Shear stress regulation of stemness, chemoresistance and tumor-mesothelial interaction in ovarian cancer

Abstract

The deaths from ovarian cancer are primarily due to treatment-refractory metastatic cancer, which is characterized by widespread peritoneal dissemination and malignant ascites. Understanding the underlying molecular mechanisms of these processes are important. It is known that tumor cells metastasize and settle onto secondary sites under dynamic flow but not static condition. However, the role of shear stress in the growth and dissemination of tumor cells remains poorly understood. In this study, a three-dimensional microfluidic platform which is relevant to the tumor spheroids with continuous flow rate to emulate the physiological conditions in the malignant ascites of ovarian cancer patients was developed. Using this model, it showed for the first time that shear stress that resembles the ascitic fluid flow rate played a significant role in inducing cancer stem-like phenotypes (Oct-4, CD117) and enhanced the resistance to front-line chemotherapeutic agents cisplatin and paclitaxel. These activities were mediated through a phosphatidylinositol 3-kinase/Akt-microRNA-199a-3p pathway and the subsequent multidrug transporter ATPase binding cassette G2 and P-glycoprotein activation. To delineate the key molecules mediating metastasis, an isogenic pair of highly metastatic (HM) and non-metastatic (NM) ovarian cancer cell lines with opposite metastatic properties was first used. Adhesion to the peritoneal mesothelium is a key rate-limiting step in the formation of metastasis. Using a coculture system, it has been found that HM tethered and rolled more slowly and adhered more stably onto the peritoneal mesothelium than NM under ascitic shear flow. Functional blocking and recombinant protein binding assays showed that this initial tumor-mesothelial interaction was mediated by P-selectin, but not other selectins. The requirement of P-selectin was further confirmed in a subset of cancer stem/tumor-initiating cells (M-CSCs) which possessed the distinct capability to support metastasis initiation. M-CSCs xenograft in P-selectin-deficient (Selp-/- Rag2-/-) mice confirmed a role for P-selectin in ovarian cancer metastasis in vivo. P-selectin could bind heparan sulfate (HS) proteoglycan in a sulfation-dependent manner. Moreover, another ligand on M-CSCs for P-selectin binding was found to be an O-glycoprotein decorated with the sialyl Lewis x (sLex) glyocoepitope, in a sialic acid- and fucose-dependent manner. Insulin-like growth factor receptor-1 may confer the sLex oligosaccharide on M-CSCs. 1,3-fucosyltransferase V was responsible for the elevated sLex expression and enhanced adhesive capability of M-CSCs. Tumor xenograft models and clinical data analysis further corroborated the clinical relevance of these findings. Taken together, these findings reveal the P-selectin-HS/sLex binding in ovarian tumor-mesothelium interaction in early metastasis, and may potentially lead to a new avenue for efficacious therapies to cancer metastasis.