**2. The GI cancer microenvironment**

#### **2.1 The gastrointestinal tract**

The gastrointestinal tract starts at the mouth, extends to the anus and includes the oesophagus, stomach, small intestine, large intestine, liver and pancreas. Its main functions are primarily the disruption and digestion of food, absorption of nutrients and elimination of waste products. With the diverse functions of the GI tract, it is not unsurprising that it has a number of diverse environments which are contributed to by various types of immune cells and the multiple bacteria that reside in the GI tract.

Movement of food down the GI tract is facilitated by muscular contractions. Much of the tube that makes up the GI tract is muscle lined to enable this to occur, with sphincters at particular junctures to enable control of food passage. The muscle layers are coated by a mucous membrane which varies depending on the function of that section of the GI tract.

The epithelium that lines the GI tract can be broadly divided into three subtypes, primarily based on their function. Squamous epithelium is found at the start (mouth and oesophagus) and end (anus) of the GI tract providing a protective covering. Secretory epithelium is found in the stomach. Absorptive epithelium is found in both the small and large intestines. The small intestine has numerous fingerlike projections, called villi, that increase the surface area to facilitate absorption of nutrients with interspersed crypts, or glands, which contain the stem cells that give rise to the epithelial cells. The absorptive epithelium of the large intestine is more closely packed with glands specialised for water absorption and mucus-secreting cells to lubricate the passage of faecal material down the GI tract.

The tube that forms the GI tract has a number of layers that lie between the outer muscular wall (the muscularis propria) and the innermost epithelium. The epithelium forms the innermost layer of the mucosa, which has two additional components, the lamina propria (composed of supportive connective tissue) and a thin layer of smooth muscle, the muscularis mucosae. Underneath the mucosa is the submucosa, which contains connective tissue, nerves and lymphatic and blood vessels. The submucosa is surrounded by the outer muscularis propria, the muscle layer whose contractions facilitate passage of material down the GI tract. The supporting tissue surrounding the GI tract is called the adventitia or serosa and contains major nerves and blood vessels.

**77**

*The Influence of Microbial Metabolites in the Gastrointestinal Microenvironment on Anticancer…*

Our lifestyle, including diet, exercise, childhood microbial exposure and the use of antibiotics strongly, influences the composition of our microbiota [9, 11–14]. Two phyla of bacteria dominate the human gut *Bacteroidetes* and *Firmicutes*. Over decades the ability to classify bacteria into their genus and species has evolved with technology resulting in numerous reclassifications. Bacteria can be additionally classified into subspecies on the basis of small but relevant differences within a species. Further classifications into strains or serovars, indicating variable immune antigens present on their surface, can be allocated outside nomenclature rules. This level of complexity demonstrates the purpose of studying microbial metabolites in the context of gut immunity, thereby avoiding the complexities of bacterial species,

Epidemiological data initially made links between bacteria and cancer development. However, identifying the role of bacteria in cancer development has been challenging due to the importance of host factors in cancer susceptibility combined with the ubiquitous nature of bacteria and the prolonged period between introduction of a bacterium and development of overt cancer [15]. This is further complicated by environmental factors which are thought to play a much larger role than genetic makeup in determining the makeup of an individual's microbiota [16].

While the knowledge of outcomes from bacterial interactions with human cells is growing, there is enormous potential for further discovery when accommodating other microbes that populate different levels of the gastrointestinal tract such as

Gastrointestinal (GI) cancers are as diverse as the environments of the GI tract and the various cell types found in the GI tract. Squamous cell carcinomas arise in the squamous epithelium of the oral cavity, oesophagus and anus. Those that arise in the oral cavity are considered head and neck cancers rather than GI cancers. Adenocarcinomas are cancers that arise from glandular epithelium and can arise in the oesophagus, stomach, small intestine, pancreas and large intestine. Other cancers that can arise from the GI tract include cholangiocarcinoma, with origin from bile duct cells; hepatocellular carcinoma (HCC), originating from hepatocytes (liver cells); gastrointestinal stromal tumours, originating from the interstitial cells of Cajal which have a role in the control of peristaltic contractions [18]; and neuroendocrine cancers which can arise from neuroendocrine cells throughout the GI tract. Multiple studies examining these GI cancers have demonstrated diverse molecular alterations within cancers that arise from the same cell type in the same

