Análisis de los perfiles de expresión génica obtenidos a partir de líneas celulares de cáncer de cuello uterino cultivadas en monocapa y en esferoide multicelular

Abstract

Traditional two-dimensional (2D) cell cultures have limitations such as the impossibility of reproducing the morphology, biochemical properties, and microenvironment of cells in the living organism. Therefore, various protocols have been developed for the cultivation of cells in three dimensions (3D), forming three-dimensional structures called tumor spheroids, which have allowed the in vitro study of the relationships between cell structure and function in normal or pathological situations. Spheroids reflect many important properties of solid tumors, including the development of extracellular matrix (ECM), cell junctions between epithelial cells, nutrient and oxygen concentration gradients, and cell proliferation from the periphery to the center. In the tumor, the aberrant proliferation of tumor cells generates regions of necrosis, hypoxia, and lack of nutrients that induce changes in gene expression levels, altering the microenvironment to favor tumor development. These alterations constitute the main barrier to the action of antitumor agents. The development of 3D culture models has allowed the study of tumors ex vivo because they recreate more accurately the conditions in which cells are found in the living organism. In order to validate the use of three-dimensional cultures as a tool to study individual gene expression profiles for personalized cancer therapy, to determine the penetration power of different immunotherapies, and of gene immunotherapy using the recombinant adenoviruses AdEasy/CMV/HLA-A*02:01 and AdEasy/CMV/HLA-B*44:02 constructed in the laboratory, two cervical cancer cell lines (HeLa infected with HPV-18 and SiHa infected with HPV 16) were cultured in 2D and three-dimensional cultures were established with them to evaluate, using protein electrophoresis techniques, the differences in protein expression between the two cell lines and the two cell culture techniques (2D and 3D). In this work, the optimal conditions for the 3D culture of the SiHa and HeLa cell lines were established. Once these procedures were standardized, the samples were processed to obtain the tumor lysate, which was used for SDS-PAGE, where differences in protein expression were observed in both types of culture and between the two cell lines. In addition, total RNA was extracted from the two cell lines cultured in 2D and 3D. This RNA will be used to create libraries and sequence the entire genome to determine the differential expression between the two types of culture. Unfortunately, this assay, which would allow us to more accurately characterize the differential gene expression between the groups studied, could not be performed.