Establishment of primary culture of canine melanoma for testing new antineoplastic agents

• Grant number: 15/25158-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: July 01, 2016
• End date: December 31, 2016
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Pathology
• Principal Investigator: Maria Lucia Zaidan Dagli
• Grantee: Andreia Caringi Miraldo
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Canine melanoma is a highly aggressive and metastatic neoplasm, and currently there are no effective treatments for this disease. The objective of this project is to generate a strain in vitro canine melanoma. Therefore, canine oral melanoma fragment will be harvested, the cells will be dissociated and placed in culture flask containing DMEM supplemented with 10% fetal bovine serum, 200U / ml penicillin, 200¼g / ml streptomycin and 50 mcg / ml amphotericin B. The fragmented tumor tissue will be incubated in a humidified incubator with atmosphere containing 5% CO2 at 37 ° C. The cells gradually detached tissue fragments of forming a monolayer on the bottle cultivation. The growth of neoplastic cells will be quantified at 24, 48 and 72 hours by MTT. After the initial processing of the tumor cells are maintained in culture, allowing the exit of tumor cells fragment into the bottle cultivation. The cells will be monitored periodically for evaluation of cell growth and check for possible contamination. Coming to a 90% confluence the cells are divided into more bottles to expansion thereof. For this purpose, cells will be trypsinized with trypsin EDTA 0.05% (Gibco) for 10 minutes at 37 ° C. After detachment from the bottle trypsin is inactivated cells with culture medium containing fetal bovine serum. The cell suspension is centrifuged at 1500 rpm for 5 minutes. The supernatant is discarded and the cells resuspended in half again and divided into new bottles. The freezing of the cells is carried out with a mixture of 90% fetal bovine serum and 10% dimetilsulfoxóxido (DMSO) and these cells will be maintained in liquid nitrogen or -80 ° C freezer. Melanoma cells are identified by immunohistochemistry. It is expected thereby obtaining melanoma strain canine, allowing its further use for testing of new anticancer agents.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

NISHIYA, ADRIANA TOMOKO; NAGAMINE, MARCIA KAZUMI; MACKOWIAK DA FONSECA, IVONE IZABEL; MIRALDO, ANDREA CARINGI; SCATTONE, NAYRA VILLAR; GUERRA, JOSE LUIZ; XAVIER, JOSE GUILHERME; SANTOS, MARIO; MASSOCO DE SALLES GOMES, CRISTINA OLIVEIRA; WARD, JERROLD MICHAEL; et al. Inhibitory Effects of a Reengineered Anthrax Toxin on Canine Oral Mucosal Melanomas. TOXINS, v. 12, n. 3, MAR 2020. (16/20479-7, 15/25158-1, 15/16776-3, 17/12855-1)