Identifying cancer stem cells in an in vitro engineered model of cell-generated hypoxic gradients using adaptive thresholding of fluorescent reporters

Authors

• G Vitale
• B Tang
• J Minin
• S Lockett
• L Wakefield
• W Heinz

Abstract

Triple negative breast cancer (TNBC) is an aggressive, metastatic, and recurrent breast cancer subtype with 5-year survival rates as low as 13%. TNBC has a complex tumor microenvironment (TME) characterized by a spatial oxygen (O2) gradient formed by cellular consumption of O2 diffusing from the vasculature. This gradient drives phenotypic changes that encourage cancer cell survival and metastasis. We hypothesize that hypoxia upregulates Sox2 and Oct4, master transcription factors of cancer stemness, promoting cancer stem cell (CSC) development.

Restricted exchange environment chambers (REECs) mimic the cell-generated gradients of oxygen of solid tumors in 2D cell culture. To identify CSCs, we used a lentiviral-based red fluorescent protein (RFP) reporter activated by the binding of Sox2 and Oct4 to an artificial enhancer element (SORE6) in MBA-MD-231 cells, a human TNBC cell line. A parallel cell line lacking the Sox2/Oct4 response elements (mCMV) was used to find an RFP intensity threshold for SORE6 positivity. Because the RFP signal is affected by cell density and O2-dependent maturation, we used live-cell imaging to develop a standard curve of cell density versus SORE6+ and an adaptive thresholding technique to account for these effects, respectively.

We cultured SORE6 and mCMV cells in REECs for up to 7 days and imaged every 2 hours. At 68 hours, hypoxic regions of the REEC contained a significantly higher percentage of SORE6+ cells than normoxic regions. We conclude that hypoxia is an integral part of the CSC niche.

This project has been funded in part under contract 75N91019D00024.

Scientific Focus Area: Cancer Biology

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