Microbiomics: Novel Biomarkers of Colorectal Cancer Diagnosis and Prognosis

1. Introduction

Newly diagnosed colorectal cancer (CRC) cases achieved more than 150 thousands and ranked third most in both sexes in America, 2024 [1]. The predicted death number of CRC is about 53,000 in the USA and 153,000 in the European Union during 2024 [1,2]. CRC casts persistent heavy burden on both individual family expenditure and worldwide hygiene. One of the most powerful factor in prejudging the disease outcome is tumor stage. Most CRC developed from benign polyps in a long term of 10 to 15 years. Proper adenoma detection and excision do break the incidence of CRC [3]. In England, Duke A stage CRC reached a 5 year overall survival (OS) as high as 98%, with a gradual decrease from 85%, 63% to 7.5% for Duke B, C and D stage diseases [4]. This result was identical to another research concluded stage III CRC 65-70% 5 year survival and steep reduction to stage IV, 13-14% [5,6]. A huge scale study based on Martinique Cancer Registry between 1993 and 2012 reported a median OS of 2.0 year for stage III to IV CRC while stage I to II diseases had not reach that outcome until program terminated [7]. Efficient test is in urgent need to advance diagnosis and prognosis.

So far, the most widely accepted screen methods include colonoscopy, fecal immunohistochemical test (FIT). Colonoscopy provided magnified view and appended clarification based on directly acquired sample [8]. Although colonoscopy is the golden standard test in CRC diagnosis, the trauma and high expense refused it in some patients. Researches concluded FIT displayed sensitivity of 74% to 94% and specificity of 85% to 95% [9,10]. There are still large unknown area for particular anticipation. For example, as for patients undergoing watch-and-wait procedure after their lesion assured complete remission with CT or colonoscopy, a novel screen test feasible to predict its progress instead of just waiting until in some cases relapse would definitely be more safety. Another occasion is therapeutic predication. These further demands call for more markers other than just 100% accurate colonoscopy alone. Circulating tumor DNA (ctDNA) test and tumor mutation burden (TMB) are both innovative and derivative tumor examination in these decades.

About 100 trillion microorganisms reside in human gut, of which Firmicutes taking the lead proportion of approximately 67% and Bacteroidetes as the second of 21% [11,12]. Previous evidences supported their role in some tumors, such as oral, breast cancer and malignant melanoma [13,14,15,16]. Same as them, microbiomic test has a great potential in predicting CRC outcomes, including therapy related or not. Recent studies demonstrated high level of Fusobacterium nucleatum and Bacteroides fragilis indicates poor prognosis [17]. The former is also reported to closely relevant to chemotherapy resistance and distant metastasis [18].

Based on the accumulating assays on the microorganisms, we conducted this narrative review to summarize existing evidences, differences, limitations and expectations of them as CRC biomarkers and associative mechanisms.

2. The Roles of Microbiomes Play in CRC Formation and Progression

Human gut microbiomes affects colorectal carcinogenesis and progression in intricate pathways. To summarize, we collected fetched information listed as below: metabolism, immune, inflammation, proliferation or apoptosis, genetoxin and barrier.

