Fig 2: Cancer cells in physioxia and ambient air display different levels of additional cell surface markers including CD274.(A) CD274/EpCAM staining pattern of cancer cells under physioxia and ambient air (n = 3 to 6, one-way ANOVA). (B) Quantitation of CD274+ cells. (C) Culturing under physioxia increased CD274+/EpCAM+ Her2/Neu but not PyMT cancer cells. (D) CD24/CD29 staining patterns (n = 3 to 6, one-way ANOVA). (E) O2 tension had no effect on CD29high/CD24medium basal cells. (F) Physioxia increased the levels of CD24high/CD29medium luminal cells. (G) O2 tension affected the ratio between CD24high/CD29medium luminal and CD29hi/CD24medium basal cells. (H and I) Mammospheres derived from PyMT tumor cells under physioxia contained higher levels of LGR5+ cells compared to mammospheres under ambient air. (J) Cells processed under physioxia display higher levels of nuclear ROS (n = 4, one-way ANOVA). (K) Quantitative differences in nuclear ROS in cells collected and processed under physioxia and cells processed under ambient air. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by ANOVA.

Fig 3: Cells obtained from ascites fluids of patients with cancers such as serous ovarian cancer show O2 tension–dependent variability in cell surface marker profiles.Cells for flow cytometry analysis were cultured for 5 days, and only lineage-negative (CD31-PE, CD45-PE, and CD140b-PE negative) cells were gated for the analysis. (A) CD49f/EpCAM staining patterns (n = 9). (B) The number of CD49f+/EpCAM- cells varied on the basis of O2 tension and tumor type. (C) EpCAM positivity also showed tumor-specific variability under physioxia compared to ambient air. (D) CD24/CD44 staining patterns of cells from ascites fluid under physioxia and ambient air (n = 9). (E) Percentage of CD44+ cells in each sample under physioxia and ambient air. (F) Percentage of CD24+ cells under physioxia and ambient air. CD24+ cells were lower under physioxia compared to ambient air. (G) Cells from ascites fluids collected under physioxia contained elevated levels of BRD4 protein compared to cells collected under ambient air.