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The Impact Of Bacterial Contamination In Bile Spillage During Pancreaticoduodenectomy On Pancreatic Cancer Cell Growth
*Hannah R. Shrader, *Ann M. Miller, *Ann Tomanek-Chalkley, *Ashley McCarthy, *Kristen L. Coleman, Carlos H.F. Chan
University of Iowa Hospitals and Clinics, Iowa City, IA

OBJECTIVE(S): Introduction of gut flora into the biliary system is common due 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 growth.
METHODS: Under approved IRB protocol, human bile samples were collected during pancreaticoduodenectomy. Human bile (50 υl/mouse) was co-injected intraperitoneally with 105 Panc02 mouse pancreatic cancer cells in C57BL6/N mice for evaluating the impact of bile on peritoneal metastasis 3 weeks after tumor challenge. Bacterial strains were isolated from contaminated (stented) bile and identified using 16S rRNA sequencing. Human pancreatic cancer cells (AsPC1, CFPAC, and Panc1) were treated for 24 hours with sterile (non-stented) bile and sterile bile pre-incubated with 106 CFU of live or heat-killed bacteria isolated from contaminated bile for 24 hours at 37C, and evaluated using CellTiter-Blue Cell Viability Assay. Note all bile samples were sterile filtered prior to cell culture. Student t-tests were used for statistical analysis.
RESULTS: While all bile samples significantly reduced peritoneal metastasis of Panc02 cells in mice, some contaminated bile samples had less anti-tumor effect (Figure 1). Sterile bile (GI0724 and GI0730) reduced viability of pancreatic cancer cells with variable sensitivity in vitro (9-18% (AsPC1), 21-36% (Panc1), and 80-84% (CFPAC) at 1:50 dilution). Pre-incubation of sterile bile GI0724 with live bacteria isolated from bile samples GI0857 (Enterococcus faecalis) and GI0867 (Streptococcus oralis) reduced the anti-tumor effect of sterile bile GI0724 (Figure 2). These changes were not observed with sterile bile GI0724 pre-incubated with heat-killed bacteria or culture media pre-incubated with live or heat-killed bacteria, suggesting live gut bacteria can modify the anti-tumor components present in bile.
CONCLUSIONS: While bile spillage, stented or not, may not have negative oncologic impact after pancreaticoduodenectomy, alteration of bile microbiome from biliary stenting will likely modify tumor microenvironment.


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