Transmembrane distribution of phosphatidylethanolamine in plasma membrane of ovarian cancer cells under conditions mimicking tumour microenvironment

In healthy cells rather than being evenly distributed phosphatidylserine (PS) and phosphatidylethanolamine (PE) are found preferentially in the inner leaflet of the plasma membrane. Unlike normal cells, tumor cells lose the ability to maintain aminophospholipid asymmetry and expose PS naturally which promotes tumor growth and immune suppression by mechanisms that are not established. Since both PS and PE are co-regulated by the same transporters, in the case of PE translocation it might also serve as a specific lipid biomarker diagnostic the prognostic value of which is yet to be determined. We aimed to investigate whether hypoxia and low pH characteristics of tumor microenvironment (TME) might promote PE translocation on the surface leaflet of cancer cells membranes. Ovarian cancer cells OVCAR-4 were incubated under standard (pH=7.5, O2=21%) and TME modeling conditions (pH=5.8, О2=1%) following with a two-step staining with PE-specific biotinylated Ro09 and streptavidin conjugated with Alexa Fluor 405. Propidium iodide (PI) staining was conducted to justify plasma membrane integrity. Visualization of PE on living OVCAR-4 cells was performed with the use of laser confocal microscopy. Under normoxic conditions a signal from PE was not detected on the surface of intact OVCAR-4 cells. However, under TME conditions cells were positive for PE staining, but not for PI, demonstrating that PE is translocated from the inner to the outer leaflet of cancer cell membrane. Our results show that non apoptotic externalization of aminophospholipids in cancer cells has become a subject of great interest due to correlation with innate immune suppression and promotion of tumor growth. We propose that TME-driven surface exposure of PE, a second abundant phospholipid in cell membrane, coincides with constitutive PS externalization and can serve as a potential TME-specific therapeutic target and/or diagnostic tool for ovarian and other types of cancer.