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.

Mechanistic Basis of pH-Dependent 5-Flucytosine Resistance in Aspergillus fumigatus

“…Despite 326 studies demonstrating the efficacious, monotherapeutic use of 5FC in murine models of 327 aspergillosis, this drug is typically not applied in clinical settings (9, 40). To our 328 knowledge, no data is available to indicate the pH in the lung during aspergillus infection 329 however from studies of other pathological states it has been shown that cellular 330 damage, inflammation and infection can lead to a localised reduction of pH below pH 7 331 (41)(42)(43). This coupled with the ability of Aspergillus sp.…”

Applications of pHLIP Technology for Cancer Imaging and Therapy

“…Recent studies using a fluorophore with a pH-dependent emission spectrum have shown that the pH in the vicinity of the plasma membrane of cancer cells is about 0.3–0.7 pH units lower than the bulk extracellular pH [12]. In experiments in vitro, where the pH of the solution in which the cancer cells are growing (bulk pH) is maintained at pH 7.4, the cell surface pH for highly metastatic cells has been shown to be around pH 6.7.…”

“…The conjugation of the pH-sensing fluorescent dye SNARF to a pHLIP results in a compound that can measure pH at the surface of individual cells, which differs greatly from the more easily and commonly measured bulk pH [12]. Currently, approaches for cell surface pH mapping using SNARF-pHLIP in liquid and solid biopsy samples are being developed, which might result in an opportunity to get information about the metabolic status of tumors, ultimately aiding in the prediction of tumor aggressiveness and the tailoring of therapy.…”

pH-sensitive pHLIP® coated niosomes

“…We choose to treat cells with constructs at pH7.8, which is slightly above than normal physiological pH7.4, since it was shown that pH at the surface of cancer cells, especially highly metastatic cancer cells such as 4T1, is lower even when pH of media is normal (Anderson et al, 2016). We also choose to treat cells with constructs at pH5.5, which is slightly lower than mean pH established at the surface of cancer cells within tumors, pH6.0, (Anderson et al, 2016) with main goal to enhance difference in cellular uptake of niosomes in this model experiment. The fluorescent signal from the cells treated with niosomes was analyzed using cellometer (Figure 2).…”

“…Family of pH (Low) Insertion Peptides (pHLIP ® peptides) are under development as novel agents, which target tumor acidity (Andreev et al, 2009, Weerakkody et al, 2013). The peptides sense pH at the surface of cancer cells, where it is the lowest (Anderson et al, 2016), and insert into cellular membranes (Reshetnyak et al, 2006, Reshetnyak et al, 2007, Reshetnyak et al, 2008, Andreev et al, 2010). Nanocarriers decorated with pHLIPs are biocompatible, can target tumor and demonstrate enhanced cellular uptake by cancer cells (Du et al, 2014, Wijesinghe et al, 2013, Yao et al, 2013b, Arachchige et al, 2015, Yao et al, 2013a).…”