Microbiome in GIT Cancers: New Frontiers in Personalized Medicine – A Review of Literature

Every person has a characteristic “enterotype,” the gut components and the environment. Recently, a strong biological correlation has emerged among the microbiome of the gut, the immune system, cancer development and pharmacological effects of chemotherapy. In this review, we outline the role of gut microbiota in the gastrointestinal tract (GIT) cancers pathogenesis and their implications for enhancing the efficacy of GIT cancer management in clinical practice. We also summarize the molecular pathways linking gut microbiota and GIT cancers and the effectiveness of manipulating microbiota in GIT cancer therapies such as personalized cancer therapy. © 2020 Maha Saad. Hosting by Science Repository. All rights reserved.


Introduction
Gut microbiome is the total amount of microorganisms, bacteria, viruses, protozoa, fungi, and their totality of genetic material found in the gastrointestinal tract (GIT). The microbiome help digesting the food, regulate, train our immune system, protect against other bacteria that cause disease, and produce vitamins including B vitamins (B12, thiamine and riboflavin) and Vitamin K, which is important for blood coagulation [1]. The gut microbiota has a vital role in the absorption of minerals and nutrients, the production of amino acids and enzymes, and the synthesis of short-chain fatty acids (SCFAs). Propionate, butyrate, and acetate are important for gut health, promote the integrity of the epithelial barrier, and yield immunomodulation and protection against pathogens and supply energy for epithelial cells. These are the byproducts of the fermentation [2].
Autoimmune diseases such as diabetes, rheumatoid arthritis, muscular dystrophy, multiple sclerosis, and fibromyalgia are strongly related to microbiome dysfunction. Disease-causing microbes' accumulation over time causes changing gene activity and metabolic processes and that result in an abnormal immune response against substances and tissues present in the body. Autoimmune diseases appear to be passed in families by inheriting the family's microbiome not by DNA inheritance [3,4].
Gut microbiota in humans is divided into different groups each is called phyla. It consists basically of four major phyla including Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria [5]. However, bacteria settle down in our body, they live in the GIT, skin, oral cavity, vagina, and placenta, most of the bacteria inhabit the GIT, with most of the anaerobic bacteria inhabit the colon. Our bodies have 20,000 eukaryotic genes whereas the gut microbiota has 3.3 million prokaryotic genes [6].
Numerous studies have elucidated that the carcinogenicity is mainly attributed to microbial dysbiosis like chronic inflammation with DNA damage in the epithelium, aberrant DNA methylation, increased interleukin, and tumor necrosis factor-α (TNF-α) levels with the activation of mitogen-activated protein kinases (MAPK) pathway, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and Wnt signaling and, the inhibition of apoptosis, and increase oxidative stress, thus activating proto-oncogenes (e.g., KRAS mutation) also inactivate tumor-suppressor genes (e.g., P53 mutation), triggers a number of innate immune responses involved in the tumor formation process by recognizing the structural components of bacteria, such as lipopolysaccharide (LPS), and flagellin through Toll-like receptors (TLRs) and Nod-like receptors (NLRs) modulating gut microbial composition and enhancing dysbiosis mediated by the inflammasome [10][11][12][13]. Also, B cells, T helper (Th) cells, and T regulatory (Treg) cells participate in tumorigenesis through the adaptive immune system, of note, microbial metabolites like lipoteichoic acid, and short-chain fatty acids (SCFAs) have critical roles in GIT cancer through binding to TLR2 [14,15]. These aforementioned mechanisms may contribute to GIT malignant transformation.
In this review of literature, we have tried to highlight the role of microbiota in the pathogenesis of GIT cancers. Moreover, we have addressed the scientists' efforts to uncover the mystery of oncomicrobiomes as a promising strategy in GIT cancer diagnosis and treatment. We have searched using pub med and google scholar databases using the following keywords, microbiome, GIT, cancer, diagnosis, and treatment in the period between 2000 and 2020. (Table 1) i

