealed enrichment for CD63 and Alix in the iEV frac-tion, but not in melanoma cell lysates. Super resolution and confocal microscopy revealed that purified zebrafish iEVs were GFP+ indicating that they integrate the oncogene, GFP-HRASV12G within their vesicular membrane. Analysis of RNA-Seq data revealed that 118 ncRNAs are differentially distributed between zebrafish melanoma and their iEVs, with only 18 of them be-ing selectively enriched in iEVs. Among these, the RNA components of RNAses MRP and P, which process ribosomal RNA precursors, mitochondrial RNAs and some mRNAs, were enriched in iEVs. We found that melanoma iEVs induce an inflammatory response when injected in larval blood stream with increase of macrophage and induction of Interferon Responsive Genes. To clarify whether MRP and P contribute to the inflammation induced by melanoma iEVs we inject-ed larvae with MRP or P RNAs and found an inflammatory response similar to that induced by melanoma iEVs. This suggests that zebrafish melanoma iEVs are a source of MRP and P RNAs that can trigger inflammation in cells of the tumor-microenvironment.

ORGANISM(S): Danio rerio

PROVIDER: GSE189352 | GEO | 2022/06/01

REPOSITORIES: GEO

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Project description:High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. Tumor cells release extracellular vesicles (EVs) that can polarize macrophages towards tumor-associated macrophages (TAMs), which play a role in tumor progression. However, there is a lack of understanding of IMP2’s effect on TAMs. EVs were isolated from colorectal cancer HCT116 parental (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells via ultracentrifugation and tangential flow filtration. EVs were characterized according to MISEV guidelines and microRNA cargo was assessed by microRNA-Seq. Primary human monocyte-derived macrophages were polarized towards a TAM-like phenotype by EVs and the expression of genes and surface markers was assessed by qPCR and flow 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 metabolic activity of macrophages were quantified in live cells in a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish embryos, and macrophages were isolated by fluorescence-activated cell sorting. EV samples from both WT and KO EVs had a similar size and concentration and were positive or CD9, CD81, CD63, and ALIX. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs, while the expression of tumor-suppressing TNF and IL6 was reduced. 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. They exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate. microRNA-seq revealed a significant difference in the microRNA composition of WT and KO EVs. Macrophages transfected with miR-181a-5p mimic showed a lower level of the phosphatase DUSP6. Zebrafish macrophages upon polarization with WT EVs showed lower expression of IL6 and TNF. Our results show that IMP2 can determine the cargo of EVs released by cancer cells, thereby modulating the EVs’ actions on macrophages. Expression of IMP2 is responsible for the secretion of EVs that polarize macrophages toward a tumor-promoting phenotype.

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Characterization of zebrafish melanoma-derived interstitial EVs and their ncRNA content

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