Isolation, establishment, and characterization of ex vivo equine melanoma cell cultures

Abstract

Gray horses spontaneously develop metastatic melanomas that resemble human disease, and this is often accompanied with metastasis to other organs. Unlike in other species, the establishment of primary equine melanoma cultures that could be used to develop new therapeutic approaches has remained a major challenge. The purpose of the study was to develop a protocol for routine isolation and cultivation of primary equine melanocytes. Melanoma tissues were excised from 13 horses under local anesthesia, mainly from the perianal area. The melanoma cells were isolated from the melanoma tissue by serial enzymatic digestion using dispase and collagenase. Out of the 13 excised melanomas, cell cultures from eight melanomas were established, which corresponded to a success rate 62%. These cells showed different degrees of melanin pigmentation. Characterization of these cells using confocal microscopy, FACs analysis and western blotting showed that they expressed melanoma-associated antigens; Melan-A, MAGE-1, and MAGE-3, and PCNA expression was higher in fast-proliferating isolates. The protocol we developed and established proved successful for routine isolation and cultivation of primary equine melanoma cells. This method provided a large number of primary equine melanoma cells that could be used to study new therapeutic approaches for treatment of equine melanomas.

Copy number expansion of the STX17 duplication in melanoma tissue from Grey horses

“…The three horses showing a higher copy number in melanoma tissue than expected from their zygosity for Grey , were all euthanized possibly due to what might be internal melanoma metastases (ID1, ID2, ID3; Table 1, Figure 2B). mRNA from the tumour samples ID1 and ID2 were used for expression analysis in our previous study [19] and both samples had upregulated expression of STX17 and NR4A3 consistent with our interpretation that the copy number expansion detected in the present study may be relevant for tumour development. The melanoma cell lines showing a higher copy number than expected from the Grey genotype, Ho-Mel-A1 (Seltenhammer et al, in preparation) and M14 [20], have both been characterized as malignant, with M14 showing a high expression of the proliferating cell nuclear antigen PCNA [20], previously correlated with aggressive behaviour of human cutaneous malignant melanoma [19].…”

“…The present study has provided strong genetic support for our previous conclusion that the 4.6 kb duplication in intron 6 of STX17 causes Greying with age [19]. Firstly, the copy number expansion seen in tumours suggests that it may promote tumour progression.…”

Immunohistochemical Expression of Melanocytic Antigen PNL2, Melan A, S100, and PGP 9.5 in Equine Melanocytic Neoplasms

“…13 Melan A expression in 1 equine melanocytic neoplasm and melanocytic cell lines has been reported. 3,25 PNL2 is a monoclonal antibody that recognizes a fixativeresistant melanocytic antigen in humans, dogs, and some laboratory animals, 2,7,8,16,18,24 the exact structure of which has yet to be identified. 10 In humans, PNL2 also labels angiomyolipomas and mature myeloid cells, including neutrophils, but not other nonmelanocytic tumors, including various carcinomas and sarcomas.…”

“…13 Melan A expression in 1 equine melanocytic neoplasm and melanocytic cell lines has been reported. 3,25…”

Culturing of Melanocytes from the Equine Hair Follicle Outer Root Sheath

“…Although cosmetic concerns do not play an important role in daily veterinarian routine, there are implications for practice and research: tumors of melanocytic origin, the socalled white horse melanoma, present an area of intense clinical research interest. Here, tumors and melanoma cells derived from them have been characterized [28][29][30]. The underlying mutation has been looked into by Rosengren et al 2008 and identified as a 4.6 kb duplication in Syntaxin 17 on chromosome 25.…”