Oncobiosis and Microbial Metabolite Signaling in Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma is one of the most lethal cancers in both men and women, with a median five-year survival of around 5%. Therefore, pancreatic adenocarcinoma represents an unmet medical need. Neoplastic diseases, such as pancreatic adenocarcinoma, often are associated with microbiome dysbiosis, termed oncobiosis. In pancreatic adenocarcinoma, the oral, duodenal, ductal, and fecal microbiome become dysbiotic. Furthermore, the pancreas frequently becomes colonized (by Helicobacter pylori and Malassezia, among others). The oncobiomes from long- and short-term survivors of pancreatic adenocarcinoma are different and transplantation of the microbiome from long-term survivors into animal models of pancreatic adenocarcinoma prolongs survival. The oncobiome in pancreatic adenocarcinoma modulates the inflammatory processes that drive carcinogenesis. In this review, we point out that bacterial metabolites (short chain fatty acids, secondary bile acids, polyamines, indole-derivatives, etc.) also have a role in the microbiome-driven pathogenesis of pancreatic adenocarcinoma. Finally, we show that bacterial metabolism and the bacterial metabolome is largely dysregulated in pancreatic adenocarcinoma. The pathogenic role of additional metabolites and metabolic pathways will be identified in the near future, widening the scope of this therapeutically and diagnostically exploitable pathogenic pathway in pancreatic adenocarcinoma.

Changes to the oral microbiome 11,328 individuals in a prospective study. Dental health was monitored between  Periodontitis increases the risk for pancreatic adenocarcinoma. [81] 10 resectable patients with pancreatic cancer and 10 matched healthy controls for oral microbiome assay, 28 resectable pancreatic cancer, 28 matched healthy controls, and 27 chronic pancreatitis samples for validation
Neisseria elongata and Streptococcus mitis were validated as biomarkers for pancreatic adenocarcinoma. [63] Pre-diagnosis blood samples from 405 pancreatic cancer cases and 416 matched controls, collected as part of the European Prospective Investigation into Cancer and Nutrition study High serum antibodies against Porphyromonas gingivalis ATTC 53978 showed a two fold increase in risk for pancreatic adenocarcinoma. Those individuals who had high antibody titer against the commensal flora had a lower risk for pancreatic adenocarcinoma as compared to those with low titer. [82] 8 pancreatic adenocarcinoma patients and 22 healthy controls 16S rDNA was amplified and sequenced The pancreatic cancer group had higher levels of Leptotrichia, and lower levels of Porphyromonas, and Neisseria. No difference in diversity. Leptotrichia to Porphyromonas ratio was significantly higher in pancreatic adenocarcinoma patients. [83] Among 149 orodigestive cancers 6 pancreatic adenocarcinoma cases Treponema denticola chymotrypsin-like proteinase that can induce matrix metalloproteinases, was found in pancreatic adenocarcinoma using immunohistochemistry. [84] 361 incident pancreatic adenocarcinoma patients and 371 matched controls from two prospective cohort studies, the American Cancer Society Cancer Prevention Study II and the National Cancer Institute Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.
DNA was isolated from oral wash samples; 16S rRNA gene V3-V4 was amplified and sequenced using Roche 454 FLX Titanium Pyrosequencing system Carriage of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were associated with a higher risk for pancreatic adenocarcinoma. Fusobacteria and its genus, Leptotrichia decreased the risk of pancreatic adenocarcinoma. The genus Alloprevotella increased the risk for pancreatic adenocarcinoma. [85] 30 stage I pancreas head adenocarcinoma patients and 25 healthy controls Tongue scrapes were collected and the V3-V4 16S rDNA was amplified and sequenced Saliva samples from 280 pancreatic adenocarcinoma cases (29 stage I, 160 stage II, 37 stage III, and 54 stage IV pancreatic tumors) of which 273 was used in the study and 285 controls V4 region of the 16S rRNA gene was PCR amplified and sequencing was performed on the Illumina MiSeq.
