T. Alfieri1, A. Macleod1, H. Talbott1, J. Markert1, S. Galandiuk1 1University Of Louisville, Price Institute Of Surgical Research, Hiram C. Polk Jr. MD Department Of Surgery, Louisville, KY, USA
Introduction:
Colorectal Cancer(CRC) is one of the most common cancers diagnosed in the United States. Immunotherapy is a promising treatment option that targets immune proteins on cancer cells, such as programmed death-ligand 1(PDL1). Mismatch-repair deficient(MMRd) cancers which make up 10-15% of CRC, express PDL1 and have shown some promising responses to immunotherapy; however, response has been poor in MMR proficient CRC. MMRd CRC contain a higher abundance of immune cells within the tumor microenvironment(TME). Macrophages make up a significant portion of the TME and can exist between pro-(M1) or anti-(M2) inflammatory states. Macrophages can also express PDL1, which contributes to tumor PDL1 expression and immunotherapy response. The aim of this study was to measure macrophage PDL1 gene and cell surface protein expression, comparing M1 vs M2 macrophages and changes in PDL1 expression following co-culture with either MMRd or MMRp colon cancer cell lines.
Methods:
THP-1 monocytes were differentiated into M0 non-polarized macrophages. M0 macrophages were then polarized into M1-like macrophages with lipopolysaccharide(LPS) or into M2-like macrophages using Interleukins-4/13. Polarized macrophages were co-cultured with HT29(MMRp) or HCT116(MMRd) colon cancer cell lines. Following 24-hours in either single or co-culture with cancer cells, macrophages were harvested, and total RNA extracted to measure PDL1 gene expression using TaqMan qRT-PCR. PDL1 cell surface protein expression was measured using flow cytometry (% cells expressing PDL1). For qRT-PCR results, mean ΔCT±SD values were calculated using 18S as a housekeeping gene. Results were compared using the Kruskal-Wallis test and significance set at p<0.05.(N=12 samples)
Results:
In single culture, M1-like macrophages expressed more PDL1 gene expression (ΔCT=15.9±0.3) than either M0 (ΔCT=22.9±0.6) and M2-like macrophages (ΔCT=20.6±0.4) p<0.05. Following co-culture with cancer cell lines, there was a slight decrease in PDL1 gene expression in M1-like macrophages (HT29 ΔΔCT: 2.01±0.7; HCT116 ΔΔCT: 1.11±0.12, p<0.05). In single culture, flow cytometry demonstrated that 21.1±7.5% of M1-like macrophages expressed PDL1. When co-culturing with the HT29 cell line, the percentage of M1-like macrophages expressing PDL1 on their surface increased to 52.1±11.7% (p<0.05). The M0 and M2-like macrophages did not express PDL1 cell surface protein in the single or co-culture.
Conclusion:
In single culture, M1-like pro-inflammatory macrophages had higher PDL1 gene and protein expression compared to M0 and M2-like macrophages. When co-culturing with HT29 and HCT116 cancer cell lines, PDL1 cell surface protein expression increased. We hypothesize that MMRd CRC have higher proportion of M1 macrophages within the TME compared to MMRp CRC, which contributes to the overall tumor PDL1 expression and response to immunotherapy.