2.1. Metabolism

Microbiomes take carbon hydrates, proteins, lipid, water and other nutrition from intraluminal contents, produce substances they need and excrete metabolic waste. Some natural or metabolic intermediate sulfur-containing proteins (like disulfide bonds) discharge hydrogen sulfide along degradation under particular enzymes catalysis [19]. This process is halfly adjusted by intestinal microbes [20]. Hydrogen sulfide acts a dual role in gastrointestinal micro-ecology. It enables electric transmission in mitochondrial electron transport chain in complex II [21]. However, excessive produced hydrogen sulfide inactivated complex IV and inhibited mitochondrial respiratory chain [22,23]. This further caused triphosadenine loss and reinforced reactive oxygen (ROS) [24,25]. Hydrogen sulfide exceeds mitochondrial permeability transition (MPT) and triggers hepatocyte apoptosis [26]. Overload ROS strain injures both mitochondrial DNA (mtDNA) and nuclear DNA, contributing to genetic instability in CRC to some extents [23,27]. It suppresses short chain acyl-CoA dehydrogenase and thus downregulating butyrate β-oxidation, which is deemed to be one of the main energy supply for colonic epidermal cells [28]. Desulfovibrio, a gram staining negative obligate anaerobic bacillus, belongs to Proteobacteria phylum. As one of the most currently studied sulfate-reducing bacteria (SRB), Desulfovibrio degrade sulfate and extract hydrogen sulfide through membranous and soluble sulfite reductase [29]. Contrary to most other microbiomes in bowel generating hydrogen sulfide in assimilatory sulfate reduction pathway, Desulfovibrio operated that in dissimilatory path [30]. This process synthesized hydrogen sulfide was proved to be harmful to colonocyte respiratory. Following accumulated ROS stress interrupted DNA stability and act like a genetoxin [31]. Additionally, colonic epithelial tight junction was broken by Desulfovibrio via activating transcription factor Snail 1 and altering occludin localization [32].

Bile acid is a group of hepatocyte synthesized cholesterol derivatives promoting lipid digestion and absorption. Primary bile acids (PCAs) include cholic and chenodeoxycholic acids. They are delivered into duodenum after combined with glycine or taurine. Some PCAs are modified right after secretion into secondary bile acids (SBAs). Deoxycholic Acid (DCA), Lithocholic Acid (LCA), isoLCA, alloLCA and other derivatives composed SBAs family [33]. Classic bild acid receptors consist of Farnesoid X Receptor (FXR) and Takeda G Protein-Coupled Receptor 5 (TGR5). Apart from bile acids physiological administrated glucolipid metabolism, enterohepatic circulation and cholecystic systemic function, excessive load of them deteriorates inflammation, damages DNA double chains and arouses cellular proliferation [34]. A recent experiment elucidated mice exposed to high level DCA for a long interval suffered raised risk of colonic adenoma converting into adenocarcinoma [35]. DCA enhances arachidonic acid excretion and promptes their transformation into pro-angiogenic prostaglandin and ROS, injuring DNA and curb restore process. It also augments cyclooxygenage 2 (COX-2) via stimulate epidermal growth factor receptor (EGFR), further impels colonic carcinogenesis or distant metastasis [36]. An animal experiment conducted microbiomic test and founded that DCA modulated APC gene and cause bowel dysbiosis. Facilitate opportunistic plantation of Shigella and Desulfovibri and reduce Bifidobacterium and Lactobacillus [37]. That disruption on entero-ecology thus changed equilibrium to pro-tumor side. In a high fat diet fed Apc^min/+ CRC rodent model, SBAs stimulated colonic cell stemness and tumourogenesis by combination with FXR, leading to ribosomal instability partly due to ROS production [38]. High fat diet extends the proportion of colonic Clostridium and increase DCA level. The latter upregulates hepatic stellate cell caducity-related expression and pro-flammatory phenotype, which advanced into hepatocellular carcinoma in final [39]. Similarly, Clostridium was revealed to induce CRC development by other independent researches [40,41]. It is safe to draw a conclusion that high fat diet potentially elected PCA producing microorganisms, accelerating tumor formation and process [42]. SBAs attach their pressure on digestive ecology in various aspects including immune, inflammatory and microbial composition. LCA inhibit hepatic infiltrated macrophages glycolysis and facilitate intracellular oxidative phosphorylation, reshaping macrophages from anti-tumor M1 into immune suppressive M2 phenotype [43]. This adaption is synergized by activating mannose receptor followed M2 recruitment in CRC microenvironment [44]. M1 is more aggressive as an inflammatory biomarker maintaining immune clearance. M2 typed tumor associated macrophages (TAMs) oppositely establish an immune privilege. Physiological bile acid synthesis and metabolism do behave in intestinal micro-ecological balance. Pathological suppression of FXR ruined that rhythm, which restrained secretory immunoglobulin A (sIgA) and elevated Enterotoxigenic Bacteroides Fragilis (ETBF) colonization, causing colitis associated CRC [45].