The GI tract includes two large glands, the liver and pancreas. Both develop from the primitive foregut embryonically and have functions that contribute to digestion by generating digestive fluids. The liver produces bile, which can be stored and concentrated in the gallbladder. When lipids enter the duodenum, neuroendocrine cells of the duodenal mucosa are stimulated to release cholecystokinin-pancreozymin (CCK) causing contraction of the gallbladder releasing bile into the duodenum. Bile acids are emulsifying agents which aid in lipid digestion. Pancreatic secretions reach the duodenum via the pancreatic duct and contain a high content of alkaline bicarbonate ions which assist in neutralising the acidic fluid that has come from the stomach. The pancreas also produces a number of enzymes including trypsin, chymotrypsin, amylase, lipase and carboxypeptidases which are involved in the

*DOI: http://dx.doi.org/10.5772/intechopen.88137*

breakdown of proteins, carbohydrates and lipids.

focusing instead on their metabolic output.

fungi and viruses [17].

**2.3 Gastrointestinal cancers**

**2.2 Microbiota in the GI tract**

*The Influence of Microbial Metabolites in the Gastrointestinal Microenvironment on Anticancer… DOI: http://dx.doi.org/10.5772/intechopen.88137*

The GI tract includes two large glands, the liver and pancreas. Both develop from the primitive foregut embryonically and have functions that contribute to digestion by generating digestive fluids. The liver produces bile, which can be stored and concentrated in the gallbladder. When lipids enter the duodenum, neuroendocrine cells of the duodenal mucosa are stimulated to release cholecystokinin-pancreozymin (CCK) causing contraction of the gallbladder releasing bile into the duodenum. Bile acids are emulsifying agents which aid in lipid digestion. Pancreatic secretions reach the duodenum via the pancreatic duct and contain a high content of alkaline bicarbonate ions which assist in neutralising the acidic fluid that has come from the stomach. The pancreas also produces a number of enzymes including trypsin, chymotrypsin, amylase, lipase and carboxypeptidases which are involved in the breakdown of proteins, carbohydrates and lipids.

### **2.2 Microbiota in the GI tract**

*Current Cancer Treatment*

particular anticancer immunity.

**2.1 The gastrointestinal tract**

reside in the GI tract.

that section of the GI tract.

nerves and blood vessels.

**2. The GI cancer microenvironment**

Microbial metabolites, as the sum of products modified and synthesised by microbiota, can be a useful tool to understand microbiota-driven immune modulation when analysis of bacterial lineages proves difficult. Diversity and abundance of microbial communities varies greatly amongst healthy individuals, whereas metabolic pathways are conserved and stable [9, 10]. Therefore, assessing changes of metabolic pathways and how they affect immunity may provide crucial insights into the role of the GI microenvironment in health and disease. Microbial metabolites are commonly divided into three categories, (1) metabolites produced by bacteria, derived from host products; (2) metabolites modified by bacteria, derived from

In the following sections, we will briefly describe GI cancers and components of the GI tract that shape the tumour microenvironment. Furthermore, we will discuss the evidence for connecting changes in the microbiome and its metabolites with carcinogenesis and the role of bacterial metabolites in shaping immunity and in

The gastrointestinal tract starts at the mouth, extends to the anus and includes the oesophagus, stomach, small intestine, large intestine, liver and pancreas. Its main functions are primarily the disruption and digestion of food, absorption of nutrients and elimination of waste products. With the diverse functions of the GI tract, it is not unsurprising that it has a number of diverse environments which are contributed to by various types of immune cells and the multiple bacteria that

