Microbiota in the Natural History of Pancreatic Cancer: History
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Pancreatic cancer is still burdened with a severe prognosis, despite advances in the diagnosis and surgical management of this disease. The microbiota is a true organ, capable of several interactions throughout the digestive system. Microbiota can influence the development of precancerous disease predisposing to pancreatic cancer (PC). At the same time, neoplastic tissue shows specific characteristics in terms of diversity and phenotype, determining the short- and long-term prognosis.

  • oral microbiota
  • pancreatic microenvironment
  • chronic pancreatitis (CP)
  • autoimmune pancreatitis (AIP)
  • pancreatic cystic neoplasm (PCN)

1. Introduction

Pancreatic cancer (PC) is an aggressive disease with an increased worldwide incidence. Currently, it is the fourth cause of cancer-related deaths, but it is expected to become the second one by 2030 [1]. Pancreatic ductal adenocarcinoma (PDAC) is the histotype corresponding to approximately 90% of cases of pancreatic malignancy and originates from pancreatic ducts. The current standard of care for patients with PDAC consists of curative surgery, but only 20% of PDACs are diagnosed within resectability criteria [2]. Therefore, the current overall prognosis remains poor (5-year survival at 9%) [3,4].
Only 10% of PDACs are linked to genetic mutations such as BRCA2, STK11/LKB1, CFTR, and PRSS1 or to familial syndromes such as Von Hippen-Lindau disease (VHL), multiple endocrine neoplasia syndrome type 1 (MEN-1), and neurofibromatosis type 1 (NF-1). Accordingly, the majority (90%) of PDACs are sporadic and related to several risk factors such as age, gender, alcohol, smoking, obesity, and lack of physical activity [5].
The human microbiota consists of the totality of commensals (archeobacteria, bacteria, fungi, and viruses) within our body and above all our gut [11]. This complex structure represents a real continuous changing organ related to multiple factors such as diet, drugs, old age, and even mental status [9,12]. Growing evidence has suggested an independent relationship between microbiota dysbiosis and pancreatic diseases such as chronic and autoimmune pancreatitis (CP, AIP), pancreatic cystic neoplasms (PCNs), and even PDAC [1,13].
In addition to the involved microorganisms themselves, there are also several relationships with respect to the molecular contribution encoded by the commensals. Metabolites derived from the microbiome could influence molecular processes in cells, including in pancreatic ones. Several mechanisms linked to PDAC oncogenesis have been involved in chronic flogosis promotion through Toll-like receptors (TLRs) and nuclear factor kappa B (NF-kB) cascade activation [4] by lipopolysaccharide (LPS) release. Moreover, polyamines could be able to directly promote the development of PDAC because they are necessary for cell growth; indeed, elevated polyamine concentrations have been detected in animal models of PDAC, whereas trimethylamine-N-oxide (TMAO) and its derivatives could be able to influence indirectly the onset of PDAC, promoting metabolic syndrome, obesity, and a chronic subacute inflammatory state [17,18,19]. Short-chain fatty acids (SCFAs), in particular butyrate, could modulate immunoregulation, promoting antimicrobial peptide (AMP) production in pancreatic cells, which results in a pro-inflammatory status [20].

2. Microbiota Pancreatic Diseases and Pancreatic Oncogenesis

The relationship between pancreatic diseases and gut microbiota (GM) is determined by the interaction among the immune system, inflammatory state, and dysbiosis [28]. Dysbiosis is characterized by a decrease in bacterial species diversity and an imbalance between bacteria with pro-inflammatory and anti-inflammatory features that can influence immunological equilibrium, e.g., an increase in segmented filamentous bacteria linked to higher levels of Th1 and Th17 cells instead of a decrease in SCFA-producing bacteria connected to higher levels of Treg cells [29]. Moreover, this compromises the integrity of the mucosal barrier with subsequent bacterial translocation, which causes the development of gastrointestinal diseases, including pancreatic diseases [30].
Normally the GM and the immune system work in symbiosis to maintain human body homeostasis, regulating the processes of cell proliferation and the vascularization, as well as blocking the excessive growth of pathogens [31,32,33,34,35]. Once homeostasis is interrupted, some microorganisms appear to be able to translocate and colonize the pancreas through the gastrointestinal lumen and blood circulation, inducing the activation of the pattern recognition receptors (PRR) of the innate immune system that are present in pancreatic acinar cells [36,37]. Secretion of pro-inflammatory cytokines and activation of the immune system is subsequently triggered. As a final result, an inflammatory process is created that, over time, may be capable of initiating pancreatic oncogenesis [38,39,40].
The role of inflammation in creating a favorable environment for the onset of cancer has been widely confirmed over the years, but the underlying molecular mechanisms are still unclear [41]. Ren et al. confirmed the role of chronic inflammation and oxidative damage in the development of pancreatic cancer. They compared the microbiota in 57 healthy people and 85 pancreatic cancer patients, highlighting in the latter an increase in lipopolysaccharide (LPS)-producing bacteria (including Prevotella; Hallella; Enterobacter; and other pathogens such as Veillonella, Klebsiella, and Selenomonas). In parallel, a reduction of different commensals and butyrate-producing bacteria was highlighted. Evidence of an increase in LPS-producing bacteria confirms the role of dysbiosis and oxidative damage in inducing chronic inflammation through the production of pro-inflammatory cytokines and activation of the NF-kB pathway [42].

