Construction of fluorescently-labeled cell lines to evaluate cancer-associated processes in live cells

Abstract

Chemotherapy resistance arises from few remaining cells that behave differently from most cells in the population which are killed by chemotherapy.Thus, to better understand the molecular mechanisms leading to resistance it is paramount to investigate such processes in individual cells. Several experimental approaches have been used for that, including the use of fluorescently labeled probes to study such process as DNA damage and repair, autophagy, apoptosis, senescence, endoplasmic reticulum stress and cell cycle arrest. The groups involved in this project have extensively studied these molecular mechanisms in cancer cells, but mostly in cell populations. Previous results from our groups indicated that when we analyze more than one of these processes, some individual cells do not behave as the average cell in the population, highlighting the need for understanding variant phenotypes in the process of acquiring resistance.The main goal of this project is to engineer cell lines expressing fluorescent indicators that can measure, in individual cells, the cellular processes and signaling pathways involved in cell survival after chemotherapy. These cellular models will then be used by the different research groups involved in the project to evaluate specific biological questions in the several tumor models proposed.The experimental design proposes to construct cellular models stably expressing the labels using lentiviral or retrotransposon methodologies. Combinations of three labels will be engineered into glioma, breast, prostate and lung cancer, osteosarcoma and melanoma. Each combination of 3 labels will then be used by the groups to study one or more specific pathways. As most chemotherapeutic agents induce DNA damage, we focus on DNA dam-age formation and repair and in the biological consequences of damage accumulation, including cell cycle arrest, apoptosis, autophagy and senescence, mitochondrial dysfunction, and ER stress, and in the epithelial-mesenchymal transition. The mechanistic evaluations will be carried out by 5 groups from São Paulo (SP) and 7 groups in Rio Grande do Sul (RS). In addition, 3 other groups in RS will work on developing specific strategies for automated detection and machine learning, and statistical analysis and modeling of the large volume of data obtained. This project will also generate a plasmid and cell line bank, which will then be made available for biotechnological use in diagnostics and drug development.The collaborative network is well establish between the RS and SP groups, who have been working together for over 10 years and have several joint publications. The subject areas covered by the different groups include biotechnology, molecular and cellular biology and medicine, but also statistics, mathematics and physics for the development of new algorithms for data analysis.We expect that the cellular tools generated here will allow for further mechanistic studies to unveil the functional relationships between the pathways investigated, specially in regards to chemotherapy resistance. Moreover, the efforts to develop the aims proposed here will contribute to consolidate the ongoing collaborations and to establish a solid research network. (AU)