The anti-tumor effects of intestinal microbiota metabolites have attracted much attention in recent years (Table 1, Ref. [28, 47, 48, 49, 50, 51, 57, 58, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76]). Protein metabolism occurs mainly in the intestine. Bacteria in the colon have relatively high efficiency in degrading endogenous and exogenous proteins [44]. Colonic microbiota has rich proteolytic activities. Bacterial peptidases, proteases, and endopeptidases from the microbiota hydrolyze dietary proteins, thus generating polypeptides and various amino acids, such as aromatic amino acids, methionine, cysteine, cysteine, and taurine [45]. Many bacterial commnunities in the intestine, such as Clostridioides, Bacillus, Lactobacillus, Streptococcus and Proteobacteria, can decompose and transform a small amount of undigested proteins, unabsorbed amino acids and other digestive products, a process known as “fermentation” [46]. Amino acid fermentation can produce several by-products, such as amines, phenols, indoles and its derivatives and other substances. SCFAs are the most abundant end-products of metabolism [43]. These intestinal flora metabolites, including SCFAs, indoles, amino acids, and polyamines can be used to prevent tumor development.

2.1.1 Short-Chain Fatty Acids (SCFAs)

Anaerobic bacteria produce short-chain fatty acids (SCFAs, volatile fatty acids) in colon fermented from undigested and absorbed carbohydrates in human body. Short-chain fatty acids mainly contain acetate, propionate, and butyrate. Concentrations of these components are highest in the cecum and proximal colon. Acetic acid and propionic acid are mainly produced by Bacteroidetes, whereas butyric acid is mainly produced by Firmicutes [30].

Short-chain fatty acids are key metabolites of intestinal micro-organisms which have diverse effects on the host. For instance, SCFAs participate in energy metabolism of body. Butyric acid significantly affects cells and is the main energy provider for colon epithelial cells [31]. However, it can inhibit the growth of cancer cells. Intestinal flora disorder is common in patients with CRC. Short-chain fatty acids can regulate intestinal flora [32]. Recent studies have found that SCFAs, such as butyric acid can also inhibit the proliferation of cancer cells or participate in immune regulation, hence achieve anti-tumor effects [44].

The acids can regulate the function of immune cells [45], such as monocytes, dendritic cells (DCs) , and macrophages. They can also regulate the differentiation of T and B cells and antigen-specific adaptive immunity [29, 46], thus participating in tumor immunosuppression. Therefore, it is evident that, SCFAs can be used as intracellular agents [44] to inhibit the proliferation, differentiation, and migration of cancer cells by inhibiting histone deacetylation. It can also be used as extracellular regulators [57, 58, 59] to immunomodulate tumors through participation in various signaling pathways and thereby inhibiting tumor occurrence as well as development (Fig. 2, Ref. [29, 77, 78, 79, 80]).

Fig. 2.

Anti-tumor mechanisms of SCFAs. (1) DNA demethylation inhibits cancer cells proliferation [29]. (2) GPR41/GPR43 receptors exist on immune cells [29]. They interact with butyric acid. Through signal pathways, they regulate T cells, cytokines and chemokines to attack and inhibit cancer cells [77]. (3) Receptor tyrosine kinase stimulates PI3K under the activation of growth factors, which can change the AKT protein structure to activate tumor suppressor factors [78]. (4) mTOR, as a downstream target, can produce P70 ribosomal protein S6 kinase (S6K), which plays an important role in the apoptosis of cancer cell apoptosis. However, when mTOR is inhibited for a long time, the activity of S6K1 is reduced, and feedback inhibition loop of S6K1 to receptor tyrosine kinase is removed. This results are in promotion of tumor growth through more compensation circuits [79]. Butyric acid activates mTOR to inhibit the fatty acid synthase gene [29]. (5) Secretory glycoprotein regulates the activation through butyrate acid. Accumulated $\beta$-Catenin in the cytoplasm enters the nucleus and binds the TCF transcription factor family to initiate the transcription of downstream target genes and inhibit cancer cells [80].

