Cancer, the deadly disease that sends shivers down the spine of whole humankind, involves abnormal cell growth that can spread throughout the body of an individual; however, not all cancers have the ability to spread itself or metastasize. Global Cancer Observatory (GCO) estimates the number of new cases of cancer inclusive of all kinds will go from 19,292,789 in 2020 to 28,887,940 in 2040. From ages 0–85+ years, including both sexes the numbers are estimated to increase by 59.2% from 2020 to 2040 alone in Asia. In Europe and North America, the rise in all kinds of cancer cases is estimated to be 21% and 37.9% respectively [1]. Cancer development is a slow and multistep process and the initiation depends on several factors. Some of the factors include diet, smoking, consumption of alcohol, physical inactivity whereas the other factors that are on the far side one’s direct management embrace exposure to carcinogenic pollutants, chronic stress, radiation and microbiota within one’s body [2]. In the process of development of cancer, gene-environment interactions play a key role. Exposure to certain environmental factors can influence the susceptibility of developing this deadly disease. Such an environmental factor is the microbiota present inside our body. Humans are often called ‘superorganisms’ due to the presence of a huge variety of microorganisms in our bodies. As the environment changes, the microbiota is subject to change throughout the life of an individual, although it stays quiet stable during the adult life [3]. Microbiota is related to every aspect of our body functions and alteration of which may result in several abnormalities. Every anatomical niche inside a human body shelters its own population of microbes even a tumor microenvironment has its own microbial population. The role of microbiota in cancer development was largely overlooked, until recently the attention of researchers shifted towards it. The arrival of next-generation sequencing techniques has made it relatively easy to study the epigenetic changes and mutations that occur throughout cancer development. Scientific communities from all over the world are trying to figure out the link between microbiota and the development of cancer which can lead us to new ways of preventing, detecting and treating cancer.

The sixth most common cancer kind worldwide is liver cancer, accounting for 830,200 fatalities worldwide in 2020 [4]. The most common primary liver cancer type is Hepatocellular Carcinoma (HCC) [5]. There are several risk factors that cause HCC, namely Hepatitis B (HBV) or Hepatitis C virus (HCV), diabetes, consumption of alcohol, metabolic-dysfunction associated steatotic liver disease (MASLD) and several other genetic diseases also [6, 7]. HCC typically occurs from chronic liver disease (CLD) through stages like fatty liver disease, steatosis, steatohepatitis, fibrosis and cirrhosis [8]. HCC is often detected at advanced stages of the disease where the liver already shows symptoms of dysfunction and failure [5]. Out of all the HCC therapies available, one of the major options is surgical resection for primary or metastatic liver tumors, but the success rate depends on early-stage surgery where chances of metastasis are less [9]. Where surgery is not an option immunotherapy, chemotherapy and targeted therapy are preferable. Sorafenib, a multi-kinase inhibitor having anti-proliferative and anti-angiogenic effects, is one of the first line treatment options. Because of their low cost, Sorafenib and Doxorubicin are the two primary drugs used to treat advanced HCC in under developed countries where instances of liver cancer are more [10, 11]. In case of both primary and metastatic tumors; microwave ablation (MWA), irreversible electroporation (IRE), radiofrequency ablation (RFA), and high-intensity focused ultrasound (HIFU) are the minimal invasive therapies that are available for treatment [12, 13]. Recently FDA has approved five new agents for HCC treatment as first line and second line therapies. Lenvatinib is approved as a line first treatment and other four second line treatment options are Regorafenib, Nivolumab, Pembrolizumab, and Cabozantinib [14]. Application of these drugs is dependent of the stage and dimensions of the tumor. Depending on the condition of the disease, recurrence of cancer and/or therapeutic resistance, combination therapy i.e., using two or more treatment options, is often preferable [15].

If HCC is detected early, there are many different treatment options available, and results are frequently positive, although early detection is very rare in this case. The biomarkers that are unique to HCC include des-gamma-carboxy prothrombin (DCP), alpha-fetoprotein (AFP), and Lens culinaris agglutinin A-reactive fraction of AFP (AFP-L3) [16, 17]. Aldo-keto reductase family is one of the possible novel biomarkers that has been researched to diagnose HCC and forecast its prognosis [18]. Since gut microbiota is related to the development of liver diseases like cirrhosis/fibrosis and cancer, gut microbial changes may act as early indicators of HCC, where disease diagnosis will benefit from the non-invasiveness of gut microbiota [19]. Gut microbiota has been shown to be related with the efficacy of anti-cancer therapies as well as with the adverse effects of radiotherapy, immunotherapy and chemotherapy [20]. Combining the therapeutics approaches with antibiotics, probiotics or prebiotics to modulate the gut microbiota may prove to be fruitful in side effect management or combating cancer as a whole. In this regard, this review aims to explore recent research on the connection between cancer and the microbiome, to shed light upon recent microbiome mediated therapeutic approaches with a special focus on HCC and to point out the little explored or unexplored portions of this newly emerging field of science. We might be on the verge of the advent of a new paradigm in cancer research.

