Viral evolution of T1L mammalian orthoreovirus enhances breast cancer stem-like cell killing

Breast cancer stem-like cells (BCSCs) drive metastasis and therapy resistance, yet selective therapeutics to eliminate this quiescent population are lacking. We explored the potential of mammalian orthoreovirus (MRV) as an oncolytic agent against paclitaxel-resistant (TaxR) ER+ BCSCs. Through serial passaging of the MRV strain T1L in TaxR tumorspheres, we developed novel viral clones (T1L SP B and C) with enhanced oncolytic potency. Notably, these evolved clones demonstrated superior killing of TaxR BCSCs despite reduced infectivity and viral protein synthesis compared to the parental strain. Whole-genome sequencing identified specific viral protein mutations correlating with this enhanced potency. Mechanistically, while all strains induced apoptosis, T1L SP B triggered markers of immunogenic cell death (ICD), significantly increasing the release of the damage-associated molecular pattern (DAMP) extracellular ATP. Our findings demonstrate that directed viral evolution can generate potent oncolytic agents capable of both eliminating resistant BCSCs and potentially stimulating anti-tumor immunity.