68Ga-NODAGA-E[c(RGDyK)]2

Shank-associated RH domain-interacting protein (SHARPIN) is a cytosolic protein that plays a key role in activation of nuclear factor κ-light-chain enhancer of activated B cells and regulation of inflammation. Furthermore, SHARPIN controls integrin-dependent cell adhesion and migration in several normal and malignant cell types, and loss of SHARPIN correlates with increased integrin activity in mice. Arginyl-glycyl-aspartic acid (RGD), a cell adhesion tripeptide motif, is an integrin recognition sequence that facilitates PET imaging of integrin upregulation during tumor angiogenesis. We hypothesized that increased integrin activity due to loss of SHARPIN protein would affect the uptake of α_vβ₃-selective cyclic, dimeric peptide ⁶⁸Ga-DOTA-E[c(RGDfK)]₂, where E[c(RGDfk)]₂ = glutamic acid-[cyclo(arginyl-glycyl-aspartic acid-D-phenylalanine-lysine)], both in several tissue types and in the tumor microenvironment. To test this hypothesis, we used RGD-based in vivo PET imaging to evaluate wild-type (wt) and SHARPIN-deficient mice (Sharpin^cpdm , where cpdm = chronic proliferative dermatitis in mice) with and without melanoma tumor allografts. Methods:Sharpin^cpdm mice with spontaneous null mutation in the Sharpin gene and their wt littermates with or without B16-F10-luc melanoma tumors were studied by in vivo imaging and ex vivo measurements with cyclic-RGD peptide ⁶⁸Ga-DOTA-E[c(RGDfK)]₂ After the last ⁶⁸Ga-DOTA-E[c(RGDfK)]₂ peptide PET/CT, tumors were cut into cryosections for autoradiography, histology, and immunohistochemistry. Results: The ex vivo uptake of ⁶⁸Ga-DOTA-E[c(RGDfK)]₂ in the mouse skin and tumor was significantly higher in Sharpin^cpdm mice than in wt mice. B16-F10-luc tumors were detected 4 d after inoculation, without differences in volume or blood flow between the mouse strains. PET imaging with ⁶⁸Ga-DOTA-E[c(RGDfK)]₂ peptide at day 10 after inoculation revealed significantly higher uptake in the tumors transplanted into Sharpin^cpdm mice than in wt mice. Furthermore, tumor vascularization was increased in the Sharpin^cpdm mice. Conclusion:Sharpin^cpdm mice demonstrated increased integrin activity and vascularization in B16-F10-luc melanoma tumors, as demonstrated by RGD-based in vivo PET imaging. These data indicate that SHARPIN, a protein previously associated with increased cancer growth and metastasis, may also have important regulatory roles in controlling the tumor microenvironment.

Introduction: The advent of the Germanium-68 (Ge-68)/Gallium-68 (Ga-68) generator has contributed enormously to a plethora of molecular imaging approaches for in vivo identification of tumor characteristics. The present study compares the effect of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane,1-gluteric acid-4,7-acetic acid (NODAGA) bifunctional chelators on radiolabeling of arginine-glycine-aspartic acid (RGD) dimer, an antagonist of integrin α_vβ₃ with Ga-68 and their biodistribution in C57BL/6 mice bearing melanoma and in patients with breast carcinoma. Methods: Radiolabeling parameters for DOTA-(RGD)₂ and NODAGA-(RGD)₂ with Ga-68 were optimized in-house. After quality control procedures, preclinical studies were done in C57BL/6 mice bearing melanoma. The percent radioactivity associated with per gram of various organs and tumor (% ID/g) was analyzed. Positron emission tomography-computed tomography patient imaging was performed in clinically diagnosed locally advanced breast carcinoma patients (n = 30). The uptake of various organs and lesions for both radiotracers was compared. Results: Radiolabeling yield >95% was obtained by heating 15-20 μg of peptide at 95°C for 5-10 min and 3.5-4.0 reaction pH. NODAGA-(RGD)₂ could also be radiolabeled at room temperature, but 40-50 μg peptide was required. Animal biodistribution study revealed the kidney as the major excretory organ for both the radiotracers. Maximum counts were observed in tumor at 45 min. During the clinical study, liver, spleen, bilateral brain ventricles, salivary glands, and intestines were the organs with physiological uptake of both Ga-68-DOTA-(RGD)₂ and Ga-68 NODAGA-(RGD)₂. The major excretory route was through kidneys. All primary lesions were picked by both the radiotracers. Additionally, in 5 patients, metastatic lesions were also picked up. Conclusion: DOTA- and NODAGA-chelated RGD₂ were successfully radiolabeled with Ga-68. Good tumor to background contrast exhibited by Ga-68-DOTA-(RGD)₂ and Ga-68 NODAGA-(RGD)₂ in both preclinical and clinical studies suggested that both radiotracers can be used as potential molecular tools for imaging angiogenesis.