In Vitro Investigation of Therapy-Induced Senescence and Senescence Escape in Breast Cancer Cells Using Novel Flow Cytometry-Based Methods

Abstract

This study explores the phenomenon of therapy-induced senescence (TIS) in breast cancer cells and the subsequent escape from this state, which can contribute to cancer recurrence and treatment resistance. Researchers developed novel flow cytometry-based methods using Ki-67 and CellTrace CFSE staining to detect and isolate senescence-escaped cells in MCF-7 and MDA-MB-231 breast cancer cell lines. The study identified DPP4/CD26 as a potential marker for senescence and investigated the senolytic effects of azithromycin, both alone and in combination with the DPP4 inhibitor sitagliptin. Findings suggest that targeting DPP4/CD26 could reduce senescence escape, offering new avenues for therapeutic intervention.

Key Findings

• DPP4/CD26 expression increases in senescent breast cancer cells, serving as a potential marker for senescence.
• Silencing or inhibiting DPP4/CD26 reduces senescence escape in MCF-7 cells but has a variable effect on MDA-MB-231 cells.
• Azithromycin exhibits senolytic activity, decreasing the viability of senescent cells, with enhanced effects when combined with sitagliptin.
• The combination therapy of azithromycin and sitagliptin synergistically reduces the number of senescence-escaped cells in MCF-7 lines.
• Developed flow cytometry-based methods enable efficient detection and isolation of senescence-escaped cells for further analysis.

Methodology

The study utilized MCF-7 and MDA-MB-231 breast cancer cell lines to model therapy-induced senescence using agents such as bromodeoxyuridine (BrdU), gemcitabine (GEM), and palbociclib (PALBO). Senescence was confirmed through SA-β-galactosidase staining, cytokine secretion assays (IL-6 and IL-8), and cell cycle analysis. Senescence escape was assessed by monitoring Ki-67 expression and CFSE dilution via flow cytometry. Functional assays, including mammosphere formation, migration, and colony formation, were conducted on isolated senescence-escaped cells to evaluate their proliferative and metastatic potential.

Conclusion

The research highlights the dynamic nature of therapy-induced senescence and its role in cancer progression. The identification of DPP4/CD26 as a marker and potential therapeutic target offers promising strategies to mitigate senescence escape. The novel methodologies developed provide valuable tools for future studies aimed at understanding and combating cancer recurrence due to senescence escape.