br Materials and Methods br Results br Discussion

Materials and Methods
Results
Discussion Until recently, many internal organs were believed to be sterile environments. Recent studies have however changed that view, showing that even healthy placentas consistently harbor microbial communities (Aagaard et al., 2014). This study serves to shed light on the complex microbial communities’ resident in parts of the hepatobiliary system. Crosstalk between these communities and those in the intestine could be mediated by the process of enterohepatic circulation, influencing key processes in the host such as nutrient acquisition and drug metabolism (Yip et al., unpublished). Tumor microenvironment, defined as the Calcein AM of host and microbial cells associated with tumors, is known to be critical in regulating carcinogenesis (Swartz et al., 2012). There has been increasing evidence that supports a role for microbiota in shaping the microenvironment through its metabolic output and interaction with host cells (Louis et al., 2014; Swartz et al., 2012). The biliary system is prone to microbial infections (Carpenter, 1998) and the interactions between bacteria and bile profoundly impact human health (Begley et al., 2005). However, little is known about the biliary tissue microbiome and its contribution to bile duct tumorigenesis. Description of the biliary tissue microbiome has been typically generalized from results originating from bile fluid cultures and may not be reflective of the biliary tissue microenvironment. This study serves to shed light on the role of the bile duct tissue microbiome in CCA development based on extensive 16S rRNA profiling of O. viverrini associated and non-associated paired tumor-normal tissues. To the best of our knowledge, this is the first study to profile tumor and adjacent normal tissue microbiomes of the biliary tree and serves to further our understanding of the diversity and functional capacity of the resident community, complementing earlier studies that used bile fluids or unmatched samples (Aviles-Jimenez et al., 2016; Wu et al., 2013). Overall, we found systemic differences in tumor and adjacent normal CCA tissue microbiomes compared to normal hepatic tissue, but limited divergence in paired tumor vs normal microbiomes. In fact, O. viverrini associated tissues did not exhibit any significant microbiome alterations between tumor-normal pairs. The systemic alterations in CCA tissue microbiomes suggest that the tumor and tissue-resident microbiomes influence each other using far-acting mechanisms. For non-O. viverrini associated tissue samples, the genus Stenotrophomonas was found to be more abundant in tumor tissue. This is similar to the distribution profile for Fusobacterium in colorectal carcinoma (Kostic et al., 2012). Stenotrophomonas is known to be involved in bile duct infections (Perez et al., 2014) and shown to elicit proinflammatory cytokine production in vitro (Roscetto et al., 2015). Furthermore, CCA development has been strongly linked to an inflammatory phenotype (Sia et al., 2013). Consequently, an inflammation mediated mechanism tying the enrichment of Stenotrophomonas to non-OVa CCA tumorigenesis deserves further investigation. Compared to non-OVa tissues, there was notable enrichment of specific enteric microbes such as Bifidobacteriaceae and Enterobacteriaceae in the OVa tissue microbiome. Bifidobacteriaceae was also seen as the major constituent of the O. viverrini microbiome (with Enterobacteriaceae at much lower abundance), providing a link between O. viverrini infection and CCA tissue microbiome alteration in humans, and extending on previous findings based on rodent models (Plieskatt et al., 2013). As O. viverrini infection in humans requires the consumption of infected raw fish, we hypothesize that it triggers the observed microbiome alterations and not vice versa. While an earlier study detected the presence of Helicobacter pylori in the bile of liver fluke infected CCA patients (Boonyanugomol et al., 2012), we did not detect H. pylori in our tissue samples, possibly due to the transient nature of bile fluids and their differences in comparison to bile duct tissue microbiomes as noted in this study.