Synthesis and initial biological evaluation of GRP78 targeted peptides for molecular imaging of metastatic melanoma

Objectives GRP78 is a 78 kDa protein that plays major roles as a molecular chaperone and in the unfolded protein response in the endoplasmic reticulum. Emerging evidence suggests that cellular stress in the tumor microenvironment (e.g., oxidative stress) leads to a remarkable increase in the expression of GRP78 within cells and at the cell surface (1-4). This study aims to establish a backbone to develop cell-surface GRP78 targeted peptides for PET imaging of melanoma.

Methods Variation in GRP78 expression was studied in B16 melanoma tumors, HepG2 and melanocytes by immunochemistry (IHC). Seven GRP78 targeted peptide derivatives with micromolar affinity (8 to 13-mer in length) were synthesized based on previous studies in which peptides with were selected by bacteriophage techniques for other applications (2-4). Three linear and four cyclized derivatives were modified with a DOTA chelator and a fluorescent (fluorescein) moiety. DOTA conjugates were labeled with Ga-68 in acetate buffer at 99 °C.

Results IHC staining of melanoma tumor cross sections shows a remarkable difference in the expression of GRP78 in tumors vs adjacent tissue. Immunofluorescence studies show expression of GRP78 on the surface of HepG2 cells increases upon incubation with 50 µM hydrogen peroxide - evidence that enhanced expression can be induced by oxidative stress. Fluorescein-labeled-cyclized derivatives were internalized and binding affinity did not depend on the position of DOTA/fluorescein. DOTA derivatives were efficiently labeled with Ga-68.

Conclusions GRP78 is emerging as a target for PET imaging. Cell surface expression appears to be mediated by stress mechanisms. DOTA conjugated GRP78 targeted peptides can be labeled with Ga-68 for PET. Further studies are needed to improve the affinity of GRP78 targeted peptides for PET imaging of melanoma.

Research Support NRC-HQ-12-G-38-0041; NIH 1R01CA167632-01, K25CA172218-01A1; UI HCC.