Abstract LB-364: Mesothelial cells promote ovarian cancer stemness and chemoresistance through osteopontin paracrine signaling
Authors: Qian Jin , LeSavage Bauer , Ma Chenkai , Natarajan Suchitra , Eggold Joshua , Hubka Kelsea , Xiao Yiren , Fuh Katherine , Krishnan Venkatesh , Enejder Annika , Heilshorn Sarah , Dorigo Oliver , Rankin Erinn 
Published: September 2020

Abstract
Aims: As mesothelial cells are a major stromal component of the ovarian cancer metastatic microenvironment, in this study we investigated the role of ovarian cancer-associated mesothelial cells (CAMs) on cancer responsiveness to chemotherapy and intrinsic-related cancer stemness properties. Methods: Indirect co-culture system that enables medium exchange between human primary serous-type ovarian cancer cells and primary CAMs isolated from patient ascites was employed, and these co-cultured cancer cells were subsequently grafted subcutaneously to NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and received serial cisplatin challenges, or checked by Annexin V apoptosis assays and cell viability assays to determine chemosensitivity. In addition, patient derived tumor organoid cultures were compared between the addition of control and its paired primary CAM conditioned medium. In vivo limiting dilution assays and sphere formation assays were utilized to evaluate CAM regulated cancer stemness properties. Finally, a cytokine array on CAM conditioned medium was performed to identify the secreted factors mediating phenotypical changes, and RNA-sequencing analysis of co-cultured cancer cells was conducted to uncover the major events that underlie changed chemo-responsiveness. Results: Cancer cell response to platium-based chemotherapy was inhibited after indirect CAM co-culture or CAM conditioned medium priming, as evidenced both in vivo and in vitro. Organoid formation was drastically enhanced with the supplement of paired CAM conditioned medium, while no tumor organoid was well formed in control medium. These organoids in CAM conditioned medium group are positive for cancer stem cell markers. Correspondingly, CAMs induced a cancer stemness phenotype, as shown by increased tumor formation rate in limiting dilution assays and by strengthened sphere formation abilities in sphere formation assays. Through a cytokine array and functional validations, we pinpointed soluble osteopontin as a key mediator of CAM-responsive cancer stemness and chemoresistance, and it initiates paracrine signaling through the CD44 receptor on cancer cells. RNA-sequencing of ovarian cancer cells primed with CAMs revealed overexpression of multi-drug resistance-related ABC-binding cassette transporters (ABC transporters). Lastly, genetic inhibition of osteopontin in CAMs with shRNA constructs and therapeutic targeting with an anti-osteopontin neutralizing antibody reduced cancer stemness and chemoresistance. Conclusions: We demonstrated that CAMs promote ovarian cancer chemoresistance and tumor formation by establishing a cancer stem cell niche. Importantly, our study identified paracrine osteopontin signaling as a critical mediator of chemoresistance and stemness, and targeting osteopontin from CAMs holds the potential to restore ovarian cancer chemosensitivity.
Citation Format: Jin Qian, Bauer LeSavage, Chenkai Ma, Suchitra Natarajan, Joshua Eggold, Kelsea Hubka, Yiren Xiao, Katherine Fuh, Venkatesh Krishnan, Annika Enejder, Sarah Heilshorn, Oliver Dorigo, Erinn Rankin. Mesothelial cells promote ovarian cancer stemness and chemoresistance through osteopontin paracrine signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-364.