The acidity is associated with development of various pathological states such as solid tumors, ischemic stroke, neurotrauma, epileptic seizure, inflammation, infection, wounds, cystic fibrosis and others. Normal cell could be distinguished from highly glycolytic cell (for example, metastatic cancer cell) by transmembrane pH gradient and value of pH at surface of plasma membrane. We propose to develop novel tool to map pH at the extracellular and intracellular surfaces of individual cell in highly heterogeneous environment of cells in vivo. The tool would allow opening an opportunity to contribute in understanding of diseases progression and development of approaches of pH-based image-guided intervention. We will employ optical spectroscopic and imaging approaches, which allow achieving cellular resolution. Our strategy is based on use of peptides of pHLIP (pH Low Insertion Peptide) family. pHLIPs are water-soluble membrane peptides, which insert and fold in lipid bilayer of membrane only at slightly acidic conditions. Since the equilibrium is strongly shifted toward membrane inserted form at low pH, pHLIP injected into blood, circulates in body and accumulates in acidic tissue of tumors, site of inflammatory arthritis and ischemic regions. At 24 h after i.p. or i.v. administration of pHLIP, it is washed out completely from the blood and stays in plasma membrane of cells with low extracellular pH. pHLIP labeled with optical, PET or SPECT probes is considered to be first acidity markers, which are currently under development for clinical uses. We plan to conjugate pHLIP peptides of different pKa of insertion into membrane ranging from 4.5 to 6.5 with pH-sensitive fluorophore, SNARF-1. The main goal of using pHLIPs is to deliver and tether optical probe to the outer or inner leaflet of bilayer of plasma membrane. The SNARF-1 was selected, since it demonstrates shift of the emission spectra in response to pH, which solves the problem of calibration for the probe concentration. The probe will be attached to the N- or C- terminus of pHLIPs. In first case, SNARF-1 will stay in the extracellular space being tethered to the cell surface. On the other hand, when SNARF-1 would be conjugated with the peptide inserting end (C-terminus), pHLIP would """"""""flip"""""""" SNARF-1 across the bilayer and expose it to the intracellular space, while keeping it close to the inner leaflet of membrane. Thus, we propose to measure pH from the outer and inner leaflets of plasma membrane and identify transmembrane pH gradient. Experiments in solution, 2D and 3D cell culture, as well as on mouse cancer models will be performed. Our goals are: - to map pH at the surface of cancer cells in a process of cell division and migration in 3D culture; - to map pH on the surface of individual cells in tumors implanted into mice; - to monitor kinetics of pH changes at the surface of cancer cells in real time induced by the glucose infusion; - to establish the minimal size of metastatic and non-metastatic tumors, which can acidify microenvironment below pH 7.0.

Public Health Relevance

The acidity is associated with development of various pathological states such as solid tumors, ischemia, stroke, inflammation, infection, wounds, cystic fibrosis and others. We propose to develop a novel tool to map pH at the extracellular and intracellular surfaces of individual cell.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1-CB-D (51))
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Knowlton, John R
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University of Rhode Island
Schools of Arts and Sciences
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