Microbiome and Oral Cancer
It is of great importance to determine which bacterial species are responsible for driving oral tumorigenesis. Alterations in the oral commensal microbial sets have potential application in diagnosis, to predict oral squamous cell carcinoma (OSCC ) [16].
The most frequently affected molecular mechanisms by the microbiota to induce carcinogenesis involve epithelial-mesenchymal transition -(EMT) dependent barrier changes and tumor-inducing inflammation [17]. Bacterial infection leads to the emergence of oral cancer and it's the main cause of chronic inflammation that favours mutagenesis, oncogene activation, cell proliferation, and angiogenesis [18]. It leads to increased expression of matrix metalloproteinase-(MMP-1) and (MMP-10) and activation of (EMT) factors Zeb1 Snail, and Slug without requiring inhibition of miR-200b or E-cadherin [19]. Moreover, it increases the expression of the cancer stem cell markers such as CD133 and CD44 [20]. Many bacterial species are involved in the pathogenesis of oral cancer, Mager et al. reported that Fusobacterium periodonticum was found in the saliva of OSCC patients. Capnocytophaga gingivalis, Prevotella melaninogenica, and Streptococcus mitis were much abundant in saliva of OSCC patients and may serve as a potential marker in the diagnosis of oral cancer [21,22]. Infection with human papillomavirus (HPV) is strongly correlated with malignant oral lesions. Yet, it may not be enough for the neoplastic transformation of normal epithelial cells. This could be explained by the high prevalence of HPV infections, the prolonged incubation period between initial HPV infection and the emergence of cancer, and the regression of most HPV-induced dysplasias. Chronic inflammation is suggested to promote oral carcinogenesis with HPV infection. Tumor necrosis factor α (TNFα) which is main mediator of chronic inflammation, reinforces inflammation-associated tumorigenesis by nuclear factor-κB signaling activation, which suppresses precancerous cells' death through the evolution of inflammation-associated cancers in HPV infected cases [27]. The microbiome may offer signatures that can be used as potential biomarkers for early detection, discriminating oral cancer subtypes and predicting clinical behaviour as metastasis and oral cancer recurrences [28]. Using saliva may avoid biopsies and serves a non-invasive, inexpensive, and easily accessible diagnostic and prognosis tool in oral cancer patients [29].
ii Microbiome and Esophageal Cancer

a. Esophageal Adenocarcinoma
It is of high importance to better comprehend the function of the microbiome in esophageal adenocarcinoma. Previous studies used surgically resected samples and culture-based techniques of esophageal adenocarcinoma and squamous cell carcinoma and found the same matching microbiota in both normal and cancerous tissues. Yet, these investigations were mainly interested in recognizing microbes related to infections postoperatively, not in confronting the non-pathogenic bacteria in esophageal cancer patients and control cases, Narikiyo et al. lately used the 16S sequencing technique to distinguish microbiota of both normal and cancerous esophageal tissues [30][31][32]. Cancerous tissue specimens were collected from 20 patients who experienced surgical resection for cancer esophagus.
The oral periodontopathic spirochete Treponemadenticola, Streptococcus anginosus and Streptococcus mitis were dominant in both microbiota, but the pathological classification of the cancers was not given [33]. Blackett et al. used a culture-independent and mixed culturedependent strategies to compare the microbiota in reflux-asymptomatic controls and patients with GERD, Barrett's esophagus, and esophageal adenocarcinoma. Significant enrichment of Campylobacter was noticed in Barrett's esophagus and GERD patient's comparative with the controls and esophageal adenocarcinoma patients. Furthermore, cytokines involved in tumorigenesis (e.g. IL-18) were more up-regulated in tissues colonized with Campylobacter [34].
Considering the potential pathogenicity of Campylobacter species, its role in the emergence of esophageal adenocarcinoma may mimic the Helicobacter pylori role in cancer stomach [35]. The rapport between the esophageal adenocarcinoma development and microbiome has been investigated experimentally. Sawada et al. investigated whether altering microbiome with antibiotics would impact the emergence of esophageal adenocarcinoma in a rat model with esophagojejunostomy [36]. By using terminal restriction fragment length polymorphism analysis, the esophageal microbiomes were found to be different between the two groups; for example, the proportions of Lactobacillales were reduced in the antibiotics group, but Clostridium were elevated in the antibiotics group. Whilst the incidence of esophageal adenocarcinoma wasn't impacted by the alteration of the microbiome. In a rat model with esophagojejunal anastomosis, Zaidi et al. reported a predominance of Escherichia coli in Barrett's esophagus and esophageal adenocarcinoma. This indicates an association between E. coli and TLR signaling pathway, furthermore, TLR 1-3, 6, 7 and 9 were notably up-regulated in esophageal adenocarcinoma comparative with normal epithelium, proposing that these early molecular changes are mediated by microbes in the rat model of esophageal adenocarcinoma tumorigenesis [37].
A significant correlation was found between transcriptionally active HPV, Barret's esophagus, and esophageal adenocarcinoma, proposing a potential role in esophageal tumorigenesis. Moreover, when transcriptional activity markers (E6/E7 mRNA and p16INK4A) and HPV DNA were all positive, a very strong correlation between disease severity along the Barrett's metaplasia-dysplasia-adenocarcinoma sequence was found [38].

