The pulmonary metastasis assay (PuMA), a tool to study novel therapies for ovarian cancer: example, EO3001 in clear cell carcinoma
Yuchen Ding1,2, Shary Yuting Chen2, Lucy Yuqin Li1,2, Joyce Yuhan Zhang1,2, Farhia Kabeer1,2, Forouh Kalantari1,2, Jeffrey Bacha3, Dennis Brown3, Sarath Kanekal3, Neil Sankar3, Michael Lizardo2, Amal M EL-Naggar1,2, David G Huntsman1,2.
1Department of Pathology and Laboratory Medicine, The University of British Columbia, Vancouver, BC, Canada; 2Department of Molecular Oncology, BC Cancer Research Institute, Vancouver, BC, Canada; 3Edison Oncology Holding Corp, Menlo Park, CA, United States
Introduction
In Clear Cell Ovarian Cancer (CCOC) loss of ARID1A induces reactive oxygen species (ROS) accumulation, making CCOC cells dependent on oxidative phosphorylation (OXPHOS) for energy. EO3001, a synthetic drug that selectively transports extracellular Cu(II) to mitochondria, induces mitochondrial ROS, thereby triggering cuproptosis. Recent studies showed enhanced sensitivity of ARID1A-deficient cells to EO3001. To study EO3001's therapeutic effectiveness for ARID1A-deficient CCOC we employed an ex vivo model, the Pulmonary Metastasis Assay (PuMA). Through this assay metastasis are seeded through tail vein injections and lung explants can be harvested and studied immediately or after metastasis are fully established. PuMA provides a richer microenvironment than traditional 2D and 3D culture models and enables single lungs to be treated with several experimental regimens.
Methods
Fluorescent-labelled isogenic CCOC cell lines (RMG-1, OVCA429, and JHOC-5 +/- ARID1A) were generated using CRISPR-Cas9, and therapeutic effects of EO3001 were evaluated on the tumorigenic potential and cell growth of these cells in vitro and ex vivo using fluorescent microscope for signal intensities and colony size, cell viability, proliferation, total and mitochondrial ROS levels, OXPHOS activities under ambient and stress conditions. Both early-and late-harvest PuMA were employed to investigate the effects of EO3001 on cancer cells within the lung environment.
Conclusions
While EO3001 showed promising therapeutic value in ARID1A-deficient cells, both in vitro and ex vivo models, hypoxia significantly diminishes the effects of EO3001, potentially attributed to alterations in glycolysis and OXPHOS further supporting in the hypothesis of EO3001 selective activity in OXPHOS dependent cancer phenotypes. By exploiting the dependence of ARID1A-deficient CCOC on OXPHOS, the use of EO3001 could offer a viable therapy approach. This assay is ideal for assessing other treatment approaches.