An enormous number of microbes reside within the human body including bacteria, archaea, eukaryotes, and viruses. The collection of all microbes that reside on or within the human body is referred to as the human ‘microbiota’, including the skin, placenta, lung, saliva, oral mucosa, seminal fluid, mammary glands, uterus, ovarian follicles, conjunctiva, biliary tract, and gastrointestinal tract [21]. The term ‘microbiome’ refers to the combined genome of the microbiota [22]. The gastrointestinal (GI) tract has been estimated to shelter more than 10${}^{14}$ number of microorganisms. Compared to the human genome, the microbiome contributes more than 100 times as much genetic content [23]. In 2016, a study by Sender et al. [24], suggested that the total number of bacteria that are present inside the human body is estimated to be ~3.9 $\times$ 10${}^{13}$, with a ratio of almost 1:1 with the total number of cells in the human body. The majority of these microorganisms are bacteria residing within the GI tract. The aggregate of bacteria, archaea, and eukarya residing in the GI tract is termed as the ‘gut microbiota’. The microbiota has co-evolved with the host over the evolutionary time scale which formed a mutually beneficial relationship [25]. Recently, our capacity to scan the gut microbiota has been substantially enhanced by high-throughput and inexpensive sequencing techniques, such as targeting of the bacterial 16S ribosomal RNA (rRNA) gene and shotgun metagenomics [26].

The bacteria living in our guts are protected from their predators and also get a consistent supply of nutrients from our diets which help them survive. In return, the microbiota helps the host in various ways, such as harvesting energy [27], regulating host immunity [28], and maintaining the intestinal barrier, thus restricting harmful organisms from breaching the epithelium and causing illness [29]. The symbiotic bacteria produce disaccharides and monosaccharides by digesting glycans for the host’s and their own source of energy. Symbiotic relationship with these microbes improves our ability to absorb nutrients from our diets and get the most energy out of it [30]. The variation of microbiota at levels of genus, species, and strain is enormous between human individuals [31]. This occurs due to environmental factors namely diet, lifestyle, health situation and host genetics [32]. The microbiota inside a human individual also varies over the lifespan of the individual and in response to diet, use of chemotherapeutics, antibiotics, stress, and several other factors [33, 34, 35]. Gene-environment interaction is the primary reason for human disease susceptibility, and it is now believed that the microbiome is an important factor also. Currently, it is understood that gut microbes primarily fall under the phylogenetic gates of Bacteroidetes, Firmicutes, Proteobacteria, Verrucomicrobia, Actinobacteria, Fusobacteria, and Cyanobacteria, the predominant bacteria being the first and second. The bulk of “good” bacteria in the gut of a human are Firmicutes and Cytophaga-Flavobacterium-Bacteroides [36, 37].

‘Gut dysbiosis’ is referred to as a state where the gut microbial composition of the host gets unbalanced in comparison to a healthy individual. This might trigger inflammatory signaling pathways which can affect the immune system as a whole, rather than being limited to the gut environment. Gut microbiota dysbiosis is linked with many inflammatory and autoimmune conditions including rheumatoid arthritis, multiple sclerosis, type I diabetes, and inflammatory bowel disease [34]. Metagenomics is the tool that scientists use to study the microbiome. However, metagenomic studies are not enough to determine whether a certain microbiome difference causes a disease state or is itself a consequence of it. Thus, manipulating the microbiota in a precisely regulated manner is essential for finding out how the microbiota is connected to a particular disease. Because of this, gnotobiotic mouse models are essential for experiments in this field of research. Completely germfree laboratory mice can be raised maintained in specialized gnotobiotic facilities; if their foods are autoclaved and vital nutrients that the microbiota usually produces are given [38]. Germfree mice show metabolic anomalies such as hypoglycemia, reduced insulin and glycogen levels, as well as resistance to obesity even if it is provided with a high-fat diet. Studies have compared germ-free mice to genetically identical controls that have a complete but unidentified microbiota. Compared with controls, mice that are devoid of germs eat more, but are leaner and have around 35% less fat [39]. It can be said that, these experiments indicate that the host’s capacity to obtain calories is enhanced by the gut microbiota. Additionally, the microbiota is crucial for regulation of the immune system and inflammatory reactions and germfree mice have been employed to show that [40]. Mice lacking transforming growth factor-1 display colorectal cancer and inflammation, which can be prevented by keeping them in a germ-free state [41]. A colitis phenotype is exhibited in IL-10 (interleukin 10, a potent immunosuppressive cytokine) knockout mice and it is reduced by maintaining them in a germ-free state [42]. So, it can be suggested that IL-10 has a primary function of the prevention of an unwarranted inflammatory response towards the commensal gut microbiota. The roles of microbiota in cellular energetics, immune response, and inflammation are very much relevant to cancer as these are recognized as the hallmarks of cancer [43]. Thus, many researches are shedding light upon how the microbiome can influence the host’s susceptibility to various kinds of diseases including cancer.

Bile acids (BAs), short chain fatty acids (SCFAs), and other key metabolites are produced by the gut bacteria. Cholic acid (CA) and chenodeoxycholic acid (CDCA) are main components of primary BAs, whereas lithocholic acid (LCA) and deoxycholic acid (DCA) are key components of secondary BAs [44]. Conjugated-BAs can be created by mixing BAs with taurine or glycine. A sequence of liver enzymes produces primary BAs, which the gallbladder then excretes into the intestinal lumen. The generation of secondary BAs is significantly aided by the expression of bile salt hydrolase and 7-dehydroxylase by the gut microbiota [45]. The majority of BAs in the colon create enterohepatic circulation when they travel back to the liver via the portal vein system. BAs can facilitate lipid uptake and regulate energy metabolism. Additionally, bile acids play a significant role in immune modulation by mediating a number of downstream signal pathways. Microbes in the gut transform these chemicals into a variety of forms, considerably increasing their diversity and activity. SCFAs, as the name indicates, acidify the colon environment. That low pH promotes the development of helpful microbes while inhibiting disease colonization and improving mineral absorption. Furthermore, an acidic environment caused by SCFA synthesis reduces the quantity of secondary bile acids, that are shown to have negative impacts on gut health. SCFAs give colonic cells energy while also supporting in regeneration and maintaining optimal gut barrier impermeability. Butyrate does, in fact, induce the creation of tight connections between cells, forcing them to pack firmly together thereby rendering the gut increasingly impermeable [46]. Studying the gut microbiota’s metabolites may therefore yield crucial information for aetiology and treatment of a disease. Via the biliary tract, the portal vein, and the systemic circulation, the gut and liver maintain close bidirectional connections that make up the gut-liver axis ,shown in the Fig. 1 [47]. Normal gut bacteria can collaborate with the host’s immune system to form the gut barrier, which works to keep pathogens and toxins out and safeguard the body against sickness. However, microbial dysbiosis can disrupt gut barrier function, and a number of detrimental microbial related substances, along the gut-liver axis, reach the liver causing the development of HCC [48].

