A simple and inexpensive in vitro enrichment of ovarian cancer stem cells for the identification of effective anti-cancer stem cell drugs

Subpopulations of ovarian cancer cells in ascites exhibit cancer stem-like phenotypes that possess an enhanced resistance to chemotherapeutic drugs and have the potential for distant metastatic spread and recurrent disease. The isolation of cancer stem cells (CSCs) from ovarian cancer cell culture required a defined culture medium with different growth factors which in the long term is not cost-effective. Here, we describe for the first time a cost-effective method for the isolation of ascites-derived CSCs without using a cocktail of growth factors. In this study, ascites fluid samples were collected from patients (n=۳) with high grade serous ovarian cancer (HGSOC) and cultured with MCDB۱۰۵/M۱۹۹ (۵۰:۵۰) media supplemented with ۱۰% fetal bovine serum (FBS) to reach a monolayer. For CSCs enrichment, EOC cells were cultured in ۱۰% patient-derived ascites fluid and ۸۰% of MCDB۱۰۵/M۱۹۹ without FBS using ۳D spheroid culture. We found that ascites-derived EOC cells expressed high levels of epithelial markers: CK-۷, CK-۱۸, and EpCAM. Our results showed remarkably and significantly increased expression levels of CD۱۳۳, CD۴۴, Oct-۴, Nestin, Nanog, and ALDH۱ in secondary and tertiary spheroids cultured with ۱۰% ascites fluid compared to monolayer cell culture. Moreover, we found a higher Paclitaxel (PTX)-resistance spheroids obtained from culture condition with ascites fluid compared to the monolayer. Treatment of tertiary spheroids with Galunisertib as a TGFBR۱ inhibitor and Verteporfin an inhibitor of YAP۱/TAZ signaling showed a significant and strong decrease of CSCs markers. This method could be a simple and cost-effective cell culture method for ovarian CSCs enrichment for the identification and targeting of key signaling pathways and molecules for ovarian CSCs maintenance.

کلیدواژه ها:

epithelial ovarian cancer ، cancer stem cells ، ascites fluid ، chemoresistance ، signaling pathways