2.1.1.1 SCFAs Mediate Histone Deacetylation Inhibition

According to the Warburg effect, most of the tumor cells produce the required energy in aerobic glycolysis. Butyrate cannot be metabolized in cancer cells because it is the energy source of colon epithelial cells. Consequently, there is increased accumulation of non-oxidized butyrate, inversely promoting the inhibition of histone deacetylation and thus increasing the expression of tumor apoptosis-related as well as cell cycle-related genes [81]. A study conducted by Eslami et al. [52] demonstrated that dietary fiber has tumor-suppressive effect using a mouse model colonized by butyrate-producing bacteria. They found that butyrate produced by a high dietary fiber diet can inhibit the proliferation of CRC cells and induce their apoptosis as histone deacetylation inhibitors.

Studies have shown that SCFAs mediate histone deacetylase inhibition. SCFAs are histone deacetylase inhibitors that regulate the transcription of genes [47]. For instance, butyrate deacetylase has a significant antagonistic effect. It can non-specifically promote DNA methylation and histidine acetylation, affect DNA replication and transcription, increase the transcription of P21 gene, and inhibit the transformation of cells from G1 to S phase, thus inhibiting the proliferation of colon cancer cell [70]. Acetate-derived acetyl groups can enter T lymphocytes to form cellular acetyl-CoA pools, thus affecting the proliferation of cancer cells by influencing their histone acetylation and cytokine gene expression [43].

Recent studies have shown that SCFAs regulate cellular immune metabolism. Butyrate can intrinsically alter Foxp3 gene in the T cells, thereby induce changes in the cells. It can inhibit histone deacetylase by enhancing histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus [82].

2.1.1.2 GPR41/GPR43 Signaling Pathway

GPR41/GPR43 is an orphan type of receptor in G protein-coupled receptors which is activated by SCFAs. GPR43 is highly expressed in immune cells [77]. Butyrate can diffuse to the intestinal epithelium and interact with the main SCFAs receptors in lamina propria (GPR41 and GPR43). Therefore butyrate can regulate gene expression in immune cells to control the production of T cells, cytokines, and chemokines, hence play a role in tumor immune regulation [43].

2.1.1.3 mTOR Signaling Pathway

Unlike the GPR42/GPR43 signaling pathway, mammalian target of rapamycin (mTOR) is an effector protein that functions by forming a complex with other proteins. The mTOR signaling pathway plays different roles in different diseases. It can modulate metabolic processes, apoptosis, and autophagy. Inhibition of fatty acid synthase can inhibit malignant phenotype of colorectal cancer by down-regulating energy metabolism and activating the mTOR cell signaling pathway [43]. Besides butyric acid activating the mTOR path, valeric acid can also induce changes in T cells by regulating the activity of mTOR complexes and regulating their glucose metabolism, hence affecting proliferation of the cancer cells.

2.1.1.4 Wnt Signaling Pathway

The Wnt signaling pathway is a complex protein action network commonly used to regulate embryonic development and cancer [59]. It has been found that butyrate regulates Wnt activity in CRC cells [83]. Butyric acid affects apoptosis, cell cycle arrest, and differentiation of CRC cells by altering Wnt activity. In addition, it has been shown that butyric acid-induced apoptosis is directly related to the increased activity of Wnt which is induced by clonal growth inhibition in CRC cell lines of 10 individuals [84, 85].

2.1.1.5 PI3K/AKT Signaling Pathway

The phosphatidyl inositol 3-kinase (PI3K)/AKT pathway regulates different biological processes. The PI3K/AKT signaling pathway can directly or indirectly regulate several key epigenetics. The carcinogenicity of the PI3K cascade involved in cancer is abnormally activated in human cancers [57]. Sitosterol can maintain the balance of intestinal flora and significantly increase acetic acid and propionic acid in intestinal flora metabolism. Sitosterol can also weaken the signal transduction of PI3K/AKT and change the expression level of several apoptotic-related proteins, eventually leading to tumor apoptosis [86]. Therefore, SCFAs can induce tumor cells apoptosis both in vivo and in vitro.