Movement of food down the GI tract is facilitated by muscular contractions. Much of the tube that makes up the GI tract is muscle lined to enable this to occur, with sphincters at particular junctures to enable control of food passage. The muscle layers are coated by a mucous membrane which varies depending on the function of

The epithelium that lines the GI tract can be broadly divided into three subtypes, primarily based on their function. Squamous epithelium is found at the start (mouth and oesophagus) and end (anus) of the GI tract providing a protective covering. Secretory epithelium is found in the stomach. Absorptive epithelium is found in both the small and large intestines. The small intestine has numerous fingerlike projections, called villi, that increase the surface area to facilitate absorption of nutrients with interspersed crypts, or glands, which contain the stem cells that give rise to the epithelial cells. The absorptive epithelium of the large intestine is more closely packed with glands specialised for water absorption and mucus-secreting

The tube that forms the GI tract has a number of layers that lie between the outer muscular wall (the muscularis propria) and the innermost epithelium. The epithelium forms the innermost layer of the mucosa, which has two additional components, the lamina propria (composed of supportive connective tissue) and a thin layer of smooth muscle, the muscularis mucosae. Underneath the mucosa is the submucosa, which contains connective tissue, nerves and lymphatic and blood vessels. The submucosa is surrounded by the outer muscularis propria, the muscle layer whose contractions facilitate passage of material down the GI tract. The supporting tissue surrounding the GI tract is called the adventitia or serosa and contains major

cells to lubricate the passage of faecal material down the GI tract.

host products; and (3) metabolites synthesised by bacteria directly.

**76**

Our lifestyle, including diet, exercise, childhood microbial exposure and the use of antibiotics strongly, influences the composition of our microbiota [9, 11–14]. Two phyla of bacteria dominate the human gut *Bacteroidetes* and *Firmicutes*. Over decades the ability to classify bacteria into their genus and species has evolved with technology resulting in numerous reclassifications. Bacteria can be additionally classified into subspecies on the basis of small but relevant differences within a species. Further classifications into strains or serovars, indicating variable immune antigens present on their surface, can be allocated outside nomenclature rules. This level of complexity demonstrates the purpose of studying microbial metabolites in the context of gut immunity, thereby avoiding the complexities of bacterial species, focusing instead on their metabolic output.

Epidemiological data initially made links between bacteria and cancer development. However, identifying the role of bacteria in cancer development has been challenging due to the importance of host factors in cancer susceptibility combined with the ubiquitous nature of bacteria and the prolonged period between introduction of a bacterium and development of overt cancer [15]. This is further complicated by environmental factors which are thought to play a much larger role than genetic makeup in determining the makeup of an individual's microbiota [16].

While the knowledge of outcomes from bacterial interactions with human cells is growing, there is enormous potential for further discovery when accommodating other microbes that populate different levels of the gastrointestinal tract such as fungi and viruses [17].

#### **2.3 Gastrointestinal cancers**

Gastrointestinal (GI) cancers are as diverse as the environments of the GI tract and the various cell types found in the GI tract. Squamous cell carcinomas arise in the squamous epithelium of the oral cavity, oesophagus and anus. Those that arise in the oral cavity are considered head and neck cancers rather than GI cancers. Adenocarcinomas are cancers that arise from glandular epithelium and can arise in the oesophagus, stomach, small intestine, pancreas and large intestine. Other cancers that can arise from the GI tract include cholangiocarcinoma, with origin from bile duct cells; hepatocellular carcinoma (HCC), originating from hepatocytes (liver cells); gastrointestinal stromal tumours, originating from the interstitial cells of Cajal which have a role in the control of peristaltic contractions [18]; and neuroendocrine cancers which can arise from neuroendocrine cells throughout the GI tract. Multiple studies examining these GI cancers have demonstrated diverse molecular alterations within cancers that arise from the same cell type in the same

organ of the GI tract, highlighting the multitude of malignancies that can arise in the GI tract [19–27].