Fig. 1.

The gut-liver axis. The gut-liver axis is the bidirectional interaction between the gut, its microbiota, and the liver due to a complex combination of signals generated through genetic, nutritional, and environmental variables. Short chain fatty acids (SCFAs), various microbial metabolites, secondary bile acids, and numerous drug metabolites produced in the gut by the gut microbiota aid in the regular operation of the liver. Several xenobiotic compounds, as well as microbial antigens and lipopolysaccharides (LPS) generated by the gut microbiota, might, nevertheless, have a deleterious impact on liver function. The liver also contributes to intestinal homeostasis by generating various bile acids and immunoglobulins. Although certain antibacterial compounds generated by the liver can impair proper intestinal function.

Carcinogenesis or oncogenesis is the process through which normal cells turn into cancerous cells and causes the onset of the disease. This is a multistep process and is characterized by various changes at the cellular, genetic, and epigenetic levels. In this process of development of cancer, a key player is inflammation. A study in 2012, showed that IL-10 knockout mice with colitis have up to 100-fold higher levels of the Enterobacteriaceae family of bacteria in their colons compared with control mice without colitis. Escherichia coli and Enterococcus faecalis commensal bacteria strains were individually able to cause colitis when they were mono-associated with IL-10 mutant mice. However, only the E. coli strain was capable of causing colorectal cancer in the same mice after treatment with azoxymethane which is a procarcinogen. Therefore, in this case, carcinogenesis may not have been possible without inflammation, but it also wasn’t necessary. The next step, then, was to determine which bacterial-triggered processes contributed to carcinogenesis. The polyketide synthases (pks) pathogenicity island, which is about 54 kb in size and codes for the enzymes needed to produce the genotoxin known as ‘colibactin’, was discovered through additional investigation. This colibactin was absent in the E. faecalis strain. The next objective was to show that colibactin is crucial for the development of cancer. To do that, a pks-deleted ($\mathrm{\Delta }$pks) isogenic strain of E. coli was mono-associated with IL-10 knockout mice. Both the +pks strain and the $\mathrm{\Delta }$pks strain mice promoted inflammation. But the $\mathrm{\Delta }$pks strain was unable to induce carcinogenesis [49]. A study was conducted using RAG-2 knockout mice, where they were infected with H. hepaticus and a link between bacteria-mediated inflammation and DNA damage was found [50]. Activated macrophages and neutrophils infiltrated the inflammatory sites which boosted the synthesis of cytokines, chemokines, nitric oxide, superoxide, and other reactive species. This led to DNA and RNA damage and ended up causing colorectal cancer. Certain bacterial species can promote carcinogenesis by stimulating an inflammatory state through induction of proinflammatory toxins, increased generation of reactive oxygen species (ROS), and alterations in signaling pathways in human and mice model [51, 52].

A well-recognized risk factor for the occurrence of cancer is obesity and is one of the most thoroughly researched causes of dysbiosis [39]. In the guts of both humans and mice, obesity has been linked to higher Firmicutes populations and lower Bacteroidetes populations [53]. Inflammation, along with many other factors contributes to obesity-associated cancer, and recent studies are suggesting that bacterial metabolites can also play a role in it. In the case of biliary cancer, some of the known cancer-causing gut bacteria include Salmonella typhi and Helicobacter pylori. [54]. H. pylori is associated with gastric adenocarcinoma and also cancer of mucosa-associated lymphoid tissue (MALT lymphoma). It has been categorized as a class I carcinogen by the World Health Organization (WHO) [55]. In the case of the development of colorectal cancer, there are many shreds of evidence suggesting that gut dysbiosis is a key player. The tumor microbiota and the adjacent healthy mucosal microbiota are distinctly different, and stool transplants from colorectal cancer patients compared to healthy controls, can cause the production of polyps, pro-carcinogenic signals, and can also change the local immunological environment in mice [56, 57]. Gallbladder cancer and mucosa-associated lymphoid tissue (MALT) lymphomas provide examples of how certain bacterial infections might trigger cancer. Clonal expansion of T helper cells and B cells are the identifying characters of gastric MALT lymphoma. These cells react to antigens derived from H. pylori and regression occurs after the eradication of H. pylori [58]. There is no known microbiome for the pancreas. Recent studies suggest pancreatic cancer is promoted by inflammatory microbe-associated molecular patterns (MAMPs) and its receptor toll-like receptor 4 (TLR4) [59]. However, the mechanism through which the microbiota promotes pancreatic cancer remains unclear. The prevalence of skin cancer in mice deficient in bacterial MAMP receptors and germ-free mice is reduced which suggests that in skin carcinogenesis there may be a role for the microbiota [60, 61, 62].