Short chain fatty acids (SCFAs) refers to a saturated fatty acid with a limited length of carbon chain less than 6. Acetic acid, propionic acid and butyric acid are common SCFAs within human body. Peroxisome proliferator-activated receptor γ (PPAR-γ) is activated by butyrates, remodeling to β-oxidation, a peculiar energy supply for colonic epithelium, and blocks some harmful germs colonization [46]. More than 90% SCFAs are rapidly absorbed in proximal colon [47]. Merely about 5% are later discharged in feces [48]. CRC development is correlated to colonic Bacteroides and Firmicutes proportion deduction. They both synthesize SCFAs. SCFA producing flora enrichment in turn restrain PI3K/AKT pathway, triggering apoptosis related genes and decrease tumor volume [47,49]. Those phenomena are corresponded to colonic butyric acid level reduce in CRC patients [50]. According to animal and cellular experiments, SCFAs inhibited pro-imflammatory factors, TNF-α, IL-18, IL-1β, IL-6 and iNOS, while simultaneously raise IL-10, repolarizing macrophages into M2 type [51,52]. Besides, butyrates enhanced Lysine 9 on histone H3 (H3K9) methylation and downregulated histone deacelylases (HDAC) methylate STAT6, which also transformed macrophages into M2 [53]. SCFAs is a group of anti-inflammatory substances downsizing potential energy associated damages.

In addition to above discussed metabolites, literature mentioned some others. Aromatic amino acid abnormal synthesis would disturb mucosal barrier integrity. Colonic tissue samples collected from volunteers and patients supported a gradual elevation of some specified amino acids, L-alanine, glycine, L-valine, and myristic acid, from healthy controls, adenoma to CRC [54]. Knockout of monocarboxylate transporterprotein 2 (MCT2) gene interrupted Firmicutes and Bacillus spp. Following Lactic acid excessive gathering would drive macrophages into M2 phenotype, thus promoting pro-tumor microenvironment in respiratory and upper digestive malignant diseases [55,56]. In short, specified or common productions are core intermediate nodes for large quantities of microorganisms contributing to pro- or anti- CRC process. Microbial metabolites effects and mechanisms are depicted in Table 1. Figure 1 illustrated microbial metabolites and products influences on CRC.

2.2. Immune

Microbiomes intervene immune microenvironment through various metabolic poisons or modifications. Fusobacterium nucleatum is a gram staining negative obligate anaerobion colonizing in human mouth, gastrointestinal and urogenital tract, encoding Fusobacterium adhesin A (FadA). It combines to T cell immune receptor immunoglobulin and immuno receptor tyrosine-based inhibitory motif (ITIM) domains and dampens T cells and natural killer (NK) cells via downstream signals [57]. Fap2 protein is a fusobacterial Gal-GalNAc-binding lectin. It manages F. nucleatum adheres to CRC membranous E-cadherin and propels proliferation [58,59]. At the same time induce over expression of toll-like receptor 4 (TLR4), actuating the failure of immunological surveillance and chemotherapeutic resistance [60]. F. nucleatum expands CD11b⁺ myeloid-drived suppressor cells (MDSCs) consisting of TAMs, dendritic cells (DCs), and granulocytes within tumor immune microenvironment [61,62]. The abundance of F. nucleatum is positively correlated with CCL20 expression. MiR-1322/CCL20 axis expanded TAMs and MDSCs recruitment via NF-κB signal pathway, giving rise to M2 polarization markers, such as CD206, ARG1, IL-10 and TGF-β [63,64]. F. nucleatum reinforced suppressive T cells, TAMs, DCs and MDSCs, contributing to immune escape and motivate angiogenesis in CRC.

