Abstract
Background: Programmed death-ligand 1 (PD-L1) is a key immune checkpoint molecule involved in tumor immune evasion. Its expression is highly heterogeneous across cancer types and subtypes, influencing therapeutic response. Understanding how different immunomodulatory agents influence PD-L1 expression in breast cancer cells could inform novel therapeutic strategies. This study aimed to investigate the temporal and dose-dependent effects of Bacillus Calmette-Guérin (BCG) and Toll-like receptor (TLR) agonists on PD-L1 expression in two breast cancer cell lines: MCF7 (luminal) and MDA-MB-231 (triple-negative). Methods: MTT (thiazolyl blue tetrazolium bromide) assays were conducted to determine non-cytotoxic concentrations of the immunomodulatory agents: 25 µM IMQ (imiquimod), 10 µg PPG (peptidoglycan), 1 mg LPS (lipopolysaccharide), and two BCG doses (200 µg/mL and 800 µg/mL). Flow cytometry assessed anti-PD-L1 (CD274) antibody expression at 24- and 48 hours post-treatment. Results: In MCF7 cells, BCG induced a dose-dependent upregulation of PD-L1 at 24 hours, which was not sustained at 48 hours, while TLR agonists had minimal or slightly suppressive effects. In contrast, MDA-MB-231 cells exhibited a time-dependent modulation of PD-L1, with an increase at 24 hours followed by a reduction at 48 hours in response to BCG, while TLR agonists consistently decreased PD-L1 levels compared to controls. Conclusion: These findings suggest distinct immunomodulatory responses between cancer subtypes, emphasizing the need for tailored approaches targeting the PD-1/PD-L1 axis. Further studies should explore the molecular mechanisms underlying these differential effects and assess the potential for combinatorial immunotherapeutic strategies in cancer.