Standard chemotherapy impacts on in vitro cellular heterogeneity in spheroids enriched with cancer stem cells (CSCs) derived from triple-negative breast cancer cell line.

Triple-negative breast cancer is a heterogeneous disease with high recurrence and mortality, linked to cancer stem cells (CSCs). Our study characterized distinct cell subpopulations and signaling pathways to explore chemoresistance. We observed cellular heterogeneity among and within the cells regarding phenotyping and drug response. In untreated BT-549 cells, we noted plasticity properties in both CD44 ⁺ /CD24 ⁺ /CD146 ⁺ hybrid cells and CD44 ^- /CD24 ⁺ /CD146 ⁺ epithelial cells, enabling phenotypic conversion into CD44 ⁺ /CD24 ^- /CD146 ^- epithelial-mesenchymal transition (EMT)-like like breast CSCs (BCSCs). Additionally, non-BCSCs may give rise to ALDH ⁺ epithelial-like BCSCs. Enriched BCSCs demonstrated the potential to differentiation into CD44 ^- /CD24 ^- /CD146 ^- cells and exhibited self-renewal capabilities. Similar phenotypic plasticity was not observed in untreated Hs 578T and HMT-3522 S1 cells. BT-549 cells were more resistant to paclitaxel/PTX than to doxorubicin/DOX, a phenomenon potentially linked to the presence of CD24 ⁺ cells prior to treatment. Under the CSCs-enriched spheroids model, BT-549 demonstrated extreme resistance to DOX, likely due to the enrichment of BCSCs CD44 ⁺ /CD24 ^- /CD146 ^- and the tumor cells CD44 ^- /CD24 ^- /CD146 ^- . Additionally, DOX treatment induced the enrichment of plastic and chemoresistant cells, further exacerbating resistance mechanisms. BT-549 exhibited high heterogeneity, leading to significant alterations in cell subpopulations under BCSCs enrichment, demonstrating increased phenotypic plasticity during EMT. This phenomenon appears to play a major role in DOX resistance, as indicated by the presence of the refractory cells CD44 ⁺ /CD24 ^- /CD146 ^- BCSCs EMT-like, CD44 ^- /CD24 ^- /CD146 ^- tumor cells, and elevated STAT3 expression. Gene expression data from BT-549 CSCs-enriched spheroids suggests that ferroptosis may be occurring via autophagic regulation triggered by RAB7A, highlighting this gene as a potential therapeutic target.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Milene Pereira Moreira has patent #BR 10 2022 011155 3 pending to no. Luciana Maria Silva has patent #BR 10 2022 011155 3 pending to no. Eliza Pereira Franco has patent #BR 10 2022 011155 3 pending to no. Leticia da Conceicao Braga has patent #BR 10 2022 011155 3 pending to no. Geovanni Dantas Cassali has patent #BR 10 2022 011155 3 pending to No. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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