Controlling Stemness in 2D and 3D Models of Triple‐Negative Breast Cancer Using a Ruthenium(II) Arene Complex

ABSTRACT Triple‐negative breast cancer (TNBC) spheroids serve as a robust in vitro model to study tumor heterogeneity, drug resistance, and cancer stem cell (CSC) dynamics, which are critical factors contributing to the aggressiveness of TNBC. This study explores the therapeutic efficacy of ruthenium(II) arene complex, [(η 6 ‐ p ‐cymene)(2‐bis(quinolin‐2‐ylmethylene)hydrazine)RuCl]PF 6 , named as pCYRuL , using both two‐dimensional (2D) cell cultures and three‐dimensional (3D) tumor spheroid models of TNBC. The complex demonstrates potent antiproliferative activity by arresting TNBC cells in G2/M phase, and induces apoptosis through activation of caspase‐9. Additionally, pCYRuL treatment significantly impaired the clonogenic capacity and migratory potential of TNBC cells, while simultaneously upregulating the expression of the tumor suppressor p53 and p21, further supporting its antimetastatic potential. The complex effectively targeted the CSCs population within the spheroids, as evidenced by downregulation of key stemness markers and diminished clonogenic capacity and sphere‐forming ability, particularly in 3D spheroids, which mimic the tumor microenvironment. The pCYRuL disrupts spheroid integrity and enhanced drug penetration contributes to its efficacy. These findings underscore the potential of ruthenium(II) arene complexes as dual‐action agents capable of inducing apoptosis and eradicating both the tumor bulk and stem‐like cancer cell populations, providing a novel approach to combat the aggressive and recurrent breast cancer.