Gut dysbiosis can promote cancer via long-distance mechanisms. The liver, which does not contain any known microbiome, is an example of it. Through bacterial metabolites and pro-inflammatory MAMPs that enter the liver through the portal vein, the gut microbiome may contribute to liver cancer [63]. This portal vein is responsible for the direct exposure of different components and metabolites from the gut microbiota to the liver. During the past decade a plethora of evidences are accumulating indicating the influence of gut-liver axis on the progression of liver disease from simple inflammation to fibrosis, cirrhosis and cancer as a major one [64]. Administration of clinically isolated strain of Klebsiella pneumoniae was shown to cause MASLD in mice model. This bacterium has also been reported to be associated with MASLD in human patients. Patients of HCC differ in their gut microbiota content from patients of liver cirrhosis without HCC. Bacteroides spp. and Ruminococcaceae were found to be more abundant in HCC patients whereas Bifidobacterium abundance was lower [65]. Germ-free status in mice and non-absorbable antibiotics minimize hepatic inflammation and checks the growth of HCC [66].

In a study, the carcinogen 7, 12-dimethylbenz(a)anthracene was used to initiate oncogenesis in mice. HCC developed in those obese mice which were either leptin-deficient or were wild-type but provided with a high-fat diet, whereas cancer did not develop in genetically wild-type lean mice provided with a standard diet. Bacteria that were capable of transforming primary bile acids into DCA, were particularly overrepresented. Cluster IX of the genus Clostridium contains these bacteria. DCA being a carcinogen can cause DNA damage by free radical production and has been shown to be involved in liver and colorectal cancers. The obese mice had higher serum levels of DCA. DCA and Clostridium are functionally important, and to establish that some experiments were done. Firstly, when obese mice treated with 7,12-dimethylbenz(a)anthracene received a variety of antibiotic treatments, serum DCA levels were significantly reduced as well as the liver cancer phenotype. The antibiotics included Vancomycin, which kills Gram-positive bacteria including the overrepresented Clostridium spp. Secondly, two different strategies were used to diminish the production of DCA without the use of antibiotics. One of them was decreasing 7$\alpha$-dehydroxylation activity using difructose anhydride III and the other one was stimulating bile acid secretion by ursodeoxycholic acid. The tumor phenotype was attenuated as a result of both [67].

Many studies have confirmed that as cancer develops in an organism; its normal microbiome gets affected. A study found that in the case of oral squamous cell carcinoma, three out of 40 studied microorganisms were elevated in the patients in comparison to healthy individuals [68]. The frequency of Fusobacterium nucleatum is increased in patients of colorectal cancer [69]. As mentioned in the previous section, it is well known that H. pylori is a cause of gastric carcinoma. It can also be said that the presence of H. pylori can indicate the risk of developing gastric cancer. So, as cancer progresses, the changes in microbiota can be detected as the studies throughout this review reveal. Discussed below are some possible mechanisms of transformation of the normal microbiome to cancer microbiome.

Microbes attach to their host cells with the help of adhesion molecules bound to their surfaces and their receptors present on the host cells [70]. Examples of such cell surface receptors include integrins, glycosaminoglycans, glycolipids, etc. Integrins are glycoproteins that are used by certain microbes such as Treponema pallidum [71]. Glycolipid receptors are used by Mycoplasma hypopneumoniae, glycosphingolipids are a type of glycolipid receptor which also act as a bacterial attachment site on cell surfaces. Studies have found that cell surface receptors of cancer cells differ significantly from the receptor found on normal cells. For example, fructose-containing glycopeptides are present on cancer cells surfaces more in number than that of normal cells. These alterations in cell surface receptors lead to alterations in normal microbe attachment to a cell. These changes enhance the attachment of certain microbes (which are not able to colonize in that specific site on normal cells surfaces) while it reduces or inhibits the attachment of other microbes which are actually present on normal cell surfaces. The electrical properties of cancer cells are also a factor in this regard. Cancer cells show more negatively charged surfaces than normal cells because they accumulate more negatively charged components [72, 73]. This may lead to alterations in microbe attachment as well. Another important factor in this context is the heavily glycosylated, high molecular weight family of proteins called Mucins. Invading bacteria first interact with these mucins that line the epithelial cells of the host, causing inflammation. A transmembrane mucin glycoprotein called Mucin 1 (MUC1) is found on the surface of nearly all epithelial cells that interacts with invading microorganisms. This interaction has the potential to cause pro- or anti-inflammatory reactions. MUC1 functions as an immunomodulatory switch, acting as either pro- or anti-inflammatory during infection. The negatively charged extended sugar residues create a physical barrier, imparting an anti-adhesive characteristic on MUC1, preventing pathogen entry. Oligomers are formed by the chains of glycosyl residues, which give rise to a lubricating mucinous gel. This protects the epithelia from desiccation, pH changes, and microbial infection [74]. MUC1’s heavily glycosylated extracellular domain functions as a barrier; yet, the intracellular cytoplasmic tail phosphorylation can activate downstream signaling pathways. MUC1 extracellular domain has the ability of bacterial attachment and thereafter getting shed from the epithelial surface. A signal might be triggered by this shedding that causes the cytoplasmic tail of MUC1 to be phosphorylated, controlling inflammatory reactions, epithelial cell adhesion, differentiation, and death. MUC1 therefore functions as a signaling receptor, sensing the external environment and activating internal signal transduction pathways. MUC1 is abundant on the mucosal surface of the stomach and is increased by H. pylori infection [75]. H. pylori has produced MUC1 oligosaccharide-specific adhesins and attaches to MUC1 on cultivated gastric epithelial cells. Despite this binding, there are fewer long-lasting adhesion events when MUC1-expressing gastric cells are cocultured with H. pylori because MUC1 is shed from the cell surface and covers the exterior of the bacterium. If the bacterial adhesins are deleted, the bacteria no longer adhere to the cells. MUC1 is not lost in this situation, but steric hindrance prevents non-MUC1-binding ligands from adhering to the cell surface [76]. Mice missing MUC1 had a fivefold increase in H. pylori colonization within one day of infection and developed atrophic gastritis with parietal cell loss [77]. According to one research, MUC1 is an intestinal receptor for enteroaggregative E. coli and interacts with the bacterial adherence fimbriae to promote attachment. Furthermore, it has been demonstrated that enteroaggregative E. coli infection increases MUC1 expression in inflamed human intestinal tissues [78]. In the event of E. coli infection, MUC1 works as a proinflammatory molecule by enabling E. coli access into gastric epithelial cells. The gut bacteria and the host epithelial mucus layer both play important roles in the large intestine’s maintenance and defense. According to an increasing body of evidence, the breakdown of the gut’s protective mucosal barrier plays an important part in the onset of colorectal cancer [79]. The disruption of the colonic barrier could increase colonocyte exposure to toxins from the colonic environment, hence boosting inflammatory processes and the generation of ROS, which can lead to carcinogenesis.

