e18072 Background: Carcinoma-associated mesenchymal stem cells (CA-MSCs) are mesenchymal stem cells (MSCs) within the tumor microenvironment (TME). We demonstrated that CA-MSCs promote ovarian cancer chemotherapy resistance through paracrine signaling with ovarian cancer cells, interact with ovarian cancer cells to form mixed-cellularity complexes which enhance metastasis, and arise from cancer cell and TME reprogramming of normal tissue MSCs. To identify mediators of the CA-MSC:tumor cell interaction, we investigated the role of tetraspanins, which are membrane-spanning proteins that have been implicated in cancer development and metastasis by influencing cell adhesion and cell-cell interactions. The tetraspanins CD9, CD81, CD151, and CD63 were identified as potential mediators of cell surface interactions between ovarian cancer cells and CA-MSCs through homo- and heterodimerization. Methods: Td-labeled OVCAR3 cancer cells were co-cultured with 3 patient-derived MSC (derived from normal tissue) and 3 patient-derived CA-MSC (derived from malignant tissue) cell lines. Flow cytometry was performed to measure surface protein expression of the tetraspanins CD9, CD81, CD151, and CD63, and was compared to control cells (not co-cultured) and their median fluorescence intensity (MFI). We next separated OVCAR3 cells co-cultured with MSCs or CA-MSCs using fluorescent activated cell sorting (FACS) based on Td expression. Following FACS, tetraspanin expression in OVCAR3 cells, MSCs, and CA-MSCs was assessed via qRT-PCR and western blotting, and compared to cell lines that were not co-cultured. Results: Flow cytometric analysis revealed an increase in MFI of CD9 and CD151 in co-cultured OVCAR3 cells, as well as CD81 and CD63 in co-cultured CA-MSCs and MSCs when compared to non-co-cultured matched cells. Increased RNA expression of CD9, CD151, and CD63 was seen in OVCAR3 cells that were co-cultured with CA-MSCs or MSCs when compared to non-co-cultured OVCAR3 cells. Increased RNA expression of CD81 and CD63 was noted in both co-cultured CA-MSCs and MSCs compared to non-co-cultured cells. Lastly, western blotting demonstrated increased protein expression of CD9 and CD151 in co-cultured OVCAR3 cells, as well as CD81 and CD63 in co-cultured CA-MSCs and MSCs compared to non-co-cultured matched cells. Conclusions: These results indicate that direct interactions between OVCAR3 cells and CA-MSCs or MSCs lead to overexpression of specific tetraspanins at the RNA and protein levels, implying that they may be facilitating tumor cell:CA-MSC and tumor cell:MSC binding.
Cord Blood Mesenchymal Stem Cells Conditioned Media Suppress Epithelial Ovarian Cancer Cells in Vitro
