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Published in final edited form as: Surgery. 2020 Oct 22;169(3):617–622. doi: 10.1016/j.surg.2020.09.029

Effect of bacterial contamination in bile on pancreatic cancer cell survival

Hannah R Shrader 1,5, Ann M Miller 1,5, Ann Tomanek-Chalkley 1, Ashley McCarthy 4, Kristen L Coleman 4, Po Hien Ear 1,4, Ashutosh K Mangalam 2,4, Aliasger K Salem 3,4, Carlos HF Chan 1,4
PMCID: PMC7870517  NIHMSID: NIHMS1632072  PMID: 33268071

Abstract

Background:

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 survival.

Methods:

Human bile samples were collected during pancreaticoduodenectomy. Bacterial strains were isolated from contaminated (stented) bile and identified using 16S rRNA sequencing. Human pancreatic cancer cells (AsPC1, CFPAC, Panc1) were treated for 24 hours with sterile (non-stented) bile, contaminated (stented) bile and sterile bile pre-incubated with 106 CFU of live bacteria isolated from contaminated bile or a panel of bile acids (BAs) for 24 hours at 37°C, and evaluated using CellTiter-Blue Cell Viability Assay. Human bile (30–50 μl/mouse) was co-injected intraperitoneally with 105 Panc02 mouse pancreatic cancer cells in C57BL6/N mice to evaluate the impact of bile on peritoneal metastasis 3–4 weeks after tumor challenge.

Results:

While all bile samples significantly reduced peritoneal metastasis of Panc02 cells in mice, some contaminated bile samples had diminished anti-tumor effect. All sterile bile (N=4) reduced pancreatic cancer cell survival in vitro. Only 40% (2/5) of contaminated bile samples had significant effect. Pre-incubation of sterile bile with live Enterococcus faecalis or Streptococcus oralis modified the anti-tumor effect of sterile bile. These changes were not observed with culture media pre-incubated with live bacteria, suggesting live gut bacteria can modify the anti-tumor components present in bile. Conjugated BAs were more potent than unconjugated cholic acid in reducing pancreatic cancer cell survival.

Conclusions:

Alteration of bile microbiome from biliary stenting has direct impact on pancreatic cancer cell survival. Further study is warranted to determine if this microbiome shift alters tumor microenvironment.

Keywords: Bile, Bile salts, Biliary stenting, Microbiome, Pancreatic cancer, Pancreaticoduodenectomy, Peritoneal carcinomatosis

TOC Statement- 20-csa-22

Treatment with human bile was found to reduce pancreatic cancer cell survival in vitro and peritoneal metastasis in vivo and the anti-tumorigenic property of bile was altered by bacterial contamination. The importance of this report is to suggest that pre-operative biliary stenting introduces foregut microbiome into the biliary system altering pancreatic cancer cell survival.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the third most common cause of cancer related deaths in the United States.1 Based on the Surveillance, Epidemiology, and End Results (SEER) data between 2009 and 2015, the 5-year overall survivals of PDACs were 37% and 12% for localized and regional disease, respectively.1 While the standard treatment for PDACs is often comprised of surgery, chemotherapy and radiation, surgical resection of the primary tumor is the essential component for curative intent treatment.2 Pancreaticoduodenectomy is the standard approach for PDACs arising from the pancreatic head and uncinate process. Since patients with PDACs of the pancreatic head often present with obstructive jaundice, percutaneous or endoscopic biliary stenting procedures are commonly performed prior to surgical consultation.3,4 This trend has been increasing from 30% in 1992–1995 to 59% in 2000–2007 based on SEER data.3 Nearly 80% of patients underwent pancreaticoduodenectomy between 2000 and 2011 in the United States received pre-operative biliary stents.4 While severe jaundice with serum bilirubin ≥ 300 μmol/L has been shown to be an independent predictor of poor long-term survival after pancreaticoduodenectomy for PDACs without pre-operative biliary stents5 and resolution of jaundice may improve early survival after pancreaticoduodenectomy6, pre-operative biliary stents increase post-operative surgical site infections7,8, thereby creating a conundrum. The impact of pre-operative stenting on oncological outcome of PDACs remains unclear, particular in the era favoring total neoadjuvant therapy when biliary stenting becomes an essential procedure.9