Haemophilus genus showed a marginal association with pancreatic cancer risk. Enterobacteriaceae, Lachnospiraceae, Bacteroidaceae, and Staphylococcaceae showed a positive correlation with pancreatic cancer risk. [88] Helicobacter pylori colonization (seropositivity) Cases with pancreatic cancer (n = 87) were matched to controls (n = 263) using age, sex and time for baseline investigation as matching variables H. pylori serology was analyzed in stored serum samples using an enzyme-linked immunosorbent assay H. pylori seropositivity was not associated with pancreatic cancer in the total cohort (adjusted OR 1.25 (0.75-2.09)). However, a statistically significant association was found in never smokers ( Ligation of mannose-binding lectin (MBL), which binds to glycans of the fungal wall to activate the complement cascade, was required for oncogenic progression. [68] Long-term surviving (n = 22) and short-term surviving (n = 21) pancreatic adenocarcinoma patients.
From the tumor and feces 16S rDNA V4 region was amplified by PCR and sequenced in the MiSeq platform (Illumina).
The microbiome that provides long-term survival can be transplanted. [91]  50 patients with pancreatic adenocarcinoma were enrolled. In cases where a biliary stent was inserted prior to surgery, the stent was removed and cultured. In other cases, swabs of bile or pancreatic fluid and tissue from the bile duct or pancreas were obtained and cultured.
Classical culture 96% of the specimens demonstrated the presence of microbes, 90% of all cases were polymicrobial. The most frequent species found were Enterobacteriaceae, Enterococcus species, Candida species, and Streptococcus milleri [92] 152 Italian patients of which 72 had pancreas head adenocarcinoma patients were present Classical culture The most common bacteria among pancreas head adenocarcinoma patients were E. coli, K. pneumoniae, and P. aeruginosa, and less frequently, Alcaligenes spp., Serratia spp., and Enterococcus spp.
Although pancreas head carcinoma patients were not assessed separately, only such patients were present in the shortest survival cohort enabling the assessment of that patient population. E. coli, K. pneumoniae, and P. aeruginosa showed a high percentage of resistance to third-generation cephalosporins (3GCs), aminoglycosides class, and quinolone group, especially to levofloxacin, but the same bacteria were sensitive to carbapenems. [93] 50 patients with pancreatic adenocarcinoma, 34 other organs (i.e., controls). In total, 189 tissue samples (pancreatic duct, duodenum, pancreas), 57 swabs (bile duct, jejunum, stomach), and 12 stool samples.
The 16S rRNA V3-V4 hypervariable regions were amplified using Illumina MiSeq Lactobacillus ssp. was significantly higher in noncancer subjects compared with cancer subjects and the relative abundance of Fusobacterium spp was higher in cancer subjects compared with noncancer subjects. [94]

Changes to stool microbiome
Prospective study, 85 pancreatic cancer (PC) and 57 matched healthy controls (HC) MiSeq sequencing Phylum Bacteroidetes was significantly increased, while Firmicutes and Proteobacteria were decreased in PC patients versus healthy controls.
Gut microbial diversity decreased in pancreatic adenocarcinoma. Alpha diversity decreased. The abundance of certain pathogens and lipopolysaccharides-producing bacteria increased.
Probiotics and butyrate-producing bacteria decreased. Changes to the microbiome can be used as markers to detect pancreatic adenocarcinoma and the obstructive and non-obstructive forms. [43] Patients with pancreatic adenocarcinoma (n = 32), and matched healthy individuals (n = 31). stool and pancreatic tissue were assessed Sequencing of the V3-V4 hypervariable region of the 16S RNA gene after PCR amplification Proteobacteria are more abundant in patients with pancreatic adenocarcinoma as compared to healthy controls. [41] Long-term surviving (n = 22) and short-term surviving (n = 21) patients. Sequencing of intratumor and stool microbiomes.
16S rDNA V4 region was amplified by PCR and sequenced in the MiSeq platform (Illumina).
