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

Abstract

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 v β 3-selective cyclic, dimeric peptide 68 Ga-DOTA-E[c(RGDfK)] 2 , where E[c(RGDfk)] 2 5 glutamic acid-[cyclo(arginyl-glycylaspartic acid-D-phenylalanine-lysine)], both in several tissue types and in the tumor microenvironment. To test this hypothesis, we used RGDbased in vivo PET imaging to evaluate wild-type (wt) and SHARPIN-deficient mice (Sharpin cpdm , where cpdm 5 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 68 Ga-DOTA-E[c(RGDfK)] 2. After the last 68 Ga-DOTA-E[c(RGDfK)] 2 peptide PET/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 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 68 Ga-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.

Keratinocyte-specific deletion of SHARPIN induces atopic dermatitis-like inflammation in mice

“…Increased expression of SHARPIN was reported in different types of cancers in human patients and appears to be associated with increased malignant behavior [ 36 – 41 ]. This may be in part due to its role in increasing neovascularization in cancer models [ 42 ].…”

[68Ga]Ga-DOTA-Siglec-9 Detects Pharmacodynamic Changes of FAP-Targeted IL2 Variant Immunotherapy in B16-FAP Melanoma Mice

“…Previously, using a mouse B16 melanoma model, we demonstrated that [ 68 Ga]Ga-DOTA-Siglec-9 can be used to image tumor-associated inflammation. In that study, [ 68 Ga]Ga-DOTA-Siglec-9 could detect subcutaneous B16 melanoma tumors 7 days and 9 days after tumor cell inoculation (6). Moreover, blood vessels within melanoma tumors express VAP-1 in humans and mice (31,32).…”

“…Indeed, VAP-1 mediates binding of T cells to sinusoidal endothelial cells in hepatocellular carcinoma, and adhesion of tumor-infiltrating lymphocytes, lymphokineactivated killer cells, and NK cells to tumor vasculature (3,4). We have previously shown that sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is a VAP-1 ligand, and that a labeled Siglec-9 motif-containing peptide can be used for PET imaging of inflammation and B16 melanoma tumors (5,6).…”

Evaluation of [68Ga]Ga-NODAGA-RGD for PET Imaging of Rat Autoimmune Myocarditis

“…The radiochemical purity was ≥ 99% and the molar activity was 18 ± 7.3 GBq/μmol ( n = 9). A non-targeted peptide [ 68 Ga]Ga-DOTA-E[c(RGEfK)] 2 , where E[c(RGEfk)] 2 = glutamic acid-[cyclo(arginyl-glycyl-glutamic acid- D -phenylalanine-lysine)], was used as a negative control as previously described ( 28 ).…”