The immune response plays a major role in the colonization of the microbiome in the human body. The immune system discriminates between pathogenic and non-pathogenic bacteria using TLRs. For example, the bacteria Bacteroides fragilis produce a symbiotic factor that causes immunologic tolerance via the TLR2 receptor on the helper T cells [80]. A recent study has also established a correlation between TLR4 levels and lung cancer malignancy. In that study, TLR4 and TLR9 expression have been shown to increase in lung cancer [81]. Activation of certain TLR mediated pathways increase acute inflammation as well as wound healing and epithelial proliferation. If due to an altered microbiota the inflammation becomes chronic then this can lead to cancer. So, the expression of TLRs can alter the microbiota during cancer by mounting immune responses against certain bacteria.

Growth promoting hormones are always associated with the transformation of a normal cell to a cancerous one. Several studies have shown that these hormones are related to cancer cell proliferation in animal models as well as in cancer cell lines [82]. A study showed that hormones can alter the normal microbiome. Ovariectomized rats were compared with normal rats to see the differences in vaginal microbiota. Indeed, significant differences were found in the microfloral compositions of the two rat groups [83]. Another study showed that human gut microbiota can metabolize estrogen thus altering the hormone level in the body [84]. It is known that estrogen through estrogen receptors can drive neoplastic growth. Estrogen concentration is directly related to increasing the risk of breast cancer. As tumorigenesis progresses and the mass of the solid tumor increases, hypoxic conditions are generated due to an insufficient supply of oxygen. This condition can be favourable for the growth of certain bacteria over others [85]. Studies have revealed that more than 30 different types of intestinal microorganisms that are present in individuals with HCC might change in the early stages of the disease, demonstrating the importance of the gut microbiota in the formation and progression of HCC through various stages from a simple liver disease [86]. Alteration of gut microbiota associated to various liver diseases including liver cancer is show in Table 1 (Ref. [87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111]). Some of the above-mentioned mechanisms by which the eubiotic gut gets altered into dysbiotic gut is shown in the Fig. 2.

Fig. 2.

Some of the possible mechanisms responsible for the alteration of eubiotic gut into dysbiotic gut. Alterations in cell surface attachment molecules, structural and physiological changes such as hypoxia and electrical charge changes, immunological changes via pattern recognition receptors (PRRs) such as toll-like receptors (TLRs), and hormonal changes can all contribute to gut dysbiosis.

Microbiome can directly or indirectly promote cancer by various mechanisms. The immune system detects microbes by pattern recognition receptors (PRRs). These control the microbial population in our body by initiating the innate immune response and by producing antimicrobial substances. These substances not only kill cancer-promoting bacteria and suppress cancer, but can also prevent cells from dying thus trigger inflammation which is a major factor of carcinogenesis [112]. The microbiota modulates the development of cancer by releasing genotoxins which are carcinogenic in nature. Fig. 3 summarizes the mechanisms through which microbes may promote carcinogenesis.

Fig. 3.

Mechanisms of carcinogenesis by human microbiota. Changes in hormones and energy metabolism, for example, can cause breast cancer. Chronic inflammation and genotoxic chemicals can cause lung, stomach, and colon cancer. H. pylori infestation can also result in stomach and colon cancer. Toll-like receptors (TLRs) identify microbe-associated molecular patterns (MAMPs), which can cause liver and pancreatic cancer. Bacterial metabolites, secondary bile acids, ROS production, acetaldehyde formation from ethanol, and hepatitis B and C virus infection can all cause liver cancer.

