Análise fenotípica das vias de pluripotência, transição epitélio-mesenquimal e ferroptose em resposta à Doxorrubicina em modelo tridimensional de câncer de mama triplo-negativo enriquecido de células-tronco do câncer

Abstract

Triple-negative breast cancer (TNBC) accounts for 10–20% of all breast cancer diagnoses worldwide. It is considered more aggressive than other forms of invasive breast cancer due to its ER-/PR-/HER2- phenotype, which offers patients few treatment alternatives other than chemotherapy and a poor prognosis. Cancer stem cells (CSCs) are a small subpopulation of the tumor microenvironment (TME) involved in differentiation, migration, self-renewal, and thriving. They are resistant to induction of programmed cell death, such as ferroptosis, post-chemotherapy and tend to expand after treatment, leading to recurrence, metastasis, and a more aggressive tumor phenotype. The objective of this project is to understand the mechanisms by which CSCs regulate biological processes and cause chemoresistance, through the cultivation of the triple negative breast cancer cell lines MDA-MB-231 and Hs 578T in three-dimensional culture for the formation of tumor spheroids enriched with CSC, for analysis of gene expression by qPCR of different genes of pluripotency signaling pathways and cell death by ferroptosis, epithelial-mesenchymal transition panel, morphology by Confocal Laser Microscopy, immunophenotyping with CD24, CD44 and CD146 markers and lipid peroxidation assay by Flow Cytometry. The treatment used to perform the assays was doxorubicin IC50 118µM for MDA-MB-231 and 15.9µM for Hs 578T. Gene expression by RT-qPCR with targets related to pluripotency and tumor development, ferroptosis and autophagy such as OCT4, MYC, KLF4, AKT family, NCOA4, GPXR4, SLC7A11 and the ATG5-12 complex was performed in MDA-MB 231 and Hs 578T spheroids treated with Doxorubicin (DOX) and indicates that there is modulation of pluripotency, increased tumor progression in vitro and sensitization to ferroptosis after chemotherapy treatment. An EMT panel assay performed demonstrates the mesenchymal profile of the spheroids after DOX treatment, with upregulation of genes such as CDH2, COL1A2, COL3A1, GNG and MMPs. Structural analysis shows the organization of the cells, the presence of CSCs by the CD44 marker and their tendency to cluster in the international regions of the spheroids. Flow cytometry evaluation shows that the treatment induces lipid peroxidation in spheroids of the Hs 578T cell line but not in MDA-MB-231. The results obtained demonstrate heterogeneity among cell lines of the same tumor subtype and highlight the extreme importance of CSCs in tumor control and progression. Taking into account the various mechanisms by which the tumor can avoid cell death, it is necessary to understand different subpopulations and their respective roles in the tumor microenvironment to improve existing treatments and develop alternative therapies.