Bacteroides fragilis toxin (BFT) is a metalloproteinase toxin synthesized from some detrimental Bacteroides Fragilis. It is a vital medium in B. fragilis drived colitis and CRC occurrence [65,66]. Studies manifested recruited Th17 type regulatory T cell (Treg) and IL-17 play a role in B. fragilis drived CRC [67,68]. Inconsistent with sporadic CRC, intensive MDSC infiltration were spotted within BFT constructed CRC microenvironment [69]. IL-17 aroused δγT cells attracted CXCR2⁺ polymorphic nuclear innate myeloid cells and impelled the latter differentiate into pro-tumor MDSCs [69,70]. B. fragilis tends to establish an immune privilege microenvironment as soil for tumorigenesis centered on regional high-level Th17 and IL-17.

More than above discussed representative bacterium, evidences indicated immune regulation a momentous step of most microorganisms attaching influences on CRC. Kikuchi et al. founded more Bacteroides and Bacillus faecalis linked with incremental Tregs infiltration in CRC [71]. Lopes team detected regional CD3⁺ T cell decline were exhibited in Colibactin-Positive Escherichia Coli (CoPEC) colonized CRC, whether when investigate on patients or murine animal models [72]. Compared with control and adenoma group, colonic samples acquired from rats allocated to colitis associated CRC group exhibited higher abundance of Streptococcus gallolyticus. Zhang and colleagues illustrated their recruitment on MDSCs and laid of carcinogenetic microenvironment [73]. Probiotics defend local pro-tumor factors through their own immune intervention, too. Nontoxigenic Bacteroides Fragilis (NTBF) secrets SCFAs, suppressing NLRP3 induced inflammatory pathways and refraining macrophages from uncontrollable activation. Colitis associated CRC is repressed as a result [52]. NTBF and Akkermansia muciniphila synergy in expedition of dendritic cells (DCs) development and maturity. They upregulate IL-12 and enlarge cytotoxicity T cells group. Strengthened immune elimination has been ensured to multiply therapeutic efficiency of immune checkpoint inhibitors (ICIs) in clinical practice [74,75]. B. adolescent regulates GAS1 via Wnt signal pathway. Improved CD143⁺ cancer associated fibroblasts (CAFs) is then engaged in anti-tumor process [76]. Bifidobacterium strain raises CD4⁺ and CD8⁺ T cells, NK cells, and the ratios of CD4⁺/Treg, CD8⁺/Treg, and effector CD8⁺/Treg to inhibit tumor [77]. To summarize, carcinogenetic flora facilitate suppressive immune microenvironment while probiotics promote anti-tumoral immune cells.

2.3. Inflammation

Various metabolic products from intestinal microbes take part in inflammation control. Hydrogen sulfide not only break mitochondrial respiratory chain, but also increase risk of CRC in virtue of inflammatory NF-κB pathway [23]. Inflammatory recession on basis of HDAC inhibition and G protein-coupled receptor expression enable SCFAs minimize metabolic damage [78,79]. SCFAs interactively cast negative regulation on inflammatory factors, TNF-α, IL-1β/6, iNOS and stimulate anti-inflammatory IL-10 expression. These adaptions modify macrophages to M2 phenotype and drive T cells differentiation to manage inflammation [51,80].

Organic generic hurt from B. fragilis is realized with BFT. BFT increases the level of IL-17 and IL-23, boosts spermine oxidase (SPO) to produce ROS, both directly added inflammation [49,81]. The evoked IL-17 and Th17 cells would plus trigger chemokines, CXCL1, CXCL2, and CXCL5, appending secondary inflammatory cascade. S. gallolyticus execerbates CRC invasiveness by means of COX-2, IL-1 and IL-8 expansion [82]. Samples collected from CRC patients and those suffered from other colorectal diseases supported abundances of Faecalibacterium prausnitzii and A. muciniphila sank down from that of healthy controls. Studies explained the two potential protective mechanism with inflammatory restrictions from Treg cells and butyrates [83,84].