Microbiome promotes cancer by stimulating cytokine and chemokine production through inflammation. TLRs are a kind of PRRs which sense the MAMPs and mount immune responses leading to inflammation. TLR4 is the receptor for sensing bacterial lipopolysaccharides (a constituent of Gram-negative bacteria’s cell wall). Mice which are deficient of TLR4 show reduced tumor development as compared to controls, which suggest that TLR4 has some role in tumor development [113]. A similar kind of receptor named TLR2 senses peptidoglycan and lipoteichoic acid on the bacterial cell wall. It has been shown that this TLR2 promotes the development of gastric cancer [114]. TLRs promote cancer through myeloid differentiation primary response 88 (MYD88) pathway which in turn triggers the nuclear factor-$\kappa$B (NF-$\kappa$B) and signal transducer and activator of transcription 3 (STAT3) signaling pathways [113, 114, 115]. TLR2 present on the surface of Treg cells when senses ‘polysaccharide A’ present on the cell wall of Bacteroides fragilis, it causes immunogenic tolerance, and thus these bacteria can live within our gut. But when polysaccharide A is lacking from the cell wall of B. Fragilis, no tolerance is generated and T helper 17 response eliminates the bacteria from the system. This is an example of the mechanism of discriminating between ‘good’ and ‘bad’ bacteria by our immune system [80]. Another example showing the importance of the TLR signaling pathway in carcinogenesis is from TLR5 signaling. TLR5 is responsible for the recognition of bacterial flagellin. To confirm that the microbiome can alter the metabolism of an organism, a study showed that, TLR5 knockout mice showing symptoms of metabolic disorders such as hyperphagia had significant changes in their microbiome. As the microbiota from these mice were transplanted into germ-free mice which were wild-type, the symptoms developed in the later [116]. Like TLRs, NOD-like receptors (NLRs) are also a kind of PRRs that have a nucleotide-binding oligomerization domain or NOD at its center. NOD2 is an NLR that senses muramyl dipeptide which is derived from peptidoglycan and is present on the cell wall of both Gram-negative and Gram-positive bacteria. The deficiency of NOD2 in mice leads to an increased incidence of colorectal cancer. NOD2 is very important for bacterial immunity because studies show that NOD2 knockout mice are very much susceptible to bacterial infections. Recently a study showed that NOD2 deficiency promotes dysbiosis which eventually causes cancer [117].

Bacteria can contribute towards carcinogenesis via specific toxins. These toxins can either produce chronic inflammation which generates reactive oxygen species (ROS), thus causing genomic instability, or can directly damage DNA, both of which promote the development of cancer. Two examples of bacterial genotoxins are cytolethal distending toxin (CDT) produced by gram-negative bacteria and colibactin produced by the members of the Enterobacteriaceae family. Both of these toxins can induce genomic instability by damaging DNA directly causing double-strand DNA damage which activates the ATM-CHK2 signaling pathway [118, 119, 120]. The bacteria present in the lumen can establish direct contact with the host cells if the function of the barrier somehow gets altered and thus can deliver toxins directly. The most studied genotoxin is Cytolethal Distending Toxin (CDT) and is produced by several bacteria including Helicobacter sp. and E. coli [119]. CDT is made up of 3 subunits namely CdtA, CdtB and CdtC. CdtA and CdtC subunits bind to the host cell-surface and CdtB is delivered into the cytoplasm. The CdtB subunit exhibits DNase activity and induces double-strand DNA breaks. Thus, CDT arrests the host cells in the G2/M phase of the cell cycle, resulting in enlarged cells [120]. Along with genotoxins, metabolites that are derived from bacteria may cause genomic instability. Enterococcus faecalis can promote colorectal cancer in IL-10 knockout mice by generating a hefty amount of superoxide which causes double-strand DNA damage and instability of chromosomes. $\mathrm{\Delta }$menB, a mutant strain of E. Faecalis which cannot produce extracellular superoxide, is not able to promote the development of cancer in IL-10 knockout mice [121].

The microbiome is responsible for the fermentation of proteins as well as carbohydrates inside our gut. Ammonia, phenols, nitrosamines, sulfides, etc are generated via protein fermentation by microbes mainly in the distal colon which are toxic and potential carcinogens [122]. Microbiota plays key roles in bile acid metabolism via many kinds of hydrolases, thereby changing bile acid composition in our gut. A study suggesting how a high-fat diet can increase the levels of DCA which is a secondary bile acid and can lead to cancer has been discussed in section 3. On the other hand, short-chain fatty acids are generated as a result of the fermentation of carbohydrates such as butyrate. Cancer preventing the role of butyrate as a prebiotic will be discussed in section 7.1 of this review. Lastly, alcohol metabolism is also related to microbiota which is a major causative agent of cancer worldwide. Alcohol is metabolized into acetaldehyde and ROS is also generated in this process, both acetaldehyde and ROS contributes to cancer. Germfree mice have a significantly lower level of acetaldehyde compared to normal mice which indicates that there might be a relation between microbiota and acetaldehyde production [123].

From cholesterol, Bile acids (BAs) are made. The enterohepatic circulation of BAs plays a crucial part in preserving the gut-liver axis’ homeostasis. The transformation, conjugation, reabsorption, and deconjugation of primary BAs into secondary BAs are all processes that the gut microbiota participates in throughout the metabolism of BAs. By altering the content and quantity of BAs, important bile acid metabolic stages can affect homeostasis [124]. When BAs build up to large concentrations, they directly impact liver cells [125]. The breakdown of BA hemostasis is seen as a negative aspect. Through fibrosis, oxidative stress, and resistance to apoptosis, the altered BA metabolism can encourage carcinogenesis [126]. Secondary BAs encourage hepatic inflammation and carcinogenesis in MASLD-related HCC via the mTOR signaling pathway [127]. Shen et al. [128] in mouse model discovered that in case of both in vitro and in vivo studies, reducing gut bile salt hydrolase (BSH)-rich bacteria (i.e., Bacteroidales Clostridiales, Lactobacillales, Bifdobacteriales) significantly reduced serum conjugated DCAs, a result that may be linked to the development of HCC. SCFAs exhibit metabolic and immunologic effects on carcinogenesis, similar to BAs. Acetate, propionate, and butyrate make up the majority of SCFAs, which are by-products of gut microbiota’s colonic fermentation of dietary fiber [129]. Through G-protein-coupled receptors GPR43 and GPR41, SCFAs affect the genes involved in fatty acid oxidation, ROS genesis, lipogenesis, and insulin sensitivity to change metabolism [130, 131]. By improving epithelial integrity, SCFAs reduce systemic endotoxemia. Additionally, SCFAs prevent histone deacetylases (HDAC), which has an impact on gene transcription. In immunomodulation, SCFAs have a complex role to play [132]. The fermentation of glucose in the intestines is able to make ethanol. Klebsiella pneumonia, Saccharomyces cerevisiae and Candida spp. are among the organisms that have the ability to create endogenous ethanol [131]. The prevalence of ethanol-producing bacteria is higher in MASLD and obese patients, and it has been discovered that serum ethanol levels are much higher in these patients than in healthy individuals, even in the absence of alcohol consumption [133, 134]. The well-known carcinogen ethanol interacts with other risk factors in the liver to cause cancer. It has been determined that increased free radicals and ROS caused by the metabolism of alcohol, are crucial factors in the development of hepatocarcinogenesis. In the development of HCC, Hepatitis B and C virus have a major role to play. Compared to healthy controls, patients with persistent liver cirrhosis, HBV infection and HCC had less dysbiosis, notably a reduced abundance of Firmicutes and a greater abundance of Bacteroidetes. In the meantime, persistent HBV infection was associated with an increase in genes involved in glycan production, metabolism, and lipid metabolism. In comparison to healthy controls, individuals with HCV and HBV infection showed greater plasma levels of lipopolysaccharides (LPS), IL-6, sCD14 generated upon LPS activation of monocytes, and intestinal fatty acid binding protein [134].

