Abstract 1803: Exosome-encapsulated PD-L1 siRNA reduces tumor growth by modulating the tumor-associated macrophages

Abstract Immunotherapy, a rapidly advancing cancer treatment, influences the patient’s immune system to combat tumors. However, cancer cells evade T-cell immunosurveillance by overexpressing PD-L1 on their surfaces. Additionally, tumor-associated macrophages (TAMs) further complicate the therapeutic efficacy of immune checkpoint inhibitors (ICIs) within the tumor microenvironment. The success of immunotherapy in solid tumors is limited to 20-45%, primarily due to tumor heterogeneity, an immunosuppressive tumor microenvironment, and inefficient tumor-specific trafficking of therapeutics. In this study, we developed an exosome-polyethyleneimine matrix (EPM) nanoplatform functionalized with a tumor-targeting ligand, folic acid (FA-EPM), for the targeted delivery of PD-L1 siRNA to the lung tumor. This approach efficiently silences PD-L1 expression on both tumor cells and TAMs. Exosomes were isolated from bovine colostrum through rehydration and differential centrifugation processes, resulting in exosomes with an average particle size of 66±2.5 nm, a zeta potential of -9.6 mV, and a polydispersity index (PDI) of 0.28. The entrapment efficiency of siPD-L1 within the EPM matrix exceeded 90%. siPD-L1 sequences with specificity for mouse and human PD-L1 were screened in vitro against LLC and A549 cells, respectively. Protein lysate analysis revealed that EPM-siPD-L1 significantly downregulated PD-L1 expression in LLC-1 cells (up to 80%) and in A549 cells (>80%). The most effective siPD-L1 candidate from the in vitro studies was tested in vivo in C57BL/6 mice bearing orthotopic lung tumor xenografts established with LL/2-Luc2 cells (50 × 103 cells/mouse). Tumor-bearing mice were divided into three groups (n = 7-9) and treated intravenously with vehicle, FA-EPM-scrambled siRNA, or FA-EPM-siPD-L1 (15 µg/dose). Tumor growth was monitored twice weekly using bioluminescence imaging. FA-EPM-siPD-L1 treatment significantly reduced tumor growth (>70%; p < 0.01) compared to vehicle or scrambled siRNA-treated mice. Additionally, a significant reduction in tumorous lung weight (∼60%; p < 0.01) was observed in the FA-EPM-siPD-L1 group. At euthanasia, tumorous lungs were processed into single-cell suspensions and analyzed for myeloid and T-cell markers using flow cytometry. The tumor reduction was found to be associated with the marked reduction in PD-L1 expression, a significant depletion of immunosuppressive TAMs and increased levels of anti-tumoral gamma delta T cells. In summary, these results demonstrate that EPM-mediated delivery of siPD-L1 enhances immune surveillance, effectively inhibits lung tumor progression, and establishes EPM as a promising nanoplatform for delivering siRNA-based therapeutics. Funding: Supported by Immunotherapy CCII CoBRE pilot project (P20GM135004) and the Agnes Brown Duggan Endowment. Citation Format: Raghuram Kandimalla, Omar S. Sarkar, Disha N. Moholkar, Margaret Wallen, Chuanlin Ding, Kavitha Yaddanapudi, Ramesh C. Gupta, Farrukh Aqil. Exosome-encapsulated PD-L1 siRNA reduces tumor growth by modulating the tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1803.