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_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.

Keywords: PET; RGD; SHARPIN; melanoma; αvβ3 integrin.

FIGURE 1.

Increased tissue uptake of ⁶⁸Ga-DOTA-E[c(RGDfK)]₂ in Sharpin^cpdm mice. (A) Alopecia on dorsal skin of Sharpin^cpdm mouse, with wt littermate for comparison. (B) Ex vivo uptake of ⁶⁸Ga-DOTA-E[c(RGDfK)]₂ in Sharpin^cpdm and wt mice without tumors. (C) Competition with nonlabeled DOTA-E[c(RGDfK)]₂ peptide and imaging with control peptide ⁶⁸Ga-DOTA-E[c(RGEfK)]₂ revealing specific binding of tracer. Ex vivo results are expressed as percentage of injected radioactivity dose per gram of tissue (%ID/g). n = 4–9/group. *P < 0.05. **P < 0.01. ***P < 0.001.

FIGURE 2.

SHARPIN deficiency increases metastasis but not growth in tumor microenvironment. (A) Growth curves of B16 melanoma tumors during follow-up period (n = 8–9/group). (B) Tumor volume at end of experiment in wt and Sharpin^cpdm mice. (C) Pie-chart presenting lymph node metastasis (red) vs. no metastasis (black) in B16 melanoma tumor–bearing wt and Sharpin^cpdm mice. (D) Quantification of blood flow in B16 melanoma tumors.

FIGURE 3.

⁶⁸Ga-DOTA-E[c(RGDfK)]₂ binding is enhanced in SHARPIN-deficient tumor microenvironment. (A) Representative autoradiographs (ARG) and corresponding hematoxylin–eosin (HE) staining of B16 melanoma tumors (scale bar, 2 mm). (B) Quantification of autoradiographs showing distribution of ⁶⁸Ga-DOTA-E[c(RGDfK)]₂ radioactivity concentration in tumor, skin, and muscle (n = 12/group). (C) Representative coronal PET/CT images of wt and Sharpin^cpdm tumor–bearing mice and in vivo tumor uptake of ⁶⁸Ga-DOTA-E[c(RGDfK)]₂ in wt and Sharpin^cpdm mice. Bars show SUV_mean 45–60 min after injection. (D) In vivo tumor uptake of ⁶⁸Ga-DOTA-Siglec-9 in wt and Sharpin^cpdm mice. Bars show SUV_mean 20–30 min after injection. PSL = photostimulated luminescence.

FIGURE 4.

Stromal SHARPIN regulates tumor vascularization. (A–C) Representative cryosections of B16 tumors from wt and Sharpin^cpdm mouse immunolabeled with VAP-1 (A), CD31 (B), and β₃ integrin antibody (C). Scale bar, 200 μm. Bars show VAP-1–positive, CD31-positive, and β₃ integrin–positive tumor areas from B16 tumors implanted into wt and Sharpin^cpdm mice.