Chronic inflammation and infection are intimately associated with the development of cancer, with 15% of global cancer cases in 2012 being attributed to a carcinogenic infection [28]. Examples from the GI tract include HCC with hepatitis B and C virus infections contributing to more than 70% of global HCC diagnoses in 2012 [28]. In gastric adenocarcinoma four molecular subtypes were described by The Cancer Genome Atlas (TCGA) in 2014, one is characterised by Epstein-Barr virus positivity and shows extreme DNA hypermethylation [29]. *Helicobacter pylori* (*H. pylori*) is considered a class I carcinogen by the World Health Organization due to the association of chronic infection with the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma, a form of B cell lymphoma in the stomach [30, 31]. The risk of developing gastric cancer with *H. pylori* is dependent on the virulence factors of the strain causing infection, other environmental factors and host genetics [32–34].

In colorectal cancer studies, *Fusobacterium* subspecies were consistently identified as being differentially present in tumour samples; however, these findings are still limited by small sample sizes [35]. A study of the microbiome in a hereditary form of CRC has implicated oncotoxins produced by co-colonisation with *Bacteroides fragilis* (*B. fragilis*) and *Escherichia coli* (*E. coli*) subspecies in mucosal biofilms. Further animal studies have revealed the bacterial synergy involved in carcinogenesis whereby the *B. fragilis* toxin increases expression of the proinflammatory cytokine interleukin (IL)-17 enabling the oncotoxin-producing *E. coli* to invade the mucosa inducing DNA damage in epithelial cells [36].

In addition to bacteria and viruses being implicated in carcinogenesis, parasitic infections have also been implicated in cancer development in the GI tract, with liver fluke infection, particularly *Opisthorchis viverrini,* being associated with the development of cholangiocarcinoma [37].

#### **2.4 Inflammation in the GI tract**

Acute inflammation is an integral part of the host defence against pathogens and tissue damage and is also required for the initiation of beneficial antitumour immunity [38, 39]. In contrast, it is ongoing 'smouldering' inflammation that contributes to tumour development, progression, invasion and metastasis [40]. Low-grade inflammation affects the function of immune cells and promotes an immune-suppressive, tumour-promoting phenotype [41, 42]. This in turn is associated with reduced immune surveillance and clearance of tumour cells by the immune system.

Chronic inflammation can be induced through a variety of mechanisms, including chronic infections [43], autoimmunity [44], metabolic disorders [45] and altered microbiota [46, 47]. In the GI tract in particular, the host immune system has to maintain a delicate balance, and pathogens and malignant cells need to be cleared, whereas normal flora has to be tolerated. Disruption of immune tolerance or dysbiosis may result in loss of epithelial barrier function and overstimulation of immune cells, leading to tissue damage and chronic inflammation.

Conditions associated with recurrent or chronic inflammation, such as inflammatory bowel disease (IBD), have been shown to contribute to the risk of developing small and large intestine cancers [48, 49]. Mechanistically, this has been related to increased stimulation of inflammation-promoting immune cells by altered microbiota [46]. As a result, pro-inflammatory cytokines and chemokines are secreted, attract further immune cells into the tissue and polarise them towards tumour-promoting functions [50–52]. Particularly the presence of pathogenic

**79**

reviewed extensively elsewhere [56–59].

*The Influence of Microbial Metabolites in the Gastrointestinal Microenvironment on Anticancer…*

Cytokines, such as IL-12, promote the development of Th1 cells, which are efficient at secreting IFN-γ and TNF-α, important cytokines for antitumour

environment high in IL-6 and TGF-β, they develop into Th17 cells, which promote autoimmunity and are a negative prognostic marker for colorectal cancer [49]. Th cells can develop into immunesuppressive Tregs in the presence of TGF-β when expressing the transcription factor *FOXP3*. **Mucosa-activated invariant T cells (MAIT cells)** MAIT cells reside at mucosal surfaces in the lung and the intestine [163] and are widely distributed in tissue and the systemic blood circulation [164, 165]. They have innate immune cell features but also express a semi-invariant T-cell receptor, which can recognise antigens presented on a monomorphic MHC class 1-related protein (MR1) expressed by antigen-presenting cells [166]. MAIT cells are first responders to a variety of infections caused by bacteria, fungi and viruses through detection of microbial B vitamin antigens.