The microbiome that provides long-term survival can be transplanted. [91] 30 patients with pancreatic adenocarcinoma, 6 patients with pre-cancerous lesions, 13 healthy subjects, and 16 with non-alcoholic fatty liver disease 16S RNA was PCR amplified and was sequenced using the Illumina MiSeq platform and LEfSe linear discriminant analysis (LDA) was performed Patterns of the microbiome can separate pancreatic adenocarcinoma patients from healthy subjects and patients with comorbidities (NAFLD, etc.) and can discriminate between the etiology of pancreatic adenocarcinoma.
Some bacterial species showed a strong association with pancreatic adenocarcinoma. In the oral microbiome, Porphyromonas gingivalis increased in pancreatic adenocarcinoma [64,82,83,85,101]. Helicobacter pylori [62,69,70], Enterobacter, Enterococcus [64,90,92,93], Fusobacteria [89,94,102], and E. coli [64,93] were also shown to increase in pancreatic adenocarcinoma patients in multiple studies. In a study assessing intratumor DNA and serum cell-free DNA (1000+ patients), Fusobacteria count in tumors was higher compared to the healthy, untransformed tissues [102]. Fungal species, like Malassezia, also increased in pancreatic adenocarcinoma patients [68]. The oral microbiome can be used for diagnosis [63]. In fact, different risk factors of pancreatic adenocarcinoma are associated with changes to the microbiome, including smoking [103], poor oral health or tooth loss [78], or recurrent pancreatitis. There is a lower bacterial load in pancreatitis than in pancreatic adenocarcinoma.
Besides the direct immunogenicity of the microbiome, an endocrine-like function was also described in several cancers [30,[105][106][107] including pancreatic adenocarcinoma. Bacteria can produce bacterial metabolites that enter the systemic circulation and act on distant cancer cells. This process possesses features of endocrine signaling: a chemical entity is synthesized at one location, then transferred to another anatomical site where it binds to receptors and exerts biological responses there. Hereby, we will review the bacterial metabolites with possible pro-or anti-neoplastic features in pancreatic adenocarcinoma.

Bacterial Metabolites Playing Role in Pancreatic Adenocarcinoma
The gut microbiome harbors a large number of species with an immense and diverse metabolism. Bacterial metabolites or components of bacteria can enter the systemic circulation of the host and be transferred to distant sites where the metabolites can exert hormone-like effects [19,30,108]. Bacterial metabolism is largely dysregulated in pancreatic adenocarcinoma [43]. Below, we will review the source and (possible) roles of pro-or anti-carcinogenic bacterial metabolites ( Figure 1). Some bacterial species showed a strong association with pancreatic adenocarcinoma. In the oral microbiome, Porphyromonas gingivalis increased in pancreatic adenocarcinoma [64,82,83,85,101]. Helicobacter pylori [62,69,70], Enterobacter, Enterococcus [64,90,92,93], Fusobacteria [89,94,102], and E. coli [64,93] were also shown to increase in pancreatic adenocarcinoma patients in multiple studies. In a study assessing intratumor DNA and serum cell-free DNA (1000+ patients), Fusobacteria count in tumors was higher compared to the healthy, untransformed tissues [102]. Fungal species, like Malassezia, also increased in pancreatic adenocarcinoma patients [68]. The oral microbiome can be used for diagnosis [63]. In fact, different risk factors of pancreatic adenocarcinoma are associated with changes to the microbiome, including smoking [103], poor oral health or tooth loss [78], or recurrent pancreatitis. There is a lower bacterial load in pancreatitis than in pancreatic adenocarcinoma.
Besides the direct immunogenicity of the microbiome, an endocrine-like function was also described in several cancers [30,[105][106][107] including pancreatic adenocarcinoma. Bacteria can produce bacterial metabolites that enter the systemic circulation and act on distant cancer cells. This process possesses features of endocrine signaling: a chemical entity is synthesized at one location, then transferred to another anatomical site where it binds to receptors and exerts biological responses there. Hereby, we will review the bacterial metabolites with possible pro-or anti-neoplastic features in pancreatic adenocarcinoma.