Bile is mainly composed of water and electrolytes, it also contains other components such as bile salts, cholesterol, phospholipids, and proteins.10,11 Human primary bile acids (BAs), cholic acid (CA) and chenodeoxycholic acid (CDCA), are produced in hepatocytes and conjugated either with glycine or taurine prior to their secretion into the bile duct.11 These conjugated primary BAs are deconjugated by bile acid hydrolase derived from intestinal bacterial species, such as Bacteroides, Clostridium and Enterococcus, and reabsorbed via the enterohepatic circulation.11,12 Primary BAs (cholic acid) can also be converted to secondary BAs (deoxycholic acid, DCA) after 7α/β-dehydroxylation by a limited number of bacteria usually present in the large intestine, for example, Clostridium species.12,13 The effects of BAs on cancer development and progression have been debated. While some studies showed pro-tumorigenic effects, others showed anti-tumorigenic effects.14–22 The biological effects of BAs on cancer progression seem to be dependent on their doses, molecular structures (i.e., conjugated vs. unconjugated and primary vs. secondary) and cellular context. Since pre-operative biliary stenting inadvertently introduces gut flora into the biliary system, we hypothesize that gut bacteria may alter the composition of BAs present in the bile, leading to a differential effect on pancreatic cancer cell survival. In this study, we aim to evaluate the impact of bile bacterial contamination on pancreatic cancer cell survival.

Methods

Patients and bile sample collection

Patients undergoing pancreaticoduodenectomy were approached and consented under an IRB-approved protocol (Gastrointestinal Molecular Epidemiology Resource, IRB#201202743) at the University of Iowa Hospitals and Clinics. Non-stented bile samples (GI0724, GI0730, GI1078, GI1207) and stented bile samples (GI0731, GI0835, GI0849, GI0857, GI0867) were collected intra-operatively. GI0724, GI0731, GI0835, GI0867, GI1078 and GI1207 were female patients. GI0730, GI0849 and GI0857 were male patients. Aerobic and anaerobic bacterial cultures and fungal cultures were obtained as part of the standard of care. Glycerol stocks were made by mixing crude bile samples with sterile glycerol in a 1:1 ratio and stored in −80°C freezer. Bile samples were also sterile-filtered and stored in −80°C freezer.

Bacterial isolation, identification and quantification

Human bile was streaked for isolation on Tryptic Soy Agar (Sigma) plates supplemented with 5% sheep blood (Remel) and incubated for 24 hours at 37°C. Single large colonies were selected and cultured in Brain-Heart Infusion broth (BHI, Difco) for 24–48 hours at 37°C in a shaking incubator. Liquid cultures were used for both bacterial identification and colony-formation unit (CFU) determination.

For bacterial strain identification, bacterial DNA was extracted from the liquid culture using the Qiagen DNeasy Blood Tissue Kit following manufacturer’s instructions. Two different sets of 16S rRNA bacterial primers were used to amplify bacterial DNA as previously described (Set 1: LPW57 5’-AGTTTGATCCTGGCTCAG-3’ and LPW58 5’-AGGCCCGGGAACGTATTCAC-3’; Set 2: XB1 5’-CAGACTCCTACGGGGAGGCAGCAGT3’ and PSR 5’-ACTTAACCCAACATCTCACGACAC-3’).23 Following 16S rRNA gene amplification PCR products were visualized on a 1% agarose gel stained with SYBR Safe DNA Gel Stain (Invitrogen). PCR products were then purified using the QIAquick PCR Purification Kit (Qiagen) following manufacturer’s instructions. Sanger Sequencing was performed on the purified PCR products in the forward and reverse direction using the primers listed above at the Iowa Institute of Human Genetics Genomics Division. Bacteria were identified using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST). For this study, bacterial strains (Enterococcus fecalis and Streptococcus oralis) were successfully isolated from patient bile samples (GI0857 and GI0867, respectively).

For bacterial quantification, the conversion between OD 600 nm to bacterial CFU/ml was determined as previously described.24 Briefly, liquid cultures from individual identified colonies were subcultured to the exponential growth phase in BHI broth for 4 hours at 37°C in a shaking incubator. Following subculture, bacterial cells were centrifuged at 8,000 rpm for 3 minutes and resuspended in Phosphate Buffered Saline (PBS). 100 μl of resuspended bacteria was plated in a 96-well plate (Corning) and serially diluted (2x) in PBS. The OD 600 nm of the serial dilutions was determined using a microplate reader (Tecan Infinite 200 PRO). The CFU/ml of each of the serial dilutions was determine by dilution plating on Tryptic Soy Agar plates supplemented with 5% sheep blood. The CFU/ml, calculated from dilution plating, was plotted versus the OD 600 nm. The resulting graph was used to determine the CFU from the corresponding OD 600 nm.

