Date of Award
Doctor of Philosophy in Physics
Oleg A. Andreev
Solid tumors have a microenvironment that is inherently acidic and hypoxic. Hypoxia is caused by leaky blood vessels and large diffusion distances from cells to them. It is heterogeneous throughout the tumor and while all solid tumors are hypoxic to a degree, it is difficult to predict invasiveness based on it. However, acidity is a near ubiquitous characteristic of tumors with more aggressive tumors producing greater acidity. It is important to measure pH in diseased tissue with accuracy and precision, since acidity is associated with the development of various pathological states including tumors. In this work we focus on the acidosis aspect of the tumor microenvironment by describing the development of pHLIP® (pH (Low) Insertion Peptides) targeting based tools that are capable of imaging the pH of a tumor microenvironment. pHLIP was chosen as a targeting vehicle because of its pH dependent insertion mechanism that allows it to effectively target acidic tissues, including tumors.
We used pHLIP® to study the roles of carboxyl groups in transmembrane (TM) peptide insertion. pHLIP binds to the surface of a lipid bilayer as a disordered peptide at neutral pH; when the pH is lowered, it inserts across the membrane to form a TM helix. Peptide insertion is reversed when the pH is raised above the characteristic pKa (6.0). A key event that facilitates membrane insertion is the protonation of aspartic acid (Asp) and/or glutamic acid (Glu) residues, since their negatively charged side chains hinder membrane insertion at neutral pH. In order to gain mechanistic understanding, we studied the membrane insertion and exit of a series of pHLIP variants where the four Asp residues were sequentially mutated to nonacidic residues, including histidine (His). Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ~ pH 4 driven by the protonation of carboxyl groups at the inserting end of the peptide. We expect that our understanding will be used to improve the targeting of acidic diseased tissue by pHLIP.
Looking from the lipid bilayer’s perspective, small angle x-ray scattering studies showed membrane thinning by 18% induced by insertion of short-pHLIP (truncated version of pH Low Insertion Peptide) into bilayer. Thinning allows to reduce stress on membrane associated with negative hydrophobic mismatch. Also we observed 12% of membrane thinning when long-pHLIP partitions into outer leaflet of bilayer at high pH adopting coil conformations. The long-pHLIP at high pH creates an asymmetric inclusion in the bilayer, which results in increase of tension leading to the bilayer thinning. The tension and thinning is released when long-pHLIP inserts into bilayer as a transmembrane helix at low pH.
The first tool developed is a new 64Cu-pHLIP peptide for targeting, imaging and quantifying acidic tumors by positron emission tomography, and our findings reveal utility in assessing prostate tumors. The new pHLIP version limits indiscriminate healthy tissue binding, and we demonstrate its targeting of extracellular acidification in three different prostate cancer models, each with different vascularization and acid-extruding protein carbonic anhydrase IX (CAIX) expression. We then describe the tumor distribution of this radiotracer ex vivo, in association with blood perfusion and known biomarkers of acidity such as hypoxia, lactate dehydrogenase A and CAIX. We find that the new probe reveals metabolic variations between and within tumors, and discriminates between necrotic and living tumor areas.
The second tool introduced is a novel approach of extracellular pH measurements at the surface of cells, which is based on the use of a pH-sensitive fluorescent dye SNARF conjugated to a pH Low Insertion Peptide (WT-pHLIP), which targets plasma membranes of cells in acidic diseased tissue. Our experimental set up includes two different approaches, one is based on acquisition of fluorescent spectra, and other one is based on recording of images via two emission filters. By using appropriate calibration curves obtained on liposomes and tumor spheroids in the presence of 2-deoxyglucose, both approaches give the same values of surface pH. The developed tool was validated on cancer cells grown in tumor spheroids, in mice and excised tumors ex vivo. We establish that highly metastatic cancer cells have lower pH at their surface compared to non-metastatic cells. Our approach was sensitive enough to detect pH changes in vitro and in vivo induced by glucose, which leads to the enhancement of cancer cells metabolism and acidification of the extracellular space. The introduced tool could be developed for clinical application of surface pH measurements in biopsy samples. It might provide important clinical information about tumor stage and invasiveness, and can guide in the choice of treatment approach.
Anderson, Michael D., "Development of New Tools for Study of Tumor Microenvironment" (2015). Open Access Dissertations. Paper 359.