Targeting PD-L1 with BMS-202 Enhances Antitumor Cytokine and Cytotoxic T-Lymphocyte Responses in C57BLx/6 Mouse Lung Carcinogenesis

Mustafa Fahmi Rajab,1 Abdullah Abubaker Shareef,1 Shukur Wasman Smail,1,2 Shang Ziyad Abdulqadir,1 Kovan Faidhalla Jalal,3 Christer Janson,4 Arash Abdolmaleki,5 Mohammad B Ghayour5 1Department of Biology, College of Science, Salahaddin University-Erbil, Erbil, Kurdistan Region, Iraq; 2College of Pharmacy, Cihan University-Erbil, Erbil, Kurdistan Region, Iraq; 3Medical Analysis Department, Tishk International University, Erbil, Kurdistan Region, Iraq; 4Department of Medical Science, Respiratory, Allergy and Sleep Research, Uppsala University, Uppsala, Sweden; 5Department of Biophysics, Faculty of Advanced Technologies, University of Mohaghegh Ardabili, Namin, IranCorrespondence: Christer Janson, Department of Medical Science, Respiratory, Allergy and Sleep Research, Uppsala University, Uppsala, Sweden, Email [email protected] Shukur Wasman Smail, Department of Biology, College of Science, Salahaddin University-Erbil, Erbil, Kurdistan Region, Iraq, Email [email protected]: Lung cancer remains a leading cause of cancer-related mortality, and although immune checkpoint inhibitors have improved outcomes, their use is often limited by immune-related adverse effects and treatment resistance. This study therefore investigates the effects of the small-molecule PD-L1 inhibitor BMS-202 on lung carcinogenesis using a subcutaneous tumor model in C57BL/6 mice. Specifically, we aimed to evaluate its cytotoxic activity, induction of apoptosis, and impact on antitumor immune modulation.Methods: In this study, we utilized in vivo mouse models and in vitro CMT167 cancer cells to evaluate the anti-tumor effects of BMS-202. Tumor growth inhibition was assessed through caliper measurements and histological analysis. Apoptosis was analyzed using AO/PI staining, Annexin V-FITC/PI flow cytometry, Caspase-3 activity assays, and Real-Time PCR to evaluate the expression of apoptosis-related genes (Bad, Bax, Apaf1, Bcl2, and Bcl-xl). Additionally, flow cytometry was employed to analyze lymphocyte infiltration in the tumor microenvironment. Finally, enzyme-linked immunosorbent (ELISA) was used for measurement of cytokines.Results: Specifically, cell viability decreased from 98.1% to 30.1% at concentrations ranging from 0 μM to 40 μM (P< 0.001). Apoptosis assays revealed distinct fluorescence patterns indicating increased apoptosis, with late apoptotic cells becoming prominent at higher concentrations. In vivo, results showed that treatment with BMS-202 significantly reduced tumor weight, with mean weights of 609 ± 41.5 mg and 371.88 ± 47.5 mg at 30 mg/kg and 60 mg/kg, respectively (P< 0.001). Flow cytometry analysis indicated a marked increase in CD3+CD8+ cytotoxic T cells, rising from 6.8% to 26.2% (P< 0.001), and a reduction in PD-1 expression, suggesting enhanced immune activation. Additionally, BMS-202 treatment significantly elevated levels of pro-inflammatory cytokines, including IFN-γ and TNF-α, indicating a robust immune response (P< 0.001).Conclusion: These findings suggest that BMS-202 effectively promotes apoptosis and enhances immune responses in lung cancer, underscoring its potential as a therapeutic agent in treating lung carcinogenesis. Keywords: BMS-202, lung carcinogenesis, apoptosis, cytotoxic T lymphocytes, PD-1 inhibition, C57BL/6 mouse model