2.1. Microbiota and Chronic Pancreatitis (CP)

CP is a fibro-inflammatory disease characterized by progressive destruction of the pancreas acinar and islet cells that are replaced by fibrous scar tissue. The perpetuation of the inflammatory stimulus causes progressive and irreversible damage to the pancreatic tissue that puts patients at risk of developing pancreatic cancer. The causes of CP are numerous, and the most common are alcohol; smoking; and metabolic, autoimmune, and genetic diseases; however, the pathogenesis of CP still remains unclear today [48].
The disease manifests with a highly variable clinical picture and evolution, which over time leads to the loss of the exocrine and endocrine function of the pancreas. Exocrine pancreatic insufficiency (EPI) manifests itself with symptoms such as diarrhea, flatulence, and abdominal bloating that may be caused by a state of dysbiosis of the small intestine [49,50]. In fact, it is well known that roughly one-third of CP patients undergo the development of small intestinal bacterial overgrowth (SIBO), a syndrome characterized by excessive growth of GM that leads to an immeasurable fermentation and inflammation of the small intestine [51]. The high frequency of SIBO in CP patients appears to be a consequence of reduced intestinal motility, reduced pancreatic synthesis of AMP, impaired formation of chyme in the lumen of the intestine, and reduced alkalization resulting from the poor pancreatic secretion of bicarbonate. This bacterial overgrowth in the small intestine is thought to exacerbate EPI and underlie the symptoms, malnutrition, and morbidity of CP patients [40,52].
Frost et al. analyzed the GM composition and diversity in CP patients, and they found strong dysbiosis with reduced GM diversity that appeared independent of exocrine pancreatic function. In detail, Enterococcus overgrowth, an opportunistic pathogen that makes the CP patient at greater risk of systemic infections, was observed. The study also highlighted an abundance of facultative pathogens, such as Streptococcus and Escherichia-Shigella, and a reduction of Faecalibacterium and Fusicatenibacter, bacteria that exert an important anti-inflammatory action through the production of SCFA [49,54].
Jandhyala et al. analyzed the composition of the GM of 30 CP patients by comparing it with that of 10 healthy controls. The study showed in CP patients, especially in those with endocrine insufficiency and EPI, an important reduction of Faecalibacterium prausnitzii, a commensal producer of SCFA that performs important anti-inflammatory functions. Therefore, Faecalibacterium prausnitzii would seem to represent a factor capable of promoting the onset of diabetes and its worse progression. Similarly, a reduction in Bifidobacterium and Ruminococcus bromii was found, also responsible for an impaired glucose metabolism [52].

2.2. Microbiota and Autoimmune Pancreatitis (AIP)

AIP is a form of chronic pancreatitis sustained by a fibro-inflammatory process on an autoimmune basis, characterized by inflammatory lymphoplasmacytic infiltrate, fibrosis, and consequent organ dysfunction. The activated lymphocyte infiltration is mainly localized around the pancreatic ducts with subsequent periductal fibrosis that obliterates the lumen and causes an obstruction to the outflow of the pancreatic secretion. Two types of AIP are recognized: (1) type 1, characterized by high serum levels of IgG4 immunoglobulins (IgG4-RD), systemic involvement, extra pancreatic lesions, and a histopathological pattern of lymphoplasmacytic sclerosing pancreatitis; (2) type 2, characterized by a histopathological pattern of idiopathic ductocentric pancreatitis, in the absence of systemic involvement and without extra pancreatic lesions [59].
Although genetic factors are considered behind AIP, the pathogenesis of the disease remains unknown. However, even in this context, a strong correlation between microbiota, the innate immune system, and autoimmune diseases is emerging.
In fact, although AIP and IgG4-RD are characterized by a production of IgG4 Ab, with involvement of adaptive immunity, recent studies highlight the role of innate immunity in the development of the disease [60], as demonstrated by an increased expression of TLR in the pancreas of AIP patients [46,61]. GM may contribute to the activation of the innate immune system. In fact, numerous studies have shown that the activation of the immune system in AIP is a consequence of some microbial antigens and that LPS of Gram-negative bacteria can activate the immune response through TLRs. There are several TLRs involved in the development of AIP (TLR2, TLR3, TLR4, TLR5, and TLR7) and, among these, the most implicated are TLR3 and TLR7 [45,62]. All this induces a subsequent activation of antigen-presenting cells (APC), such as M2 macrophages and pancreatic dendritic cells (pDC), which ends with the triggering of pro-inflammatory cytokine responses.

