Investigating the transcriptional regulation of intratumoral hypoxia in CTCs

Using a hypoxia-fate mapping system that causes a fluorescent switch from DsRed to GFP expression, we isolated CTCs from the blood of 2 NSG mice 40 days post orthotopic tumor implantation. The CTCs were sorted into DsRed+ or GFP+ populations by fluorescence-activated cell sorting (FACS). Mice were inoculated with hypoxia fate-mapping MDA-MB-231 cells (described in Godet et. al, Nat Comm, 2019) orthotopically to generate breast tumors. After 40 days, whole blood was collected, lysed to eliminate red blood cells and washed. Samples were immediately sorted into DsRed+ or GFP+ populations and submitted to RNA extraction.