**4. Microbiome Research and Engineering in HCC Metabolism**

Physiological responses in the host are maintained and coordinated by metabolites produced by the microbiota. The liver cancer processes that HCC may impact through bacterial metabolism, such as these metabolic transportation pathways and their efficacy, must be understood [91]. It is important to comprehend and describe the variety of metabolites that the gut microbiota excretes. Metabolomics has been widely used to illustrate the metabolites produced by gut microbes, particularly in relation to the disease states of the host that they may affect. Metabolomics generally no longer needs to be defined, as we have thoroughly examined this technology [92–94].

In an early study, Nicholson's team discovered significant metabolic differences between germ-free mice and their healthy counterparts for so-called 'cometabolites' such as hippuric acid, which is produced when benzoic acid and glycine are conjugated [95,96]. The gut bacteria produce benzoic acid by converting chlorogenic acid into quinic acid, which is then aromatized [97]. The liver receives benzoic acid via the portal supply, where it is conjugated in the mitochondria by first forming a CoA intermediate and then adding glycine [98].

Phenylacetylglutamine is another instance of a metabolism in which phenyl acetic acid is formed from phenylalanine by the gut microbiota [36] and conjugated with glutamine in the host's hepatic mitochondria [99]. It has been documented that the three aromatic amino acids phenylacetic acid, tyrosine, and tryptophan are all converted into nine aromatic acids via the gut symbiont *Clostridium sporogenes* and circulate in human plasma. These compounds, such as tryptophan-derived indol-3-ylacetic acid, are almost certainly cometabolized by the host [99].

Dietary flavan3-ol polyphenols, epigallocatechin gallate, epigallocatechin, epicatechin, and catechin may also be sources of aromatic acid metabolites by gut microbiota through intricate oxidation and dihydroxylation pathways that ultimately result in phenylacetic

acid, benzoic acid, and catechol [100]. An examination of 143 organic acids frequently found in the urine of healthy individuals revealed that a sizable portion of these were created by the host's microbiota, with some of them being further digested by the host [101]. Figure 2 shows the three-way relationship between the host, the tumor microenvironment, and the microbiota.

**Figure 2.** A three-way relationship between the host, tumor microenvironment, and microbiota. The gut microbiome-derived metabolomics target in HCC has been summarized. The host microbiome and metabolic reprogramming for cancer cells and their microenvironment may be related. An altering gut microbiota may boost propionate synthesis, which may reduce the risk of HCC both through a cAMP level-dependent mechanism and by interacting with GPR43. The gut microbiota alteration may result in an anti-HCC impact by boosting the amount of hepatic CXCR6+, NKT cells, and IFN-γ production. Primary-to-secondary bile acid conversion, which is controlled by the gut microbiota, influenced the CXCL16 expression of liver sinusoidal endothelial cells, which in turn affected the accumulation of CXCR6+ and NKT cells. cAMP, cyclic adenosine monophosphate; GPR, G protein-coupled receptor; IFN, interferon; IL, interleukin.

An important group of acidic metabolites generated by the gut microbiota is shortchain fatty acids (SCFAs: acetate, propionate, and butyrate), which are created by gut bacteria during the anaerobic fermentation of plant structural polysaccharides such as cellulose or fiber [102]. The lipopolysaccharide (LPS)-producing genera (*Neisseria, Enterobacteriaceae,* and *Veillonella*) were more abundant among liver cancer–HCC, and butyrateproducing genera (*Clostridium, Ruminococcus,* and *Coprococcus*) were less abundant. Three more biomarkers—*Enterococcus*, *Phyllobacterium,* and *Limnobacter*—can also be used to reliably detect liver cancer.

In cellular environments, SCFAs represent a sizable proportion of energy metabolites, as it is estimated that they provide 60–85% of an animal's energy needs [102]. In HCC patients with dysregulated fatty acid metabolism, the β-oxidation process of fatty acids is associated with a worse prognosis. The proximal colon of a human contains the largest concentration of SCFAs, where they are both absorbed into the bloodstream and utilized locally by enterocytes [103,104].

In mice with altered gut commensal bacteria, the increase in NKT cells and the suppression of liver tumor growth may both be reversed by colonizing bile acid-metabolizing

bacteria (*Clostridium scindens*) and supplementing with secondary bile acids (lithocholic acid or muricholic acid).

The G-protein-coupled receptors GPR41 and GPR43 (free fatty acid receptors) and the niacin receptor GPR109A are only a few of the receptors that can recognize SCFAs, which are present in the colon at concentrations of 50–200 mM. SCFAs can regulate gene expression by inhibiting histone deacetylases in this location and act as signaling molecules that are recognized by particular receptors [105].