A biomarker is an indicator of an organism’s or disease’s presence, severity, or a physiological condition. Biomarkers, in cancer biology, are used as diagnostic tools to identify diseases, prognostic markers to forecast outcomes, and predictive markers to forecast the effects of therapy. Certain of these markers have prompted paradigm shifts in patient management in favor of personalized therapy. The potential of a few metagenomic indicators for cancer diagnosis will be discussed in this section.

New evidence points to dysbiosis of the gut microbiota to be used as a potential non-invasive tool for the early detection of a number of malignancies. Even though Helicobacter pylori infection is linked to 70% of gastric cancer, this bacterium is not a useful screening sign. In fact, only 1–4 percent of those with H. pylori infection go on to acquire gastric cancer. Faecalibacterium, Desulfovibrio, Escherichia, or Oscillospira were intriguingly found by Liu et al. [135] as fecal biomarkers to accurately predict gastric cancer with a 90% or higher rate of success. Patients with cancer in other parts of their bodies have been reported to have gut microbiota dysbiosis. A gut microbiota imbalance has been linked to breast cancer in increasing numbers of studies [136]. One potential application of the gut microbiota may be as an important biomarker in breast cancer research, according to several studies that found that women with breast cancer had a variable composition of gut microbiota, with elevated levels of Faecalibacterium, Ruminococcaceae, and Clostridiaceae, and decreased levels of Lachnospiraceae and Dorea in comparison to paired healthy controls [137]. In a study by Zhuang et al. [138] comparing the gut microbiota of 30 people with lung cancer to 30 healthy controls, it was discovered that controls had higher concentrations of the bacterial phylum Actinobacteria and the genus Bifidobacterium, while lung cancer patients had elevated levels of Enterococcus, suggesting that these bacteria could be used as potential biomarkers for the disease. One very interesting study by Bhandari et al. [139] showed a strong relationship between the volatile metabolites in exhaled breath and the fecal microbiota, as they varied between cancer patients and healthy controls. Since gastric cancer has been linked to changes in the volatile metabolic profiles and dysregulation of the gut microbiota, its composition, and the associated metabolic pathways, the exhaled breath analysis and fecal microbiome analysis were combined, and they may now be used as non-invasive diagnostic and screening tools for the disease.

Ren et al. [108] showed that transition of cirrhosis to early HCC with cirrhosis increased fecal microbial diversity. Actinobacteria, as opposed to cirrhosis, were more prevalent in early HCC. In contrast, early HCC was enriched in 13 taxa, including Gemmiger and Parabacteroides. In early HCC compared to controls, butyrate-producing genera were downregulated whereas LPS-producing genera were upregulated. Through fivefold cross-validation on a random forest model, the ideal 30 microbiological markers were found, and they produced an area under the curve of 80.64% between 75 early HCC and 105 non-HCC samples. Gut microbial indicators confirmed a substantial propensity for early and even advanced HCC [108]. According to Komiyama et al. [140], the microbiota that is associated with tumors includes Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Ruminococcus gnavus was also discovered to be a distinctive taxon for people with viral hepatitis-related HCC. Despite the fact that the level of dysbiosis was independent to the HCC stage, Ni et al. [141] discovered that patients with HCC had more pro-inflammatory gut bacteria and dysbiosis than healthy controls. However, other researchers have noted links between the HCC stage, gut microbiota alterations, and dysbiosis. Escherichia coli counts in the feces were greater in patients with HCC, according to a prospectively matched analysis of 30 cirrhotic patients reported by Grat et al. [141] which had a sensitivity of 66.7% and a specificity of 73.3% when predicting the existence of HCC based on Escherichia coli counts. Piero et al. [142] used a comparable sequencing apparatus to study 407 patients. Comparing the HCC group to cirrhotic individuals without HCC, the HCC group had a three-fold increase in Erysipelotrichaceae and a five-fold drop in the Leuconostocaceae. Patients with HCC had significantly lower levels of the Lachnospiraceae bacteria Fusobacterium and Dorea, but Odoribacter and Butyricimonas were reported to be more significantly enriched in HCC.

One critical point to consider while conducting these gut microbiota based biomarker researches is; age, gender, BMI, food, and antibiotic use are some of the variables that affect the gut microbiome’s inter-individual variance and macro- and microscopic morphological variability. So, the research outcome may not be the same in every case, and further research is necessary to standardize these in the hope for developing a potential gut microbiota based biomarker for cancer.