Inflammation and immune imbalance are key factors that cause tumors. Chronic inflammation promotes tumorigenesis and development of cancer. Inhibition of inflammation in vivo can prevent the occurrence and further development of tumors, enhance normal immune functions in the body, thus eliminating the tumors. The function of immune system is important for tumor removal and self-defense. Therefore, it is necessary to control the tumor-related inflammatory environment and promote the development of immune function for maximum efficacy of the tumor therapy.

Intestinal flora can be crucial in regulation tumor-associated inflammation. The gut microbiome exists in a dynamic equilibrium with the human immune system. Changes in the number, type, proportion, and biological function of intestinal flora causes inflammatory response, immune system dysfunction, and cancer development [101].

Intestinal microbiota can regulate inflammatory responses by reducing the expression of inflammatory cytokines and chemokines in intestine. Previous studies have shown that B. longum alleviates colorectal colitis in mice by improving the level of interleukin-10 (IL-10), down-regulating IL-12, IL-17, and IL-23 in the serum, and regulating regulatory T cells (Tregs) [102]. In addition, B. longum KACC 91563 can reduce the IL-2, IFN-$\gamma$, IL-4, IL-6, IL-10, and TNF-$\alpha$ production in cells hence promoting inflammation [36]. Besides, the transcription factor NF-$\kappa$B is a key mediator of the inflammatory response [103]. Dextran Sulfate Sodium Salt-induced colitis is effectively alleviated by B. longum through regulation of the NF-$\kappa$B signal pathway. Similarly, Pseudotreptavirus-pseudo stretched Bifidobacterium reduces the levels of TNF-$\alpha$ and IL-6 in mice with colitis, while increasing the levels of IL-10 as well as peroxisome proliferator-activated receptors. However, Bifidobacterium significantly inhibits the activation of TLR4/NF-$\kappa$B pathway [35] .

The concentration of colonic metabolites, conjugated linoleic acid (CLA) produced by different intestinal strains, such as B. longum and Bifidobacterium pseudocatenulatum, are positively correlated with the effectiveness of the strains in alleviating colitis [35]. CLA improves colitis by down-regulating the concentrations of interferon-gamma (IFN-$\gamma$), IL-1$\beta$, and IL-12 in the colon [104]. The exposure of antigen-presenting cells to CLA can modulate the response of subsequent Th cells, thus improving the intestinal immune barriers.

Moreover, the effective exercise of tumor immunity is crucial for anti-tumor process. Intestinal flora can promote the anti-tumor effect of immune cells hence affecting the intestinal barrier and the efficient development of the immune function.

Intestinal flora is closely related to immune cells. Intestinal colonization of specific microbiota promotes the maturation of intestinal mucosa-associated lymphoid tissues [105]. Over 80% of plasma cells secrete IgAs in the lamina propria of the intestine. IgA can promote the homeostasis of intestinal and colon flora [38]. Human intestine has abundant memory IgM B cells. These cells are associated with abundant IgM plasma cells [39]. Moreover, intestinal microbes positively regulate adaptive immune cells, promote the differentiation of B lymphocytes, Th0, Th1, Th17, Treg cells, and other important immune cells, thus promoting the initiation of anti-tumor adaptive immunity as well as immune checkpoint blockade therapies [17]. The systemic immune functions can be boosted by B. longum KACC 91563, thus enhancing IgE production and regulation of Th1 (IL-2, IFN-$\gamma$)/Th2 (IL-4, and IL-10) balance [36]. In addition, intestinal flora can also participate in the regulation of immune cells by producing metabolic substances [9, 10, 16, 17, 18, 19, 36, 38, 39, 41, 42, 103, 106, 107].

Recent studies have also shown that intestinal flora can play a positive role in tumor immunotherapy by activating specific tumor immune responses. Cancer cells develop various defense mechanisms to evade immune responses [40] through three stages (elimination, equilibrium, and escape) based on the immune editing theory [108]. Tumor cell proliferation and cancer development occur in presence of weak immune system [40, 109]. Therefore, immunotherapy should be used to stimulate the immune functions of patients to attack tumor cells until they are eliminated [106]. ICIs are one of the most promising and effective tumor immunotherapies [9, 10]. They mainly activate specific tumor immune responses by targeting specific molecules. Reactivating cytotoxic T lymphocytes to attack tumor cells [11] can prevent tumor cell escape and reconstruct the anti-tumor immune response.

