Tumor-Derived Extracellular Vesicles Require ?1 Integrins to Promote Anchorage-Independent Growth.
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ABSTRACT:
SUBMITTER: DeRita RM
PROVIDER: S-EPMC6461598 | BioStudies | 2019-01-01
REPOSITORIES: biostudies
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Project description:The ability of small extracellular vesicles (sEVs) to reprogram cancer cells is well established. However, the specific sEV components able to mediate aberrant effects in cancer cells have not been characterized. Integrins are major players in mediating sEV functions. We have previously reported that the ?V?3 integrin is detected in sEVs of prostate cancer (PrCa) cells and transferred into recipient cells. Here, we investigate whether sEVs from ?V?3-expressing cells affect tumour growth differently than sEVs from control cells that do not express ?V?3. We compared the ability of sEVs to stimulate tumour growth, using sEVs isolated from PrCa C4-2B cells by iodixanol density gradient and characterized with immunoblotting, nanoparticle tracking analysis, immunocapturing and single vesicle analysis. We incubated PrCa cells with sEVs and injected them subcutaneously into nude mice to measure in vivo tumour growth or analysed in vitro their anchorage-independent growth. Our results demonstrate that a single treatment with sEVs shed from C4-2B cells that express ?V?3, but not from control cells, stimulates tumour growth and induces differentiation of PrCa cells towards a neuroendocrine phenotype, as quantified by increased levels of neuroendocrine markers. In conclusion, the expression of ?V?3 integrin generates sEVs capable of reprogramming cells towards an aggressive phenotype.
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Project description:BACKGROUND:Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. METHODS:To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for ?v?3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. RESULTS:We find that SEVs secreted from MDA-MB-231 breast cancer cells carry ?v?3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of ?v?3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. CONCLUSION:In summary, our findings demonstrate for the first time a key role of ?v?3 integrin in cell-cell communication through SEVs. Video Abstract.
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Project description:Prostate cancer (PrCa) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sEVs). sEVs, as well as large extracellular vesicles (LEVs), isolated via iodixanol density gradients from PrCa cell culture media, express the epithelial-specific ?v?6 integrin, which is known to be induced in cancer. In this study, we show sEV-mediated protein transfer of ?v?6 integrin to microvascular endothelial cells (human microvascular endothelial cells 1 - HMEC1) and demonstrate that de novo ?v?6 integrin expression is not caused by increased mRNA levels. Incubation of HMEC1 with sEVs isolated from PrCa PC3 cells that express the ?v?6 integrin results in a highly significant increase in the number of nodes, junctions and tubules. In contrast, incubation of HMEC1 with sEVs isolated from ?6 negative PC3 cells, generated by shRNA against ?6, results in a reduction in the number of nodes, junctions and tubules, a decrease in survivin levels and an increase in a negative regulator of angiogenesis, pSTAT1. Furthermore, treatment of HMEC1 with sEVs generated by CRISPR/Cas9-mediated down-regulation of ?6, causes up-regulation of pSTAT1. Overall, our findings suggest that ?v?6 integrin in cancer sEVs regulates angiogenesis during PrCa progression.
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