Isolation and characterization of Cancer Associated Fibroblasts from canine Mast Cell Tumors and its influence on the malignancy of neoplastic cells: a combined molecular, pathologic and in vitro approach

• Ano de defesa: 2019
• Autor(a) principal: Pulz, Lidia Hildebrand
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: Cão; Co-cultivo; Co-culture; Dog; Extracellular matrix; Matriz extracelular; Miofibroblastos; Myofibroblast; RNA-Seq
• Link de acesso: http://www.teses.usp.br/teses/disponiveis/10/10133/tde-29082019-110458/

Mast cell tumours (MCTs) are common neoplasms in dogs and are considered potentially malignant. Several researches have attempted to identify biomarkers to better predict biological behavior for this tumor. In addition, studies with primary culture of canine MCTs coud be a valuable tool for the analysis of the cells functional properties. The objetive of this study was to identify molecular pathways connected to histopathological malignancies, shorter survival time and poor prognoses associated with MCTs. Moreover, we aimed to investigate the in vitro behavior of mast cells obtained from canine cutaneous MCTs of different histopathological grades and the type of interaction with the stromal fibroblasts. We performed genome-wide gene expression analyses on tissues obtained from 15 dogs with single MCTs, and identified two distinct tumour subtypes - high-risk and low-risk - associated with differences in histological grades, survival times, Ki67 indices, and occurrence of death due the disease. Comparative analyses of RNA sequence profiles revealed 71 genes that were differentially expressed between high and low-risk MCTs. In addition to these analyses, we examined gene co-expression networks to explore the biological functions of the identified genes. The network construction revealed 63 gene modules, 4 of which were significantly associated with the more aggressive tumour group. Two of the gene modules positively correlated with high-risk MCTs were also associated with cell proliferation and extracellular matrix-related terms. At the top of the extracellular matrix module category, genes with functions directly related to those of cancer-associated fibroblasts (CAFs) were identified. Immunohistochemical analyses also revealed a greater number of CAFs in high-risk MCTs. Culture experiments were made immediately after surgical resection and cells were cultured in complete DMEM-F12 for 4 to 7 passages. Mast cells was stained with Romanowsky and toluidine blue and showed progressive loss of their granules in culture. The presence of fibroblasts as adherent cells was confirmed by use of qRT-PCR for Fibroblast-specific Protein 1 (FSP1) gene. The characterization of cultured fibroblasts as myofibroblasts was performed by immunofluorescence for -smooth muscle-actin (SMA) and vimentin. The percentage of viable cells in the supernatant was determined in each passage. During 410 weeks of culture without any addition of growth factors or cytokines, living mast cell population decreased 11 progressively and adherent fibroblasts and myofibroblasts continue to grow until senescence. High-grade MCTs samples were viable for shorter periods in culture (P=0.0442) and lower number of passages (P<0.0001). We also have examined the short-term effects of stroma fibroblasts on neoplastic mast cells in different cultures conditions. The cell-cell contact co- culture was the best condition in which canine neoplastic mast cells remained viable in highest proportion during the experiment compared to all other conditions, i.e. with the transwell condition and mast cells isolated cultures (P<0.05). We also found that isolated neoplastic mast cells are not viable for more than 4 days in the absence of fibroblasts or their soluble factors. These results indicate an important interaction between mast cells and fibroblasts, which may also occur in the tumor microenvirnomental setting.