Ex vivo bile modification

Enterococcus fecalis and Streptococcus oralis were inoculated in the BHI broth and quantified as described above. Non-stented bile samples (GI0724, GI0730 and GI1078), which had no bacteria identified in standard of care clinical cultures and no growth in the laboratory culture media, were incubated with or without 106 CFU/ml of live Enterococcus fecalis or Streptococcus oralis at 37°C for 24 hours. These bile samples were then sterile-filtered to remove bacteria prior to cell culture experiments.

Cell lines

Panc02 mouse pancreatic adenocarcinoma cells were obtained from Dr. Xinhui Wang (Massachusetts General Hospital, MA). AsPC1, CFPAC and Panc1 human pancreatic adenocarcinoma cells were obtained from Dr. Mezhir’s laboratory (University of Iowa, IA). Panc02, AsPC1 and Panc1 cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Thomas Scientific), 100 U/ml penicillin G and 100 μg/ml streptomycin (Gibco). CFPAC cells were cultured in IMDM (Gibco) supplemented with 10% FBS, 100 U/ml penicillin G and 100 μg/ml streptomycin.

Bile treatment on cell culture

AsPC1, CFPAC and Panc1 cells (20,000 cells in 100 μl media/well) were plated in the 96-well tissue culture plates (Corning) and incubated in the CO2 incubator at 37°C for 2 hours. For dose response curve, non-stented bile samples (GI0724, GI0730, GI1078, GI1207) and stented bile samples (GI0731, GI0835, GI0849, GI0857 and GI0867) were added to each well at 1:10 and 1:50 dilutions and incubated for another 24 hours in the CO2 incubator at 37°C. Cellular activity was determined using CellTiter Blue (CTB) Cell Viability Assay (Promega) and measured at OD 590 nm using a microplate reader (Tecan Infinite 200 PRO). The background control reading was media with CTB reagent. The baseline control reading was cells without bile treatment. Relative cellular activity was calculated as the ratios between treatment groups over the baseline controls after subtracting the background signal. For experiments using bacteria-modified bile, all bile samples were added to each well at 1:50 dilutions. Bacteria-treated media were used as controls. Cellular activity was determined using CTB Cell Viability Assay and measured at OD 590 nm using a microplate reader. The background control reading was media with bile and CTB reagent. Relative cellular activity was calculated as the ratios between modified bile over unmodified bile after subtracting the background signal.

Bile acid treatment on cell culture

AsPC1 (A), CFPAC (B) and Panc1 (C) cells (10,000 cells in 100 μl media/well) were plated in the 96-well tissue culture plates (Corning) and incubated in the CO2 incubator at 37°C for 2 hours and treated with various concentrations (0 to 5 mM) of cholic acid (CA), glycol-cholic acid (GCA), tauro-cholic acid (TCA), chenodeoxycholic acid (CDCA), tauro-chenodeoxycholic acid (TCDCA), glyco-chenodeoxycholic acid (GCDCA) and deoxycholic acid (DCA) for another 24 hours in the CO2 incubator at 37°C. Cellular activity was determined using CellTiter Blue (CTB) Cell Viability Assay (Promega) and measured at OD 590 nm using a microplate reader (Tecan Infinite 200 PRO).

Mouse pancreatic cancer peritoneal metastatic model and bile treatment

Animal study was approved by the University of Iowa IACUC. 6–8 week-old C57BL/6N female mice (Charles River Laboratories) were injected intra-peritoneally (i.p.) with 1×105 Panc02 in 200 μl PBS and 30–50 μl of patient-derived sterile-filtered bile samples. Mice were euthanized 3–4 weeks after tumor challenge for ascites fluid and tumor harvest. Total tumor weights and ascites volumes were measured as previously described.25

Statistical Analysis

Statistical analysis was performed using GraphPad Prism software. Statistical significance was determined by unpaired 2-tailed Student’s t test for continuous variables. P-values of less than 0.05 were considered significant.

Results

Patient-derived bile samples reduce peritoneal metastasis of pancreatic cancer cells in vivo

Panc02 mouse pancreatic cancer cells have been shown to grow in the mouse peritoneal cavity following i.p. injection.25 Three weeks after i.p. injection of 105 Panc02 cells in C57BL6/N mice, the mean total peritoneal tumor weight was 0.67 g (range, 0.29 – 1.02 g) and the mean ascites volume was 1.8 ml (range, 0.7 – 2.5 ml). Co-injection of 50 μl patient-derived bile samples significantly reduced both total peritoneal tumor weights (0.02 – 0.27 g, p < 0.05) and ascites volumes (0.1 – 0.3 ml, p < 0.05) (Fig. 1). Of note, there was no reduction of tumor burden when only 10 μl of bile samples were injected, suggesting a dose-dependent tumor response (data not shown). When comparing GI0730 (non-stented bile sample) to stented bile samples (GI0731, GI0835 and GI0849), GI0835 had significantly less impact of peritoneal metastasis of Panc02 cells (0.27 g vs. 0.02 g of tumors in GI0730 bile treated mice, p = 0.014) (Fig. 1), suggesting the presence of gut bacteria in stented bile samples may have altered their effects on pancreatic cancer peritoneal metastasis.