In recent years, key immune checkpoint molecules, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein-1/programmed cell death protein ligand-1 (PD-1/PD-L1) pathways have been found to be key targets for the treatment of different cancer immunotherapies [12, 13]. Some clinical studies have shown that intestinal flora, such as symbiotic Bifidobacteria, can also potentiate the anti-tumor immunotherapy effects of PD-1 blockade [14, 106] whereas Bacteroides can promote anti-tumor immunotherapy for CTLA-4 blockade [9].

For instance, DCs, as specialized antigen presenting cells, activates T cells in anti-tumor immunity [10]. The immune system can then recognize this exogenous antigen by interacting with its T cell receptor (TCR) and the peptide epitopes presented by MHC-I molecules on tumor cells [106]. The long-term interaction between CTLA-4 on the surface of effector T cells and CD80/CD86 expressed by DCs may be depleted [15]. In vitro experiments have demonstrated that Lactobacillus from intestinal tract can increase the expression of mature CD80 and CD86 in DCs. Bifidobacterium or other symbiotic bacteria can stimulate the maturation of DCs [16]. Therefore, specific bacterial species in ICIs can enhance DC activation, increase the infiltration of CTLs, and reduce the number of Tregs in TME thereby increase the sensitivity of ICIs to tumor cells [17].

Intestinal microbiota plays a key role in tumor immunotherapy. It promotes development, differentiation, and functions of immune cells through the intestinal microbiome as well as its metabolites. Intestinal microbiome, as the signal center of the body, can also combine environmental input, such as drugs and diet with immune signals to affect the normal immune function of the host [18]. Recent evidence has indicated that intestinal microbiome can modulate toxic side effects, drug resistance, and therapeutic effect in tumor immunotherapy [19, 41]. Intestinal flora has an indirect bidirectional effect on immunotherapy by influencing the immune system of the host [20, 42]. Therefore, studying the role of intestinal micro-organisms in blocking immunomodulatory effects, such as PD-1 and CTLA-4 is important towards the development of tumor immune checkpoint blockers.

Studies have suggested that adverse intestinal microbiota may also play a role in promoting the occurrence and development of tumors. How to overcome these factors remains to be further explored.

Clostridioides perfringens (C. perfringens) enterotoxin promoted the malignant progression of sessile serrated adenoma/polyp with dysplasia into colon cancer by activating the transcriptional co-activator yes-associated protein, suggesting that abnormal intestinal flora, such as C. perfringens, can increase the possibility of colon cancer [110]. Therefore, controlling adverse intestinal flora may prevent the occurrence of tumors. Besides, changes or imbalance of intestinal flora may lead to changes in the micro-environment in vivo, such as inflammation or the decline of immune function, thus causing the occurrence and metastasis of tumors [102]. For example, enterotoxigenic Bacteroides fragilis (ETBF), a common human symbiotic bacterium, induces colitis, colonic hyperplasia, and tumor formation through the activator of transcription-3- and T-helper type 17 (Th17) - dependent pathway, showing a new mechanism for the occurrence of the human colon cancer. Intestinal flora is also involved in extra-intestinal cancer, causing inflammation originally present in the intestine to metastasize to other parts of the host body [111]. Consequently, it leads to the emergence of infection and inflammation on healthy tissues and organs, as well as carcinogenesis by affecting the occurrence, progression, and spread of cancer in epithelial barrier and sterile tissues [112, 113]. Therefore, a well-balanced intestinal flora homeostasis is crucial for human health and improves the treatment of patients with tumors.

