J. B. George1, A. Macleod1, K. Scheurlen1, D. Snook1, C. Barbour1, S. Galandiuk1 1University Of Louisville, Price Institute Of Surgical Research, Hiram C. Polk Jr. MD Department Of Surgery, Louisville, KY, USA
Introduction: Colon cancer is the third most prevalent cancer and the second leading cause of cancer related death in both men and women in the United States. In late-stage colon cancer, the 5-year survival rate is 10% due to the limited response to standard treatments. Immunotherapy is an effective treatment option for many cancers such as melanoma, renal and lung cancer. Anti-PDL1 therapy is a type of immunotherapy that targets the PDL1/PD1immune proteins on the cell surface. In colorectal cancer, anti PDL1/PD1 therapy has been a promising treatment in mis-match repair deficient (MMRd) cancers. However, in mis-match repair proficient (MMRp) cancers, which accounts for 85% of colorectal cancers, its success has been poor. MMRp cancers have low PDL1 expression. PDL1 can be upregulated in cells by Interferon Gamma (IFNγ) treatment. The aim of this study is to characterize PDL1 expression in colon cancer cell line SW480 (MMRp) and measure its response to IFNγ treatment.
Methods: SW-480 (Stage II) colon adenocarcinoma cells were plated in 24-well plates. Cells were treated with 0, 10, 100 or 500 ng/ml of IFNγ and harvested after 0, 3, 6, 18, 24, 48 hours (n=5 for each dose and time point). Following cell and supernatant harvest, qRT-PCR was performed on mRNA. PDL1 ELISA was used to measure cell lysate and supernatant protein levels. Following staining with anti-PDL1 antibodies flow cytometry analysis was performed with a FACS Calibur flow cytometer. Data were analyzed using the Kruskal-Wallis Test (p<0.05).
Results: IFNγ upregulated PDL1 gene expression at all times and doses. The optimal time for increased gene expression was 6 hours for both 100ng/ml (9.7±1.7-fold-change, p=0.032) and 500ng/ml (14.1 ± 1.9 fold-change, p=0.026). Cell surface protein expression was also increased following treatment. At baseline 18.4 ± 4.9 % of SW480 cells expressed PDL1. This increased to 89.8 ± 2.94% following treatment with 500ng/ml IFNγ and was maintained for 48 hours (p=0.02). No difference was seen in PD-L1 protein expression in cells treated with 100 vs 500ng/ml (p=0.592). Extracellular PDL1 expression was unaffected by IFNγ.
Conclusion: SW-480 colon cancer cells express PDL1. IFNγ treatment upregulates PDL1 gene expression, as well as PDL1 cell surface protein levels. Increased cell surface expression is maintained for at least 48 hours. This provides an important baseline upon which further cell culture studies can be based to examine the effect of cancer cell PDL1 expression on other cell types within the tumor micro-environment. In the future we will compare SW-480 (MMRp) PDL1 expression and upregulation to that of the (MMRd) cell line, Hct-116.