Figure 1:

Figure 1:

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Bile treatment in pancreatic cancer peritoneal metastasis model. Mice were injected i.p. with Panc02 pancreatic cancer cells and co-injected with different patient-derived bile samples (50 μl/mouse). Injections with PBS were done in the control group. Necropsy was performed 3 weeks after tumor challenge. Total peritoneal tumor weights in grams (A) and total ascites volumes in ml (B) are shown. Each point represents a mouse. Bars denote mean+/−SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, when compared to PBS controls.

Stented bile has less effect on pancreatic cancer cell survival than non-stented bile

While treating human pancreatic cancer cells in vitro with patient-derived non-stented bile samples at 1:10 dilutions uniformly reduced cancer cell survival in all three cell lines (AsPC1, CFPAC and Panc1), three of the 5 stented bile samples had minimal effects (Fig. 2), suggesting the bacterial content may have altered the anti-tumor properties of bile. At lower doses (1:50 dilutions), some bile samples could also promote cancer cell survival. Of note, the effects on cancer cell survival in vitro mirrored the anti-tumor effects in vivo. GI0731 and GI0835 stented bile had less effects in comparing to GI0849 stented bile and GI0730 non-stented bile.

Figure 2:

Figure 2:

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Dose response of bile treatment in vitro. AsPC1 (A), CFPAC (B) and Panc1 (C) pancreatic cancer cells were treated with patient-derived non-stented bile samples (GI0724, GI0730, GI1078 and GI1207) and stented bile samples (GI0731, GI0835, GI0849, GI0857 and GI0867) at 1:10 and 1:50 dilutions. Cellular activity was determined using CTB assay and measured at OD 590 nm. Relative cellular activities are shown using cells untreated with bile samples as references. Bars denote mean+/−SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, when compared to untreated controls.

Gut bacteria can modify the effect of bile on pancreatic cancer cell survival

In this experiment, 1:50 dilutions of bile samples were chosen since it is a concentration when all non-stented bile (GI0724, GI0730 and GI1078) reduced pancreatic cancer cell growth to a certain extent. With a 24-hour pre-incubation of these bile samples with live Enterococcus fecalis or Streptococcus oralis, the inhibitory effects of GI0724 bile samples were reduced in all three cell lines (Fig. 3). However, the inhibitory effects of GI0730 bile samples were enhanced in AsPC1 and Panc1 cells. For GI1078, only Enterococcus fecalis reduced the inhibitory effects in both CFPAC and Panc1 cells. It is important to note that the observed changes of cancer cell survival were not due to contamination of bacterial content. Experiments using media pre-incubated with live Enterococcus fecalis or Streptococcus oralis did not have any impact of cell survival at all (Fig. 3). This finding was further tested in the in vivo peritoneal metastasis model. Enterococcus-treated non-stent bile sample GI0730 enhanced its anti-tumor property (Fig 3D). These findings support the concept of bile modification by introducing gut bacteria into the biliary system; hence altering the bile composition and biological property.

Figure 3:

Figure 3:

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Effect of bile modification by gut bacteria. AsPC1 (A), CFPAC (B) and Panc1 (C) pancreatic cancer cells were treated with patient-derived non-stented bile samples (GI0724, GI0730, and GI1078) or media alone pre-incubated with or without 106 CFU of live Enterococcus fecalis or Streptococcus oralis at 1:50 dilutions. Cellular activity was determined using CTB assay and measured at OD 590 nm. Relative cellular activities are shown using cells treated with unmodified bile samples as references. Bars denote mean+/−SD. *p<0.05, **p<0.01, ****p<0.0001, when compared to controls. (D) Mice were injected i.p. with Panc02 pancreatic cancer cells and co-injected with GI0730 non-stented bile sample with or without pre-incubation of Enterococcus fecalis (30 μl/mouse). Injections with PBS were done in the control group. Necropsy was performed 4 weeks after tumor challenge. Total peritoneal tumor weights in grams are shown. Each point represents a mouse. Bars denote mean+/−SD. *p<0.05, **p<0.01.

