title: Probe for the measurement of cell surface pH in vivo and ex vivo.
abstract: 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.
authors.affiliation: Physics Department, University of Rhode Island, Kingston, RI 02881;
authors.forename: Michael
authors.initials: M
authors.lastname: Anderson
authors.affiliation: Physics Department, University of Rhode Island, Kingston, RI 02881;
authors.forename: Anna
authors.initials: A
authors.lastname: Moshnikova
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511 andreev@mail.uri.edu donald.engelman@yale.edu.
authors.forename: Donald M
authors.initials: DM
authors.lastname: Engelman
authors.affiliation: Physics Department, University of Rhode Island, Kingston, RI 02881;
authors.forename: Yana K
authors.initials: YK
authors.lastname: Reshetnyak
authors.affiliation: Physics Department, University of Rhode Island, Kingston, RI 02881; andreev@mail.uri.edu donald.engelman@yale.edu.
authors.forename: Oleg A
authors.initials: OA
authors.lastname: Andreev
mesh_terms: D000818:Animals; D001578:Benzopyrans / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry*; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009369:Neoplasms / Q000737:chemistry; D018874:Spheroids, Cellular / Q000737:chemistry
chemical_list: D001578:Benzopyrans; D005456:Fluorescent Dyes; D008565:Membrane Proteins; C063917:SNARF dye; C533201:pHLIP protein
keywords: SNARF; fluorescence; pH low insertion peptide; tumor acidity