2.1 Microbiota and increased genomic damage

2.2 Microbiota and DNA repair

Throughout the cell cycle, human DNA undergoes repetitive DNA damage events. A complex network of cellular systems exists to maintain the integrity of DNA. This biological system detects DNA damage and promotes DNA cell cycle repair checkpoints or apoptosis, or both. The process of DNA mismatch repair (MMR) is a well-studied and widely used biological route that helps keep genes intact and the genome stable. DNA replication and recombination both generate base mismatches and insertion/deletion mismatches, which are specifically targeted by MMR.¹⁰² For instance, enzyme activation-induced cytidine deaminase (CDD) converts cytosine to uracil, creating a G:U mismatch detected and treated by MMR.^{103, 104} MutL homolog 1 (MLH1) and mutS homolog 2 (MSH2) play central roles in the MMR. Both form heterodimeric complexes with all other MutL and MutS homologs, respectively, and deletion of MSH2 or MLH1 results in complete inactivation of the MMR in humans and other species.¹⁰⁵

E. coli depletes the MMR proteins MSH2 and MLH1 in cultured colon cells in a T3SS-dependent manner, inducing host mutagenesis.^{106, 107} Similar results were seen in Helicobacter (H.) pylori-infected GEpiC.¹⁰⁸ This may be due to specific CagA EPIYA motifs and vacA genotypes.¹⁰⁹ H. pylori suppress MMR protein (MLH1, MSH2, MSH3, and MSH6) expression through miR-155-5p and miR-3163 upregulation.¹¹⁰ F. nucleatum increases miR-205-5p expression through TLR4 and myeloid differentiation primary-response protein 88 (MyD88)-dependent innate immune signaling pathways and suppresses MLH1, MSH2, and MSH6 expression, leading to DNA MMR damage and cell proliferation in HNSC.¹¹¹ Among the 30 cancer types, validation revealed 21 different mutational signatures, with C>T at NpCpG mutations predominating in Signature 6. This pattern of indels (mostly of 1 bp) is commonly referred to as “microsatellite instability (MSI).”¹¹² MSI is a hypermutated phenotype resulting from deficient DNA MMR activity, prevalent in around 15% of CRC. Out of these cases, Lynch syndrome accounts for only 3%, while the remaining 12% are linked to sporadic acquired hypermethylation of MLH1 gene promoter observed in tumors with CpG island methylator phenotype.^{113, 114}

In addition to changes in gut microbiota richness and diversity, elevated levels of Proteobacteria were associated with decreased HR of thyroid carcinoma by typing analysis, a mechanism that needs to be further investigated.¹¹⁵ HFD leads to disruption of the gut microbiota, causing innate immune dysregulation, producing an inflammatory environment, decreasing DSB repair, and being reversed with antibiotic treatment.¹¹⁶ The microbiota in a hypoxic microenvironment increases tumorigenesis as demonstrated in Figure 2.

2.3 Microbiota and signaling pathways in TME

Long-term settlement of microbiota in tumor tissue, on the one hand, causes an inflammatory response, which can result in damage to the vascular endothelium, dysfunction of vascular endothelial cells, impaired blood transport, leading to hypoxia. On the other hand, some pathogenic bacteria can rob oxygen from the TME, or microbial metabolites, such as short-chain fatty acids (SCFAs), increase cellular O₂ consumption through β-oxidation of butyrate and oxidative phosphorylation (OXPHOS), thereby stabilizing hypoxia-inducible factor (HIF).^117-119 Moreover, microbiota-recruited innate and adaptive immune cell infiltrates, most notably infiltrating neutrophils and eosinophils, consume local oxygen via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase during respiratory burst.^{120, 121} A large amount of ROS produced by these inflammatory responses stimulates HIF-1α expression and becomes stable under hypoxic conditions.¹²² TLRs are a type of PRR that has a crucial function in the immunological host defense system by identifying pathogen-associated molecular patterns.¹²³ After LPS stimulation, splicing protein like MyD88 is recruited to the TLR cytoplasmic structural domain.^{124, 125} LPS-induced ROS production and TLR4 or MyD88 are required for HIF-1 activation.¹²⁵ LPS stimulates macrophages to activate HIF-1α in TLR4-dependent rather than hypoxia manner, increases HIF-1α mRNA level, decreases prolyl hydroxylase (PHD) mRNA production, and promotes glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF) expression.^{126, 127} LPS increases HIF-1α mRNA expression by stimulating the monocytes NF-κB, while hypoxia stabilizes HIF-1α protein after translation.¹²⁸

LPS activates the TLR4-NF-κB pathway to upregulate HIF-1α, which in turn promotes pancreatic cancer (PC) progression.¹²⁹ Furthermore, LPS promotes PC cell migration by decreasing the levels of two tumor suppressors, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and MAP2K4, through the TLR4-miR-181a signaling pathway.¹³⁰ PTEN deletion promotes HIF-1-mediated gene expression,¹³¹ Siderophores (yersiniabactin, salmochelin, aerobactin) are secreted by Yersinia enterocolitica, S. enterica subsp enterica, and Enterobacter aerogenes, induce dose-dependent HIF-1 stabilization in epithelial and endothelial cells, and independently with hypoxia.¹³² Pathogenic Gram-negative bacteria secretes enterobactin that stabilizes HIF-1α, which binds to HIF-1β and enters the nucleus to bind hypoxia response element, stimulating the expression of HIF-1α-regulated genes.^{133, 134} CagA produced by H. pylori is injected into host cells via the type IV bacterial secretion system (T4SS), which activates β-catenin, leading to the upregulation of transcriptional genes associated with gastric cancer.¹³⁵ Insertion of AvrA secreted by S. typhimurium into host cells upregulates β-catenin signaling, characterized by increased Bmi1, matrix metalloproteinase-7 (MMP-7), and cyclin D1.¹³⁶ BFT induces E-calmodulin cleavage, β-catenin nuclear localization, upregulation of c-Myc transcription and translation, and cancer cell proliferation.^{71, 137} FadA on the surface of F. nucleatum binds to E-cadherin and activates β-catenin signaling.¹³⁸ F. nucleatum activates TLR4 signaling to MyD88, which activates NF-κB and increases the expression of miR21, a miRNA that reduces the level of the RAS GTPase RASA1.¹³⁹ F. nucleatum increases cytochrome P450 2J2 and 12,13-epoxyoctadecenoic acid through activation of the TLR4/Kelch-like ECH-associated protein 1/NF-erythroid 2-related factor 2 axis to promote epithelial–mesenchymal transition (EMT) and metastasis in CRC.¹⁴⁰ Porphyromonas (P.) gingivalis can invade CRC cells and promote their proliferation by significantly activating the MAPK/ERK signaling pathway.¹⁴¹ Mannose-binding lectin binds to glycans of the Malassezia wall and triggers C3 convertase, which can cleave C3 to C3a, and then C3a binds to C3aR on the surface of the tumor cells, promoting proliferation of PC cells in vitro and tumor growth in vivo.³⁷ Staphylococcus, Lactobacillus, and Streptococcus penetrate the cell membrane and localize in the cytoplasm, inhibit RhoA and ROCK activation, remodel the cytoskeleton to resist mechanical stress, and promote lung metastasis of murine breast cancer cells.¹⁴² Tumor cell signaling pathways altered by microbiota are showed in Figure 3.

