Abstract TMEM-032: MULTIDISCIPLINARY CHARACTERIZATION OF OVARIAN CANCER SPHERES
Authors: Liu Shubai , Yang Junzheng , Choi Pui-Wah , Kwok Jamie Sui-Lam , Hasselblatt Kathleen , Nomura Daniel , Fong Wing Ping , Tsui Stephen KW , Vitonis Allison , Cramer Daniel , Welch William R. , Cravatt Benjamin , Berkowitz Ross , Ng Shu-Wing 
Published: June 2017

Abstract
Sphere-forming cultures have been widely used in stem cell biology. Ovarian cancer sphere culture is also a good model to study ovarian cancer ascites spheroids, which propagate without attachment to a substratum. We studied non-adherent ovarian cancer sphere cultures derived from ovarian cancer cell lines and epithelial cancer cells isolated from clinical samples. Western blot analysis and immunofluorescence showed that ovarian cancer spheres expressed elevated levels of stem cell markers CD133, c-Myc, Nanog, and Oct4. Metabolically, ovarian cancer sphere cells showed decreased ATP levels and were under constant oxidative stress, with significant depletion of the endogenous antioxidant glutathione. Accordingly, supplementation with antioxidant supplement N-acetylcysteine (NAC) in cancer sphere cultures relieved oxidative stress and improved growth of the sphere cultures in a dose-dependent manner. In contrast, adherent cancer cell cultures did not show any significant growth enhancement. Furthermore, microarray gene expression profiling and activity-based proteomic profiling using a sulfonate ester chemical probe revealed that the ovarian cancer spheres demonstrated increased level of FOS/JUN expression, which activated the expression and activity of the omega class of glutathione-S transferase GSTO1, an enzyme involved in the metabolism of xenobiotics and cisplatin resistance. Knockdown of FOS expression using siRNA reduced the levels of GSTO1 and abrogated chemoresistance of the sphere cultures. Separately, gene set enrichment analysis of the cancer sphere expression profiles revealed elevated expression of endosomal pathway genes for exosome secretion in the sphere cultures. Knockdown of one elevated gene, Rab27B, significantly reduced the exosome number in the spent medium of ovarian cancer sphere cultures. Clinically, measurement of the exosome number in patient body fluids using the NanoSight instrument and a flow cytometry method indicated that plasma and urine samples from a panel of ovarian cancer patients had increased number of exosomes compared with samples derived from benign patients.
CONCLUSIONS: Our multidisciplinary studies have discovered a plethora of properties of ovarian cancer spheres that may promote ovarian cancer growth. Elevated expression and activity of GSTO1 in ovarian cancer spheres may represent a novel mechanism by which ovarian cancer spheroids respond to heightened oxidative stress and chemotherapeutic agents. Antioxidant supplements may not have the intended benefits to cancer survivors. Instead, they may promote cancer growth by relieving the oxidative stress of ovarian cancer spheroids. In contrast, inhibitors that target GSTO1 may have better clinical value in prolonging survivorship of ovarian cancer patients with malignant ascites. Ovarian cancer spheres also have increased expression of endosomal pathway genes for exosome secretion, which may promote the establishment of tumor microenvironment for cancer propagation, and is consistent with the increased exosomes in the plasma and urine samples of ovarian cancer patients.
Citation Format: Shubai Liu, Junzheng Yang, Pui-Wah Choi, Jamie Sui-Lam Kwok, Kathleen Hasselblatt, Daniel Nomura, Wing Ping Fong, Stephen KW Tsui, Allison Vitonis, Daniel Cramer, William R. Welch, Benjamin Cravatt, Ross Berkowitz, and Shu-Wing Ng. MULTIDISCIPLINARY CHARACTERIZATION OF OVARIAN CANCER SPHERES [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-032.