Abstract 2649: A novel approach to fate-map hypoxic cells during tumor progression uncovers metastatic potency of post-hypoxic cells
Authors: Godet Inês , Shin Yu Jung , Ju Julia A. , Bhoyar Soumitra , Ye I Chae , Wang Guannan , Sukumar Saraswati , Gilkes Daniele M. 
Published: August 2019

Abstract
Hypoxia occurs in 90% of solid tumors due to inefficient vasculature and has been demonstrated to promote a more aggressive phenotype, correlating with worse patient prognosis. Current methods to measure hypoxia in human tumors only identify cells that are hypoxic at the time the tissue is collected and do not delineate cells that were transiently exposed to hypoxia but survived and adapted. To overcome this challenge, we generated a Lox-Cre dual-vector oxygen-dependent reporter system. Under hypoxia, Cre expression leads to the cleavage of a DsRed reporter and permanent expression of a GFP reporter. We stably transduced breast cancer cell lines and demonstrated an irreversible reporter-switch that occurs when cells are exposed to 0.5% O2, the typical O2 level in human breast tumors, but does not occur under physiological conditions (8% O2). To explore the utility of our system, we embedded spheroids derived from MDA-MB-231 reporter cells in collagen and cultured them under 20% O2 conditions. A defined hypoxic core (GFP+) was visible after 15 days in culture and it correlated with hypoxic O2 measurements. We then generated orthotopic breast tumors by transplanting MDA-MB-231 reporter cells into the mammary fat pad of mice. Hypoxic cells (GFP+) were localized in the peri-necrotic region of the tumor and co-localized with hypoxyprobe and HIF1α labeling. RNA sequencing of GFP+ compared to DsRed+ tumor cells demonstrated that GFP+ cells were enriched for the hallmark signature of hypoxia. Additionally, we developed a transgenic mouse expressing the hypoxia-driven construct that was bred to a tdTomato-floxed GFP mouse. A female double fluorescent/hypoxia-reporter mouse was then bred to a male FVB/N-Tg(MMTV-PyMT) mouse in order to generate triple-transgenic mice that form breast tumors that spontaneously metastasize. We systematically studied hypoxia in spontaneous breast tumors from the development of ductal hyperplasia to late stage invasive carcinoma. Tumor hypoxia was first detected in early stage ductal carcinoma in situ (DCIS) lesions. Similarly to the spheroid model, mammary organoids derived from triple-transgenic mice tumors cultured under 20% O2 conditions also exhibited a defined hypoxic core after 15 days in culture. Both orthotopic and transgenic mouse models revealed an increased potency of post-hypoxic cells at arriving to distant sites in early stage metastasis. We utilized RNA sequencing to identify potential mechanisms of enhanced metastatic capacity. Ultimately, our system allowed the fate-map of hypoxic cells during tumor progression to metastasis. We show for the first time that 3D in vitro organoids derived from PyMT tumors develop regions of hypoxia under normal tissue culture conditions. In vivo, we show that hypoxic cells are more potent at arriving at distant sites and that they retain a different gene expression that potentially confers a metastatic advantage.
Citation Format: Inês Godet, Yu Jung Shin, Julia A. Ju, Soumitra Bhoyar, I Chae Ye, Guannan Wang, Saraswati Sukumar, Daniele M. Gilkes. A novel approach to fate-map hypoxic cells during tumor progression uncovers metastatic potency of post-hypoxic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2649.