Effect of bacterial contamination in bile on pancreatic cancer cell survival

Abstract

Background: Introduction of gut flora into the biliary system is common owing to biliary stenting in patients with obstructing pancreatic head cancer. We hypothesize that alteration of biliary microbiome modifies bile content that modulates pancreatic cancer cell survival. Methods: Human bile samples were collected during pancreaticoduodenectomy. Bacterial strains were isolated from contaminated (stented) bile and identified using 16S ribosomal RNA sequencing. Human pancreatic cancer cells (AsPC1, CFPAC, Panc1) were treated for 24 hours with sterile (nonstented) bile, contaminated (stented) bile, and sterile bile preincubated with 106 colony forming unit of live bacteria isolated from contaminated bile or a panel of bile acids for 24 hours at 37°C, and evaluated using CellTiter-Blue Cell Viability Assay (Promega Corp. Madison, WI). Human bile (30–50 μl/mouse) was coinjected intraperitoneally with 105 Panc02 mouse pancreatic cancer cells in C57BL6/N mice to evaluate the impact of bile on peritoneal metastasis 3 to 4 weeks after tumor challenge. Results: While all bile samples significantly reduced peritoneal metastasis of Panc02 cells in mice, some contaminated bile samples had diminished antitumor effect. All sterile bile (n = 4) reduced pancreatic cancer cell survival in vitro. Only 40% (2/5) of contaminated bile samples had significant effect. Preincubation of sterile bile with live Enterococcus faecalis or Streptococcus oralis modified the antitumor effect of sterile bile. These changes were not observed with culture media preincubated with live bacteria, suggesting live gut bacteria can modify the antitumor components present in bile. Conjugated bile acids were more potent than unconjugated cholic acid in reducing pancreatic cancer cell survival. Conclusion: Alteration of bile microbiome from biliary stenting has a direct impact on pancreatic cancer cell survival. Further study is warranted to determine if this microbiome shift alters tumor microenvironment.