b. Esophageal Squamous Cell Carcinoma
Esophageal microbiome, it is more prominent in adenocarcinoma when compares to squamous cell carcinoma (SCC). A study by Yu et al. reported an inverse relationship between the complexity of the microbiota population and the liability to develop squamous dysplasia [39]. Contamination of the esophageal epithelium with Porphyromonas gingivalis was not only implicated in the pathogenesis of esophageal SCC but also in the tumor cells differentiation and metastasis, suggesting it's potential role as a prognostic biomarker [40]. Gastric dysbiosis specially Erysipelotrichales and Clostridiales rich population is implicated in the progression of esophageal squamous cell carcinoma from dysplasia [41]. Catonella, Peptococcus, Moryella and Cardiobacterium responsible for good oral health was less prominent in SCC when compared with healthy controls [42]. Fusobacterium nucleatum, oral microbiota that has been associated with multiple periodontal diseases. Previously linked to colon cancer, Fusobacterium nucleatum, has been abundantly found in 23% esophageal SCC in a study including 325 samples, recently linked to esophageal SCC [43].
Future detailed studies to determine different microbiome populations implicated in the development and progression of SCC paves the road for better understanding of esophageal SCC etiology and pathogenesis and developing de novo therapeutic and preventive strategies against esophageal SCC.
iii Microbiome and Gastric Cancer a.

H. pylori and Gastric Cancer
H. pylori have been adapted to live in the harsh, acidic environment of the stomach. These bacteria reduce its acidity so they can survive and change the environment around them. The spiral shape of H. pylori permits them to penetrate the stomach lining, where they are protected by mucus and thus the body's immune cells are not able to reach them. The bacteria can inhibit the immune response resulting in several gastric problems [8]. The direct impact of H. pylori on tumorigenesis is exerted primarily through two virulence factors: CagA and VacA [44]. H. pylori is estimated to be responsible for 5.5% of all cancer cases and over 60% of gastric cancer cases [45].
Paik et al. found that H. pylori-induced expression of LC3, one of the major markers of autophagy, was more evident than that observed in the cells subjected to starvation for 48 h. Therefore, it is more possible that H. pylori may be one of the strong autophagy inducers like Nrf2 transcription factor that regulates the autophagic signaling pathway by modulating the adaptor protein p62/ SQSTM. These findings suggest the existence of a positive feedback loop between Nrf2 and p62/SQSTM1. One of the key target molecules of Nrf2 is the stress-responsive enzyme, HO-1. These findings elucidate the role of HO-1 in H. pylori-induced autophagy through Nrf2 activation [46].
β-catenin is a proto-oncogene that has been found to accumulate inside the nucleus in precancerous lesions of gastric cancer. H. pylori activates β-catenin expression, such that β-catenin activates its expression via H. pylori as a positive feedback mechanism to induce intestinal metaplasia [47].

b. Epstein-Barr Virus (EBV) and Gastric Cancer
Epstein-Barr virus (EBV) is involved in gastric carcinogenesis as about 9% of gastric tumors harbor monoclonal viral episomes. The Cancer Genome Atlas project identified EBV-positive tumors as one of four molecular subtypes of gastric cancer. EBV-positive tumors are characterized by recurrent PIK3CA mutation, absence of TP53 mutation, JAK2 amplification, and extreme DNA hypermethylation. These serologic patterns are consistent with viral reactivation in the presence of EBV positive tumors as three (anti-VCA, anti-EA-D, and anti-ZEBRA) of the four studied antibodies target proteins expressed during lytic replication. This pattern of viral reactivation could represent lytic phase infection in either EBV-positive gastric cancer cells or benign lymphocytes related to reduced immune function [48].