Fungus affected inflammatory condition, too. Malik’s research group discovered colonic symbiotic fungus modulated spleentyrosine kinase (SYK)-caspase recruitment domain 9 (CARD9) pathway and elevated inflammasome and IL-18. Mild uplift of inflammation heightened regional immune elimination and curbed CRC development [85]. However, extensive and longtime inflammatory infiltration will oppositely promote CRC.

2.4. Proliferation and Apoptosis

Cell cycle adaptation from intestinal microorganisms is put into effect under the alteration of some correlative signal pathways. NF-κB is a hot node located in that regulatory network. It is a family of transcription factors exert influences on a wide range of biological activities. As one aspect of core functions, maintenance of cell survival and uncontrollable proliferation permit malignant cellular multiplication. Aberrant activation of NF-κB evokes diversified driving genes of cell cycle, cyclin D1 and c-Myc for example, sustaining consistent dynamic cell division [86,87]. Excessive stimulation of NF-κB was detected in lung and breast cancer [88,89]. Tumor of APC gene knockdown mice gained diversity with the aid of F. nucleatum advanced on NF-κB [63]. In E. coli enema transplanted murine animals, tumor microenvironment generated lipopolysaccharide binded to histone-degrading enzymecathepsin K (CTSK), initiated TLR4 and vitalize CRC expansion and metastasis [90]. BFT also motivates Wnt, NF-κB, MAPK and STAT3 pathways by Th17 and IL-17 [67,91,92]. Peptostreptococcus anaerobius membranous signal domain PCWBR2 crosslinks with colorectal epithelial α2/β1 integrin, propelling NF-κB and proliferation through PI3K-Akt pathway [93].

Wnt/β-catenin is another significant proliferative pathway in CRC initiation and process. When Wnt ligand interrupted degradation complex, causal stable β-catenin catabolic process came to a brake, encouraging downstream cascade including c-Myc, cyclin D1 etc. and launch immoderate proliferation [94]. BFT lifts CRC proliferative rate in this pathway. Unique adhesive factor of F. nucleatum, FadA, expedites β-catenin by binding with epithelial E-cadherin to promote tumor formation [59]. In contrast, Parvimonas micra and P. anaerobius inactivate β-catenin and perturb colonic epithelial renewal homeostasis. Inordinate suppression on biological proliferation coherently brings about CRC [95]. Probiotics enable elimination based on autophage or apoptosis mechanisms. SCFAs produced from some Bacteroides and Firmicutes downregulate PI3K/AKT pathway. Following pro-apoptosis effect facilitates fade of tumor mass [47]. NTBF secreted membranous exosomes starts non-canonical autophage path and dispels established colitis associated CRC according to Hiutung et al. conducted research [96]. Delicate homeostasis on proliferation or elimination is one of the central regulatory spot by which microorganisms attached their impacts on CRC. Microbiomes adjusted proliferation or apoptosis associated signal pathways or targets is listed in Table 2.

2.5. Genotoxin and Mucosal Barrier

Some germs generated special toxins capable to unwind and break DNA. They are called genotoxins. The most common representative one is E. coli produced colibactin. E. coli could be divided into 4 phylogroups, A, B1, B2, and D [97]. Colibactin is a polyketide-peptide genotoxin encoded in pks gene island of B2 group E. coli. [98,99]. That group is correlated with inflammatory bowel disease (IBD) and tends to force out other group strains once civilized [100,101]. Persistent colitis in rodent ulcerative colitis (UC) model induced pks expression, contributing to amplified mutation frequency and consolidated colitis associated CRC [102,103]. Whole genome sequencing affirmed adenosine and thymine base pair is enriched in colibactin broken DNA target fragments [104]. Apart from former mentioned BFT and others, Campylobacter jejuni synthesize cytolethal distending toxin (CDT). Its catalyzation on DNA dissociation relys on CDT b subunit since this part mutation reversed carcinogenesis [105].

