Gemcitabine augments HLA class I expression in pancreatic cancer cells through alterations in transcript production and surface stability

Background: Pancreatic adenocarcinoma, or PDAC, is the fourth leading cause of cancer-related deaths in the United States. Gemcitabine, a nucleoside analog, is a primary standard of care in pancreatic cancer. In addition to its normative cytotoxic function, evidence suggests that this chemotherapy drug also harnesses immunomodulatory capabilities in the form of increasing human leukocyte antigen (HLA) class I expression in lung, breast, colon, and cholangiocarcinoma cells. HLA class I is a complex (alpha heavy chain and beta 2-microglobulin light chain) located at the surface of nearly all cells where it presents peptides, including cancer-associated peptides, to cytotoxic T cells. Recognition of these peptides as atypical leads to T-cell mediated lysis of the presenting cell. Subsequently, understanding the ability of gemcitabine and the mechanisms by which it influences the HLA class I complex are of great importance. Methods: To investigate the effect of gemcitabine treatment on HLA class I expression in pancreatic cancer, alterations in HLA class I protein levels were monitored via western blot analysis, flow cytometry, and quantitative polymerase chain reaction (qPCR) in three pancreatic cancer cell lines. Changes in surface stability of the HLA class I complex were evaluated through brefeldin A (BFA) assays at the 72-hour time point. For western blot and flow cytometry experiments, impacts on the alpha heavy chain were further assessed for all three types of alpha heavy chains (HLA-A, HLA-B/C) at 72 and 96 hours. In qPCR experiments, alterations were analyzed for the HLA-A, HLA-B, and beta 2-microglobulin transcripts after a 48-hour gemcitabine exposure period. Results: Administration of gemcitabine to pancreatic cancer cell lines (S2-013, Capan-1, PANC-1) increased total protein levels of both HLA class I constituents (alpha heavy chain and beta-2-microglobulin light chain). All PDAC cell lines evaluated demonstrated enhanced surface expression of HLA-A2 and HLA-B/C with maximal increases of 3 and 2.5-fold respectively, as indicated by flow cytometry. Our qPCR analysis of the Capan-1 and S2-013 cell lines revealed increases in the levels of HLA-A, HLA-B, and beta 2-microglobulin transcripts (3-12-fold). BFA assays suggested that gemcitabine treatment also enhances stability of the surface HLA class I in the S2-013 and PANC-1 cell lines. Conclusion: In summary, gemcitabine exhibits an immunomodulatory ability to stimulate expression of HLA class I in pancreatic cancer cells. We have demonstrated that this increase in HLA class I is seen at the mRNA, total protein, surface, and stability level. Characterizing the ability of gemcitabine to influence the HLA class I complex and defining the mechanisms by which it does so will increase the potential for identification of suitable immunotherapies, including novel peptide-based cancer vaccines with enhanced treatment efficacy for PDAC patients. Citation Format: Alaina C. Larson, Shelby M. Knoche, Joyce C. Solheim. Gemcitabine augments HLA class I expression in pancreatic cancer cells through alterations in transcript production and surface stability [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P071.