iv Microbiome and Hepatocellular Cancer
The role of the microbiome in hepatic tumorigenesis is mostly through inflammatory pathways that start by the interaction between the intestinal bacteria, liver, and immune system. The process mainly includes the interaction between Kupffer cells, macrophages, and PAMPs in the liver. In the cascade of eradicating microorganisms, most populations of Kupffer cells and macrophages respond to very low concentrations of PAMPs, endotoxins, or LPS via the activation of NF-κB by binding to TLRs, mostly TLR-4 and -9, and nucleotide-binding oligomerization domain-like receptor (NOD-like receptor). This accordingly leads to a chain of inflammatory reactions that induces inflammation and cytokine release [49,50].
One study by Grat et al. investigated the intestinal microbiota (IM) of 15 patients with HCC undergoing liver transplantation and compared them to 15 patients who did not have HCC but had a similar etiology of cirrhosis and a similar stage. The study reported that HCC was strongly correlated with increased fecal Escherichia coli [51].
Another study assessed liver tissue samples in patients with HCC and found the presence of Helicobacter spp., proposing intestinal translocation as a potential mechanism for carcinogenesis. However, Helicobacter could not be found in patients with viral-induced HCC [52].
In another study, they introduced the dysbiosis (Ddys) index to measure dysbiosis and found that the Ddys was markedly boosted in patients with primary hepatocellular carcinoma comparative with the healthy controls. Additionally, the development of primary HCC is associated with the increase of the Ddys, although no significant difference was detected between patients with different stages of primary HCC, However, The increase in the bacterial genera that were closely correlated with dysbiosis in primary HCC occurs by Ddys. Further studies are needed to verify the application of this index for use in other diseases [53].
In a study by Pinero et al. Patients with HCC showed a more diverse gut microbiome than without-HCC group. First, HCC patients showed specific changes in family members of Firmicutes including a 5-fold decrease in family Leuconostocaceae and a 3-fold increase in abundance of Erysipelotrichaceae comparative with controls. Second, genus Fusobacterium was 5-fold less abundant in HCC patients comparative with controls. Third, the Bacteroides/Prevotella ratio showed an increase in patients with HCC due to the remarkable decrease in the genus Prevotella. This pattern has been correlated with an inflammatory medium and was recognized as HCC potential biomarkers, including genus Odoribacter and Butyricimonas of Odoribacteraceae family that showed an increased abundance, while Lachnospiraceae family genus Dorea showed a decreased abundance in HCC patients [54].
Hepato-tropic viruses, as HCV and HBV, are highly correlated with the emergence of HCC. Hepatic virus-induced hepatotumorigensis is usually a complex process that may comprise the induction of oxidative stress, cellular inflammation, and involvement of signaling pathways, resulting in selective activation of oncogenic pathways and integration of the viral genome into host DNA via host DNA deletion. Without entire viral elimination from the host, uncompromising replication stimulates inflammation that precipitates chronic liver disease and so resembles a risk factor for HCC. Limited information is available about the viruses involved in the dysbiosis of microbiome-mediated HCC. Yet, some studies have proposed that the pathophysiology of viral hepatitis is aggravated by the gut microbiota and may develop to advanced stages of HCC. The incorporation of the gut microbiome highly impacts the liver's immune response, resulting in either viral eradication or persistence [55].