Antibiotics are widely used to treat cancer. Anti-tumor is the function of antibiotics found in the process of fighting infections. Antibiotics are natural or derived chemicals produced by environmental microorganisms, including bacteria, fungi, and actinomycetes. For instance, Doxorubicin (DOXO, ADM), produced by Streptomyces caesius subspecies, is a traditional and common anthracycline antibiotic that has a broad anti-tumor spectrum and a decades-long history of clinical treatment of cancer [114].

Based on different targets of anti-tumor drugs on each phase of the cell proliferation cycle, the drugs can be divided into two categories: cell cycle specific and cell cycle non-specific drugs. The former only acts on a specific phase of the cell proliferation cycle. Their effects are weak and slow and takes a limited period of time to exert their killing effects. Pingyangmycin, which acts on the G2 phase belongs to this category. The latter directly acts on various phases of the cell proliferation cycle, without selection. Their effects are strong and fast, and can quickly kill tumor cells. Most anti-tumor antibiotics belong to this category.

Antibiotics are produced by many microorganisms and are known to exert anti-tumor effects through different mechanisms. Anthracycline antibiotics, such as Daunorubicin, DOXO, Epirubicin and Mitoxantrone, some of which are derived from Streptomyces peucetius, inhibit DNA replication and mRNA synthesis by forming stable complexes between base pairs embedded in DNA double-strands. Glycopeptide antibiotics, such as Bleomycin A5 (Pingyangmycin) and Bleomycin A6 (Boanmycin) are mainly produced by Streptomyces and Actinomyces. They inhibit DNA polymerase activities by breaking the single strand of DNA to interfere with the transcriptional process. The glycopeptide antibiotic (actinomycin D) and the anthracycline antibiotic (Aclacinomycin) inhibit RNA polymerase activities by embedding in the DNA double strand to bind guanine groups on DNA, thereby interfering with the transcriptional process. Some anti-tumor antibiotics derived from plant endophytic bacteria, marine fungi and soil bacteria, can inhibit the proteins associated with tumor cell division, proliferation, and migration. Midostaurin, which is derived from Actinomycetes and Rapamycin from Streptomyces inhibit important proteases in tumor cells. Antilekemic activities of anti-infectious antibiotic tigecycline are associated with inhibition of mitochondrial protein translation [115].

Therefore, anti-tumor antibiotics have a wide range of microbial sources and important tumor suppressive properties, thus, they are an important option for malignant tumor treatment.

Various antibiotics, including Pingyangmycin, DOXO, Daunorubicin and Mitoxantrone among others are important in cancer treatment, however, they are associated with various side effects. They have been shown to lead to imbalanced intestinal flora and decreased microbial diversity. Suppressed microbial diversity correlates with reduced abundances of SCFAs (important metabolic compounds from intestinal flora) and may also induce damage to the TJ barrier of the intestinal epithelium [115]. They also affect the efficacies of ICIs via dysbacteriosis. In addition, they have a risk for cancer induction. In a previous meta-analysis [116], antibiotics were established to be an independent risk factor for cancer development (OR 1.18, 95% CI 1.12–1.24, p 0.001), and excess long-term use of antibiotics was associated with slightly increased risks of various cancers, such as lung cancer, lymphoma, pancreatic cancer, renal cell carcinoma and multiple myeloma among others.

Toxic side effects limit the applications of anti-tumor antibiotics. The current clinical cytotoxic anti-tumor antibiotics do not exhibit ideal selective effects on tumor cells and normal cells. Since the drug kills malignant tumor cells, there is a certain degree of damage to some normal tissues, resulting in various adverse reactions. Common toxic reactions include bone marrow suppression and gastrointestinal reactions among others. Long-term and excess use of Bleomycin is associated with specific toxic side effects, such as interstitial pneumonia, pulmonary fibrosis and tumor resistance [117]. DOXO can induce acute cardiotoxicity, resulting in multifocal vacuole degeneration of myocardial cells, myoblast lysis, myocardial fibrosis and myocardial interstitial edema [118]. Actinomycin-D causes acute injury to human hepatic sinusoidal endothelial cells, hepatic sinusoidal obstruction and acute toxic damage to hepatocytes [119]. Most of the non-absorbable antibiotics, like DOXO, may cause central nervous system diseases, such as brain dysfunction through the microbiome-gut-brain axis [120, 121].

