Abstract
BackgroundThe role of exosomes in the pathogenesis and metastatic spread of cancer remains to be fully elucidated. Recent studies support the hypothesis that the release of exosomes from cells modifies local extracellular conditions to promote cell growth and neovascularisation. In addition, exosomes may modify the phenotype of parent and/or target cell. For example, sequestration of signaling mediators into exosomes may reduce their intracellular bioavailability to the parent cell thereby altering cell phenotype and metastatic potential. The fusion of released exosomes with target cell and delivery may also modify cell function and activity. In this study, to further elucidate the role of exosomes in ovarian cancer, the release of exosomes from two ovarian cancer cell lines of different invasive capacity and their miRNA content of exosomes were compared. The hypothesis to be tested was that ovarian cancer cell invasiveness is associated with altered release of exosomes and discordant exosomal sequestration of miRNA.MethodsHigh (SKOV-3) and low (OVCAR-3) invasive ovarian cancer cell lines were used to characterize their exosome release. SKOV-3 and OVCAR-3 cells were cultured (DMEM, 20% exosome-free FBS) under an atmosphere of 8% O2 for 24 hours. Cell-conditioned media were collected and exosomes were isolated by differential and buoyant density centrifugation and characterised by Western blot (CD63 and CD9). Exosomal microRNA (let-7a-f and miR-200a-c) content was established by real-time PCR.ResultsExosomes were identified with by the presence of typical cup-shaped spherical vesicle and the expression of exosome markers: CD63, CD9. SKOV-3 cells released 2.7-fold more exosomes (1.22 ± 0.11 μg/106 cells) compared to OVCAR-3 (0.44 ± 0.05 μg/106 cells). The let-7 family miRNA transcripts were identified in both ovarian cancer cell lines and their exosomes. The let-7 family transcripts were more abundant in OVCAR-3 cell than SKOV-3 cells. In contrast, let-7 family transcripts were more abundant in exosomes from SKOV-3 than OVCAR-3. miR-200 family transcripts were only identified in OVCAR-3 cells and their exosomes.ConclusionsThe data obtained in this study are consistent with the hypothesis that the releases of exosomes varies significantly between ovarian cancer cell lines and correlates with their invasive potential.
Highlights
The role of exosomes in the pathogenesis and metastatic spread of cancer remains to be fully elucidated
Characterisation of human epithelial ovarian cancer cell lines Initially, studies were conducted to confirm the invasiveness of the two ovarian cancer cell lines (OVCAR-3 and SKOV-3 cells) used in these studies
Characterisation of ovarian cancer cell line-derived exosomes Exosomes derived from OVCAR-3 and SKOV-3 were isolated from cell-conditioned cultured media by differential and buoyant density centrifugation
Summary
The role of exosomes in the pathogenesis and metastatic spread of cancer remains to be fully elucidated. The keystone to improving health outcomes remains the timely and accurate diagnosis of the predisposition to, or early detection of disease. In the context of ovarian cancer, despite recent progress in chemotherapeutic treatments, the diagnosis of late stage disease is associated with a five-year survival rate of ~30%. The development of more accurate and earlier detection tests for this disease is undoubtedly the number one priority for achieving long- term reduction of mortality from ovarian cancer. To date, such tests have not been developed and the use of traditional biomarker approaches and screening methods have failed to deliver significant clinical outcomes. New approaches to the development of in vitro diagnostics that deliver greater diagnostic sensitivity are required
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