Microbiota can alter the malignant phenotype of tumors by inducing inflammation, modulating the antitumor immune system, altering TME, and influencing cellular metabolism, and is strongly linked to tumor patients' prognosis.^{143, 144} TLR activated by microbiota can cause derangements of multiple tumor suppressor proteins (e.g., p16, p21, p27, p53, pRb, PTEN, and MAP2K4), induce STAT3 activation, and enhance EMT and oncogene-induced senescence.¹⁴⁵ Microbiota, on the other hand, can generate ROS by inducing a prolonged inflammatory response via changes of essential substance as lipids, nucleic acids, and preproteins that result in protein misfolding, membrane disintegration, and DNA fragmentation and accumulation over time,¹⁴³ which may dominate cellular senescence.¹⁴⁶ Senescent cells, in contrast to quiescent and apoptotic cells, are nonetheless highly viable and perform metabolic functions efficiently.¹⁴⁷ SASPs are a group of paracrine signaling molecules secreted by senescent cells that include cytokines, chemokines, growth factors, and proteases.¹⁴⁸ In an autocrine or paracrine fashion, senescent cells promote the emergence of age-related disorders, particularly cancers, while also aiding in tissue formation, suppressing tumors, tissue healing, and cell proliferation.¹⁴⁴ Evidence suggests that xenograft tumor growth can be accelerated by a dysregulated SASP that sustains inflammatory conditions in TME that promote cancer proliferation, migration, invasion, and EMT.¹⁴⁴ For instance, exposure to the harmful DCA, produced by intestinal bacteria, stimulates cellular senescence and secretes SASP, which increases tumor-promoting inflammation and promotes the development of HCC.⁷⁹ There are now experiments to identify the molecular mechanisms of hypoxia-associated senescence onset. For example, elevated miR-125a-5p is implicated in senescence and SASP secretion across lung epithelial cells via specificity protein 1 (Sp1)/sirtuin 1 (SIRT1)/ IF-1α.¹⁴⁹ Inhibition of HIF-1α may inhibit SASP secretion.¹⁵⁰

3.1 Glycolysis

Even under aerobic settings, tumor cells glycolyze glucose into lactate, called the Warburg effect, to meet their energy requirements.¹⁵¹ HIF-1α is the primary transcription factor responsible for promoting Warburg-like metabolism.¹⁵¹ Some bacteria also affect the glycolysis of tumor cells. Aspergillus fumigatusinduces aerobic glycolysis in colon cancer cells.¹⁵² Increased ENO1-IT serve as a guide module for KAT7 histone acetyltransferase, specifying histone modification patterns of its target genes (as ENO1) and thereby varying CRC glycolysis and tumorigenesis. This is accomplished by enhancing Sp1's binding efficiency to lncRNA ENO1-IT1 promoter region.¹⁵³ However, some bacterial metabolic wastes such as microbiota-derived Staphylococcal superantigen-like protein 6 (SSL6) downregulate PI3K/AKT-mediated glycolysis by hindering CD47, which contributed to enhanced susceptibility of HCC to sorafenib.¹⁵⁴ Microbiota can regulate immune cell glycolysis through TLR signaling. When TLR was activated, the glycolysis of macrophages increased significantly, and mitochondrial activity was inhibited. HIF-1α stabilization with ROS after LPS stimulation significantly upregulated GLUT1, hexokinase 3, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, phosphoglucomutase-2, and enolase 2 (ENO2) confirming the increase in glycolysis.¹⁵⁵ This shift in metabolic activity, called glycolytic reprogramming, leads to altered mitochondrial function, increased ROS production, and increased secretion of proinflammatory cytokines.¹⁵⁶ LPS-stimulated TLR signaling in immune cells (e.g., monocytes, dendritic cells [DCs], regulatory T [Treg] cells) increased PI3K–AKT–mTORC1 signaling, glycolysis, and GLUT1 expression.^157-159 TLR also induces a metabolic shift from OXPHOS to aerobic glycolysis in DC via PI3K/Akt pathway, mTOR–iNOS–nitric oxide (NO), because NO can inhibit mitochondrial function.^160-162 Other bacteria, such as B. uniformis, have a strong glycolytic capacity and produce butyrate.¹⁶³ Butyrate accumulates in tumor cells because of the Warburg effect, where it functions as an histone deacetylase inhibitor to promote histone acetylation, which in turn suppresses cell proliferation and induces apoptosis, so helping to prevent CRC.¹⁶⁴

3.2 Lipid metabolism

The study findings revealed that several bacterial taxa in the oral and intestinal tracts, such as Fusobacterium, Erysipelotrichaceae, and Lachnospiraceae families in the gut, were strongly connected to low-density lipoprotein and total cholesterol.¹⁶⁵ Compared with germ-free mice, conventionally housed mice had significantly higher levels of pyruvate, citrate, fumarate, and malate and lower cholesterol and fatty acid (FA) levels.¹⁶⁶ Microbiota provide raw materials for lipid metabolism synthesis through their own metabolites or stimulate lipid synthesis in host cells. For example, SCFAs serve as substrates for various metabolic processes, including cholesterol synthesis, lipogenesis, and gluconeogenesis.¹⁶⁷ Microbiome-derived metabolite δ-valerobetaine activates genes encoding mitochondrial energy production and FA oxidation via the transcription factor PPAR-α, thereby reducing mitochondrial FA oxidation and increasing lipid accumulation.¹⁶⁸ Microbes induce monounsaturated FA production via stearoyl-CoA desaturase 1 and poly-unsaturated FA elongation with FA elongase 5, resulting in significant glycerophospholipid acyl-chain profile alterations, as acetate from intestinal microbiota breakdown of dietary fiber is a precursor for hepatic production of C16 and C18 FAs and their related glycerophospholipid species, which are likewise transferred into circulation.¹⁶⁹ Small bowel C. bifermentans selectively induces participation in diacylglycerol O-acyltransferase 2, which increases duodenal and jejunal oleic acid uptake.¹⁷⁰ Activation of TLR-4 signaling may promote increased lipid synthesis.¹⁷¹ However, controversy exists.¹⁷² Bile acids help absorb dietary lipids and fat-soluble vitamins. Synthesized from cholesterol in the liver, they are stored within the gallbladder before being released into the intestine postingestion. Primary bile acids combine with taurine or glycine and undergo further metabolism by C. (clusters XIVa and XI) as well as Eubacterium to produce secondary bile acids.^173-175 About 95% of bile acids that are secreted into the intestine are taken back up, mostly as bound bile acids located in the distal ileum by a protein known as ASBT or IBAT. These reabsorbed bile acids go through the portal vein and return to the liver where they are once again released. This cycle, known as hepatic-intestinal cycle, takes place approximately six times daily in humans.¹⁷⁶ Bile acids have the capacity to serve as signaling agents by binding with farnesoid X receptor (FXR), that is a nuclear receptor predominantly present in the ileum and liver.¹⁷⁷ FXR has a role in the control of lipid metabolism, namely the transportation, production, and usage of triglycerides.¹⁷⁸ Chenodeoxycholic acid (CDCA) is the strongest FXR ligand, followed by cholic acid, DCA, and lithocholic acid,¹⁷⁶ and in an FXR-dependent manner, gut microbiota stimulates excess weight and hepatic steatosis,¹⁷⁹ whereas in hepatic FXR absence, elevated LXR expression and induction of LXR's lipogenic target genes, Scd-1 and Fas, and elevated triglyceride and bile acid levels are observed.^{180, 181} This has important implications in bile acid-induced HCC.¹⁸² The gut microbiota has the ability to metabolize nutrients that contain methylamine, namely choline, lecithin, and l-carnitine. As a result of this process, trimethylamine (TMA) is produced, which then undergoes a further transformation into TMAO through the action of flavin monooxygenases (FMO) located in the liver.¹⁸³ Bile acids by gut microbiota activate FXR-induced FMO3 expression and increase the development of hyperglycemia, hyperlipidemia, and atherosclerosis.^{184, 185} In other experiments, it was discovered that bile acids hindered the ability of DU-145 migratory prostate cancer cells to adhere, migrate and invade. Both CDCA and DCA were found to destabilize HIF-1α across all cells while effectively impeding crucial phenotypes related to cancer progression. This novel observation implies that bile acids have significant physiological implications in targeting hypoxic tumor advancement.¹⁸⁶ With the rapid expansion of gut flora research, future studies will elucidate how the flora interacts with intestinal lipid metabolism pathways, undoubtedly leading to additional therapeutic options.

