Canine malignant melanoma alpha-3 integrin binding peptides

Abstract

There is a need to develop novel targeted imaging and therapeutic agents that can aid in early diagnosis, detection of metastasis and treatment of melanoma. Alpha-3 integrin is overexpressed in 82% of metastatic melanomas in humans and may be a potential target for peptide ligands carrying therapeutic agents. Five melanoma cell lines were generated from canine primary oral and metastatic canine tumors, grown in mice, and validated with melanoma markers Melan A, S-100, Micropthalmia transcription factor (MITF), Tyrosinase, and MART-1. The melanoma cell lines were tested for binding affinity to previously published alpha-3 integrin-binding peptides containing the cdGXGXXc motif. Fluorescent conjugates of the alpha-3 integrin binding OA02 peptide were used to quantify receptor affinity in the cell lines, a specimen of canine primary oral melanoma, and melanoma xenografts. Alpha-3 integrin was expressed by all 5 canine melanoma cell lines. Four of the 5 lines as well as the primary canine tumor showed affinity to alpha-3 integrin binding peptides with the cdGXGXXc motif. Optical imaging of canine melanoma xenografts in nude mice indicates rapid, strong uptake of the optical tracer in the tumor with an average persistence of approximately 48 h. Ex vivo images showed high tumor-to-background ratio, with tumor signals more than twice that of the kidney and other vital organs. We propose that integrin alpha-3 integrin binding ligands could potentially become useful probes for imaging and delivery of cytotoxic agents for the treatment of melanoma.

Figure 1

Western Blot analysis of canine melanoma cell line expression of the alpha3 integrin A subunit. The cell lines were uniformly positive. The human melanoma cell line A2058 was used as a positive control. β-actin was used as a positive loading control.

Figure 2

Cell surface expression of primary canine melanoma and cell lines. A. Canine tumor tissue specimen labeled with OA02-Streptavidin-Alexa 488. The tumor cells on the mucosal surface showed strong staining. The DAPI image shows the densely packed tumor nuclei which correspond with the stained tumor in the overlaid image. Higher magnification of the tissue shows similar affinity to the ligand. B. The sample was positive for Tyrosinase, S100, MITF, and MelanA.

Figure 3

In vivo imaging of canine melanoma xenograft tumors in nude mice with OA02-Cy5.5. The color scales represent fluorescent intensity detected with optical imaging, with the greatest fluorescence to the right of the scale. There is a high uptake of peptide in the tumor tissue in whole body images and ex-vivo images. The tumor remains strongly fluorescent up to 168 hours after injection.

Figure 4

Biodistribution of OA02-Cy 5.5 in tissues. Mice were sacrificed at 18 hours, 48 hours, 72 hours, and 7 days, and the fluorescent intensity for each organ and tissue was measured to display affinity of the peptide to tissues over time. Results of representative amelanotic tumors are displayed as organ to tumor ratio, and as optical intensity in semiquantitative arbitrary units (AU). The kidney and bladder had a high tumor:organ ratio and is the assumed route of excretion of the tracer.