Análise da imunoexpressão de marcadores da transição epitelial-mesenquimal em espécimes e linhagem celular do carcinoma adenoide cístico

Abstract

Adenoid Cystic Carcinoma (ACC) characterized by persistent and invasive growth, with a high tendency to local recurrences and distant metastases. It is believed that the mechanism responsible for tumor progression is based on the collective or individual cell invasion, kwon as Epithelial-Mesenchymal Transition (EMT), where occur a decrease in the expression of epithelial biomarkers, such as E-cadherin, and increased expression of mesenchymal biomarkers, such as Twist, Snail and Vimentin. Thus, the present research aimed to carry out an investigation on the immunoexpression of Twist1, Snail1, Vimentin and E-cadherin of TEM, in specimens of ACC and the influence of cisplatin on the expression of these markers and on the migratory potential and ability to form spheres tumors, in a ACC lineage. Expressions of Twist, Snail, E-cadherin and Vimentin were evaluated in 20 tissue specimens of ACC in a quantitative and semi-quantitative manner and analyzed for cellular compartment. In order to look for an association with clinicopathological parameters, survival and two histopathological grading systems, the cases were divided into low expression and high expression, with a score of 4 as the cutoff point considered. In the in vitro assays, the staining of EMT markers by immunofluorescence using cisplatin (stock: 10mg/ml; IC50: 4μg/m), in migration assay (wound healing) and in tumorsphere assay were evaluated. There were no significant differences between clinicopathological parameters and grading systems (p>0.05). The immunohistochemical analysis revealed significant differences between the immunoexpression of vimentin, which was statistically significant when compared with the clinical variables: presence of lymph node metastasis (p=0.020), distant metastasis (p=0.007), late clinical staging (p=0.008) and death (p=0.004). The statistical analysis showed no significant associations between immunohistochemical variables and specific survival and disease-free survival (p>0.05). In in vitro assays, the migratory capacity of cells significantly decreased in the first 6h (p=0.050) and 12h (p=0.050) after exposure to chemotherapy. In immunofluorescence, the immunopositivity of Twist (p<0.001), Snail (p<0.001) and Vimentin (p<0.001) was significantly decreased when exposed to cisplatin. In the tumorsphere assay, there was a statistically significant decrease in the types of spheres when compared between the vehicle and cisplatin groups (holospheres: p=0.034; merospheres: p=0.050; paraspheres: p=0.050) and the number of paraspheres was significantly higher than merospheres (p=0.001) and holospheres (p=0.001) in the vehicle group. Through the results of this study, it was possible to conclude that EMT is present in an expressive way in the development of ACC. We also observed that ACC cells that exhibit EMT markers are sensitive to the chemotherapy drug cisplatin, affecting the expression of these markers, their migration and the formation of tumorspheres.