4.1 Microbiota and tumor stromal cells

Fibrosis in some solid tumors is an important factor affecting the therapeutic effect of tumors. One of the causes of fibrosis in solid tumors (e.g., liver and pancreas) is the presence of a large number of stromal cells in TME, with stellate cells being the main component of stromal cells. Stellate cells are retinol-storing cells that are quiescent under normal conditions; in the pathological state, they are activated and release extracellular matrix (ECM) components, including numerous critical regulators of fibrosis.^{187, 188} Microbiota and their derived metabolites can directly or indirectly activate stellate cells. Stenotrophomonas maltophilia exacerbates liver fibrosis by activating the TLR4/NF-κB/NLRP3 pathway and promoting HSC expression of α-smooth muscle actin (α-SMA) and collagen I.³³ HCC development in mice is stimulated by a number of inflammatory and tumor-promoting mechanisms, all of which are upregulated by dysbiosis of the intestinal microbiota and hence elevated DCA levels.⁷⁹ LPS activates PC cells via TLR4, NLRP3 inflammatory vesicle-driven, expresses IL-1β, and stimulates the activation and secretory phenotype of quiescent pancreatic stellate cells (PSCs).¹⁸⁹ Microbially recruited immune cells, such as M2 macrophages, secrete transforming growth factor-β (TGF-β), which in advanced PC can effectively induce PSCs to secrete collagen α-SMA, collagen I, and IV to exacerbate fibrosis.^190-192 The TGF-β1-activated enhancer-binding protein δ/HIF-1α/hepatoma-derived growth factor axis contributes to the antiapoptosis of PSC, leading to synthesis and deposition of ECM proteins.¹⁹³ These fibrosis-associated ECM, such as type IV collagen, periostin, promote cancer cell proliferation, migration, and apoptosis resistance, confer resistance to starvation and hypoxia, and limit the delivery of chemotherapeutic drugs to cancer cells.^194-196

4.2 Microbiota and angiogenesis

Dysbiosis of the intestinal microbiota enhances intestinal permeability and chronic hypo-inflammation features of inflammation, accompanied by elevated levels of IL-6, IL-1β, tumor necrosis factor alpha (TNF-α), and VEGF-A, ultimately exacerbating pathological angiogenesis.¹⁹⁷ C. difficile generates two primary exotoxins, namely Toxin A and Toxin B, which induce VEGF-A expression in endothelial cells through the HIF-α, p38-MAPK, and MEK1/2 signaling pathways and increase angiogenesis in human intestinal microvascular endothelial cells (HIMEC).¹⁹⁸ LPS stimulates NOD-like receptors (NLR) and TLR to increase micro angiogenesis and induce motility and proliferation of HIMEC.¹⁹⁹ LPS-mediated ERK phosphorylation leads to forkhead box protein C2 (FOXC2)-ERK protein linkage, ERK-dependent phosphorylation of FOXC2 serine and threonine, and additional delta-like 4 activation (the master regulator of sprouting angiogenesis expression) gene expression to promote angiogenesis.²⁰⁰ LPS stimulates intestinal fibroblasts to produce insulin-like growth factor-1, IL-6, PDGF-BB, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein 1α, all of which may promote mucosal angiogenesis in a complementary manner.²⁰¹ In vitro, low concentrations of the microbial derivative butyrate act proangiogenically via the receptor G-protein-coupled receptor 43.²⁰² HPV-16 oncoprotein induced HIF-1α, VEGF, and IL-8 expression in vitro and significantly enhanced angiogenesis in lung cancer cells, which may be associated with the involvement of PI3K/AKT signaling pathway and c-Jun.²⁰³ Additionally, angiogenesis requires endothelial cells to interact with various cell types.²⁰⁴ The mesenchymal cells (MSCs) are in close proximity to the endothelial cells and generate factors that promote angiogenesis.²⁰⁵ TNF-α induced IL-8 production by HIMEC and intestinal fibroblasts, as well as upregulated VEGF-R2 expression on HIMEC to promote angiogenesis by initiating endothelial cells and stimulating MSCs to generate angiogenic factors.²⁰⁶ Taken together, microbiota disorders may lead to an imbalance in the levels of pro- and antiangiogenic factors; these genes are tightly regulated in normal tissues and facilitate swift yet aberrant tumor angiogenesis.²⁰⁷

However, some scholars believe that tumor vessels exhibit tortuosity, dilation, and uneven distribution. The adjacent endothelial cells demonstrate loose attachment to each other, while the pericytes that surround the vessels generally remain detached from endothelial cells. Consequently, tumor vascular leakage occurs with dysfunctional blood flow due to irregular regulation of vascular permeability by pericytes.²⁰⁸ In vitro studies showed that some fungi significantly suppress HPV-16 E7 oncoprotein-induced angiogenesis in lung cancer.^{209, 210} Tumor angiogenesis does not necessarily equate to the increased blood supply to the tumor, as discontinuous basement membrane of immature neovascularization permits extravasation of plasma and protein, further increasing intratumor interstitial pressure, prolonged vascular collapse, and inadequate nutrient delivery.²¹¹ Furthermore, some tumors are unable to sustain vascular survival, which illuminates the presence of well-formed, aggressive peripheral and central areas of necrotic hypoxia in certain highly angiogenic tumors.²¹² Thus, despite high vascular density, the neoplastic endothelium generated in the majority of malignancies is often deformed and dysregulated, lacks blood transport function, and is less effective in transporting oxygen and nutrient and drug delivery.^{213, 214}

5.1 Microbiota and innate immunity in cancer

5.2 Microbiota and adaptive immunity in cancer

6.1 Microbiota and drug resistance

The anticancer activity of chemotherapeutic drugs relies on the disruption of DNA integrity, that is, the enzymes used for DNA repair and synthesis. Generally, symbiotic microorganisms interact with chemotherapy medications by influencing drug metabolism (pharmacokinetics) and host immunity (i.e., pharmacodynamics). Microorganisms and their derivatives can transform medicines directly into nontoxic or toxicity-reducing metabolites.³²⁹ Mycoplasma (M.) hyorhinis in cancer contains many nucleoside metabolizing enzymes such as thymidine phosphatase that degrade pyrimidine nucleoside analogs, including 5-fluoro-2′-deoxyuridine (FdUrd), 5-trifluorothymidine and 5-halogenated 2′-deoxyuridine to their inactive bases.³³⁰ M. hyorhinis produces CDD and pyrimidine nucleoside phosphorylase, which can determine gemcitabine to less cytostatic metabolite 2′, 2′-difluoro-2′-deoxyuridine, which impairs anticancer effects of drugs.³³¹ Gammaproteobacteria in TME can also produce CDD, leading to gemcitabine degradation and drug resistance.³³² Besides, the microbiota antagonizes chemotherapeutic drug efficacy by activating cellular autophagy and remodeling the TME matrix. F. nucleatum targeted TLR4 and MyD88 innate immune signaling and specific miRNAs (miR-18a* and miR-4802), respectively, targeting UNC-51 like kinase 1 and autophagy-related protein 7 (ATG7) to promote autophagic activation in CRC cells to inhibit apoptosis and induce resistance to oxaliplatin and 5-FU (Figure 6).³³³ Microbiota dysbiosis induces TLR4–NLRP3 inflammasome signaling pathway and promotes IL-1β secretion by PC cells.¹⁸⁹ IL-1β induces chemo/immunotherapy resistance by activating PSCs, which can induce mesenchymal fibrosis.³³⁴