Bacterial Metabolites Playing Role in Pancreatic Adenocarcinoma
The gut microbiome harbors a large number of species with an immense and diverse metabolism. Bacterial metabolites or components of bacteria can enter the systemic circulation of the host and be transferred to distant sites where the metabolites can exert hormone-like effects [19,30,108]. Bacterial metabolism is largely dysregulated in pancreatic adenocarcinoma [43]. Below, we will review the source and (possible) roles of pro-or anti-carcinogenic bacterial metabolites ( Figure 1).

Short Chain Fatty Acids (SFCA)
Short chain fatty acids (SCFAs), namely acetate, propionate, butyrate, and lactate, are derived from non-digestible carbohydrates by bacterial saccharolytic fermentation [109,110]. The major SCFAs are acetate, propionate, and butyrate [111]. A smaller quantity of SCFA can be formed by amino acid deamination; this is the only source of branched-chain short chain fatty acids [110]. Hydrolysis, glycolysis, and the pentose-phosphate pathways are the key pathways for SCFA production [111], nevertheless, other pathways are also active. SCFAs are produced in the colon. SCFA production affects the pH of the colon and, hence, modulates the composition of the microbiome in the colon. SCFAs can reduce the proliferation of Enterobacteriaceae (e.g., E. coli, Salmonella ssp., or Clostridia ssp.) and Borrelia burgdorferi [112][113][114][115]. Furthermore, SCFAs can modulate the composition of the gut microbiome through the direct modulation of the immune system [116].
Acetate can ameliorate pancreatitis and its sequels, and, hence, protect against a risk factor of pancreatic adenocarcinoma [145]. Acetate drives the epigenetic reprogramming of mesenchymal stem cells towards cancer-associated fibroblasts that enhance the invasiveness of pancreatic adenocarcinoma cells [146]. Butyrate, at a 2 mM concentration, can reduce the proliferation of cultured pancreatic adenocarcinoma cells (Panc-1 and HPAF cells) and induce differentiation towards a secretory phenotype marked by ultrastructural changes [147]. Furthermore, a hyaluronic acid conjugate of butyrate proved to be cytostatic in a cultured pancreatic adenocarcinoma cell line [148]. Valproic acid, a branched chain synthetic SCFA, was also cytostatic in pancreatic adenocarcinoma cells when given in combination with 5-fluorouracil, suggesting similar properties for bacterial SCFAs [149]. In the pancreatic adenocarcinoma-associated oncobiome, probiotics and butyrate-producing bacteria decreased [43], suggesting that the above-detailed beneficial effects of SCFAs are largely suppressed in the disease.

Secondary Bile Acids
Chenodeoxycholic acid (CDCA) and cholic acid (CA) are primary bile acids, which are mainly synthesized in the liver; however, extrahepatic tissues (e.g., ovaries, macrophages, vascular endothelium, and brain) can contribute to this synthesis [150]. Primary bile acids are conjugated to glycine or taurine and are secreted into the bile, then, via bile, into the duodenum. Hepatic primary bile acids emulsify fats and activate lipases. The microbiome of the gut (mostly in the large bowels) dehydroxylate and deconjugate bile acids. Thus, primary bile acids are modified to produce secondary bile acids, including lithocholic acid (LCA), deoxycholic acid (DCA), and ursodeoxycholic acid (UDCA) [151]. There are 16 bile acids in early life, while in adulthood there are 20 different bile acids in humans [124,152,153]. The majority of bile acids undergo reuptake via the portal circulation and are then transported to the liver, where secondary bile acids are re-hydroxylated and re-conjugated for reuse. This cycle is called the enterohepatic circulation of bile acids. A small fraction of the reabsorbed bile acids can enter the systemic circulation [154] and systemic bile acids exert hormone-like, systemic effects [23,30,[155][156][157].
Bile acid levels were reported to increase in pancreatic adenocarcinoma. A study comprised of 15 patients with pancreatic cancer and 15 patients with benign disease showed increasing trends in all bile acid species detected in pancreatic cancer patients. Increases in unconjugated bile acid levels in pancreatic adenocarcinoma patients were significant and surprisingly large (26 fold) [178].
