Abstract 1338: Subpopulations of cancer associated fibroblasts serve as cancer stem cell niche in ovarian cancer via activation of non-canonical Wnt signaling
Authors: Xiao Xue , Fang Yiming , Pepin David , Zamarin Dmitriy , Mitra Anirban K. 
Published: June 2023

Abstract
Ovarian cancer (OC) is the most lethal gynecologic cancer and the 5th leading cause of cancer-related deaths among women in the US. One of the main reasons for the poor outcome is a high recurrence rate and development of chemoresistance. The standard care involves debulking surgery and carbo-taxol chemotherapy. While most patients respond well initially, a small fraction of OC cells survive - these persister cells are enriched in cancer stem cells (CSCs), and are capable of tumor initiation and cause relapse. Most studies on OC stem cells (OCSCs) have focused on their cell-intrinsic properties and not much is known about the role of the tumor microenvironment (TME) in regulating their growth, fate, and sustenance. Cancer-associated fibroblasts (CAFs) are a key constituent of the OC TME and have been reported to play important roles in regulating tumorigenesis, metastasis, and immune evasion. Moreover, the residual tumors following neo-adjuvant chemotherapy are highly fibrotic. Our studies using matched pre- and post-chemotherapy OC patient tumors revealed an enrichment in OCSCs and CAFs following chemotherapy. Using heterotypic 3D cocultures of patient-derived CAFs and OC cells and in vivo limiting dilution assay, we found that CAFs could induce OCSCs. CAFs stimulated symmetric division of OCSCs as well as dedifferentiation of some non-OCSCs. Using a novel interface interaction assay that we developed, we found that CAFs could influence only those OC cells that are in their immediate neighborhood. We have identified the underlying mechanism involving a non-canonical Wnt signaling pathway in the OCSCs induced by Wnt5a secreted by CAFs. Using functional rescue experiments, we determined that Wnt5a acted via its coreceptor ROR2 on the OCSCs that activated PKC and CREB1 to induce stemness. Inhibiting the CAF-OCSC crosstalk using a Wnt5a-specific inhibitor, Box5 abrogated the effects of CAFs on OCSCs both in vitro and in vivo. While Box5 treatment alone was sufficient to inhibit tumor growth in vivo, its combination with carboplatin sensitized tumors to carboplatin by eradicating residual OCSCs. CAFs are heterogenous and not all CAFs are capable of inducing OCSCs, similarly, only a subpopulation of OC cells can respond to CAFs and dedifferentiate into OCSCs. We performed single-cell RNA-seq using heterotypic 3D cocultures of patient-derived CAFs and OC cells, and characterized the pathways activated in Wnt5ahigh CAFs. Using trajectory analysis, we identified OC subpopulations that respond to CAF signals and become OCSCs. In summary, we have identified a novel mechanism by which a subpopulation of CAFs in the OC TME act as OCSC niche. Our research provides compelling evidence in support of targeting Wnt5a, in combination with the standard of care carbo-taxol chemotherapy, to prevent OC relapse and chemoresistance.
Citation Format: Xue Xiao, Yiming Fang, David Pepin, Dmitriy Zamarin, Anirban K. Mitra. Subpopulations of cancer associated fibroblasts serve as cancer stem cell niche in ovarian cancer via activation of non-canonical Wnt signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1338.