2.3. Microbiota and Pancreatic Cystic Neoplasms (PCNs)

PCNs represent a clinically complex entity and they are characterized by variable biological behavior. PCNs are divided into various types: serous cystadenoma (SCA), mucinous cystic neoplasm (MCN), intraductal papillary mucinous neoplasm (IPMN), solid-pseudopapillary neoplasms (SPN), and cystic pancreatic neuroendocrine tumors [68]. These cysts have a potential for neoplastic transformation, with a greater risk for MCN, especially for IPMN. The latter are the most common PCNs, and they are epithelial cysts, characterized by the proliferation of mucinous cells that create papillary projections within the pancreatic ducts. They can present a highly variable biological aggressiveness, ranging from low-grade dysplasia (LGD) to high-grade dysplasia (HGD) up to transformation into invasive carcinoma [69,70].
Li et al. studied the pancreatic cyst fluid, obtained by endoscopy, to evaluate the presence of bacterial DNA and to analyze the kinds of bacteria present inside it. The study highlighted the presence of a bacterial ecosystem consisting mainly of Bacteroides spp., Escherichia/Shigella spp., Acidaminococcus spp., and the less abundant Staphylococcus spp. and Fusobacterium spp. Finally, Hp was marginally detected in the cystic fluid. Therefore, these results underlined the presence of bacteria with already known pathogenic functions in the gastrointestinal system that could therefore be involved in the development of pancreatic carcinogenesis [72].

3. Microbiota and Pancreatic Cancer (PC)

The human microbiota is represented by 10–100 trillion microorganisms (archeobacteria, bacteria, fungi, and viruses) inhabiting our body and in particular the gut. A relationship between dysbiosis and human pathology seemed to be linked to a lower bacterial species diversity and an imbalance between bacteria with pro-inflammatory and anti-inflammatory features but, to date, it remains difficult to highlight common modifications and mechanisms for every disease [78,79].
To date, mounting evidence has suggested an independent relationship between microbiota dysbiosis and PDAC development via chronic flogosis stimulation and oncogene (e.g., KRAS) upregulation, as mentioned below in the text [1]. Although several studies have established the presence of microbiota within the pancreas, the exact mechanisms by which microbiota could reach the pancreas are still unknown. It is possible that microbiota could migrate from the upper and lower gastroenteric tract to the pancreas through the major ampulla in the duodenum or via the mesenteric venous and lymphatic system from the gut [16,17]. In the last assumption, a defective intestinal permeability (e.g., caderine unit downregulation) and microbe translocation are supposed to be the result.
The first commensals linked to a possible role in human pancreatic cancer were part of the oral microbiome [2,80,81]. It is now established that the association between poor oral health and the development of PC is well supported in that inflammation of the gingiva, periodontal disease, and tooth loss represent independent risk factors for PDAC [82,83,84,85,86].
Indeed, in the near future, such variations in salivary microbiota from patients with PDAC could support the possibility of using salivary microbial biomarkers for systemic disease prediction. In this context, P. gingivalis and A. actinomycetemcomitans were associated with a higher risk of PC, while conflicting evidence has been found regarding Fusobacteria genera that were decreased in some studies but increased in others [88]. To examine the relationship between host immune response, oral microbes, and pancreatic cancer risk, Michaud et al. measured antibodies to oral bacteria in blood samples from 405 PC cases, before their diagnosis, and 416 matched healthy controls [87]. They found that patients with high levels of P. gingivalis antibodies (>200 ng/mL) had a twofold higher risk of PC (OR 2.14; 95% Cl 1.05, p = 0.05). Detection of antibodies against these oral bacteria could be utilized as a biomarker to identify people with a high risk of PC, but the impact of P. gingivalis on pancreatic oncogenesis, if any, to date is unknown. Whereas the previous studies relied on the oral microbiome, increasing evidence has associated changes in the gut microbiota with pancreatic diseases as well [20].