Most bile acids have a carcinogenic role in pancreatic adenocarcinoma. Bile acids modulate risk factors for pancreatic adenocarcinoma; bile acids impact pancreatitis and bile acid efflux disorders, type II diabetes, obesity, and hyperlipidemia. Furthermore, bile acids reduce susceptibility to apoptosis, induce inflammatory mediators, and may perturb membranes and cellular movement (reviewed in [179]). Gallstones can obstruct the outflow of bile and, hence, can induce and sustain pancreatitis [180], a risk factor for pancreatic adenocarcinoma [179,181,182]. Exposure of pre-malignant pancreas ductal cells to bile may lead to carcinogenic transformation through inflammatory signaling, as demonstrated in rodent and human data [183][184][185][186]. DCA, through binding to TGR5, can activate EGFR, mitogen-activated protein kinase, and STAT3 signaling in pancreatic adenocarcinoma cells, inducing cell cycle progression [187]. Interestingly, there seems to be a selectivity among bile acids, as UDCA inhibits the epithelial-to-mesenchymal transition in pancreatic adenocarcinoma cell lines, and in that regard, acts as an anti-carcinogenic factor [188].
Expression levels for VDR [189], FXR [190], and PXR [191] are higher in tumor tissue than in the normal tissue of the pancreas. LXRβ, but not LXRα, is abundantly expressed in human pancreatic adenocarcinoma cases [192]. In the serum of PDAC patients, components of the LXR/RXR system are enriched [193]. Furthermore, higher FXR expression correlates with higher TNM stage, shorter survival, and poorer prognosis [190]. Higher PXR expression correlated with higher histological grade of pancreatic adenocarcinoma [191]. Nevertheless, unexpectedly, enhanced PXR/RXRβ expression correlated with smaller tumor size and the absence of lymph node metastases and longer survival [191]. Additionally, LXR agonist treatments disrupted proliferation, cell-cycle progression, and colony-formation in PDAC cells [194].

Polyamines
Polyamine metabolism is dysregulated in pancreatic adenocarcinoma [195]. The functional role of polyamine biosynthesis in (human) pancreatic adenocarcinoma is highlighted by the fact that the effects of the standard cytostatic therapies can be accentuated or ameliorated by modulation of the polyamine cycle [196][197][198][199][200]. Cadaverine, putrescine, spermine, and spermidine are classified as polyamines, but bacteria can produce other polyamines also [131,201].
Cadaverine is a decarboxylation product of lysine and the bacterial enzymes LdcC and CadA are responsible for cadaverine biosynthesis [202,203]. Both the human body and bacteria can produce cadaverine. Shigella flexneri, Shigella sonnei, Escherichia coli, and Streptococcus possess enzymes for cadaverine biosynthesis [132]. Putrescine can be derived from arginine through decarboxylation, as is the case in E. coli [108]. Polyamines support bacterial growth and biofilm formation and in many pathogenic species are considered virulence factors [131].
A metabolomic and metatranscriptomic study of the fecal microbiome from a murine pancreatic adenocarcinoma model [67] showed that bacterial polyamine biosynthetic capacity was upregulated and aggravated by tumor progression. The main polyamines synthesized were putrescine, spermine, and spermidine. In accordance with these results, serum polyamine levels were also higher in pancreatic adenocarcinoma-bearing mice and patients. In contrast, Ren and co-workers [43] found that polyamine biosynthesis and transport pathways were downregulated in samples from pancreatic adenocarcinoma patients. Nevertheless, the lysine and putrescine transport systems were upregulated.

Bacterial Lipopolysaccharide (LPS)
Lipopolysaccharides, lypoglycans, or endotoxins are components of the bacterial outer membrane in Gram-negative bacteria [204,205]. Although LPS is not a classical bacterial metabolite in the strict sense, LPS seems to play a crucial role in the pathogenesis of pancreatic adenocarcinoma. Lipopolysaccharides are built upon a lipid anchor to which a polysaccharide chain is attached. The inherent role of LPS is to protect bacteria against toxins, antibiotics, or bile acids. However, LPS has high immunogenic potential and is considered a member of the pathogen associated molecular patterns (PAMPs). LPS elicits its effects through TLR4 and TLR2 receptors to induce innate immunity [204,205].