v Microbiome and Pancreatic Cancer
Patients with pancreatic ductal adenocarcinoma (PDAC) showed diverse microbiome changes comparative with healthy controls at several body sites, including oral cavity, pancreatic tissues, GIT using quantitative polymerase chain reaction (qPCR) and16S ribosomal RNA (16S rRNA) gene sequencing [56].
The human oral cavity is colonized by more than 700 different microorganism species, yet the oral microbiota remains relatively stable in healthy individuals [57]. Periodontitis is the most widely recognized infectious condition prompting tooth loss and has been correlated with different malignancies of the pancreas, colorectum and other extraintestinal organs [58][59][60][61][62][63]. Of the known independent risk factors for PDAC are Poor oral health status, pathogenic oral flora, periodontal disease, and tooth loss [64][65][66][67][68][69][70]. Microbiologists have reported the propagation of oral microbes to the pancreas by translocation or dissemination in both animal models and human subjects [71]. Many oral bacteria are involved in PDAC tumorigenesis such as Fusobacterium, Porphyromonas gingivalis (P. gingivalis), Streptococcus mitis (S. mitis) and Neisseria elongata (N. elongata) [72].
HCV and HBV are of the hepato-tropic viruses which cause hepatitis and hepatocellular carcinoma (HCC). Yet, their infection is not limited to the liver and these viruses can be found in extrahepatic tissues, including the pancreas. So, they may have a role in the tumorigenesis of extrahepatic cancers, including PDAC. HBsAg and HBcAg were detected in the cytoplasm of pancreatic acinar cells, and patients with chronic HBV infection showed partially elevated serum and urinary levels of pancreatic enzymes. Researchers detected HBsAg in the pancreatic juice of patients with HBV infection and was correlated with the emergence of chronic pancreatitis, proposing that HBV-related pancreatitis could be a precursor of PDAC. Agreed conclusions drawn from meta-analyses support the positive correlation of PDAC risk with HBV infection, especially for long-lasting persistent infection, chronic/inactive HBsAg carriers and occult infection. Apparently, HBV can replicate apart from infecting in the tumor and non-tumorous pancreatic tissues of PDAC patients. The REVEAL-HBV study reported the association between HBV and PDAC in subjects with higher viral DNA loads (HBV DNA > 300 copies/mL). In patients with HBV infection, researchers confirmed the integration of HBV DNA to pancreatic tissue and pancreatic metastases to the liver. HBV infection was reported to increase the rate of concurrent liver metastasis in patients with PDAC, which could be considered as a prognostic factor. However, some studies reported inconsistent conclusions regarding the correlation between PDAC and the presence of HBV and HCV [73].

vi Microbiome and Colorectal Cancer
Colorectal tumorigenesis is a complex process, affected by genetic and environmental factors with different causative mechanisms. Many of these mechanisms, such as inflammation, immune regulation, metabolism of dietary components and genotoxin production, are strongly related to the gut microbiota and have been comprehensively investigated. We will focus on selected microbiota-associated components in CRC tumorigenesis that are potential for modulation and clinical use [74]. The gut microbiota can influence colorectal tumorigenesis through many mechanisms, including microbial-related factors such as metabolites or genotoxins. Moreover, some bacteria can be directly procarcinogenic (driver bacteria) or proliferate as opportunistic microorganisms in the tumor-associated microenvironment (passenger bacteria) [75].
Some particular bacterial species can trigger incendiary reactions or produce poisons that straightforwardly harm gut cells. For example, Bacteroides fragilis and Enterococcus faecalis produce enterotoxins (for example fragylisin) and reactive oxygen species that cause oxidative DNA harm, prompt aggravation, and harm the epithelial hindrance [76]. Also, B. fragilis modulate the expression of β-catenin and affect cell multiplication [77]. Fusobacterium nucleatum can attack colonic epithelial cells by means of the FadA surface protein, which interfaces with E-cadherin to intervene changes in β-catenin and Wnt signaling [78].
Although the fecal microbiome is just an intermediary for the gut microbiome, this non-invasive methodology has provided vital data of the alterations in the gut biological system related to CRC and is the most regularly utilized inspecting technique in gut microbiome contemplates. Various investigations looking at fecal microbiota from CRC patients and few microbes, for example, Streptococcus gallolyticus, F. nucleatum, Escherichia coli, B. fragilis and E. faecalis, higher level in CRC patients when contrasted with the healthy control. Moreover, Roseburia, Clostridium, Faecalibacterium and Bifidobacterium are commonly lower in CRC patients [79].
By sequencing the V3 V4 region of the 16S rRNA gene, Sheng et al. analysed the fecal samples of healthy controls and patients with CRC. There was a marked variety of fecal gut microbiota in CRC patients compared with the healthy controls. There was a relative richness of Prevotella, Peptostreptococcus and Collinsella, but Escherichia and Shigella was markedly decreased comparative with healthy controls. Moreover, variance in the fecal gut microbiota was investigated in patients with stage I IV CRC and healthy controls. Stage I CRC patients showed marked increase in Collinsella, Peptostreptococcus and Ruminococcus compared to the healthy controls, while stage III CRC patients showed enrichment of Alistipes compared to patients with stage IV. Furthermore, another study found that the genera Coprobacter and Veillonella showed more abundance in the proximal segments than in the distal segments of the colon [80]. Among several bacterial candidates, F. nucleatum stood out as a key marker either when being investigated alone or combined with other bacteria specifically Clostridium symbiosum, C. hathewayi and colibactin-producing clbA+ bacteria. The faecal abundance of F. nucleatum can empower the use of faecal immunochemical test (FIT) to give higher sensitivity and specificity in detecting CRC than using FIT alone [75]. (Figure 1) Four unmistakable measures can be used to modify the impacts of the gut microbiota on anticancer treatment:

II Microbiome and Cancer Treatment
Anti-infection agents, synthetic concoctions with a particular cytotoxicity for at least one bacterial animal types. ii.
Probiotics, living microscopic organisms or different microorganisms that, when directed in satisfactory sums, give a wellbeing benefit. iii.
Prebiotics, no digestible exacerbates that invigorate the development and additionally elements of specific segments of the gut microbiota. iv.
Postbiotics, nonviable results or products of the gut microbiota that apply natural or biological activities in the host [81] .

Figure 1:
Role of microbiota and different modalities in GIT cancer therapy -Probiotics have been tried in animal tumor models for their capacity to prevent carcinogenesis with promising outcomes [87]. Song et al., have used nanoparticles loaded with spores of Bacillus coagulans (BC) for GIT cancer therapy [89]. Treatment with Lactobacillus rhamnosus significantly reduced the incidence of colorectal cancer in rats [90]. Prebiotics have been examined as potential method for inactivating colorectal malignancy [89]. Immunotherapy may change the gut microbiota which can affect interleukin dependent Th1 immune response which improve tumor control with sparing intestinal integrity [98]. Chmielewski et al. used T-cells redirected for universal cytokinemediated killing (IL-18-inducing TRUCK construct) to develop a CEA-directed CAR T-cell that leads to enhanced antitumor activity in pancreatic cancer mice model [102]. The current experiments to engineer probiotic E. coli strains by CRISPR will be effectively new tools in cancer therapy [106]. Enabling the engineering of C. difficile by using the CRISPR tools could potentially be as an interesting probiotics [110]. Studies has been performed in mice to evaluate the efficacy of FMT in decreasing colon cancer with promising results [112].

III Manipulating the Microbiota for Cancer Therapy
Probiotics have been broadly tried in animal tumor models for their capacity to prevent carcinogenesis with promising outcomes. Also, genetically engineered probiotics have been effectively utilized as vectors for the delivery of tumor-associated antigens, immunestimulatory molecules, or chemicals, enzymes, that utmost the toxic side effects of chemotherapy in animal models [82].  [85].
Although prebiotics (e.g. inulin or oligofructose) and postbiotics (e.g. butyrate) have been investigated as potential methods for inactivating colorectal malignancy, yet more studies are needed to reinforce these trials [84]. Furthermore, specific synthetic substances might be effectively used to contain the negative effect of the gut microbiota on the pharmacodynamics of specific chemotherapeutics. As a proof of rule, an intense inhibitor of bacterial (however not mammalian) βglucuronidase has been appeared to shield mice from the intestinal side effects of irinotecan [86].

i Gut microbiota -Immunotherapy Crosstalk in GIT Cancer
The response to immunotherapy in GIT cancer depends on the following factors, genetic makeup of the tumor, how the immune system reacts to it, and regulatory mechanisms as the gut microbiota [81,[87][88][89]. Of note, two research groups highlight the role of gut microbiota in modulating the anticancer activity of CTLA4 and PD1 blockade. Vétizou et al. declared that the gut microbiota composition like the Bacteroides spp. can itself decrease the tumor volume and when combined with immunotherapy it more reduces the tumor size [90]. Furthermore, Lifestyle and immunotherapy may change the gut microbiota which can affect interleukin dependent Th1 immune response which improves tumor control both with sparing intestinal integrity [91]. Moreover, the oral administration of Bifidobacterium when combined with anti-PDL1 therapy nearly stops tumor growth [92]. Immune checkpoint inhibitors (ICIs) therapy is a novel procedure for cancer treatments. It is realized that heterogeneity of gut microbiome may bring about various results of treatment [93].
With the incorporation of genome-editing strategies such as CRISPR/Cas9 and transcription activator-like effector nucleases (TALENs), into synthetic biology, increasingly sophisticated and specific CAR T-cells (autologous T-cells re-directed towards a tumorspecific antigen) are being designed for clinical usage through knockout of a multiplex of inhibitory proteins like PD1, CTLA-4, and β-2 microglobulin, and intrinsic T-cell inhibitory enzymes in hematological and solid tumors [94]. Recently, Chmielewski et al. then used T-cells redirected for universal cytokine-mediated killing (IL-18-inducing TRUCK construct )to develop a CEA-directed CAR T-cell that leads to enhanced antitumor activity in pancreatic cancer mice model [95] .