Different BAs have differential effects on pancreatic cancer cell survival

BAs are major components in bile other than water and electrolytes and some BAs have been shown to have anti-tumor properties. Since gut bacteria can deconjugate TCA/GCA to CA and TCDCA/GCDCA to CDCA as well as convert primary BAs to secondary BAs (e.g., CA to DCA), pancreatic cancer cells were treated with these BAs at various concentrations to compare their anti-cancer properties (Fig. 4). When comparing conjugated to unconjugated BAs at higher doses (2.5 – 5 mM), unconjugated BAs had less anti-tumor effects (Fig. 4A and B). This was particularly significant for TCA/GCA vs. CA. At lower doses, the anti-tumor effects were more variable. When comparing secondary BAs (DCA) to primary BAs (CA), DCA had significant more anti-tumor effects (Fig. 4C). Taking together, these results suggest that composition of BA in the bile can have different impact on pancreatic cancer cell survival.

Figure 4:

Figure 4:

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Dose response curve with bile acid treatment in human pancreatic cancer cell lines AsPC1, CFPAC and Panc1. Cellular activity was determined using CTB assay and measured at OD 590 nm. Relative cellular activities are shown using cells untreated with bile acids as references. Bars denote mean+/−SD. A) Cells were treated with various concentrations (0 to 5 mM) of cholic acid (CA), glycol-cholic acid (GCA) and tauro-cholic acid (TCA). ****p<0.0001 when comparing TCA or GCA to CA. B) Cells were treated with various concentrations (0 to 5 mM) of chenodeoxycholic acid (CDCA), glycol- chenodeoxycholic acid (GCDCA) and tauro- chenodeoxycholic acid (TCDCA). **p<0.01, ***p<0.001, ****p<0.0001 when comparing TCDCA or GCDCA to CDCA. C) Cells were treated with various concentrations (0 to 5 mM) of cholic acid (CA) and deoxycholic acid (DCA). *p<0.05, ****p<0.0001 when comparing DCA to CA.

Discussion

Gut microbiome has been emerging as one of the key determinants of cancer development, progression and treatment response in many cancer types.26–30 The impact of gut microbiota on the innate and adaptive immune response has been shown to play a crucial role in tumorigenesis and cancer progression.31–33 For PDACs, Pushalkar et al. have shown that ablation of gut microbiome in the mouse KPC (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx1Cre) pancreatic cancer model protects against pre-invasive and invasive PDACs.31 Riquelme et al. have compared the microbiome compositions present in the pancreatic tumors derived from PDAC patients with short-term survival and those with long-term survival and found a tumor microbiome signature (Pseudoxanthomonas-Streptomyces-Saccharopolyspora-Bacillus clausii) highly predictive of long-term survivorship.34 In a population-based study of 361 subjects, Fan et al. have showed that oral pathogens Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans were associated with higher risk of PDACs, whereas Fusobacteria and Leptotrichia were associated with lower risk.35 These studies shed some lights on the understanding of the complexity of the interactions between different bacterial strains and the host within the tumor microenvironment.

While there is evidence that gut bacteria can migrate and colonize the pancreatic duct and be present in the pancreatic tumors31, the biliary system is often considered “sterile” prior to endoscopic or surgical manipulation. Based on our recent surgical records of patients who underwent pancreaticoduodenectomies, 8.3% of intra-operative bile cultures (n=12) were positive in patients without pre-operative biliary stents, whereas 100% of stented bile samples (n=50) were positive in aerobic and/or anaerobic bacterial cultures and occasionally fungal cultures. Our findings are consistent with other surgical series.36,37 While pre-operative biliary stenting has been associated with higher risk of post-operative surgical site infections7,8, the long-term oncological outcome of pre-operative biliary stenting with the concomitant of the introduction of foregut microbiome into the biliary system is unclear.

Our current study shows that treatment of crude human bile can reduce pancreatic cancer cell survival in vitro and in vivo. Our findings are consistent with the study conducted by Lu et al. when Panc1 human pancreatic cancer cells were treated with crude human bile.14 Our study further confirms this phenomenon with four independent non-stented “sterile” bile samples in three different human pancreatic cancer cell lines, indicating the robustness of the biological effects of human bile on cancer cells although there are some notable variations depending on the bile concentrations and cell lines used. More interestingly, we show here that three of the five stented bile samples have much less anti-cancer properties and that commonly found bacterial species in the upper gastrointestinal tract, Enterococcus and Streptococcus, isolated from the stented “contaminated” bile samples, can modify the biological effects of bile on cancer cell survival in opposing directions depending on bacterial strains, cell lines and original “sterile” bile samples. Since many studies have shown that different BAs have different effects on cancer cell growth and survival15–22, this phenomenon is most likely related to the ability of these bacterial species to deconjugate taurine or glycine-conjugated primary BAs.11–13 Similarly, Clostridium species may be able to convert primary BAs to secondary BAs in bile samples making bile more cellular toxic.11 We have directly tested this hypothesis by comparing different conjugated and unconjugated BAs and primary BAs and secondary BAs in this study and found that the anti-tumor properties were significantly different with the same molar concentrations. While sophisticated biochemical experiments including mass spectrophotometry and microbiome analysis are required to confirm our speculation, our study suggests that introduction of foregut microbiome into the biliary system via biliary stenting may alter the bile composition and its biological behavior towards cancer cells, which reside within the biliary tract or spill out during pancreaticoduodenectomy.

