Background: SHARPIN (SHANK-associated RH domain interactor) is a 45 kDa cytosolic protein

playing a key role in NF-kappa-B activation and regulation of inflammation. Furthermore,

SHARPIN controls β1-integrin dependent cell adhesion and migration in several normal and

malignant cell types, and loss of SHARPIN correlates with increased integrin activity in mice (1).

Arginyl-glycyl-aspartic acid (RGD), a cell adhesion tripeptide motif, is an integrin recognition

sequence facilitating positron emission tomography (PET) imaging of integrin upregulation e.g.

during tumor angiogenesis (2). In this study, we evaluated B16 melanoma tumors in SHARPINdeficient

mice with RGD-based in vivo PET imaging.

Materials and Methods: Mice with a spontaneous null-mutation in the Sharpin gene

(C57BL/KaLawRij-Sharpincpdm/RijSunJ, Jackson Laboratories) (n=3) and their wild-type (WT)

littermates (n=3) were subcutaneously implanted with 10^⁶ B16 mouse melanoma cells. The

progression of tumor growth was monitored at 6, 8 and 10 days after implantation of the tumor cells

by determining volume with ultrasound imaging (VisualSonics, Vevo2100). At 10 days after

inoculation, tumor blood flow was evaluated with the contrast-enhanced ultrasound (Vevo

MicroMarker), followed by in vivo PET/CT imaging (Siemens, Inveon Multimodality) and ex vivo

measurements with Gallium-68-labeled dimeric RGD peptide ([⁶⁸Ga]DOTA-E-[c(RGDfK)]₂) (2).

In addition, tumors and lymph nodes were harvested for histology.

Results: Tumors were detected 6 days after inoculation without differences between mice strains.

However, at the day 10, the volume of tumors in WT mice was on average 2-fold larger compared

to those in SHARPIN-deficient mice (Fig1A). Interestingly, the growth of B16 melanoma tumors in

the SHARPIN-deficient mice was more invasive to the tissue beneath, and 1 mouse had also

metastases in the lungs and draining lymph nodes whereas none of the WT mice had metastases.

According to ultrasound imaging, there was no difference in the tumor blood flow. Yet, RGDpeptide

PET imaging revealed significantly higher standardized uptake value in the tumors of the

SHARPIN-deficient mice (0.48±0.12) compared to WT mice (0.24±0.06, P=0.03, Fig.1B). The ex

vivo biodistribution studies showed slightly higher tracer uptake in many organs of the SHARPINdeficient

mice but the differences were not statistically significant (Fig. 1C).

Conclusion: SHARPIN-deficient mice demonstrated more aggressive melanoma tumor phenotype

and increased integrin activity as demonstrated by RGD-based in vivo PET imaging.

References:

1) Rantala JK, Pouwels J, Pellinen T et al. SHARPIN is an endogenous inhibitor of beta1-integrin

activation Nat Cell Biol. 2011;13:1315-24.

2) Dijkgraaf I., Yim C.B., Franssen G.M., et al. PET imaging of alphavbeta integrin expression in

tumours with Ga-labelled mono-, di- and tetrameric RGD peptides. Eur J Nucl Med Mol Imaging.

2011;38:128–37.