However, not all microbiota are detrimental to chemotherapy, and some can enhance the efficacy of chemotherapeutic agents. Scott et al.³³⁵ determined, using a simplified animal model of the nematode Caenorhabditis elegans, that E. coli vitamin B6 is needed for 5-FU efficacy in C. elegans. Mechanically, PLP, the active form of B6, controlled glycine cleavage system, can be synthesized by E. coli and is responsible for mediating the efficacy of 5-FU. Disruption of bacterial vitamin B6 production inhibits bacterial ribonucleotide metabolism and greatly antagonizes the efficacy of 5-FU.^{335, 336} The SSL6 microbiota promotes sorafenib-induced apoptosis of HCC cells Huh-7 and MHCC97H by blocking CD47 and thereby downregulating PI3K/Akt-mediated glycolysis.¹⁵⁴ Alkylating chemotherapeutic cyclophosphamide (CTX) depletes immunosuppressive Treg and promotes Th1 cell development, hence inducing anticancer immunity.^{337, 338} Enterococcus hirae might elevate intratumoral CD8⁺/Treg cell ratio, while Barnesiella intestinihominis could boost the infiltration of IFN-producing γδ T cells in cancer lesions and restore the CTX-induced anticancer Th1 cell or CTL response, thereby controlling tumor progression.³³⁹ Microbiota also mediates host tolerance to chemotherapeutic agents. Irinotecan is a topoisomerase I inhibitor that is used as a first treatment for advanced CRC and is effective in a variety of cancers. Irinotecan is converted by carboxylesterase into 7-ethyl-10-hydroxy camptothecin (SN-38), an inhibitor of topoisomerase I that causes DNA replication and transcription arrest in tumor cells. Inactivation of SN-38 to SN-38 glucuronide (SN-38G) occurs via glucuronidation catalyzed by various hepatic and extrahepatic diphosphoglucuronosyltransferase 1A isozymes.^{340, 341} The β-glucuronidase produced by C. clusters XIVa and IV converts SN-38G back to cytotoxic form SN-38, resulting in severe diarrhea.^{342, 343} Therefore, the researchers propose to develop bacterial β-glucuronidase inhibitors to improve the tolerance of irinotecan in cancer patients without killing the bacteria or harming mammalian cells.³⁴⁴ Such as E. coli βG-specific inhibitor pyrazolo[4,3-c]quinoline derivative (TCH-3562), uronic isofagomine, and old drugs, including N-desmethylclozapine, aspartame, and gemifloxacin, are anticipated to be a promising drug for prevention of CPT-11-induced diarrhea.^345-347 Current research has focused on the relationships between intratumor microbiota and chemoresistance, although the mechanism underlying these interactions remain obscure. Chemotherapy also poses additional problems, as it contributes to the development of antibiotic-resistant gut bacteria.³⁴⁸

6.2 Microbiota and radiation tolerance

Ionizing radiation's (IR) anticancer properties are partially mediated by the stimulation of innate and adaptive immunity.^{349, 350} The main tolerance of cancer cells to radiotherapy can be attributed to the disruption of intestinal microbiota. Gram-positive bacteria and SCFA reduce the activity of antigen-presenting cells (APCs) like DCs, inhibit their impaired antigen presentation function, limit CD8⁺ T cell infiltration and activation, and weaken radiotherapy-induced antitumor immune response (Figure 7). Vancomycin enhanced RT-mediated systemic anticancer effects by eliminating gram-positive bacteria and reducing SCFA, enhancing the activity of DCs, increasing CD8⁺ T cell infiltration, and secreting IFN-γ.³⁵¹ Overgrowth of commensal fungi can reduce antitumor immunity after tumor irradiation. Macrophages expressing C-type lectin receptor Dectin-1 (sensing fungi) are recruited into tumor tissue to suppress adiation-induced therapeutic antitumor immunity. Targeted commensal fungi enhance radiation-induced antitumor immune responses by reducing macrophage-mediated immunosuppression.³⁵² Impaired anti-HCC immunity is caused by dysbiosis of gut microbiota, which inhibits APC and suppresses effector T cell function via cGAS-stimulator of STING–IFN-I pathway.³⁵³ Oral administration of Lachnospiraceae produced butyrate suppressed STING-activated IFN-I expression in DCs by blocking phosphorylation of TANK-binding kinase 1 and IRF3, thereby abrogating IR-induced tumor-specific cytotoxic T cell immunological responses.³⁵⁴ Meanwhile, radiation treatment alters the host gut microbiota and immune response after radiation therapy. For example, irradiation therapy significantly altered the bacterial composition of large and small intestines. Elevated levels of Alistipes and Corynebacterium in large and small intestines, respectively.³⁵⁵ Total body irradiation (TBI) impairs the gastrointestinal barrier in mice, allowing bacterial translocation associated with innate immune system activation, removing cytokine sinks and Treg cells. LPS via TLR4 signal improves the functionality of adoptively transferred CD8⁺ T cells.³⁵⁶ After TBI, neutrophils migrate from the damaged intestine to MLN, express MHC-II, present host antigens, and participate in donor T cell expansion.³⁵⁷ Microbiota exacerbates tumor radiation tolerance partly by weakening the effective antitumor immunity. However, the specific molecular mechanisms by which radiotherapy interacts with the microbiota in tumors are unclear and require further study. In addition, high-dose IR used during cancer radiotherapy is associated with the induction of hematopoietic, gastrointestinal, and cerebrovascular damage.³⁵⁸ Patient tolerance to radiotherapy can be improved by altering the host microbiota and its metabolites. Propionate and tryptophan metabolites derived from bacterial taxa Lachnospiraceae and Enterococcaceae attenuate hematopoietic and gastrointestinal syndromes and reduce proinflammatory responses.³⁵⁹ FMT elevated the level of microbial-derived indole-3-propionic acid in the feces of irradiated mice, which activated pregnane X receptor/acyl-CoA-binding protein signaling and protected against gastrointestinal toxicity, demonstrating a lower system inflammatory level, recuperative hematopoietic organs, catabatic myelosuppression, improved gastrointestinal function, and epithelial integrity.³⁶⁰ Gut microbiota produced-valeric acid supplementation increased survival, protected hematopoietic organs, and enhanced gastrointestinal function and intestinal epithelial integrity in mice that had been irradiated.³⁶¹ Supplementation of microbiota-derived PGF2α via the oral route activates the F-prostanoid/MAPK/NF-κB axis, promotes cell proliferation, and inhibits apoptosis, thereby reducing lung inflammation and improving pulmonary respiratory function after local chest irradiation.³⁶²

7.1 Biocarrier applications

Some bacteria have the ability to be drawn to the hypoxic core of cancer cells, where they can proliferate and spread. The use of microorganisms to target the hypoxic zone of cancer might be a viable cancer therapy technique. Spores of C. novyi-NT significantly improved the efficacy of radiotherapy in various mouse models, such as biliary, colorectal, melanoma, and squamous cell carcinoma.³⁶⁵ Attenuated pathogenic facultative anaerobic S. typhimurium VNP20009 decorated by heptamethine cyanine dyes NHS-N782 and extra terminal domain (BET) protein inhibitor, JQ-1 derivatives, invades into tumor cells, targets mitochondria in tumor cells, and downregulates PD-L1 expression in TME to produce an effective and durable T cell immune response.³⁶⁶ Attenuated S. typhimurium (S.T.ΔppGpp) was used to engineer a new strain of bioluminescent bacteria that enhanced PDT, promoted the conversion of anti-inflammatory macrophages (M2) to proinflammatory macrophages (M1), stimulated tumor NK cells, CD4⁺ Th cells and CD8⁺ T cells, reduce immunosuppressive Treg cells in TME, and upregulate the expression of various effector cytokines.³⁶⁷ Nevertheless, colonized S. typhimurium may recruit large numbers of neutrophils, which favor tumor growth.^{368, 369} Silver nanoparticles modified with group sialic acid selectively recognize l-selectin on neutrophil surfaces, deplete neutrophils, allow migration of S. typhimurium to surviving tumor regions, and enhance tumor suppression by inducing apoptosis leading directly to tumor death.³⁷⁰ E. coli membrane vesicle is an excellent catalase delivery that delivers catalase to decompose H₂O₂ into oxygen to alleviate hypoxia and radiotherapy resistance.³⁷¹