Structural and functional intact mucosal barrier defends epithelium from varieties of mechanical and biochemical injures. The single layer epithelial cells connected by collagen IV based tight junction concurrently realize nutrition absorption and microbiomic isolation [106,107]. BFT is a exogenous zinc-dependent matrix metalloproteinase. BFT cut down E-cadherin and destroy tight junctions [108]. In human derived colonic cells this process is companied with IL-8 secretion while in rat colonoids with tight junction protein 1 (TJP1/ZO-1) reduction [109,110]. E. coli operon afa-1 encodes non-classical adhesive factor other than pilus, assuring its invasion to colonocytes [111]. F. nucleatum applies FadA binding to E-cadherin, redeploying distribution of tight junction proteins to increase mucosal permeability [112]. S. gallolyticus is a gram-positive non-motile bacteria streptococcus. S. gallolyticus adheres to mucosal collagen I and IV in a higher affinity more than S. bovis, decomposes the latter and looses epithelial barrier [113]. Proximate two thirds of invasive S. gallolyticus infected patients suffered from concomitant CRC [114,115,116]. Figure 2 demonstrated above discussed microbiomes and host interactions.

3. Contemporary Applications of Microbiological Markers in CRC Diagnosis or Prognosis

3.1. Genetic Detective Methods and Carriers

On account of the specialized genetic segments designing respective toxins in microorganisms, quantify these regions would provide a detailed landscape of microflora construction and some potential information to predict state of CRC. The most widespread adopted method of fecal microbiological test is 16S rRNA test and shotgun metagenomic sequencing [117,118]. 16S rRNA is a tiny subunit of bacteric rRNA. It possesses a highly conserved and nine hypermutable regions, insuring primer design effective and distinctive identifiability to great sorts of microbiomes [119,120]. Compared to FIT with a fair sensitivity of 79% according to a meta-analysis, multi-target DNA test ameliorates sensitivity to 92.3% accompanied with acceptable increased false positive rate [121,122]. Shotgun metagenomic sequencing get rid of targeted amplification and turned to operate on entire DNA within sample environment. Shotgun stands for randomized cut off. Obtained segments undergo high-throughput sequencing to directly analyse components and their functional information, rebuilding metagenome-assembled genomes and recognize microbes in the level of species [123,124]. Efferent results could be put into deeper researches of drug tolerance, virulence factors, biosynthetic gene cluster and other focused points [125]. Many scholars has established fecal or tissue originated metagenomic sequencing databases [41,126,127].

Organoids and gene arrays are new test vectors under investigation. Genetic mutational signature is distinctive characteristic paradigm left in particular DNA districts after exposing to respective inducer. This property props up organoids manner in gastrointestinal flora analysis [128]. Furthermore, the superiority in procedural cost reduction and facility accessibility benefited antibody test in practice [129,130]. As for the metabolites participating in CRC development, Nuclear magnetic resonance spectroscopy, chromatography and chains of advanced approaches make material authentication and quantification feasible. Table 3 summarizes methodologies in microbiological test and applications in CRC.