In conclusion, there are several limitations in this study that will need to be addressed in future studies. Major questions are whether bacterial contamination has any impact on oncological outcome clinically and which bacterial strains or bile components have the dominant or cooperative effects in this complex system. This will require a much larger cohort of patients with bile microbiome analysis, mass spectrometry of all associated bile samples and logistic regression modeling. While alteration of the bile microbiome from biliary stenting may modify tumor microenvironment and affect the overall treatment response and survival outcome, further experiments and studies will be needed.

Acknowledgments

Panc02 cells were kind gifts from Dr. Xinhui Wang (Massachusetts General Hospital, MA).

Funding/Support

This study was supported by the Central Surgical Association Foundation Turcotte Award and the Holden Comprehensive Cancer Center through funds from the National Cancer Institute of the National Institutes of Health under award number P30 CA086862 for supporting the Molecular Epidemiology Resource Core and the Genomics Core. HRS was supported by the Iowa Center for Research by Undergraduates Fellowship Awards. AMM was supported by the National Institutes of Health Free Radical and Radiation Biology T32 CA078586 training grant.

* This paper was selected to be presented at the 77th Central Surgical Association Annual Meeting in Milwaukee, Wisconsin on June 4-6, 2020.

Footnotes

Conflict of interest/Disclosures

The authors do not have any conflict of interest.