7.2 Probiotic applications

Probiotics are defined as “live bacteria that impart a health benefit on the host when administered in suitable levels” and mainly consist of Lactobacillus and Bifidobacterium species,³⁷² but also strains that reduce intestinal inflammation, induce immune regulation or enhance intestinal barrier function, such as Akkermansia muciniphila, Faecalibacterium prausnitzii, and Roseburia spp.^372-375 Available findings suggest that probiotics may regulate tumorigenesis and progression by influencing the regulation of intestinal bacteria and their metabolism, inhibition of carcinogens or carcinogenic agents, immune pathways, and apoptosis. The absence of probiotic Parabacteroides distasonis accelerates the development of CRC.³⁷⁶ Enrichment of probiotic microorganisms and depletion of pathogenic microorganisms inhibit proliferation, migration, invasion, and colony formation of tumor cells.³⁷⁷ Probiotics can improve intestinal microbiota structure, reduce carcinogen production, enhance antitumor immune response, and improve chemoradiotherapy for tumor patients.

Probiotic supplementation can improve intestinal microbiota structure, mostly by increasing other probiotics and decreasing harmful pathogenic microorganisms.^{378, 379} Pediococcus pentosaceus SL4 expresses and secretes a small protein P8, which can regulate the structure of intestinal microbiota by increasing Akkermansiaceae and Lactobacillaceae and decreasing the pathogenic bacterium Turicibacter, thereby inhibiting the proliferation of colon cancer cells and secreting antimicrobial peptides that inhibit pathogenic bacteria.³⁸⁰ C. butyricum modulates gut flora structure, changes intestinal microorganisms composition, decreases the Firmicutes/Bacteroidetes ratio, raises the relative abundance of probiotics, decreases colitis, and decreases the incidence and size of CRC.³⁸¹ Lactobacillus gasseri and Lactobacillus plantarum significantly inhibit H. pylori activity and the ability to adhere to human gastric epithelial cells through metabolites, such as organic acids and proteases.³⁸² Lactobacillus ferment, Lactobacillus acidophilus, Lactobacillus johnsonii MH-68, and Lactobacillus salivarius ssp. have similar effects.^{383, 384} L. coryniformis MXJ32 increases the abundance of Lactobacillus, Bifidobacterium, Akkermansia, and Faecalibaculum, reduced some harmful bacteria abundance, such as Desulfovibrio and Helicobacter, and reshaped the structure of antitumor intestinal microbiota.³⁸⁵

Probiotics are beneficial in reducing the production of carcinogens. Some Lactobacillus may remove carcinogenic compounds ochratoxin A, zearalenone, mycotoxins, heterocyclic aromatic amines, polycyclic aromatic hydrocarbons, and phthalic acid esters from the media environment through direct physical binding of cell wall peptidoglycan, avoiding the recontamination of toxic secondary metabolites resulting from the degradation of carcinogenic compounds.^386-389 Another reason is that Lactobacillus can inhibit the production of carcinogenic substances through metabolisms, such as by reducing ornithine decarboxylase, β-glucuronidase, β-glucosidase, nitroreductase, and azoreductase, reducing the risk of carcinogenesis.^{387, 390-393} Probiotics such as L. plantarum CRD7, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus casei, Bacillus subtilis, and Pichia anomala improve aflatoxin M1 and aflatoxin B₁ metabolism and several hepatic enzyme gene expressions.^394-396 Streptococcus thermophilus secretes β-galactosidase that suppresses cell proliferation, reduces colony formation, stimulates cell cycle arrest and apoptosis in cultured CRC, and retards the growth of CRC xenografts.³⁹⁷ Paenibacillus 79R4 has the ability to decrease the negative effects of nitrate supplementation in ruminants by boosting nitrite reduction, enhancing fermentation efficiency, and improving ruminal nitrite/nitrate detoxification.³⁹⁸

Probiotics can influence tumorigenesis and progression by regulating tumor molecular mechanisms. L. salivarius blocks rat colorectal carcinogenesis in both in vivo and in vitro by suppressing AKT phosphorylation and expressions of downstream cyclinD1 and COX-2.³⁹⁹ Lactobacillus cocktail and C. butyricum promote cancer cell apoptosis and suppress cancer cell proliferation and EMT by inhibiting Wnt/β-catenin pathway, NF-κB pathway, and Smad3 pathway.^{381, 400, 401} By downregulating the expression of antiapoptotic Bcl-2 and proto-oncogene K-ras and upregulating proapoptotic Bax and oncogenic p53, Lactobacillus rhamnosus GG decreases tumor burden and tumor multiplication.⁴⁰² Bacillus polyfermenticus inhibits the expression of ErbB2 and ErbB3 proteins and transcription factor E2F-1, thereby inhibiting the expression of cyclin D1 and preventing colon cancer development.⁴⁰³ Exopolysaccharides from L. plantarum NCU116 inhibit cancer cell proliferation and induce apoptosis through TLR2 and c-Jun dependent Fas/FasL-mediated apoptotic pathway.⁴⁰⁴ Combination of ginger extract and L. acidophilus was effective in reducing COX-2, iNOS, and c-Myc expression.⁴⁰⁵

Probiotics also modulate innate and adaptive immunity in TME. Lactobacillus bulgaricus, L. acidophilus, and L. plantarum-12 reduced the levels of proinflammatory factors IL-1β, IL-6, IL-8, IL-17, IL-23, and TNF-α and increased anti-inflammatory factor IL-10 to alleviate intestinal inflammation,^{378, 406-408} which could be linked with the suppression of Smad7 and TLR4/NF-κB signaling pathways.^{379, 409} Nevertheless, in another study, L. acidophilus could promote a Th1-dominant immune response by stimulating proinflammatory cytokines expression, including IFN-γ and suppress anti-inflammatory cytokines expression, including IL-4 and IL-10 to enhance the antitumor immune response.⁴¹⁰ Bifidobacterium longum or L. gasseri or both increase S-phase DNA such as proliferating cell nuclear antigen (PCNA) and cyclin A synthesis, significantly promoted RAW264.7 macrophage proliferation, and increased phagocytic activity of peritoneal macrophages, thereby inhibiting 1,2-dimethylhydrazine-induced colonic tumorigenesis.⁴¹¹ A. muciniphila or Amuc_1100 supplemented could reduce CTL and CD16/32 macrophages infiltration in the spleen and MLN to alleviate colitis, but significantly reduced TAM recruitment and function and increased CTL tumor infiltration in colon cancer to blunt inflammation associated tumourigenesis.⁴¹² Short-term supplementation with probiotics can increase polymorphonuclear phagocytic capacity or NK cell tumoricidal activity.⁴¹³ Feeding beneficial microorganisms are sufficient to reduce the risk of cancer and obesity across generations.⁴¹⁴ Intratumoral injection of Bifidobacterium can activate NK cells and inhibit tumor growth.⁴¹⁵ L. rhamnosus strain GG inhibits the formation of neutrophil extracellular traps.⁴¹⁶ A. muciniphila induces antigen-specific T-cell responses to regulate host immune function during homeostasis in vivo.⁴¹⁷ L. rhamnosus aerosol reduced Treg cells in the lung, increased the joining chain of immunoglobulins and IgA, and reduced the number, diameter, and area of tumor nodules.⁴¹⁸ Probiotics can induce activation of both CD4⁺ Th1 and CD8⁺ T cells in mice colon and human CRC, increase blood IFN-γ levels, promote apoptosis in colon and breast cancer cells, and suppress cancer cell aggressiveness in vivo.^{419, 420} L. casei synergistically and selectively boosted IL-12p70 expression by DCs and promoted polarization of naive CD4⁺ T cells toward a robust Th1 response without triggering Th17.^{421, 422} Bifidobacterium specifically enhances immunological checkpoint inhibitor (ICI) response and induces intratumor IFN-γ-CD8⁺ T cell accumulation.⁴²³ A. muciniphila restores the effectiveness of PD-1 inhibition by boosting CCR9⁺CXCR3⁺CD4⁺ T lymphocyte recruitment to mouse tumor beds.⁴²⁴ In addition, the fine recognition mechanism of phage M13 filamentous phage selectively kills F. nucleatum, blocks the recruitment of immunosuppressive cells MDSC, induces DC maturation, and increases M1 phenotype TAM activation.⁴²⁵ Probiotics combined with chemotherapeutic agents like 5-FU and gemcitabine can effectively boost antitumor effects through the use of polymers that are biocompatible and biodegradable with specific moieties or pH/enzyme sensitivity as carriers for targeted delivery.^426-428

