Probe for the measurement of cell surface pH in vivo and ex vivo
Significance In acidic, diseased tissues, such as tumors, cell surfaces are expected to be more acidic than the surroundings, but no means have been available to measure surface acidity specifically in such tissues. We have developed a probe that uses a pH low insertion peptide to locate a pH sensing dye, seminaphtharhodafluor, at acidic cell surfaces and find that the probe can measure surface pH, which is correlated with tumor aggressiveness. Even an individual cancer cell maintains acidity at its surface and can be detected in a well perfused area. The new probe can be used in vivo and ex vivo on tissue specimens and opens a path to both medical utility and exploration of other functions of acidity on cell surfaces. We have developed a way to measure cell surface pH by positioning a pH sensitive fluorescent dye, seminaphtharhodafluor (SNARF) conjugated to the pH low insertion peptide (pHLIP) It has been observed that many diseased tissues are acidic and that tumors are especially so. A combination of effects acidifies tumor cell interiors, and cells pump out lactic acid and protons to maintain intracellular pH, acidifying the extracellular space. Overexpression of carbonic anhydrases on cell surfaces further contributes to acidification. Thus, the pH near tumor cell surfaces is expected to be low and to increase with distance from the membrane, so bulk pH measurements will not report surface acidity. Our new surface pH measurement tool was validated in cancer cells grown in spheroids, in mouse tumor models in vivo, and in excised tumors. We found that the surface pH is sensitive to cell glycolytic activity: the pH decreases in high glucose and increases if glucose is replaced with nonmetabolized deoxyglucose. For highly metastatic cancer cells, the pH measured at the surface was 6.7 6.8, when the surrounding external pH was 7.4. The approach is sensitive enough to detect 0.2 0.3 pH unit changes in vivo in tumors induced by i.p. injection of glucose. The pH at the surfaces of highly metastatic cells within tumors was found to be about 6.1 6.4, whereas in nonmetastatic tumors, it was 6.7 6.9, possibly creating a way to distinguish more aggressive from less aggressive tumors. Other biological roles of surface acidity may be found, now that targeted measurements are possible.
Proceedings of the National Academy of Sciences