The pH low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions

Abstract

Current strategies for early detection of breast and other cancers are limited in part because some lesions identified as potentially malignant do not develop into aggressive tumors. Acid pH has been suggested as a key characteristic of aggressive tumors that might distinguish aggressive lesions from more indolent pathology. We therefore investigated the novel class of molecules, pH low insertion peptides (pHLIPs), as markers of low pH in tumor allografts and of malignant lesions in a mouse model of spontaneous breast cancer, BALB/neu-T. pHLIP Variant 3 (Var3) conjugated with fluorescent Alexa546 was shown to insert into tumor spheroids in a sequence-specific manner. Its signal reflected pH in murine tumors. It was induced by carbonic anhydrase IX (CAIX) overexpression and inhibited by acetazolamide (AZA) administration. By using 31 P magnetic resonance spectroscopy (MRS), we demonstrated that pHLIP Var3 was retained in tumors of pH equal to or less than 6.7 but not in tissues of higher pH. In BALB/neu-T mice at different stages of the disease, the fluorescent signal from pHLIP Var3 marked cancerous lesions with a very low false-positive rate. However, only ∼60% of the smallest lesions retained a pHLIP Var3 signal, suggesting heterogeneity in pH. Taken together, these results show that pHLIP can identify regions of lower pH, allowing for its development as a theranostic tool for clinical applications.

pHLIP Peptide Interaction with a Membrane Monitored by SAXS

“…The pHLIP peptides find a wide range of applications in biomedical sciences 16,20–22,43,44 and, also, they prove to be a very convenient model system for the investigation of polypeptide insertion into the lipid bilayer of a membrane triggered by pH. 12,28 Whereas various spectroscopic methods applied previously probed changes in the conformation of peptides, SAXS employed in this study allowed us, for the first time, to monitor the changes in the liposome and lipid bilayer structure.…”

Comparative Study of Tumor Targeting and Biodistribution of pH (Low) Insertion Peptides (pHLIP® Peptides) Conjugated with Different Fluorescent Dyes

“…The targeting of primary tumors by pHLIP ® peptides and delivery imaging and therapeutic agents has been demonstrated on more than 10 varieties of human and murine tumors [15–17], including lymphoma [18], pancreatic [19], breast [20–21] and prostate [22] transgenic mouse models and human head/neck biopsy samples [23–24]. It was confirmed that tumor targeting by pHLIP ® peptides is indeed pH-dependent and could be modulated by bicarbonate and glucose, which affects tumor acidity [15, 20–21, 25–26]. Three pHLIP ® variants, WT, Var3 and Va7 were selected as lead candidates for pH-specific delivery of imaging and therapeutic agents to tumors of different origins [10, 16, 19–21].…”

“…It was confirmed that tumor targeting by pHLIP ® peptides is indeed pH-dependent and could be modulated by bicarbonate and glucose, which affects tumor acidity [15, 20–21, 25–26]. Three pHLIP ® variants, WT, Var3 and Va7 were selected as lead candidates for pH-specific delivery of imaging and therapeutic agents to tumors of different origins [10, 16, 19–21]. …”

Applications of pHLIP Technology for Cancer Imaging and Therapy

“…Studies in which the pH of the surface of tumors was monitored showed that the regions of the highest tumor invasion (see Glossary) corresponded to areas of highest acidity [10]. Therefore, targeting the most acidic regions of tumors is especially beneficial because the most acidic regions are the most aggressive [11]. …”