ii Editing the Microbiome by CRISPR
While the results of the majority of therapeutic strategies that target the microbiota are usually not specific due to restricted comprehension of the ecological interactions which occur within microbes and with the host immune system. Clustered regularly interspaced short palindromic repeats (CRISPRs)and CRISPR-associated (Cas) proteins present a powerful set of tools that will enhance the study of the microbiome and may lead to the development of new techniques to alter them [96].
It may now be recognized as a promising tool to modify probiotic strains for additional therapies [97]. Probiotic E. coli strains have been engineered to express specific antigens, antimicrobial compounds, sensing molecules that regulate pathogen virulence [98]. These recent experiments to modify probiotic E. coli strains will be effectively new tools in cancer therapy [99]. An innovative and potential strategy in the genome editing area is the use of the catalytic dead form of Cas9 (dCas9) fused to a cytosine deaminase or an adenosine deaminase at specific target positions, making the DNA break not required. Kondo et al. modified this technique for E. coli and were able to optimize up to 41 loci [100].
Bifidobacteria and Lactobacilli are the most frequently used probiotic bacteria aside from E. coli. Genetically-engineered Lactobacilli are being optimized as targeted therapies against many diseases including oral mucositis, inflammatory bowel disease and viral and bacterial infections [101]. Taking into consideration that Bifidobacterium is able to replicate in solid tumors, it offers the potential to genetically modify them to produce cancer inactivating compounds [102]. Interestingly, authorizing the engineering of C. difficile by using the CRISPR tools could potentially be an interesting probiotics [103]. Gene silencing with dCas9 (CRISPRi) is much easier to be conducted in bacteria than Cas9mediated genome editing. This has resulted in the prompt and wide adoption of this strategy in various types of bacteria [104].

iii Fecal Microbiota Transplantation (FMT) in GIT Cancer
Some preclinical studies have been performed in mice to evaluate the efficacy of FMT in decreasing colon cancer with promising results, despite lack of control in this procedure because all gut microbiota is transferred with the therapeutic bacteria species [105].

Future Perspective
With increasing the precise characterization of single enteric, dysplastic, and immune cells by using high-throughput technologies like NGS sequencing and mass, many organisms have already been sequenced and become available in several databases. Thus, experimental validation of these preliminary results has become possible. Scientists shed light on "culturomics" to make more microbiota investigable, and translational bioinformatics implementations to facilitate efficient data analysis and integration. Recently, the development of three-dimensional cultures (organoids) has enabled the growth of normal intestinal units that has also been applied to study GIT diseases, gut microbe interactions, and colorectal cancer. Such organoids are amenable to gene editing by CRISPR and high-throughput small-molecule drug testing.
By throwing light on the molecular mechanisms involved in microbiome-based development and progression of GIT cancers, we hope to evolve unprecedented therapeutic strategies by modifying the microbiota. First, by modifying the combination of intestinal microbiota by prebiotics, probiotics, antibiotics, or microbiota transplants. Second, modulate the side effects and efficacy of available chemotherapy and immunotherapy using the manipulation of the microbiota. Furthermore, the microbiota may serve as a potential diagnostic or clinical outcome biomarker in GIT cancers if correct relationship between gut microbiota and poor clinical outcome has been established.

Conclusion
The host and the gut microbiota show a complex relationship that can be unbalanced in a state of gut microbiota dysbiosis secondary to environmental changes that lead to inflammation, modulation of immune checkpoint and GIT cancer. Comprehension of the microbiome composition and the different ways by which microbiome contributes to GIT tumorigenesis will offer new potentials for novel diagnostic, prognostic, and therapeutic tools.

Conflicts of Interest
None.