Cellular senescence as a prognostic marker for predicting breast cancer progression in 2D and 3D organoid models

Abstract

Breast cancer is the most prevalent malignancy in women, with its heterogeneity complicating prognosis and treatment development. This study explores the role of cellular senescence in breast cancer progression using three breast cancer cell lines-two triple-negative (HS578T, MDA-MB-231) and one luminal A (T47D)-alongside a non-tumorigenic breast epithelial cell line (MCF10a) as control. Findings were validated in patient-derived 3D organoid models (PDOs). We examined proliferation, cell cycle dynamics, and senescence markers, focusing on key regulators such as p16 and p21. Cancer cells showed increased proliferation and dysregulated senescence, particularly through p21 suppression in the G2 phase. To assess therapeutic potential, we treated cells and PDOs with Alisertib, an Aurora kinase inhibitor known to induce senescence. Alisertib treatment elevated β-galactosidase activity and senescence marker expression, confirming senescence induction. Interestingly, despite exhibiting senescent features, cancer cells partially retained proliferative capacity. We identified Plasminogen Activator Inhibitor-1 (PAI-1), a key component of the senescence-associated secretory phenotype (SASP), as a mediator in this process. PAI-1 was significantly upregulated after Alisertib exposure, particularly in cancerous samples, and correlated with reduced proliferation and a pronounced senescent phenotype. Functional experiments using a PAI-1 inhibitor (Tiplaxtinin) showed that PAI-1 actively contributes to maintaining the senescent growth arrest, as its inhibition partially restored proliferation. No senescence induction was observed in non-cancerous controls. Overall, our results reveal a complex interplay between senescence and breast cancer progression. PAI-1 emerges as a functional effector of therapy-induced senescence and a promising candidate for further investigation as a diagnostic biomarker and potential target in senescence-based breast cancer therapies.

Keywords: 3D organoid models; Breast cancer; Cellular senescence; Diagnostic biomarker; PAI-1.