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Reference

  • 1.Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. 2020;70(1):7–30. 10.3322/caac.21590 [DOI] [PubMed] [Google Scholar]
  • 2.Kamisawa T, Wood LD, Itoi T, Takaori K. Pancreatic cancer. Lancet. 2016;388(10039):73–85. 10.1016/S0140-6736(16)00141-0 [DOI] [PubMed] [Google Scholar]
  • 3.Rustgi SD, Amin S, Yang A, Kim MK, Nagula S, Kumta NA, et al. Preoperative endoscopic retrograde cholangiopancreatography is not associated with increased pancreatic cancer mortality. Clin Gastroenterol Hepatol. 2019;17(8):1580–1586. 10.1016/j.cgh.2018.11.056 [DOI] [PubMed] [Google Scholar]
  • 4.Jinkins LJ, Parmar AD, Han Y, Duncan CB, Sheffield KM, Brown KM, Riall TS. Current trends in preoperative biliary stenting in patients with pancreatic cancer. Surgery. 2013;154(2):179–189. 10.1016/j.surg.2013.03.016 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Sauvanet A, Boher J-M, Paye F, Bachellier P, Sa Cuhna A, Le Treut Y- P, et al. Severe jaundice increases early severe morbidity and decreases long-term survival after pancreaticoduodenectomy for pancreatic adenocarcinoma. J Am Coll Surg. 2015;221(2):380–389. 10.1016/j.jamcollsurg.2015.03.058 [DOI] [PubMed] [Google Scholar]
  • 6.Smith RA, Dajani K, Dodd S, Whelan P, Raraty M, Sutton R, et al. Preoperative resolution of jaundice following biliary stenting predicts more favourable early survival in resected pancreatic ductal adenocarcinoma. Ann Surg Oncol. 2008;15(11):3138–3146. 10.1245/s10434-008-0148-z [DOI] [PubMed] [Google Scholar]
  • 7.Scheufele F, Schorn S, Demir IE, Sargut M, Tieftrunk E, Calavrezos L, et al. Preoperative biliary stenting versus operation first in jaundiced patients due to malignant lesions in the pancreatic head: A meta-analysis of current literature. Surgery. 2017;161(4):939–950. 10.1016/j.surg.2016.11.001 [DOI] [PubMed] [Google Scholar]
  • 8.De Pastena M, Marchegiani G, Paiella S, Malleo G, Ciprani D, Gasparini C, et al. Impact of preoperative biliary drainage on postoperative outcome after pancreaticoduodenectomy: An analysis of 1500 consecutive cases. Dig Endosc. 2018;30(6):777–784. 10.1111/den.13221 [DOI] [PubMed] [Google Scholar]
  • 9.Oba A, Ho F, Bao QR, Al-Musawi MH, Schulick RD, Del Chiaro M. Neoadjuvant treatment in pancreatic cancer. Front Oncol. 2020;10:245 10.3389/fonc.2020.00245 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Keulemans YC, Mok KS, de Wit LT, Gouma DJ, Groen AK. Hepatic bile versus gallbladder bile: a comparison of protein and lipid concentration and composition in cholesterol gallstone patients. Hepatology. 1998;28(1):11–16. 10.1002/hep.510280103 [DOI] [PubMed] [Google Scholar]
  • 11.de Aguiar Vallim TQ, Tarling EJ, Edwards PA. Pleiotropic roles of bile acids in metabolism. Cell Metabolism. 2013;17(5):657–669. 10.1016/j.cmet.2013.03.013 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Ridlon JM, Harris SC, Bhowmik S, Kang D-J, Hylemon PB. Consequences of bile salt biotransformations by intestinal bacteria. Gut Microbes. 2016;7(1):22–39. 10.1080/19490976.2015.1127483 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Urdaneta V, Casadesús J. Interactions between bacteria and bile salts in the gastrointestinal and hepatobiliary tracts. Front Med. 2017;4:163 10.3389/fmed.2017.00163 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Lu Y, Onda M, Uchida E, Yamamura S, Yanagi K, Matsushita A, et al. The cytotoxic effects of bile acids in crude bile on human pancreatic cancer cell lines. Surg Today. 2000;30(10):903–909. 10.1007/s005950070042 [DOI] [PubMed] [Google Scholar]
  • 15.Wu Z, Lü Y, Wang B, Liu C, Wang Z-R. Effects of bile acids on proliferation and ultrastructural alteration of pancreatic cancer cell lines. World J Gastroenterol. 2003;9(12):2759–2763. 10.3748/wjg.v9.i12.2759 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ignacio Barrasa J, Olmo N, Pérez-Ramos P, Santiago-Gómez A, Lecona E, Turnay J, Antonia Lizarbe M. Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells. Apoptosis. 2011;16(10):1054–1067. 10.1007/s10495-011-0633-x [DOI] [PubMed] [Google Scholar]
  • 17.Lim S-C, Duong H- Q, Choi JE, Lee T- B, Kang J- H, Oh SH, Han SI. Lipid raft-dependent death receptor 5 (DR5) expression and activation are critical for ursodeoxycholic acid-induced apoptosis in gastric cancer cells. Carcinogenesis. 2011;32(5):723–731. 10.1093/carcin/bgr038 [DOI] [PubMed] [Google Scholar]
  • 18.Jenkins GJS, D’Souza FR, Suzen SH, Eltahir ZS, James SA, Parry JM, et al. Deoxycholic acid at neutral and acid pH, is genotoxic to oesophageal cells through the induction of ROS: The potential role of anti-oxidants in Barrett’s oesophagus. Carcinogenesis. 2007;28(1):136–142. 10.1093/carcin/bgl147 [DOI] [PubMed] [Google Scholar]
