Extracellular vesicles are candidate key factors in the communication of normal and tumor cells [56]. Metastasis formation jeopardizes the life of cancer patients worldwide [19]. Thus, the involvement of EVs in cell migration and metastasis formation is highly investigated [8].
The present study investigated the potentially different effects of EVs produced by cells with different sensitivity to vemurafenib and modelling less and more advanced stage tumors. Five pairs of syngeneic melanoma cell lines were treated with EVs isolated from their or their pair’s supernatant. Characterization of isolated EVs and determination of their effects on cell proliferation and migration were performed. Characteristics of the EVs isolated from the supernatant of the cells by ultracentrifugation were relatable with the previously described supernatant-derived EV fractions [14], [57]–[59].
Cancer cell migration, as an essential process of metastasis formation, is widely studied [60]. The effect of EV treatment on tumor cell migration is mostly investigated by transwell [10], [14], [17], [61]–[64] or wound healing assays [10], [64]–[68]. However, these assays are relatively complex and examine more aspects of the treatments’ effects simultaneously, like proliferation and invasion, not only migration. Moreover, these complex assays investigate cells migratory capacity in one direction (e.g. the closing of the scratch or migration through the membrane), without considering the dynamic characteristics of cell migration [69], [70].
Single-cell tracking based methods, as used in this study, are more direct approaches to investigate the treatments’ effect on cell migration. Two parameters MSD and TTD were calculated to quantify cell migration [50], [51]. In an earlier study, vid-eo-microscopy recording, and calculation of velocity could not prove migration-promoting effect of melanoma-derived EVs in cancer-associated fibroblasts [71].
In our results, for most of the cell lines, MSD and TTD values were elevated after EV treatments, although EVs from the more aggressive cell lines did not show an unequivocally greater effect. Since cell migration is crucial in metastasis formation, based on our results, we suggest, that EVs have a more prominent role in metastasis formation, rather than tumor growth. These results are in line with earlier findings that oral squamous carcinoma cells treated with cancer-associated fibroblasts derived EVs showed increased migration to a greater extent than proliferation [72]. Similarly, neither the non-tumorigenic and tumorigenic melanoma cell lines-derived EVs could affect the recipient cells’ proliferation, however, EVs from the more tumorigenic cells could promote the recipient cells migration [17]. Possible reason why EVs promote migration could be that EVs could transfer integrins [73] and they can promote cell adhesion [74]. Moreover, EVs can affect the phosphorylation of FAK, AKT, and ERK1/2 [75] and transfer mRNAs [18] and miRNAs [76] involved in migration and metastasis formation. Proteome analysis of metastatic melanoma cell lines-derived EVs’ by KEGG (Kyoto Encyclopedia of Genes and Genomes), BBID (Biological Biochemical Image Database), and Biocarta databases showed that EVs are enriched in proteins involved in the regulation of actin cytoskeleton and focal adhesion [17].
The selective BRAF V600E inhibitor, vemurafenib was widely used in clinics, however after the first promising period of the treatment, resistance occurred in many patients [39], that manifested in the acceleration of metastasis formation [43], [44]. Vemurafenib resistance can occur through multiple mechanisms, including EV-mediated intracellular cross-talks [59]. Our investigation revealed the migration-promoting role of EVs; therefore, we tested if these EV-related elevated metastatic potentials could be reversed by vemurafenib. However, in our experiments, we observed that the more vemurafenib-resistant cells-derived EVs could diminish the migration-inhibiting effect of vemurafenib. This is in line with the fact that EVs originating from V600E BRAF mutant PLX-4720-resistant cells could transmit resistance to the recipient cells by transporting PDGFRβ. In contrast, EVs from the sensitive parental cells were unable to promote proliferation [33]. Additionally, vemurafenib-resistant melanoma cells-derived EVs could increase the proliferation of sensitive cells during vemurafenib therapy compared to the control cells via transporting an ALK isoform [42]. Also, EVs from resistant cell lines and relapsed BRAFi-treated patient plasma are associated with BRAF splicing variants [77]. Moreover, plasma EV-associated miRNA analysis of MM patients revealed that their miRNA profile could predict the disease progression during MAPKi treatment [78]. Therefore, EVs from resistant cells could transfer specific features to the sensitive cells and help them overcome the BRAF inhibition [79].