Oxygen tension-dependent variability in the cancer cell kinome and response to combination therapies

Solid tumors are characterized by significantly lower oxygen (O2) levels. Despite this fact, routine preclinical cancer studies are performed under ambient O2 conditions. We have developed an experimental approach that allows us to collect, process and evaluate cancer and non-cancer cells under physioxia (3-5% O2), in such a way that there is very minimal exposure to air. In addition to evaluating mouse mammary tumor cell lines with respect to kinome changes due to ambient and physioxic O2 tensions, we applied single cell-RNA sequencing to determine transcriptomic changes that occur in primary human breast epithelial cells that were collected, processed and cultured in ambient air and physioxia. We demonstrate that ambient and physioxic O2 tensions induce unique transcriptomic changes in primary human breast epithelial cells that are concurrent with our observations in the tumor cells. Overall design: Breast tissue samples were collected from two female healthy donors under physioxia. The harvested tissues were then minced into tiny pieces at 3% O2 and separated into two portions. Each tissue portion was then processed into single cells via enzymatic digestion in ambient air and physioxia respectively. The derived single cells were then expanded in culture in the respective O2 tensions. Cultured cells were harvested via trypsinization, followed by determination of viability by trypan blue exclusion. We generated cDNA libraries from ambient air and physioxia-derived cells from the two donors and subjected them to sequencing a read depth of ~50,000 reads per cell.