68 Ga-DOTA-E[c(RGDfK)] 2 Positron Emission Tomography Imaging of SHARPIN-Regulated Integrin Activity in Mice.

Shank-associated RH domain-interacting protein (SHARPIN, alias SIPL1) is a cytosolic protein that plays a key role in activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) 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 positron emission tomography (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 αvβ3 selective cyclic, dimeric RGDfK peptide 68 Ga-DOTA-E[c(RGDfK)]2 , 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 (Sharpincpdm) mice with and without melanoma tumor allografts. Methods: Sharpincpdm 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 αvβ3 integrin specific cyclic-RGD peptide 68 Ga-DOTA-E[c(RGDfK)]2 After the last 68 Ga-DOTA-E[c(RGDfK)]2 peptide PET/computed tomography (CT), tumors were cut into cryosections for autoradiography, histology and immunohistochemistry. Results: The ex vivo uptake of 68 Ga-DOTA-E[c(RGDfK)]2 in the mouse skin and tumor was significantly higher in Sharpincpdm mice than in wt mice. B16-F10-luc tumors were detected 4 days post-inoculation, without differences in volume or blood flow between the mouse strains. PET imaging with 68 Ga-DOTA-E[c(RGDfK)]2 peptide at day 10 post-inoculation revealed significantly higher uptake in the tumors transplanted into Sharpincpdm mice compared with wt mice. Furthermore, tumor vascularization was increased in the Sharpincpdm mice. Conclusion: Sharpincpdm 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.