Probiotics can prevent or reduce radiotherapy-induced nausea, vomiting, bloating, liver function impairment, and cardiac dysfunction and improve patient tolerance to radiotherapy without increasing clinically serious adverse events.^429-434 However, no evidence was found that probiotics prevent diarrhea or mucositis in advanced NSCLC patients.⁴³⁵

7.3 Prebiotic and synbiotic applications

Prebiotics are nondigestible substances that affect the content or activity of intestinal microbiota through the intestinal microorganisms metabolism, resulting in beneficial physiological effects.⁴³⁶ The main prebiotics that can be used as (candidate) prebiotics are fructo-oligosaccharides, trans-galacto-oligosaccharides, soybean oligosaccharides, xylose oligosaccharides, arabinoxylan oligosaccharide, gentio-oligosaccharides, lactulose, lactosucrose, inulin, and resistant starch,^437-439 which are preferentially metabolized by probiotics into SCFAs, mainly acetate, propionate, and butyrate, and facilitate the impact on human health by improving resistance against pathogenic colonization, maintaining the integrity of mucus layer and colonic epithelium, lowering intestinal pH, modulating antitumor immunity and improving anticancer activity.^{438, 440} Among them, butyrate is the major SCFA in colonic bacteria metabolism and the preferred source of energy for colonic epithelial cells. 70–90% of butyrate is metabolized by colonic bacteria, while butyrate oxidation accounts for more than 70% of the oxygen required by human colonic tissues.^{441, 442} Butyrate is the preferred energy substrate, and it enhances the growth of normal colon gland cells while inhibiting the proliferation of colon adenocarcinoma, according to in vitro and in vivo investigations. The term “butyrate paradox” is used to describe this discrepancy.⁴⁴² Butyrate stimulates p21 protein and mRNA levels, blocks the cellular G1 cycle, and inhibits cell proliferation.⁴⁴³ Butyrate significantly suppressed glucose transport and glycolysis in CRC cells by lowering the abundance of membrane GLUT1 and cytoplasmic glucose-6-phosphate dehydrogenase (G6PD) regulated by the GPR109a-AKT signaling pathway.⁴⁴⁴ In the nucleus, butyrate acts as a histone deacetylase inhibitor to boost histone acetylation, reduce the expression of c-Myc and PCNA (a nuclear protein involved in DNA replication and double-stranded reconstruction) genes, induce apoptosis, and inhibit cell proliferation.^{164, 392, 445, 446} SCFA lowers intestinal pH, which can inhibit protease activity and thus affect protein fermentation. Limits the solubility of free bile acids and suppresses bacterial enzyme 7α-dehydroxylase, that might decrease the synthesis of secondary bile acids and tumor-promoting potential.^{447, 448} The activity and expression of nitroreductases, azoreductases, β-glucosidases, and β-glucuronidases were also inhibited in an acidic environment.⁴⁴⁸ A low pH environment inhibits the activity of enzymes responsible for ammonia release and reduces the production of ammonia, a postfermentation carcinogen for proteins and peptides.⁴⁴⁹ Furthermore, lactulose fermentation stimulates the growth of Bifidobacteria and increases the uptake of nitrogen by bacteria.⁴⁵⁰ SCFA greatly increased the generation of postswitch transcripts for IgG and IgA expression. Thus, SCFA stimulates B cells, promotes plasma cell differentiation, and facilitates the production of IgG and IgA by decreasing AMPK activity but increasing mTOR activity while enhancing B cell glycolytic activity.³¹¹ Butyrate also functions as a ligand for a specific GPR that has anti-inflammatory properties, including the capability to trigger differentiation and expansion of Treg cells.^451-453 By boosting IL-12 signaling pathway, butyrate medication directly improves anticancer cytotoxic CD8⁺ T cell responses in vitro and in vivo in an ID2-dependent manner.³⁶⁴ Pentanoate and butyrate increase the function of mTOR as a central cellular metabolic sensor and inhibit the activity of histone deacetylase class I in vitro treatment of CTL and chimeric antigen receptor T cells. This reprogramming raised effector molecules production such as CD25, IFN-γ, and TNF and dramatically enhanced anticancer efficacy of antigen-specific CTLs and ROR1-targeting chimeric antigen receptor T cells in murine melanoma and PC models.⁴⁵⁴ However, SCFA can limit the anti-CTLA-4 activity. Butyrate suppressed anti-CTLA-4-induced upregulation of CD80/CD86 on DCs and inducible T-cell costimulator on T cells, as well as accumulation of tumor-specific T cells and memory T cells, while boosting Treg cells in mice.⁴⁵⁵

Probiotics and prebiotics are ideally biocompatible and synergistic and are widely utilized as additives in food and pharmaceuticals, where they are combined to create microbiota-regulating substances that are safe.⁴⁵⁶ Prebiotics selectively increase the activity and survival of probiotic microorganisms, which help maintain intestinal homeostasis, improve liver function, enhance immune regulation, prevent bacterial translocation, and reduce the incidence of nosocomial infections in surgical patients.⁴⁵⁷ Synbiotics intervention increased glutathione-S-transferase mu-1 (GSTM1) and decreased MAPK9 gene expression in colon tumors, thereby attenuating the promotive and progressive effects of indole-3 carbinol on colon carcinogenesis and reducing the risk of colon cancer in rats.^{458, 459} Prebiotics (dextran)-encapsulated probiotic spores (C. butyricum) can effectively inhibit tumor growth in vivo by the mechanism that dextran is fermented by C. butyricum to produce SCFA, and this synbiotic can increase the overall richness of microbiota in the intestinal tract, increasing the number of SCFA-producing bacteria.⁴⁶⁰ Bifidobacterium breve strain Yakult, L. casei strain Shirota, and galacto-oligosaccharides reduced the frequencies of severe lymphopenia, febrile neutropenia and diarrhea in esophageal cancer patients receiving neoadjuvant chemotherapy (docetaxel, cisplatin, and 5-FU chemotherapy regimens) by modifying the gut microbiota compared with the probiotic Streptococcus faecalis group alone.⁴⁶¹ Our current comprehension of the mechanisms behind the beneficial effects of probiotics, prebiotics, and synbiotics is relatively superficial. Some probiotics and prebiotics cause host gastrointestinal discomfort, such as bloating, and wind up in pregnant women, newborns, and the elderly with potential probiotic infections.^{462, 463} To comprehend the interactions between microbiota, host, and prebiotic components, more clinical trials with bigger sample sizes are required.

