Fig 2: 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.

Fig 3: 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.