3.2. Microbial Biomarkers

Bacterial composition fluctuates under pathological circumstances. This drift is applicable to tumor process since adenomatous lesion set up, cancerization until tumor metastasis. The most predominant colonic microbial species includes Firmicutes, Bacteroidetes, and Actinobacteria [131]. Dysbiosis index defined as Firmicutes-to-Bacteroidetes ratio was primarily designed to reflect host gastrointestinal health status. Studies illustrated higher dysbiosis index is correlated with longer survival outcome in CRC patients [132,133]. F. nucleatum is an hot spot in this field with ample evidences supporting its predictive value on worse outcome. Fecal and colonoscopic obtained samples confirmed its higher propagation in CRC patients than others [134,135]. Comparison between CRC tissues and closely adjoining normal tissues presented extensive enrichment in the former [136]. A metagenomic analysis operated in three methods uniformly demonstrated F. nucleatum and P. micra enriched in CRC patients [137]. Higher relative abundance of F. nucleatum indicated larger CRC volume in a clinical trial [138]. FadA gene from colonic adenomas and adenocarcinomas samples expressed as 10 to 100 times as that of healthy control [59]. Another study concentrated on advanced colorectal neoplasia stated quantify F. nucleatum effectuates discrimination of adenoma from all screened individuals [139]. In 2019, a large scale research enrolled 606 patients again provided consistent conclusion that F. nucleatum abundance was positively associated with CRC clinical stage process from initial intramucosal cancer to advanced disease [41]. Dramatically, Zhang et al. published study found oral harvested F. nucleatum DNA level relevant to CRC. Diagnostic curve highlighted the superiority over traditional carcinoembryonic antigen and carbohydrate antigen 199 indeed [140].

E. coli is another attractive node deemed as a predicative factor of early onset CRC [80]. Colibactin encoding gene owned by some tributaries isolated from CRC patients emerged an overexpression condition according to Wassenaar team reported [141]. In accordance with that, a case-control study discovered gradual reinforce of colibactin-producing E. coli along with the whole development of colorectal adenocarcinoma. When united with F. nucleatum the prediction on early CRC would come up to a 63.1% specificity and 84.6% sensitivity and this is consistent with Piawah and his co-researchers study [142,143]. Other bacterium were referred in some literature. Clostridium perfringens, P. micra, E. cordens and X. perforans may be early predicative factors [54]. C. symbiosum is regarded as a stage related marker [144]. Campylobacter potentially forebodes elevated metastasis risk from a Chinese trial enrolled initial I to II stage CRC patients [145].

For patients planned to receive immunotherapy, microbial biomarkers manifested considerable efficiency in potency anticipation. A. muciniphila is a gram stained negative obligate anaerobic bacteria concerned with CRC. Studies proved higher level of A. muciniphila guided to more active therapeutic response of PD-1 block. Thus CRC mice received artificial complementary A. muciniphila exhibited improvement of PD-1 blocker curative effects [146]. This attributes to promotion on DC activation, Th1 polarization and cytotoxic T cell recruitment [75].

Microbiological biomarker not only instructed judgement on tumor present status and potential resistance against chemical therapy, but also transmit its location messages. A multi-cohort analysis completed in 2025 proposed a CRC site associated microorganic group, from which estimation on tumor location (right colon, left colon or rectum) would attain an AUC of 82.92%. They put forward successive upward α-diversity from right CRC, left CRC to RC [147]. Microbial CRC associated microbiological markers and clinical applications are presented in Table 4.

A few animo acids concentration accumulates along with CRC growth. Alanine, glutamine and glycine were examples verified in different studies [54,148,149,150,151]. Glycine offered precursors for nucleotide, lipid and protein synthesis, which is in urgent need for tumor proliferation. One carbon metabolism network consist of glycine and serine, inducing immune suppressive microenvironment [152,153]. DCA and lactate were also reported as effective CRC process predicative factors [56,154].

4. Discussion

It was estimated that in 2020 over 1.9 million new CRC cases presented all over the world. 930,000 deaths were attributed to CRC [155]. The survival outcome is negatively correlated with disease stage. To prepose diagnosis and prevent treatment opportunity loss, oncologists and clinicians explored and tested biometrical indicators under the assistance of earlier started screen tests. The most universal biomarkers are carcino-embryonic antigen (CEA) and carbohydrate antigen 199 (CA199). Nevertheless, smoking, IBD, liver diseases, pancreatitis and some other pathological alteration also pull up CEA, for CA199 relevant cases include liver cirrhosis, acute cholangitis, diabetes mellitus, endometriosis and bronchiectasis [156]. Not so ideal specificity may miss some determined while not previously exhibited CRC cases, leading to uncontrollable occasions. Microbiomes have been evaluated in a number of malignant diseases including oral, breast cancer and malignant melanoma etc. as biomarker at different degrees of validity. That is at least equally effectual in CRC as they still locate in closest site.

