The Effect of Cytotoxic CD8+ T-Cells Secretory Proteins on Hypoxic Pancreatic Cancer Cells

Abstract

Abstract Background: Pancreatic cancer is a highly progressive cancer with limited effective treatment. The tumor microenvironment in pancreatic cancer is aggressive and severely hypoxic with a high immunosuppressive effect. This has raised the attention to understanding the pancreatic cancer microenvironment and the interaction between pancreatic cancer cells and cytotoxic T-cells. Objective: This study aimed to investigate the crosstalk between hypoxic pancreatic cancer cells (PANC-1) and cytotoxic CD8+ T-cells. Methods: Pancreatic cancer cells (PANC-1) were exposed to 20 cycles of chronic hypoxic conditions for 72 hours followed by a re-oxygenation period for 24 hours. On cycles 10 and 20, PANC1 hypoxic conditioned media (CM) were harvested and the hypoxic PANC-1 cells were co-cultured with the activated cytotoxic CD8+ T-cells or with CD8+ T-cells CM. CD8+ T-cells CM was collected after 5 days of cell activation using anti-CD3/CD28 antibodies and interleukin-2 (IL-2). On the other hand, CD8+ T-cells were activated for 72 hours, and then cultured with the hypoxic PANC-1 CM. Results: Hypoxic PANC-1 cells showed a significant increase in resistance to the lytic effect of either CD8+ T-cells coculture or CD8+ T-cells CM treatment when compared with normoxic PANC-1 cells. A significant decrease in TNF-α and IFN-γ levels was also detected. Additionally, a significant increase in IL-6, P53 and TNF-α gene expression levels was observed in PANC-1 cells treated with CD8+ T-cells CM. Moreover, IL-6 gene expression level showed a significant difference between hypoxic and normoxic PANC-1 cells. Furthermore, CD8+ T-cells proliferation and cytokines production were significantly higher in cells cultured with PANC-1 CM. However, no significant differences were observed after treatment with either hypoxic or normoxic PANC-1 CM. Conclusion: Hypoxia influences PANC-1 cells' sensitivity to cytotoxic CD8+ T-cells. Several mechanisms of PANC-1 resistance were detected in this study; an increase in IL-6 production and a depletion in TNF-α and IFN-γ levels when co-cultured with PANC-1 cells. In treated PANC-1 cells, an increase in TP53 and TNF-α gene expression were observed. IL-6 activation is one of the mechanisms that may increase the resistance to CD8+ T-cells in hypoxic PANC-1 cells.