4. How the Microbiome Could Guide Systemic Therapy

4.1. Chemotherapy

The gut and intratumoral microbiome has emerged as an important factor in the treatment of PC at various levels, as it can play a role considering chemotherapy resistance, efficacy, and toxicity. The link between gut microorganisms and chemotherapy is bidirectional since they can be responsible for deep changes in the commensals’ profile.
Systemic therapy of pancreatic cancer is a cornerstone. Chemotherapy is still the main treatment adopted in cases of metastatic or locally advanced disease, whether neoadjuvant or adjuvant. It is a heavy and often poorly tolerated therapy. Gemcitabine is the main first-line drug for solid digestive tumors, including PDAC chemotherapy. There is various evidence in animal models that there is a microbiological basis for a different response to gemcitabine [123]. 
Fluoropyrimidines such as 5-fluorouracil (5-FU), capecitabine, or TAS-1 are often co-administered with gemcitabine, leading to toxicity and their being limited by resistance. A first study conducted on mouse models in Taiwan systematically analyzed the effects and safety of Lactobacillus strains on 5-FU-induced mucositis and assessed positive changes in the intestinal microbiota after probiotic intervention in terms of abundance and diversity [126]. 
Oxaliplatin is a third-generation platinum-based drug approved for PDAC first-line treatment. Dysbiosis was linked to altered response, even to this therapy. The disruption of the microbiota was shown to compromise the response of subcutaneous tumors to platinum chemotherapy in a mouse model, and furthermore microbiota mediated its effects by modulating myeloid-derived cell functions in the tumor microenvironment [131].

4.2. Immunotherapy

Growing evidence from animal models and human studies indicates that the gut microbiota may have a role not only in the occurrence of complications after pancreatic surgery but also in the effectiveness of novel targeted immunotherapies [134]. Immunotherapeutic approaches that are currently under examination for PC regard immune checkpoint inhibitors (ICIs), vaccine therapy, adoptive cell transfer, myeloid-targeted therapy, immune agonist therapy, and combinations with chemoradiotherapy or other molecularly targeted agents; in actuality, thus far, treatment with ICIs has been unsuccessful in PC [135,136,137].
The expression pattern of ICI (such as programmed cell death protein 1 (PD-1), programmed cell death protein ligand 1 (PDL-1), and citotoxyn T-lymphocyte-associated protein 4 (CTLA4)) in the PC microenvironment is not well understood, and several clinical trials are nowadays focusing on the characterization of the microbiome and its role in PC in the immunological field. One of these regards the use of pembrolizumab (NCT03637803) in association with lyophilized bacteria, showing a restricted response in otherwise ICI-refractory metastatic lung, renal, and pancreatic cancer. 
Bacteria can exercise both positive and negative reactions on the immune response. For example, Bacteroidetes spp. were shown to activate Th1 immune responses, and Listeria monocytogenes altered tumor associated macrophages from one immunosuppressive phenotype to another. The immune reaction in oncological treatment has been shown to be improved by the inhibition of T-reg cells through Bifidobacterium adolescentis, Enterococcus faecium, Collinsella aerofaciens, and Parabacteroides merdae [143].
Although the greater microbial diversity could profoundly affect carcinogenesis and the development of immune anti-tumoral response through the inflammatory activation of tumor-infiltrating immune cells, its role is not entirely clear and remains to be explored specifically in PC [131,147]. Future investigations will determine if a similar mechanism can be used by the tumor microbiota to modulate the immune system by improving or impairing the immune response against the tumor [145]. Possible future scenarios are the intentional manipulation of the gut microbiota in association with therapeutic approaches [143].

4.3. Faecal Microbiota Transplantation (FMT)

In this perspective, FMT could be the peak of this approach in counteracting dysbiosis [144]. A single pilot study is registered (NCT04975217) and is actually recruiting patients with the primary objective of assessing the safety, tolerability, and feasibility of FMT in respectable PDAC patients. The intervention is scheduled as FMT during colonoscopy and FMT capsules via OS once a week for 4 weeks before surgery. They will be followed up to 6 months after surgery to determine immunological/molecular changes, as well as to assess changes in the gut, oral, and intra-tumoral microbiome. There is also emerging evidence suggesting the beneficial role of FMT to improve immunotherapeutic outcomes in cancer patients. However, their mechanisms in enhancing or attenuating the efficacy of immunotherapies need to be identified. Through FMT or supplementation with certain prebiotics, probiotics, or antibiotics, the gut microbial composition could be manipulated to enhance host anticancer immunity and combat drug resistance.

This entry is adapted from the peer-reviewed paper 10.3390/cancers15010001

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