Abstract LT020: Cancer associated fibroblasts in the tumor microenvironment maintain ovarian cancer stem cells through non-canonical Wnt5a signaling
Authors: Fang Yiming , Xiao Xue , Wang Ji , Dasari Subramanyam , Nephew Kenneth P. , Zamarin Dmitriy , Mitra Anirban K. 
Published: March 2021

Abstract
Frequent relapse and development of chemoresistance are major causes of poor survival in ovarian cancer. Cancer stem cells (CSCs) consist of a small subpopulation in the tumor that are capable of initiation and maintenance. CSCs are typically resistant to cytotoxic chemotherapy and are a potential cause of relapse and chemoresistance. Most studies on CSCs focus on cancer cells alone while CSCs exist in a complex microenvironment in tumors. This microenvironment or CSC niche provides optimal conditions to maintain their tumor initiating potential. Hence, understanding the crosstalk between CSCs and the tumor microenvironment can potentially provide effective therapeutic targets to prevent chemoresistance and relapse. Cancer associated fibroblasts (CAFs) are a major constituent of the ovarian cancer tumor microenvironment and are highly enriched in the residual tumors following chemotherapy. Therefore, we studied the role of CAFs in promoting ovarian cancer chemoresistance and disease relapse through providing an optimal microenvironment for CSCs. CAFs isolated from ovarian cancer patient tumors were used in heterotypic 3D or 2D coculture systems with high grade serous ovarian cancer cell lines or with patient-derived ovarian cancer cells to study their effect on CSCs and chemoresistance. Matched pre- and post-chemotherapy patient tumors were used to confirm our findings. CAFs significantly increased resistance to carboplatin and enriched CSCs by increasing their symmetric division as well as dedifferentiation of bulk ovarian cancer cells. Pre-coculture with CAFs increased in vivo tumor initiation capacity of the ovarian cancer cells 10-fold. The CSC-CAF crosstalk responsible for CSC induction was found to be mediated by Wnt signaling. Inhibition of Wnt secretion by CAFs could block their effect on CSCs. Wnt5a was the most highly expressed Wnt in CAFs, which was further induced by ovarian cancer cells. CRISPR knockdown of Wnt5a in CAFs or treatment with a specific Wnt5a inhibitor abrogated the induction of CSCs by CAFs. Wnt5a was found to signal through a non-canonical Wnt pathway in the CSCs involving the coreceptor ROR2, protein kinase C (PKC), and CAMP Responsive Element Binding Protein 1 (CREB1). Inhibition of each of them prevented CSC induction and functional rescue experiments confirmed the sequence of the Wnt5a-ROR2-PKC-CREB1 axis. Treatment of mouse xenografts established by co-injection of CAFs and ovarian cancer cells, with the Wnt5a inhibitor sensitized them to carboplatin, and eliminated the CSCs in the residual tumors. Our results indicate that CAF-derived Wnt5a is instrumental in ovarian cancer CSC growth and maintenance. In the long term, our studies will broaden the understanding of the mechanism of CSC maintenance by the tumor microenvironment and contribute towards the development of novel therapeutic approaches to prevent ovarian cancer chemoresistance and relapse.
Citation Format: Yiming Fang, Xue Xiao, Ji Wang, Subramanyam Dasari, Kenneth P. Nephew, Dmitriy Zamarin, Anirban K. Mitra. Cancer associated fibroblasts in the tumor microenvironment maintain ovarian cancer stem cells through non-canonical Wnt5a signaling [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT020.