Análise da heterogeneidade celular e participação do fenômeno de transição epitélio-mesenquimal (EMT) na carcinogênese e quimioresistência de células de glioblastoma e neuroblastoma

Abstract

Cancer is one of the pathological dysfunctions that cause the most fear in society, losing only to cardiovascular disease in relation to the number of deaths. One of the models that best explains the cell heterogeneity observed in different types of tumors is the presence of cancer stem cells (CSCs). CSCs may originate from differentiated adult cells that return to an undifferentiated stage through the mechanism known as the epithelial-mesenchymal transition (EMT). EMT in cancer gives rise to cells with metastatic characteristics such as loss of extracellular matrix (ECM) adhesion and cell-cell contact, increased cell motility and resistance to apoptosis. Under normal physiological conditions, after embryogenesis, the signals that maintain the EMT are completely suppressed, however, it is observed that in central nervous system (CNS) tumors there is a reactivation of this phenomenon. The objective of this work was to evaluate the cellular heterogeneity and the participation of the EMT phenomenon in-vitro, in glioblastoma/astrocytoma and neuroblastoma tumor cell lines, in the monolayer and CSC enrichment models through the formation of neurospheres. The methodologies used were: in-vitro culture of glioblastoma (U-87 MG and LN-18) and neuroblastoma (KELLY and IMR-32) cell lines in monolayer and neurosphere models; determination of the IC50 (concentration capable of inhibiting 50% cell growth) against cisplatin chemotherapy treatment, whose dose determined in the monolayer model was used in the neurospheres; evaluation of STAT3, ZEB1, TWIST1, TGF-beta1 transcripts and long non-coding RNA, HOTAIR, by real time PCR; characterization of cell subpopulations by flow cytometry and use of the chorioallantoic membrane (CAM) model as a study of cancer recurrence and tumor aggressiveness. Among the results, the following observations were made: (i) exposure to cisplatin promote changes in expression of EMT regulatory transcripts, and the IMR-32 cell line characterized as the most sensitive to cisplatin in this work, overexpressed STAT3 and TGF-beta1 in the treated neurosphere model; (ii) glioblastoma and neuroblastoma cell lines present different cellular subpopulations expressing mesenchymal, neural and hematopoietic markers; (iii) neuroblastoma cell lines show a cell subpopulation with myeloid-derived suppressor cell characteristics (MDSCs) and (iv) tumors generated from residual cells, after exposure to cisplatin, grafted on CAM showed different patterns of organization than controls. Thus, our findings strongly support the idea that definitions of tumor phenotypic characteristics may help to establish better therapeutic strategies for the development of new drug targets.