T cells reside an

immunity. When naïve CD4+

**Innate lymphoid cells (ILCs)**

and produce IL-17 and IL-22 [109].

ILCs stem from the lymphoid lineage but have innate immune cell characteristics [109]. They are quick responders and contribute to elimination of pathogens and tissue homeostasis by producing a variety of cytokines. Based on their specific cytokine secretion, ILCs are grouped into different classes that resemble their T-cell counterparts, for example, group 3 ILCs (ILC3s) resemble Th17 cells

T-cell subsets, induced by pro-inflammatory cytokines, has been shown to be a predictor of poor prognosis in colorectal cancer patients [53]. Chronic inflammation also contributes to the expansion of oncogenic bacteria thereby re-enforcing disease progression [54]. See **Box 1** for an overview of the immune cell populations

*DOI: http://dx.doi.org/10.5772/intechopen.88137*

DCs are innate immune cells that develop from myeloid precursors in the bone marrow. They are capable of detecting pathogens or tissue disturbances and initiate an inflammatory response. In the tumour microenvironment, they are thought to engulf dead tumour cells and debris and present fragments thereof to T cells, thus initiating anticancer immune responses.

Macrophages are antigen-presenting cells that arise from either circulating monocytes or embryonic progenitors that persist into adulthood, both giving rise to tissuespecific macrophage populations that are capable of self-renewal [186, 187]. Macrophages serve important functions in immunity, cancer, metabolism and tissue repair. Macrophages play an important role in in the antitumour immune response but can also adopt a protumour phenotype in the tumour environment [188, 189].

CTLs express the CD8 receptor and recognise antigens presented on the surface of antigen-presenting cells. Once primed by this encounter, CTLs are capable of recognising the same antigens and kill target cells expressing the antigen. Tumour-specific CTL responses

Th cells express the CD4 receptor and support functions of innate and adaptive immune cells by secreting cytokines. Depending on the environment they encounter, Th cells develop into subsets with a wide range

**Dendritic cells**

**Macrophages**

**Cytotoxic T cells (CD8+ T cells)**

**Helper T cells (CD4+ T cells)**

of functions [190].

are crucial for controlling tumour growth.

involved in intestinal antitumour immune responses.

**3. Microbial metabolites that shape antitumour immunity**

**Box 1.** *Overview of immune cell populations involved in intestinal antitumour immune responses.*

Metabolites produced or modified by bacteria significantly impact health and disease by acting locally on GI tract cells but can also have systemic effects by influencing the function and activation states of immune cells. The 'metabolome' constitutes the sum of small molecules produced by a biological system and is a powerful approach to explore the current condition of that system [55]. Metabolomics refers to the analysis of metabolites using techniques, such as mass spectrometry, nuclear magnetic resonance analysis, high-performance liquid chromatography and gas chromatography coupled with mass spectrometry. Obtained peak patterns can be compared against spectral databases for identification of metabolites. Metabolomics can be combined with metagenomics, investigating the genetic material of the entire community, and metatranscriptomics, exploring which genes are expressed, to increase our understanding of microbiomes. The benefits, disadvantages and technical challenges of these omics techniques are

*The Influence of Microbial Metabolites in the Gastrointestinal Microenvironment on Anticancer… DOI: http://dx.doi.org/10.5772/intechopen.88137*

T-cell subsets, induced by pro-inflammatory cytokines, has been shown to be a predictor of poor prognosis in colorectal cancer patients [53]. Chronic inflammation also contributes to the expansion of oncogenic bacteria thereby re-enforcing disease progression [54]. See **Box 1** for an overview of the immune cell populations involved in intestinal antitumour immune responses.


**Box 1.** *Overview of immune cell populations involved in intestinal antitumour immune responses.*
