ABSTRACT
PURPOSE: To access the reliability of fine-needle aspiration biopsy in harvesting a sufficient amount of viable melanoma cells to establish a cell culture and maintain a melanoma cell line from an animal model of uveal melanoma. METHODS: For this study, fifteen male New Zealand albino rabbits had their right eye surgically inoculated with uveal melanoma cell line 92.1. The animals were immunosupressed with cyclosporine A using a dose schedule previously published. The animals were followed for 12 weeks. Intraocular tumor growth was monitored weekly by indirect ophthalmoscopy. After the fourth week, one animal was sacrificed per week preceded by fine-needle aspiration biopsy using a sharp 25-gauge, 1-inch long needle. Two separate aspirates were made from different areas of the tumor. Each aspirate was flushed to a separate cell culture media and sent for cell culture. The cells were frozen after two weeks when there were at least 1 million cells, which is enough to maintain a cell line. Cells were defrosted for HMB-45 immuno-stains to confirm the melanoma origin. RESULTS: Cell growth was observed from the samples harvested from 11 out of the 15 animals inoculated with uveal melanoma. All cell cultures, after defrost, immunoassayed positive for HMB-45. CONCLUSION: Fine needle aspiration biopsy seems to be a reliable method to harvest cells from solid intraocular melanomas in an animal model, to establish cell culture and to maintain a melanoma cell line.
Subject(s)
Biopsy, Fine-Needle/methods , Cell Line, Tumor , Melanoma/pathology , Uveal Neoplasms/pathology , Animals , Cell Culture Techniques , Disease Models, Animal , Female , Male , RabbitsABSTRACT
PURPOSE: To access the reliability of fine-needle aspiration biopsy in harvesting a sufficient amount of viable melanoma cells to establish a cell culture and maintain a melanoma cell line from an animal model of uveal melanoma. METHODS: For this study, fifteen male New Zealand albino rabbits had their right eye surgically inoculated with uveal melanoma cell line 92.1. The animals were immunosupressed with cyclosporine A using a dose schedule previously published. The animals were followed for 12 weeks. Intraocular tumor growth was monitored weekly by indirect ophthalmoscopy. After the fourth week, one animal was sacrificed per week preceded by fine-needle aspiration biopsy using a sharp 25-gauge, 1-inch long needle. Two separate aspirates were made from different areas of the tumor. Each aspirate was flushed to a separate cell culture media and sent for cell culture. The cells were frozen after two weeks when there were at least 1 million cells, which is enough to maintain a cell line. Cells were defrosted for HMB-45 immuno-stains to confirm the melanoma origin. RESULTS: Cell growth was observed from the samples harvested from 11 out of the 15 animals inoculated with uveal melanoma. All cell cultures, after defrost, immunoassayed positive for HMB-45. CONCLUSION: Fine needle aspiration biopsy seems to be a reliable method to harvest cells from solid intraocular melanomas in an animal model, to establish cell culture and to maintain a melanoma cell line.
OBJETIVO: Acessar a confiança da colheita de amostra celular suficiente através da biópsia aspirativa com agulha fina para estabelescer uma cultura celular e manter uma linhagem de células de melanoma a partir de uma modelo animal de melanoma uveal. MÉTODOS: Para esse estudo, 15 coelhos albinos da Nova Zelândia foram inoculados cirurgicamente com a linhagem celular 92.1 de melanoma uveal. Os animais foram imunosuprimidos com ciclosporina A usando uma dose previamente publicada. Os animais foram acompanhados por 12 semanas. O crescimento de tumor intraocular foi monitorado semanalmente pela oftalmoscopia indireta. Após a quarta semana, um animal foi sacrificado por semana, precedido pela biópsia aspirativa com agulha fina usando uma agulha de gauge 25 e comprimento 2,5 cm. Dois aspirados separados foram realizados a partir de áreas diferentes do tumor. Cada aspirado foi descarregado em um meio de cultura individual e enviado para cultura celular. As células foram congeladas após 2 semanas de cultura ou quando haviam pelo menos 1 milhão de células, o que é suficiente para manter uma linhagem celular. As células foram descongeladas para realizar imunocitoquímica com HMB-45 e confirmar sua origem melanocítica maligna. RESULTADOS: Crescimento celular foi observado nas amostras colhidas em 11 dos 15 animais inoculados com melanoma uveal. Todas as culturas celulares, após serem descongeladas, apresentaram imunoexpressão positiva para o HMB-45. CONCLUSÃO: Biópsia aspirativa com agulha fina parece ser um método confiável para colher células de tumores oculares sólidos em um modelo animal visando estabelecer e manter essa linhagem celular de melanoma.
Subject(s)
Animals , Female , Male , Rabbits , Biopsy, Fine-Needle/methods , Cell Line, Tumor , Melanoma/pathology , Uveal Neoplasms/pathology , Cell Culture Techniques , Disease Models, AnimalABSTRACT
BACKGROUND: Uveal melanoma (UM) is the most common primary intraocular tumor in adults, leading to metastasis in 40% of the cases and ultimately to death in 10 years, despite local and/or systemic treatment. The c-kit protein (CD117) is a membrane-bound tyrosine kinase receptor and its overexpression has been observed in several neoplasms. Imatinib mesylate is a FDA approved compound that inhibits tyrosine quinase receptors, as well as c-kit. Imatinib mesylate controls tumor growth in up to 85% of advanced gastrointestinal stromal tumors, a neoplasia resistant to conventional therapy. METHODS: Fifty-five specimens of primary UM selected from the archives of the Ocular Pathology Laboratory, McGill University, Montreal, Canada, were immunostained for c-kit. All cells displaying distinct immunoreactivity were considered positive. Four human UM cell lines and 1 human uveal transformed melanocyte cell line were tested for in vitro proliferation Assays (TOX-6) and invasion assay with imatinib mesylate (concentration of 10 microM). RESULTS: The c-kit expression was positive in 78.2% of the UM. There was a statistical significant decrease in the proliferation and invasion rates of all 5 cell lines. CONCLUSION: The majority of UM expressed c-kit, and imatinib mesylate does decrease the proliferation and invasion rates of human UM cell lines. These results justify the need for a clinical trial to investigate in vivo the response of UM to imatinib mesylate.