Tryptophan Metabolites
Tryptophan is an amino acid with a very complex and intricate metabolism, in which bacterial metabolism plays a major role. A considerable portion of tryptophan, 4-6%, is metabolized by bacteria to yield indol derivatives [206]. In germ-free mice, serum tryptophan levels increase, emphasizing the volume of bacterial tryptophan degradation [133,[207][208][209][210]. The bacterial metabolism of tryptophan has multiple branches [133,206], described as follows: (1) The decarboxylation of tryptophan yields tryptamine. Clostridium sporogenes and Ruminococcus gnavus possess enzymes for tryptophan decarboxylation [133]. (2) Tryptophanase deaminates tryptophan to indole pyruvic acid, which is then metabolized to indole. Indole can be further oxidized and the subsequent conjugation of sulphate yields indican. Tryptophanase, denoted as TnaA, can be found in the tryptophanase operon [211]. Tryptophanase expression is widespread among bacteria [212,213]. (3) Indole pyruvic acid can be decarboxylated to indole acetaldehyde. Indole acetaldehyde can be converted to tryptophol or indole acetic acid. Indole acetic acid can be decarboxylated to yield skatole or conjugated with glutamine to yield indole acetic acid-glutamine. The main genera for this pathway are Lactobacillus, Clostridium, and Bacteroides [133]. (4) The reduction of indole pyruvic acid yields indole lactate, the dehydration of which yields indole acrylic acid. This compound can be reduced to indole propionic acid. Indole propionic acid can be further converted by human enzymes.
Tryptophan-derivatives (indoles) are ligands for the aryl hydrocarbon receptor (AHR) and can also bind to the PXR receptor [214][215][216]. AHR activation is a key element in the regulation of the immune system [133,217]. A tryptophan-poor diet has immunosuppressive effects in an AHR-dependent fashion [218]. By modulating mucosal immunity through AHR, indole derivatives influence the composition of the gut microbiome. For example, indole-derivatives can facilitate the expansion of Lactobacillus reuteri and inhibit the growth of pathogenic bacteria [214,[219][220][221]. Furthermore, Lactobacillus utilizes tryptophan as an energy source [214].
Although direct data are missing for the effects of indole-derivatives in pancreatic adenocarcinoma, the invasive behavior of pancreatic adenocarcinoma cells can be modulated through the selective AHR modulators, Omeprazole and Tranilast [222]. Furthermore, as we noted above, a higher PXR expression correlates with a higher histological grade of pancreatic adenocarcinoma, while enhanced PXR/RXRβ expression correlates with a smaller tumor size, the absence of lymph node metastases, and longer survival [191].

Other Metabolites
To date, two studies reported in silico reconstruction of metabolic pathways of the microbiome in pancreatic adenocarcinoma. These data identify bacterial metabolites that potentially influence pancreatic adenocarcinoma cells or carcinogenesis itself, thus, we review these data and provide a list in Table 3.
Mendez and colleagues [67] reported a time course experiment using a murine model of pancreatic adenocarcinoma. The results of this experiment revealed that the microbiome in pancreatic adenocarcinoma shifted towards nucleotide, lipid, and polyamine biosynthesis that was accentuated during the progression of the disease. Increased polyamine biosynthesis was confirmed by direct measurement of polyamines in the serum of tumor-bearing mice and healthy controls and pancreatic adenocarcinoma patients. In addition, hexitol fermentation, carbohydrate metabolism, and vitamin biosynthesis and metabolism were upregulated.
