Paracetamol and Metformin Reduce NK-Cell Susceptibility in MCF-7 Breast Cancer Cells in Association with Enrichment of Immune-Evasive CD44+CD24− Stem-like Subpopulations

Natural Killer (NK) cell-mediated immunosurveillance is a cornerstone of anti-tumor defense, yet its efficacy can be compromised by common clinical medications. Paracetamol (APAP) and metformin (MET) are widely used for pain and metabolic management in cancer patients, but their unintended effects on the immune–tumor interface remain poorly understood. MCF-7 breast cancer cells (Luminal A subtype) were treated with APAP or MET and co-cultured with primary expanded NK cells (CD3−CD56+CD16+). We evaluated cell proliferation, cell cycle distribution, and the enrichment of the CD44+CD24− cancer stem-like cell (CSC-like) subpopulation. Transcriptional changes in immune checkpoints (PD-L1/L2), stress ligands (MICA/B), and costimulatory molecules CD80/86 were quantified via RT-qPCR. Despite inhibiting MCF-7 growth (IC50 at 48 h: 11.86 mM for APAP; 21.11 mM for MET), both drugs induced a “therapeutic paradox” by promoting an immune-evasive phenotype. APAP and MET significantly enriched the CD44+CD24− CSC-like subpopulation to 75.31% and 68.31%, respectively, compared to 11.00% in controls. Molecular analysis revealed a robust upregulation of PD-L1 (19.9-fold by APAP) and PD-L2 (16.4-fold by MET), alongside increased CD80/86 and MICA/B transcription. Consequently, drug-treated cells exhibited marked resistance to NK-mediated apoptosis and necrosis. NK cells preferentially eliminated non-stem cells (non-CSCs), inadvertently further concentrating the highly resistant CSC-like subpopulation. Additional experiments revealed that APAP directly impaired NK-cell survival and reduced the proportion of CD3−CD56+ cells, whereas MET exerted minimal effects on NK cells, suggesting distinct mechanisms underlying the observed reduction in NK-mediated cytotoxicity. Under the experimental conditions employed in this study, APAP and MET were associated with reduced susceptibility of MCF-7 cells to NK-mediated killing through distinct but partially overlapping mechanisms, including CSC-like enrichment and transcriptional activation of immune-evasion pathways. Although these findings were obtained in a mechanistic in vitro model using supra-physiological drug concentrations, they identify potential mechanisms that warrant further validation in physiologically relevant experimental systems and in vivo models.