7.4 Dietary modification

Our food impacts whether the microbiota creates metabolites that accelerate or retard tumor growth.⁴¹⁵ Based on the host health benefits of probiotics, prebiotics, and synbiotics, natural products, and synthetic preparations can be administered orally to modulate microbiota structure, improve intestinal epithelial cell function and modulate the immune response.⁴⁶⁴ Dairy products containing probiotic strains, including fermented milk, buttermilk, milk powder, and yogurt, and nondairy products, including soy products, nutrition bars, cereals, and various juices, as appropriate means of delivering probiotics to consumers.^{465, 466} The diet rich in fiber can regulate the intestinal microflora after fermentation. Fermentation of Phyllostachys edulis shoots dietary fiber caused a reduction in intestinal pH, an elevation in Alistipes and Lactobacillus, and a significant decrease in E. Shigella, Enterococcus, as well as Proteus.⁴⁶⁷ C. cluster IV and XIVa are colonic bacteria that ferment dietary fiber into SCFA, butyrate, which exert tumor-suppressing characteristics via several mechanisms.^{164, 468} Butyrate also aids in the maintenance of epithelial barrier function, which is crucial for preventing inflammation mechanistically. Butyrate may aid in the restoration of tight junction barrier in inflammatory bowel disease by modulating the expression of claudin-2, occludin, cingulin, and zonula occludens proteins (ZO-1, ZO-2).⁴⁶⁹ Another study showed that butyrate oxidation as an energy source stimulates a HIF-1α-based pathway to sustain barrier function. Bacterial-derived butyrate affects epithelial O₂ consumption and leads to the stabilization of HIF, a transcription factor that coordinates barrier protection.¹¹⁷ MC38 tumors from mice fed a high-fiber diet contained more DCs and monocytes and fewer macrophages, leading to an enhanced spontaneous anticancer response. In addition, cdAMP, a STING agonist derived from the gut microbe A. muciniphila in mice fed a high fiber diet, enhanced IFN-I production by intratumor monocytes, modulating the recruitment and activation of NK cells and improving antitumor response.²⁵¹ Oral propolis stimulates the increased cytotoxic activity of NK cells and prevents cancer progression as an adjuvant therapy to chemotherapy.⁴⁷⁰ Left ventricular mass, arterial thickness and stiffness, the risk of stroke, and the severity of heart failure, are all increased by a high-salt diet (HSD).⁴⁷¹ However, in a recent study, HSD increased NK cell-mediated tumor immunity by inhibiting PD-1 expression and increasing IFN-γ and serum hippate. Although HSD-induced tumor immunity was diminished due to a reduction in the intestinal flora, FMT from HSD mice restored tumor immunity linked with NK cell function. HSD increased Bifidobacteria abundance, which improved intestinal permeability, leading to intratumor localization of Bifidobacteria, promoting NK cell function, and resulted in tumor regression.⁴¹⁵ Conversely, a fiber-free diet leads to degradation of the mucosal barrier and increases susceptibility to pathogen ingestion.⁴⁷² Mice maintained on a high-fat and high-sugar had more severe liver inflammation, tumorigenesis, and neutrophil infiltration.^{414, 473} Compared with individuals who consumed many beans, vegetable soups, potatoes, cooked vegetables, and raw vegetables, those who consumed many French fries, snacks, dips, vegetable oils, red meat, processed meats, and potatoes had a significantly higher risk of developing PC.⁴⁷⁴ Dietary vitamin E is an effective antimutagenic agent, both against naturally occurring and NO-induced mutations. Vitamin E may provide protection through two mechanisms: elimination of NO-related genotoxic species and modification of neutrophil infiltration into tumors.⁸⁶

7.5 Antibiotic applications

Antibiotics affect tumors by altering the microbiota, modulating the immune response, and their own drug metabolism. Quinolone eliminates the specific pathogenic bacterium Klebsiella pneumoniae, which facilitates the survival rate of PC patients.⁴⁷⁵ Amphotericin B ablation of Malassezia prevents PC growth in vivo.³⁷ Ciprofloxacin increases cancer cell drug toxic response by eliminating CDD-secreting pathogenic bacteria from the mouse colon TME.³³² In the lungs of mice nebulized with vancomycin/neomycin, the reduction in bacterial load was associated with a reduction in Treg cells and an elevation in T and NK cell activation, which coincided with a significant decrease in lung metastases from melanoma B16. Treatment with bacterial isolates from antibiotic-treated lungs also reduced lung tumor metastasis.⁴⁷⁶ Administration of the antifungal terbinafine reduced fungal load, fungal-induced MDSC amplification, and tumor load. Mechanistically, terbinafine directly inhibits tumor cell proliferation by decreasing the ratio of nicotinamide adenine dinucleotide phosphate (NADP) to NADPH and inhibiting G6PD activity, thereby causing disruption of nucleotide synthesis, deoxyribonucleotide starvation, as well as cell cycle arrest.⁴⁷⁷ The chemotherapeutic drug such as gemcitabine is more effective when combined with antibiotics than when used alone.⁴⁷⁸ However, long-term antibiotic therapy significantly decreased the number of NK cells, IFN-γ production, and CD8⁺ T cells in wild-type mice lungs, hence increasing lung cancer growth and progression.⁴⁷⁹ In mice treated with antibiotics, Burkholderials families (Alcaliginaceae and Burkholderiaceae) increased, while Prevotellaceae, Rikenellacaea, and Helicobacteraceae families decreased, reducing NK cells and promoting glioma growth.⁴⁸⁰ In addition, antibiotics can prevent chemotherapy-induced diarrhea and improve patients' tolerance to chemotherapy. For example, the combination of cholestyramine/levofloxacin is effective in preventing delayed diarrhea in CRC treated with irinotecan.⁴⁸¹ Antibiotic use can cause infections with drug-resistant bacteria. Vancomycin-mediated disruption of microbiota homeostasis and subsequent loss of colonization resistance can lead to vancomycin-resistant Enterococcus faecium intestinal predominance, resulting in bloodstream infections in hospitalized patients.⁴⁸² Long-term antibiotic usage increases the risk of cancer and reduces ICI treatment efficacy. Penicillin, sulfonamides, cephalosporins, macrolides, quinolones, and tetracyclines increase the risk of esophageal, stomach, PC, lung, prostate, and breast cancer.^{483, 484} Antibiotic use may be related with unfavorable outcomes in cancer patients receiving ICI.^485-487 In mice models of sarcoma, melanoma, and colon cancer, the combined use of ampicillin, colistin, and streptomycin and the use of imipenem alone reduced the anticancer effects of anti-CTLA-4 treatment.²⁹⁵ Vancomycin pretreatment worsened CTLA-4 blockage-induced colitis in mice, which may be related to a vancomycin-induced reduction of Bifidobacterium.⁴⁸⁸ Similarly, antibiotics inhibited PD-1 blockade's effectiveness in cancer patients.^{424, 489} This may be related to antibiotic-induced dysbiosis of the intestinal microbiota.^{490, 491} Oral antibiotics cocktail alters the microbiota, decreases the abundance of probiotics, and alters microbial metabolism in the gut of oral squamous mice, increasing tyrosine and decreasing glutamate levels and promoting the development of oral squamous cell carcinoma.⁴⁹² Supplementation with probiotics can restore antibiotic-damaged gut microbiota to improve the efficacy and responsiveness of anti-PD-1-based immunotherapies.⁴⁹³