The second study was a prospective study of 85 pancreatic adenocarcinoma patients and 57 matched healthy controls in which the fecal microbiome was assessed [43]. A decrease was observed in various transport systems, amino acid metabolism, and core metabolic pathways in pancreatic adenocarcinoma patients. Among the upregulated pathways were genes for amino acid metabolism, carbohydrate metabolism, transport systems, and metabolic pathways. Lysine arginine ornithine transport system (M00225) Dipeptide transport system (M00324) Arginine transport system (M00229) Histidine transport system (M00226) carbohydrate metabolism Oligogalacturonide transport system (M00202) Entner Doudoroff pathway (M00008) transport systems Putative spermidine putrescine transport system (M00193) Microcin C transport system (M00349) Putrescine transport system (M00300) Sec secretion system (M00335)

Supporting Clinical Decision Making, Diagnostic Applications
As we noted earlier, certain studies assessing the oncobiome in pancreatic adenocarcinoma came to the conclusion that the composition of the oral [63,82,86,88,223], gut [43], pancreatic [102], and fecal [100] microbiomes differ from the corresponding healthy microbiomes. Thus, the microbiomes can be used for diagnostic applications. Diagnostics can be useful for the assessment of the risk for tumor development (i.e., tumor detection) [82], survival prediction [41,91], deducting etiology [100], predicting mortality [89], and selecting between the forms of the disease (e.g., obstructive versus non-obstructive forms [43]).
Most studies mentioned above had low patient numbers (tens of patients to a few hundred) and, therefore, did not reach the level of statistical significance. When already existing shotgun sequencing data of tumor DNA was assessed for the presence of bacterial DNA detected statistically [102], counts of bacteria in tumors differed from the corresponding non-transformed tissues. Importantly, patterns were identified that had suitable specificity and selectivity values for subsequent diagnostic applications. Furthermore, these patterns were detectable in serum in the form of cell-free DNA, suggesting that serum could be used for diagnostic applications [102]. In addition, oral swab or feces can be used as easily accessible biomaterials for the detection of pancreatic adenocarcinoma [82,88,100].
As the detection of pancreatic adenocarcinoma is difficult at early (curable) stages, early detection through detecting oncobiotic transformation has clear advantages for patients. The choice of antibiotics used for premedication before surgical excision of pancreatic adenocarcinoma can also be based on the composition of the pancreatic microbiome [92].

Future Directions
Oncobiosis in pancreatic adenocarcinoma is a complex process, involving multiple microbiome compartments, including the oral, gastric, duodenal, ductal, pancreatic, and fecal compartments. The bacterial colonization of the pancreas drives inflammation and probably facilitates the initiation and progression of the disease to determine the aggressiveness of the disease. Furthermore, there seems to be a large set of bacterial metabolites released into the circulation or tumor microenvironment that has direct effects on the behavior of pancreatic adenocarcinoma cells (Figure 2).  The involvement of bacterial metabolites is just coming of age in the microbiome field. In other cancers, the involvement of the microbiome has gained ground quickly and holds promise for new treatment modalities [224,225]. Studies on the in silico reconstruction of microbiome metabolism and other circumstantial data suggest large changes to the bacterial metabolome, making it likely that such metabolites will be identified and characterized in the future. It is important to note that in studies with bacterial metabolites, metabolites must be used in concentrations corresponding to serum or tissue reference concentrations to avoid the non-physiological effects of supraphysiological concentrations.
Establishing the causative role of oncobiosis in pancreatic adenocarcinoma will facilitate the The involvement of bacterial metabolites is just coming of age in the microbiome field. In other cancers, the involvement of the microbiome has gained ground quickly and holds promise for new treatment modalities [224,225]. Studies on the in silico reconstruction of microbiome metabolism and other circumstantial data suggest large changes to the bacterial metabolome, making it likely that such metabolites will be identified and characterized in the future. It is important to note that in studies with bacterial metabolites, metabolites must be used in concentrations corresponding to serum or tissue reference concentrations to avoid the non-physiological effects of supraphysiological concentrations.
Establishing the causative role of oncobiosis in pancreatic adenocarcinoma will facilitate the assessment of how antibiotics, probiotics, or prebiotics may modulate the behavior of the disease in analogy to other diseases and studies [59,60,[226][227][228]. Dietary intervention or special diets can be proposed to patients [229]. Thus, the study of the microbiome may change personalized medicine [100]. A better understanding of the oncobiome in pancreatic adenocarcinoma holds the promise of prolonging survival in pancreatic adenocarcinoma.
Author Contributions: All authors contributed to data collection and the writing of the manuscript. All authors have read and agreed to the published version of the manuscript.