Abstract
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Objectives Tumor acidosis is a common occurrence in cancer due to an overactive glycolytic process. Survival of these lesions relies on acid-regulating proteins to maintain an alkaline cytosol leaving the extracellular milieu with an acidic gradient. We targeted this acidic microenvironment via a PET peptide tracer called pH Low Insertion Peptide or pHLIP™ that binds to acidic cell membranes.
Methods pHLIP variants were modified with either DOTA or NOTA and labeled with Cu-64 or Ga-68. In vitro binding studies and in vivo PET imaging and biodistribution experiments were conducted on athymic mice with LnCaP and PC3 prostate cancer xenografts. Autoradiography and histology on ex-vivo tumor sections were performed to confirm specificity.
Results In vitro binding studies yielded in the selection of three variants: WT, short3E and short3D as leading candidates for in vivo experiments. Short3E and short3D demonstrate weaker binding to the surface of the cell membrane at neutral pH compared to WT while preserving pHLIP-like properties. Tumor targeting was observed via biodistribution on all WT- and short3E-DOTA chelates labeled with either Ga-68 or Cu-64. High kidney and liver retention remain problem areas. Cu-64-NOTA constructs proved promising. Tumor targeting was noted with short3E (~1.2+/-0.4 %ID/g for both tumors) and short3D (1.3+/-0.3%ID/g for LnCaP and 1.4+/-0.4%ID/g for PC3) variants at 24h p.i. Compared to the DOTA scaffolds, liver retention of the NOTA construct significantly cleared after 24h. Clearance routes include the intestines and kidneys. Autoradiography and histology studies displayed co-localization of the peptide with areas exhibiting hypoxia and CAIX, markers consistent with low locoregional pH.
Conclusions We demonstrated a marked improvement of pHLIP as imaging agents. By modifying the peptide sequence, the radionuclide or the chelate, the tracer maintained tumor retention yet cleared normal tissues. The peptide appears to target hypoxic areas and those with CAIX overexpression. Additional histology studies are underway.
Research Support NIH R01 CA13846