7.6 Defensins

Defensins are mainly synthesized by Paneth cells, neutrophils and epithelial cells, and contribute to host defense. Their roles in cancer development and progression have been a topic of intensive research.⁴⁹⁴ The study showed that 82% of prostate cancers and 90% of renal cell carcinomas exhibit cancer-specific human β-defensin-1 (hBD-1) protein deficiency.⁴⁹⁵ Overexpression of hBD-1 gene in renal cancer cells SW156 leads to caspase-3-mediated apoptosis.⁴⁹⁶ Similar results were found in basal cell carcinoma, oral squamous cell carcinoma and CRC.^{497, 498} These data suggest that hBD-1 might act as a tumor suppressor. Some probiotics can induce the production of defensins in human host cells. For example, surface layer protein (SLP) of Lactobacillus helveticus SBT2171 stimulates hBD-2 expression by activating c-Jun N-terminal kinase (JNK) signaling via TLR2 in Caco-2 human colonic epithelial cells.⁴⁹⁹ lactobacilli and the VSL#3 bacterial mixture (three bifidum and one streptococcus species) upregulates hBD-2 by inducing proinflammatory pathways including NF-κB and activator protein-1 (AP-1) as well as MAPKs.⁵⁰⁰ The flagellin of probiotic E.coli Nissle 1917 promotes hBD-2 expression through NF-κB and AP-1 pathways.^{501, 502} B. subtilis and selenium-enriched B. subtilis increase intestine BD-1 expression through the TLR2–NF-κB1 signaling pathway.⁵⁰³ Defensins are essential for gut homeostasis and restoration of the gut microbiota.^{504, 505} However, other experiments have found increased defensins expression in lung cancer tissues.⁵⁰⁶ The hBD3 promotes the proliferation and invasion of oral squamous cell carcinoma cells by regulating the expression of NF-κB p65 and its downstream c-Myc and p21.⁵⁰⁷ Overexpression of hBD-3 might have a tumor-promoting effect or contribute to lymph node metastasis.⁵⁰⁸ A deeper understanding of the interactions between probiotics and defensins will facilitate a comprehensive analysis of the crosstalk between defensins and microbiota dysbiosis in various gastrointestinal diseases, which is critical for the treatment of gastrointestinal diseases.

7.7 Oncolytic virotherapy

Oncolytic virus (OV) is a novel immunotherapy that achieves antitumor effects through the dual mechanism of selective tumor cell killing and induction of systemic antitumor immunity.⁵⁰⁹ OVs can be divided into two main categories: natural OVs (e.g., reovirus and vesicular stomatitis virus) and genetically modified OVs (e.g., adenovirus, vaccinia virus and herpesvirus).⁵¹⁰ To date, four OVs and one non-OV have been approved globally for the treatment of cancer. In 2005, the world's first oncolytic adenovirus drug, recombinant human adenovirus type 5, was approved by the National Medical Products Administration of China, in combination with chemotherapy for the treatment of patients with advanced nasopharyngeal carcinoma. In recent years, recombinant human adenovirus type 5 has made remarkable progress in the field of solid tumors.⁵¹¹ Talimogene laherparepvec (T-VEC) is a genetically engineered herpes simplex virus type 1 (HSV1) that preferentially replicates in tumor cells and induces an antitumor immune response. T-VEC was approved by the United States Food and Drug Administration (US FDA) in 2015 for the treatment of advanced melanoma.⁵¹² After ECHO-7 was approved, production was discontinued due to manufacturing issues. Teserpaturev, a third-generation HSV1-based OV, has been approved in Japan in 2021 for patients with malignant glioma.⁵¹³ In 2022, the US FDA approved nadofaragene firadenovec, the first nontumorolytic adenovirus encoding IFNα−2b, for nonmuscle-invasive bladder cancer that is unresponsive to bacillus Calmette-Guérin.⁵¹⁴ Compared with conventional chemoradiotherapy, OVs precisely lyse cancer cells by interacting with specific cellular receptors, by exploiting tumor suppressor gene defects, by downregulating antiviral pathways in tumor cells, or by designing viral vectors with specific gene knockouts, and have antitumor effects that include antiangiogenesis, reversed metabolic reprogramming, and catabolism of the ECM surrounding tumor.^{510, 515-518}

Furthermore, OV combined with small molecule inhibitors/modulators targeting oncogenic signaling pathways can effectively enhance antitumor immunity and therapeutic efficacy.⁵¹⁹ However, due to the tumor heterogeneity, the OV combination therapy strategy faces serious challenges.

7.8 Fecal microbiota transplantation

FMT, in which a healthy donor fecal microbiota is implanted and used to restore normal gut microbial community structure and function and to resist pathogen colonization, is considered a promising and effective treatment for a variety of gastrointestinal or immune-related disorders.^520-522 For instance, C. difficile, a member of the normal human intestinal flora, can cause dysbiosis of intestinal flora when antibiotics are not used in a standardized manner. Drug-resistant C. difficile can grow and multiply in large numbers, leading to diseases such as antibiotic-associated diarrhea and pseudomembranous colitis.⁵²³ Competing with C. difficile for nutritional and colonization resources, interfering with its virulence factors, and directly killing C. difficile are all capabilities of healthy gut flora. Intestinal microbiota can impede C. difficile germination and nutritional growth by activating numerous host immune defenses and restraining C. difficile with secondary bile acids.⁵²⁰ FMT with typical feces affected the immunological environment and gut homeostasis, resulting in an increase in gut length as well as epithelial proliferation and migration. This was related to dramatic alterations in microbiota composition, such as an elevation in the relative abundance of Desulfovibrio and Akkermansia. However, conventional microbiota transplantation elevated MSI in the untransformed intestinal epithelium of MSH2-Lynch mice, suggesting that microbiota composition affects the mutagenesis rate of MSH2-deficient crypts with increased oncogenic potential.^{524, 525} Therapeutic FMT decreases colonic inflammation and initiates intestinal homeostasis restoration by activating multiple immune-mediated mechanisms simultaneously, ultimately resulting IL-10 generation by innate and adaptive immune cells, such as CD4⁺ T cells, iNKT cells, and APCs, and decreases the capability of DCs, monocytes, and macrophages to present MHC-II-dependent bacterial antigens to colonic T cells.⁵²¹ Supplementation with a single bacterium, A. muciniphila, via fecal transplantation improves intestinal barrier function, reduces MDSC infiltration, and inhibits steatohepatitis activity.²⁵⁸ FMT from mice fed HSD restored tumor immunity related to NK cell function, elevated Bifidobacterial abundance, caused increased intestinal permeability, resulted in intratumor localization of Bifidobacteria, boosted NK cell function, and promoted tumor regression.⁴¹⁵ Mice receiving short-term survival FMT had increased CD4⁺ Foxp3+ and MDSC infiltration, whereas tumors from mice receiving long-term survival FMT had considerably greater CD8⁺ T cells and activated T cells as well as higher serum IFN-γ and IL-2 levels in mice.⁵²⁶ FMT and anti-PD-1 modify gut microbiota and reprogram TME to overcome anti-PD-1 resistance in a subset of advanced PD-1 melanomas.⁵²⁷ Irradiated male and female mice exhibited improved survival after FMT, increased peripheral leukocyte amounts, and enhanced gastrointestinal function and intestinal epithelial integrity. FMT maintained bacterial composition and mRNA and lncRNA expression profiles of the small intestine of host sex specifically. While boosting angiogenesis, sex-matched FMT did not increase cancer cell proliferation in vivo; therefore, FMT may act as a therapeutic method to reduce radiation-induced toxicity and enhance the prognosis of tumor patients following radiotherapy.⁵²⁸ Conventional FMT mainly transfers large intestine-derived microbes and gene functions into the recipient intestine, whereas only a small number of small intestine-derived microbes have been successfully transplanted. Compared with FMT, whole-intestinal microbiota transplantation introduces more small intestine-derived microorganisms (e.g., Proteobacteria, Lactobacillaceae, and Cyanobacteria) into the recipient intestine and associated microbial functions, which improves intestinal morphogenesis, reduces plasma concentrations of IL-5 and TNF-α and increases concentrations of anti-inflammatory cytokine IL-4 in mice, thereby reducing the systemic inflammatory response in recipients.⁵²⁹ The mechanism may be related to T-cell proliferation, gene expression, and prevention of apoptosis.⁵³⁰ Nonetheless, the possible risks of FMT, including the possibility of pathogen transmission to the recipient, should not be ignored. In order to identify more effective medications and reduce treatment complications, patients should be stratified based on their particular microbiota.