Bacteria plays dual role in cancer by supporting the tumor growth or by suppressing it. Keeping this in mind, modulating the microbial community inside the gut might be beneficial in prevention or treatment of cancer. To accomplish this goal, multiple approaches (shown in Fig. 4) have been taken into consideration such as antibiotics, fecal microbiota transplantation (FMT), probiotics, prebiotics, synbiotics, nanoparticles and bacteriophages.

Fig. 4.

Therapeutic approaches to restore eubiosis in a dysbiotic gut. There are several approaches to restoring intestinal eubiosis. Probiotics and prebiotics are being utilized to target gut microbiota restoration and have been the subject of current research, including clinical trials. Different types of nanoparticles are being studied in this area. Antibiotics are employed in this respect, with different effectiveness when administered alone or in combination with all of the aforementioned. Another promising technique is fecal microbiota transplantation (FMT). Bacteriophages may potentially be utilized to target eubiosis restoration/maintenance as a unique method.

Researchers from all over the world are conducting a sizable number of studies to learn more about the relationship between the microbiome and cancer. However, very little is known about how the microbiome really causes cancer. The majority of these studies are concentrated on identifying the evidence of the microbiome’s link with cancer. The development of this field of study has led to the discovery of numerous previously unknown fresh insights. Clarification of the link between the microbiome and cancer has prompted researchers to concentrate on how to alter gut microbiota in order to restore eubiosis for the treatment of other diseases in addition to cancer, as well as the potential function of microbiota as a biomarker to detect cancer at an early stage. By altering the gut microbiome, which is a topic of intense research right now, antibiotics may be utilized to treat cancer. Antibiotics can be harmful to our health when used excessively, but when used carefully and selectively against specific microbial populations, they can improve treatment outcomes. However, there also exists evidence indicating that using antibiotics excessively can have the opposite effect, making cancer therapy less effective for patients. Bacteriophages that have been demonstrated to be just as effective as antibiotics are now being tested for the treatment of several diseases by modifying the microbiota, and this also applies to the treatment of cancer. Without disrupting the populations of commensal bacteria that are not bacteriophage targets, these viruses destroy bacteria very selectively. Presently, phage display is being considered as a cancer therapy strategy. Phage particles that display a specific peptide of its surface may be used to identify cell-specific targeting molecules, identify cancer cell surface biomarkers, identify anti-cancer peptides, and develop peptide-based anticancer therapy [208]. Prebiotics, probiotics, and synbiotics are already being used successfully to alter the composition of the gut microbiota in order to prevent or treat cancer. Probiotics that are sold commercially are openly available and accessible to everyone on the market. Cancers can be identified using tumor microbiota signatures unique to that tumor, and they can be targeted to stop cancer from progressing and enhance treatment outcomes. Using the drug-loaded stimulus-responsive nanocarriers outlined in section 7.4, it is being investigated whether certain known bacteria that reside in particular tumors can be utilized for targeted therapeutics or can be genetically modified to either directly destroy malignant cells or create an anti-tumor immune milieu.

From simple food digestion and energy metabolism to memory and depression, gut microbiota is linked to practically every facet of human physiology. Future prospects for the study of the microbiome are very bright. Although the processes by which these two interact are largely unclear, evidence has already shown a connection between gut microbiota and cancer. Modulation of the gut microbiota may be a viable method to treat cancer. However, because the microscopic organisms are linked to other physiological functions, attempting to alter their composition in the gut can also result in additional health complications. To have a thorough understanding of how our gut microbiota and all of the physiological features are interconnected, critically designed innovative researches are required. To use the research from this sector for true human benefit, carefully monitored clinical studies are needed. The research on the relationship between gut microbiota and cancer has a number of limitations. A majority of the microorganisms found inside the human body cannot yet be grown under culture conditions that can support their growth. Gnotobiotic mice are used to explore the relationship between the microbiome and cancer, however these models lack the microbiota that would make up a person’s genuine microbiome. Although FMT suggests a viable method for altering the gut microbiota to restore eubiosis, more thorough research on the mechanisms and adverse effects is required. Prior to patient administration, careful characterization of all the microorganisms contained in the fecal sample is crucially required. The precise identification of the mediators by which bacteria induce cancer is extremely important for therapies. Meta-transcriptomic and metabolomic investigations must be employed more to identify these carcinogenic substances and their mechanisms of action, because metagenomics does not assess microbial gene expressions. By using corresponding diets and genetically modified bacteria strains with or without specific enzyme coding genes, it is possible to either raise or decrease the quantities of substances that promote tumor growth. Applications of the information gained from these studies on cancer prevention, detection and treatment are beyond imagination. The potential of microbiota getting used as a detector of cancer together with uses in treatment and surveillance is never-ending. Many questions remain unanswered as the field advances at an astounding rate, including whether early colonization by carcinogenic microbes can result in the later development of cancer, how long it takes for cancer to manifest after carcinogenic bacteria have colonized the body, and whether altering the microbiota can reduce the risk of cancer in humans. This area of cancer research offers great promise and has the ability to usher in a new era of cancer research, however, it is merely in its infancy right now and plenty of effort and hard work awaits.

SA- designed the study and wrote the manuscript. CT- provided help and advice on the review writing. Both authors contributed to editorial changes in the manuscript. Both authors read and approved the final manuscript. Both authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

Not applicable.

The author, Sancharan Acharya, gratefully acknowledges the UGC, India, for the University fellowship.

This research received no external funding.

The authors declare no conflict of interest.