  • 19.Liu R, Li X, Qiang X, Luo L, Hylemon PB, Jiang Z, et al. Taurocholate induces cyclooxygenase-2 expression via the sphingosine 1-phosphate receptor 2 in a human cholangiocarcinoma cell line. J Biol Chem. 2015;290(52):30988–31002. 10.1074/jbc.M115.668277 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Kitamura T, Srivastava J, DiGiovanni J, Kiguchi K. Bile acid accelerates erbB2-induced pro-tumorigenic activities in biliary tract cancer. Mol Carcinog. 2015;54(6):459–472. 10.1002/mc.22118 [DOI] [PubMed] [Google Scholar]
  • 21.Kaibara N, Yurugi E, Koga S. Promoting effect of bile acids on the chemical transformation of C3H/10T1/2 fibroblasts in vitro. Cancer Res. 1984;44(12 Pt 1):5482–5485. [PubMed] [Google Scholar]
  • 22.Gándola YB, Fontana C, Bojorge MA, Luschnat TT, Moretton MA, Chiapetta DA, et al. Concentration-dependent effects of sodium cholate and deoxycholate bile salts on breast cancer cells proliferation and survival. Mol Biol Rep. 2020;65(16):2461–19. 10.1007/s11033-020-05442-2 [DOI] [PubMed] [Google Scholar]
  • 23.Jenkins C, Ling CL, Ciesielczuk HL, Lockwood J, Hopkins S, McHugh TD, et al. Detection and identification of bacteria in clinical samples by 16S rRNA gene sequencing: comparison of two different approaches in clinical practice. J Med Microbiol. 2012;61(Pt 4):483–8. 10.1099/jmm.0.030387-0 [DOI] [PubMed] [Google Scholar]
  • 24.Hall BG, Acar H, Nandipati A, Barlow M. Growth rates made easy. Mol Biol Evol. 2014;31(1):232–8. 10.1093/molbev/mst187. [DOI] [PubMed] [Google Scholar]
  • 25.Miller AM, Lemke-Miltner C, Blackwell S, Tomanek-Chalkley A, Gibson-Corely KN, Coleman KL, et al. Intraperitoneal CMP-001: A novel immunotherapy for treating peritoneal carcinomatosis of gastrointestinal and pancreaticobiliary cancer. Ann Surg Oncol. 2020. 10.1245/s10434-020-08591-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Abreu MT, Peek RM. Gastrointestinal malignancy and the microbiome. Gastroenterology. 2014;146(6):1534–1546. 10.1053/j.gastro.2014.01.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.McAllister F, Khan MAW, Helmink B, Wargo JA. The Tumor Microbiome in Pancreatic Cancer: Bacteria and Beyond. Cancer Cell. 2019;36(6):577–579. 10.1016/j.ccell.2019.11.004 [DOI] [PubMed] [Google Scholar]
  • 28.Yu L-X, Schwabe RF. The gut microbiome and liver cancer: mechanisms and clinical translation. Nat Rev Gastroenterol Hepatol. 2017;14(9):527–539. 10.1038/nrgastro.2017.72 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Yang J, Tan Q, Fu Q, Zhou Y, Hu Y, Tang S, et al. Gastrointestinal microbiome and breast cancer: correlations, mechanisms and potential clinical implications. Breast Cancer. 2017;24(2):220–228. 10.1007/s12282-016-0734-z [DOI] [PubMed] [Google Scholar]
  • 30.Zitvogel L, Ma Y, Raoult D, Kroemer G, Gajewski TF. The microbiome in cancer immunotherapy: Diagnostic tools and therapeutic strategies. Science. 2018;359(6382):1366–1370. 10.1126/science.aar6918 [DOI] [PubMed] [Google Scholar]
  • 31.Pushalkar S, Hundeyin M, Daley D, Zambirinis CP, Kurz E, Mishra A, et al. The Pancreatic Cancer Microbiome Promotes Oncogenesis by Induction of Innate and Adaptive Immune Suppression. Cancer Discov. 2018;8(4):403–416. 10.1158/2159-8290.CD-17-1134 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Yu AI, Zhao L, Eaton KA, Ho S, Chen J, Poe S, et al. Gut Microbiota Modulate CD8 T Cell Responses to Influence Colitis-Associated Tumorigenesis. Cell Reports. 2020;31(1):107471 10.1016/j.celrep.2020.03.035 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Sethi V, Kurtom S, Tarique M, Lavania S, Malchiodi Z, Hellmund L, et al. Gut Microbiota Promotes Tumor Growth in Mice by Modulating Immune Response. Gastroenterology. 2018;155(1):33–37. 10.1053/j.gastro.2018.04.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Riquelme E, Zhang Y, Zhang L, Montiel M, Zoltan M, Dong W, et al. Tumor Microbiome Diversity and Composition Influence Pancreatic Cancer Outcomes. Cell. 2019;178(4):795–806. 10.1016/j.cell.2019.07.008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Fan X, Alekseyenko AV, Wu J, Peters BA, Jacobs EJ, Gapstur SM, et al. Human oral microbiome and prospective risk for pancreatic cancer: a population-based nested case-control study. Gut. 2018;67(1):120–127. 10.1136/gutjnl-2016-312580 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Bilgiç Ç, Keske Ş, Sobutay E, Can U, Zenger S, Gürbüz B, et al. Surgical site infections after pancreaticoduodenectomy: preoperative biliary system interventions and antimicrobial prophylaxis. Int J Infect Dis. 2020. 10.1016/j.ijid.2020.04.005 [DOI] [PubMed] [Google Scholar]
  • 37.Sano S, Sugiura T, Kawamura I, Okamura Y, Ito T, Yamamoto Y, et al. Third-generation cephalosporin for antimicrobial prophylaxis in pancreatoduodenectomy in patients with internal preoperative biliary drainage. Surgery. 2019;165(3):559–564. 10.1016/j.surg.2018.09.011 [DOI] [PubMed] [Google Scholar]

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