Abstract LB-256: Catena: A novel cellular entity composed of ovarian cancer stem cells

Abstract

Abstract Metastasis of cancer cells into the peritoneal cavity is a clinical manifestation of advanced-stage ovarian cancer and is associated with poor survival. In an orthotopic NOD/SCIDIL2R −/− (NSG) model of advanced ovarian serous adenocarcinoma, we have identified a novel cellular structure consisting of a chain of cells coated with an extensive pericellular glycocalyx, free-floating in the ascites of mice following intra-peritoneal engraftment with ovarian cancer cell lines (Ovcar3, Ovcar5, A2780) or primary ovarian cancer cells. We have termed these chains “catenae“ (Latin for “chains”). Ovcar3 has epithelial characteristics and expresses E-cadherin. In the inflammatory ascites of NSG mice undergoes an “epithelial to mesenchymal transition (EMT)” with E-cadherin loss, followed by a “mesenchymal to catena transition (MCT)”. Ovcar5 and A2780 are mesenchymal and do not require in vivo passage to undergo MCT and catena formation was readily obtained under our selective culture conditions. By transmission and scanning electron microscopy catenae are undifferentiated with high nuclear to cytoplasmic ratio and have amoeboid features with extensive membrane blebbing and abundant microvilli, yet retain epithelial-type ZO-1+ tight junctional connections. Time-lapse microscopy showed that a catena develops from a single cell by up to 7 rapid (16 hrs) symmetric divisions. We can expand catenae in vitro (∼10,000 fold in 7-9 days) for prolonged periods with the majority of cells retaining CSC function as determined by single cell recloning and limiting dilution engraftment in NSG mice. As few as 2 cells when injected with matrigel produce subcutaneous tumors within 1-2 weeks with the morphology of serous papillary ovarian adenocarcinoma. To our knowledge, this is the first report to show isolation of a homogeneous population of CSC, that can be expanded extensively in vitro without differentiation, yet retain the capacity to fully differentiate to all cellular components of the original tumor. At high cell density, catenae develop into spheroids by a process of “rolling up”. Spheroids contain ∼10% CSC while epithelial tumor monolayers have <1% CSC. Time-lapse studies showed that spheroids extrude fresh catenae. Microarray and massive parallel RNA-sequencing revealed a CSC gene signature with hyaluronan synthase-2 (HAS2), hyaluronan and proteoglycan link protein 1 (HAPLN1) and PDGFRA the top 3 genes differentially upregulated genes. Catenae produce PDGFC and respond to autocrine signaling via PDGFRA. HAS2 and HAPLN1 are responsible for generating the hyaluronan component of the protective glycocalyx. Treatment of catena with hyaluronidase removes the glycocalyx resulting in reversal of the MCT and EMT and loss of CSC capacity. We have adapted the catena and spheroid system for high-throughput screening for identification of compounds that specifically inhibit CSC by inducing apoptosis, proliferation inhibition or differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-256.