Abstract 4250: pHLIP® technology for imaging acidic tumors
Authors: Anna Moshnikova, Michael Anderson, Ramona-Cosmina Adochite, Donald M. Engelman, Oleg A. Andreev, Yana K. Reshetnyak
Journal: Cancer Research (2016)

Abstract Introduction: Extracellular acidosis promotes tumor development, progression and invasiveness. A combination of effects acidifies tumor cell interiors, so to maintain intracellular pH, cells pump out lactic acid and protons, acidifying the extracellular space. In addition, overexpression of carbonic anhydrases on the surfaces of cancer cells further contributes to an acidification of the environment especially near the cell surface. Thus, the pH near tumor cell surfaces is expected to be the lowest, and it increases with distance from the membrane. pH (Low) Insertion Peptides (pHLIP® peptides) pertain to the class of pH-sensitive agents able of sensing pH at the cellular surface and delivery of imaging and therapeutic agents to the cancer cells in tumors. Experimental Procedures: We have developed a way to measure cell surface pH by positioning a pH-sensitive fluorescent dye, SNARF, conjugated to the pH Low Insertion Peptide (pHLIP® peptide). The measurement tool was validated using cancer cells grown in spheroids, in mice and in excised tumors. Also, we performed imaging of primary tumors and metastatic lesions testing various fluorescent pHLIP® constructs on animal tumor models and human tissue. Results: pHLIP® is a platform technology using pH-triggered, membrane-associated folding for targeting acidic tumors. In contrast to other approaches developed to measure extracellular pH and target acidic environments (mostly in endosomes, where the pH is around 5.0-5.5), the pHLIP® technology has the capability to “sense” and target pH at the surfaces of tumor cells. We established that the pH at the surface of cancer cells is 0.2-0.4 pH units lower (in some parts of tumor it reaches values of pH6.0-6.1) than bulk extracellular pH measured up to now and demonstrated that the surface pH is sensitive to cell glycolytic activity. The approach was sensitive enough to detect 0.2-0.3 pH unit changes in vivo in tumors induced by injection of glucose. In response to low pH at the surface of cancer cells, pHLIP® peptides insert across the cell membrane and undergo coil-helix transition, which gives a high cooperativity for the transition compared with any pH-dependent diffusion-limited process, and allow targeting imaging agents to cancer cells in tumors. Even small differences in pH between normal and cancerous cells can be distinguished, and since both the pK of insertion and the cooperativity can be tuned, the system can be refined to target specific pH ranges. We have demonstrated targeting of acidic tumors in mice tumor models and in human tissue. Conclusions: Even individual cancer cell maintain acidity on its surface and could be detected in a well perfused areas. pHLIP® technology can be used in vivo and ex vivo on tissue specimens and opens both a path to medical utility and an exploration of other functions of acidity on cell surfaces. The leading fluorescent pHLIP® construct was selected for translation to clinics for diagnostic and surgical resection of primary tumors and submillimeter metastatic lesions. Citation Format: Anna Moshnikova, Michael Anderson, Ramona-Cosmina Adochite, Donald M. Engelman, Oleg A. Andreev, Yana K. Reshetnyak. pHLIP® technology for imaging acidic tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4250.