Role of the Exosome Secretion Machinery in Ovarian Carcinoma: <i>In Vitro</i> and <i>In Vivo</i> Models
Esther Channah Broner, Hadil Onallah, Tali Tavor Re’em, Ben Davidson, Reuven Reich
Abstract
Objective . We recently reported on the expression and clinical role of molecules that mediate exosome secretion in high-grade serous carcinoma. In the present study, the biological role of these molecules was analyzed. Methods . OVCAR8 and ES-2 ovarian carcinoma cells were studied using a combination of CRISPR/Cas9 technology and two 3D in vitro models—spheroids emulating effusions and alginate scaffolds representing solid lesions. Isolation of exosomes was validated by electron microscopy. TSAP6 , NSMASE2 , RAB27A , and RAB27B mRNA and protein levels were analyzed using qRT-PCR and Western blotting, respectively. Tumor aggressiveness was studied in vitro using scratch assay, invasion assay, and matrix metalloproteinase (MMP) activity assay and in vivo using a mouse model. Results . In OVCAR8 cells, mRNA expression of TSAP6 and RAB27A was significantly higher in spheroids compared to scaffolds, whereas the opposite was true for NSMASE2 mRNA. In ES-2 cells, TSAP6 and RAB27B mRNA expression was significantly higher in spheroids versus scaffolds. In addition, nSMase2 and TSAP6 protein expression was significantly higher in scaffolds compared to spheroids. CRISPR-edited cells with silencing of NSMASE2 , TSAP6 , and RAB27A/B had reduced migration, invasion, and MMP activity. Additionally, knockout (KO) of these molecules resulted in significantly diminished exosome secretion. In vivo assay showed that when injected to mice, OVCAR8 RAB27A/B KO cells, as opposed to control OVCAR8 cells, did not form ascites or visible tumor lesions and had reduced MMP expression. Conclusion . The present study provides evidence that different models for culturing ovarian carcinoma cells affect the expression of molecules mediating exosome secretion and that these molecules have a tumor-promoting role. Silencing these molecules may consequently have therapeutic relevance in this cancer.