Shree Bose1,2, Haipei Yao1, Qiang Huang1,3, Regina Whitaker4, Christopher D Kontos2,5, Rebecca A Previs4, Xiling Shen6. 1. Department of Biomedical Engineering, Duke University Pratt School of Engineering, Durham, NC, USA. 2. Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC, USA. 3. Department of Pediatric Surgery, Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China. 4. Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Duke University Medical Center, Durham, NC, USA. 5. Department of Medicine, Division of Cardiology, Duke University Medical Center, Durham, NC, USA. 6. Terasaki Institute for Biomedical Innovation, Los Angeles, CA, USA. xiling.shen@terasaki.org.
Abstract
BACKGROUND: Epithelial ovarian cancer (OC) is the most lethal gynecological malignancy and patients present with significant metastatic burden, particularly to the adipose-rich microenvironment of the omentum. Recent evidence has highlighted the importance of metabolic adaptations in enabling this metastasis, leading to significant interest in evolving the arsenal of tools used to study OC metabolism. In this study, we demonstrate the capability of genetically encoded fluorescent biosensors to study OC, with a focus on 3D organoid models that better recapitulate in vivo tumor microenvironments. MATERIALS AND METHODS: Plasmids encoding the metabolic biosensors HyPer, iNap, Peredox, and Perceval were transfected into 15 ovarian cancer cell lines to assay oxidative stress, NADPH/NADP+, NADH/NAD+, and ATP/ADP, respectively. Fluorescence readings were used to assay dynamic metabolic responses to omental conditioned media (OCM) and 100 μM carboplatin treatment. SKOV3 cells expressing HyPer were imaged as 2D monolayers, 3D organoids, and as in vivo metastases via an intravital omental window. We further established organoids from ascites collected from Stage III/IV OC patients with carboplatin-resistant or carboplatin-sensitive tumors (n = 8 total). These patient-derived organoids (PDOs) were engineered to express HyPer, and metabolic readings of oxidative stress were performed during treatment with 100 μM carboplatin. RESULTS: Exposure to OCM or carboplatin induced heterogenous metabolic changes in 15 OC cell lines, as measured using metabolic sensors. Oxidative stress of in vivo omental metastases, measured via intravital imaging of metastasizing SKOV3-HyPer cells, was more closely recapitulated by SKOV3-HyPer organoids than by 2D monolayers. Finally, carboplatin treatment of HyPer-expressing PDOs induced higher oxidative stress in organoids derived from carboplatin-resistant patients than from those derived from carboplatin-sensitive patients. CONCLUSIONS: Our study showed that biosensors provide a useful method of studying dynamic metabolic changes in preclinical models of OC, including 3D organoids and intravital imaging. As 3D models of OC continue to evolve, the repertoire of biosensors will likely serve as valuable tools to probe the metabolic changes of clinical importance in OC.
BACKGROUND: Epithelial ovarian cancer (OC) is the most lethal gynecological malignancy and patients present with significant metastatic burden, particularly to the adipose-rich microenvironment of the omentum. Recent evidence has highlighted the importance of metabolic adaptations in enabling this metastasis, leading to significant interest in evolving the arsenal of tools used to study OC metabolism. In this study, we demonstrate the capability of genetically encoded fluorescent biosensors to study OC, with a focus on 3D organoid models that better recapitulate in vivo tumor microenvironments. MATERIALS AND METHODS: Plasmids encoding the metabolic biosensors HyPer, iNap, Peredox, and Perceval were transfected into 15 ovarian cancer cell lines to assay oxidative stress, NADPH/NADP+, NADH/NAD+, and ATP/ADP, respectively. Fluorescence readings were used to assay dynamic metabolic responses to omental conditioned media (OCM) and 100 μM carboplatin treatment. SKOV3 cells expressing HyPer were imaged as 2D monolayers, 3D organoids, and as in vivo metastases via an intravital omental window. We further established organoids from ascites collected from Stage III/IV OC patients with carboplatin-resistant or carboplatin-sensitive tumors (n = 8 total). These patient-derived organoids (PDOs) were engineered to express HyPer, and metabolic readings of oxidative stress were performed during treatment with 100 μM carboplatin. RESULTS: Exposure to OCM or carboplatin induced heterogenous metabolic changes in 15 OC cell lines, as measured using metabolic sensors. Oxidative stress of in vivo omental metastases, measured via intravital imaging of metastasizing SKOV3-HyPer cells, was more closely recapitulated by SKOV3-HyPer organoids than by 2D monolayers. Finally, carboplatin treatment of HyPer-expressing PDOs induced higher oxidative stress in organoids derived from carboplatin-resistant patients than from those derived from carboplatin-sensitive patients. CONCLUSIONS: Our study showed that biosensors provide a useful method of studying dynamic metabolic changes in preclinical models of OC, including 3D organoids and intravital imaging. As 3D models of OC continue to evolve, the repertoire of biosensors will likely serve as valuable tools to probe the metabolic changes of clinical importance in OC.
Authors: Chris Jenske de Witte; Jose Espejo Valle-Inclan; Nizar Hami; Kadi Lõhmussaar; Oded Kopper; Celien Philomena Henrieke Vreuls; Geertruida Nellie Jonges; Paul van Diest; Luan Nguyen; Hans Clevers; Wigard Pieter Kloosterman; Edwin Cuppen; Hugo Johannes Gerhardus Snippert; Ronald Peter Zweemer; Petronella Oda Witteveen; Ellen Stelloo Journal: Cell Rep Date: 2020-06-16 Impact factor: 9.423
Authors: Toshiro Sato; Daniel E Stange; Marc Ferrante; Robert G J Vries; Johan H Van Es; Stieneke Van den Brink; Winan J Van Houdt; Apollo Pronk; Joost Van Gorp; Peter D Siersema; Hans Clevers Journal: Gastroenterology Date: 2011-09-02 Impact factor: 22.682
Authors: Stefanie Dietmair; Nicholas E Timmins; Peter P Gray; Lars K Nielsen; Jens O Krömer Journal: Anal Biochem Date: 2010-05-21 Impact factor: 3.365
Authors: Fabrizio Miranda; David Mannion; Shujuan Liu; Yiyan Zheng; Lingegowda S Mangala; Clara Redondo; Sandra Herrero-Gonzalez; Ruoyan Xu; Charlotte Taylor; Donatien Fotso Chedom; Mohammad Karaminejadranjbar; Ashwag Albukhari; Dahai Jiang; Sunila Pradeep; Cristian Rodriguez-Aguayo; Gabriel Lopez-Berestein; Eidarus Salah; Kamal R Abdul Azeez; Jonathan M Elkins; Leticia Campo; Kevin A Myers; Daniel Klotz; Serena Bivona; Sunanda Dhar; Robert C Bast; Hideyuki Saya; Hwan Geun Choi; Nathanael S Gray; Roman Fischer; Benedikt M Kessler; Christopher Yau; Anil K Sood; Takeshi Motohara; Stefan Knapp; Ahmed Ashour Ahmed Journal: Cancer Cell Date: 2016-07-28 Impact factor: 31.743