Reduction or elimination of potential risks of antibiotic drugs, such as cytotoxicity is a challenge in drug research. Various protective mechanisms have been explored. Bleomycin-associated lung toxicity can be controlled by deleting the sugar residue (d-mannosyl-l-glucose disaccharide) [122], or via combinations with traditional Chinese medicine antibiotics, Scutellarin [123] and Aucubin [124]. Combination with $\beta$-caryophyllene protected mice heart from DOXO-induced acute cardiotoxicity [125]. By over-expressing several specific drug transporters, the significance of pharmacokinetics of actinomycin-D were determined [64]. Chimeric molecules were formed by connecting two different pharmacophores with spacers [126], which helped in eliminating the toxic effects of anthracycline antibiotics. With advances in chemical and biosynthetic engineering, some natural active substances, such as patamycin, which have significant biological activities, but are not applied as therapeutic agents due to their strong cytotoxicity, have potential for clinical applications [127]. Toxicity reduction and efficacy enhancement in antibiotic treatment of tumors can be achieved through molecular structural modification [64, 122, 126, 127] and drug combination therapy [123, 124, 125].

Although they have certain negative effects, anti-tumor antibiotics are important for treatment of malignant tumors. Degradation of anti-tumor antibiotics and toxicity changes are worthy of attention.

Many drugs, especially natural products, are not directly absorbed by the body, but interact with intestinal microorganisms when they enter the intestines. This affects the ability of the intestinal flora to produce biologically active, inert or toxic metabolically active products, which are absorbed into the bloodstream to produce therapeutic or other effects [129]. Most of the existing anti-tumor antibiotic drugs are intravenously administered. These injected drugs are first metabolized in the liver after which they are partially excreted into the intestines with bile, where they are exposed to intestinal microbiota for further metabolism and reabsorption [130]. Therefore, intestinal flora are involved in internalization of anti-tumor antibiotics.

Some bacterial species of gut microbiome, especially enterobacteriaceae, affect the metabolism and efficacy of certain drugs [131]. Intestinal microbiota affects drug metabolism via various ways, including acetylation/deacylation decarboxylation, dehydroxylation, demethylation, dehalogenation and conjugate hydrolysis reactions on some drug-related toxicity [132]. Currently, studies on the mechanisms of intestinal bacteria on anti-tumor antibiotics are limited. DOXO can be metabolized and decomposed by Raoultela planticola, an intestinal bacterium of the enterobacteriaceae family. In anaerobic conditions, it decomposes DOXO into 7-deoxydoxorubicinol and 7-deoxydoxorubicinolone through reductive deglycosylation [133], confirming the catabolic effects of enterobacteria on anti-tumor antibiotics.

Metronidazole reduced Fusobacterium load and inhibited cancer cell proliferation as well as overall tumor growth in colon cancer xenograft mice models, implying that antibacterial intervention is a potential treatment option for patients with Fusobacterium-related colorectal cancer [134]. The metronidazole oral reagent and intestinal flora were incubated in anaerobic environments. The intestinal flora reduced metronidazole to N- (2-hydroxyethyl) oxalic acid and acetamide. The reduction degree was positively proportional to the residence time of sustained release in the intestines, while bioavailability was inversely proportional to its residence time [135]. These results imply that intestinal flora affect the metabolic outcomes of antibiotic drugs.

As natural metabolites, polysaccharides are an important member of the natural antibiotic family with anti-tumor effects. After entering the intestinal tract, polysaccharides are catabolized by intestinal flora. About 20% of the genome of Bacteroidetes is used for ingesting and decomposing complex polysaccharides [135]. Priority carbon sources for most human intestinal microbes are glycosaminoglycans (GAGs), a polysaccharide. Utilization of GAGs is based on its carbohydrate structure [136, 137]. The mechanisms underlying the preferential utilization of GAGs by intestinal microbiota were explored, and were shown to result in complete metabolism of diverse GAGs. Intestinal flora, especially Bacteroidetes, contain a large number of enzymes that can lyse polysaccharides. Comparisons of 25 Firmicutes and Bacteroides from different sources revealed that each Bacteroide genome contains a large number of genes involved in polysaccharide acquisition and metabolism. Bacteroides contain hundreds of glycosidolytic enzymes and polysaccharide lyases that allow them to up-regulate the expressions of polysaccharide utilization sites encoding multiple glycoside hydrolases [137, 138]. In this study, Pachychocetes were found to have a small number of related genomes and glycan degrading enzymes.