Microbes exert their own oncological dynamic administrations on host colorectal micro-ecology. The comprehensive influences could be classified into metabolism, immune, inflammation, proliferation/apoptosis, genetoxin and mucosal barrier associated fields. Although complicated structural network crosslinks among them, this sort manner enable us to tease out and summarize.

Too much aggregated hydrogen sulfide inactivates complex IV and cut the electron transport chain. ROS emerged soon damages intra-cellular DNA, both nuclear and mitochondrial ones. SRBs represented by Desulfovibrio engaged in this process as a role of proteins bioconversion. Excessive PCAs adhesion with FXR initiates colonic ROS and inflammation, stimulating colonocytes proliferation. One of SCAs induced effects is macrophages repolarizing into M2 phenotype. SCFAs also trigger the same change, while the theme turned to anti-inflammation other than so called anti-immune. So SCFAs adjustment of β-oxidation guards colonic epithelial from unexpected injures and carcinogenesis. F. nucleatum produced FadA influences T cells and NK cells function and attracts MDSCs. B. fragilis main set point of immune is Th17 and IL-17 related parts. BFT arouses SPO and expands ROS. SCFAs act as a anti-inflammatory factor within microenvironment. Cytokine system is broadly concerned to inflammation accommodation and under multitudinous and multiple influences from colonic microflora. B. fragilis and E. coli realize their effects via NF-κB and Wnt/β-catenin signal pathway which controlled cell cycle related proteins. E. coli encoded colibactin to damage DNA and BFT is a extracellular metalloproteinase collapsing collagen IV based tight junctions among colonic epidermis.

Traditional and mature detective methods include 16S rRNA test, shotgun metagenomic sequencing and FIT. Comparing to restricted fragment of 16S rRNA test, shotgun metagenomic sequencing expands test category to those not previously determined. Some brand new technologies like organoids and gene arrays require more verifications on efficacy or standard operation procedures (SOPs). Abundance of specified species or their ratios, predefined genic fragments and concentrations of products are dominated 3 trains of thought when trying to diving into new biomarker designment.

More than just about diagnosis and prognosis, the CRC whole developing process and most likely sites information are also contained in appropriate samples. Latest studies have switched to probe in therapeutic effective predication of leading modes represented by immunocheckpoint inhibitors predominated ones. They have already got periodical achievements. This topic calls for more clinical trials in the future, as now available facts rely heavily on animal experiments. Current evidences indicated that obvious and potential species have been analyzed a lot. Future efforts tended to be made more on further data treating and reasonable filter to deduct dimensions and simplify clinical application process. In terms of restricted predictive efficiency, suitable combinations might provide more beneficial selections. There is a to be established balance between simplification and efficacious. It might be a practical attempt to concentrate more on metabolites and their combinations as so far statistic process are too depending on microbiomic automated analysis which received statistics submitted by microorganic detectors and panels. Maybe there is a much simpler and convenient manner waiting to discover. Futhermore, though fungus, virus and phages contributed less than germs, they should not be excluded from prospective investigations. Few literatures on this topic has provided limited instructions. This part together with metabolites founded explorations has a great potential in coming new era of CRC microbiomic biomarkers. It still demands to dive much more potential useful microbiomic related biomarkers and conduct clinical translation as global CRC incidence accumulation in recent years. The already and coming to occurred lifestyle changes along with economic growth would highlight CRC correlated prejudging fields even more than instant.