Natural polysaccharides are fermented by intestinal flora to produce SCFAs. An Azomethane/Dextran sodium sulfate-induced inflammation in a tumor mice model demonstrated that Ganoderma lucidum polysaccharides modulated the intestinal microbiota, enhanced the production of SCFAs, improved intestinal barrier functions and inhibited TLR4/MyD88/NF-xB and MAPK signaling cascades, thereby suppressing tumorigenesis [139].

Through the mediating effects of intestinal microbiota on oral or injected anti-tumor antibiotics, microbiota are important for digestion, absorption and utilization of such drugs, and they may enhance the efficacy of antineoplastic treatment.

In most advanced cancer patients, cytotoxic drugs, such as anti-tumor antibiotics are the mainstay for their treatment. However, these drugs are associated with various treatment-related diseases and unpredictable toxic side effects. Specific adverse reactions and high costs are the main challenges in targeted tumor therapies [128].

Intestinal microbiota can indirectly reduce the toxicity of drugs by regulating host metabolism and producing metabolites that compete with drug receptors. Intestinal microbiota modulate host responses to chemotherapeutic drugs to promote drug efficacy, impair and eliminate anti-cancer effects, as well as to modulate toxicity [138].

DOXO is a typical example of the attenuating effects of intestinal microbes on anti-tumor antibiotics. DOXO exhibits cardiotoxic properties and may induce jejunum crypt epithelial cell apoptosis and intestinal mucosal damage [140, 141]. Intestinal microbiota are closely correlated with DOXO-induced mucosal damage [140]. Muramyl-dipeptide, a peptidoglycan motif that is common in all bacteria, has strong protective effects against oxidative stress-mediated cell death [142]. Intestinal bacteria are important in repair of DOXO-induced intestinal injury, and secretion of immunomodulatory chemokines associated with such injuries are correlated with intestinal bacteria [99]. Therefore, regulation and manipulation of intestinal microbiota to reduce damage to intestinal mucosa during DOXO therapy is associated with less damage during tumor therapy. A model of bacterial community dynamics under simulated chemotherapy pressure was developed to explore the effects of bacterial interactions on microbiome elasticity during drug detoxification. Bacteria with biotransformation resistance significantly reduced DOXO concentration in the medium, allowing drug-sensitive strains to grow in a single culture [143]. Moreover, in vivo metabolism of other antibiotics requires the regulatory effects of enterobacteria. These studies have clinical implications for development of probiotics with enteric-specific protective effects for patients treated with DOXO and other anthracycline drugs, as well as for understanding in vivo metabolism of antibiotic tumor drugs by gut microbiota.

The extent to which many oral oncology drugs are exposed and absorbed in the intestines before entering the circulatory system determines their absorption rate and bioavailability degree [129]. The microbiome regulates the body’s responses to cancer treatment by influencing the absorption degree of oral drugs in the gut and their interactions with intestinal bacteria.

Intestinal microbes influence tumor treatment responses by modulating drug metabolism and regulating susceptibility to toxic side effects [99, 129]. In addition, the loss of protective functions when the intestinal barrier interacts with the environment, such as increased intestinal permeability and changes in microbiota composition and quantity, is thought to be a mechanism that may explain the pathogenesis of immune-related toxicity [144]. Therefore, in the context of multiple therapeutics, selection of efficacious prediction markers for identifying reactions and toxicity is an important, challenging task in drug therapy.

There is a need to identify antibiotics and anti-tumor drugs that can achieve the best clinical therapeutic effects and minimize drug toxic effects.

About 15% of human cancers are due to infections and chronic inflammation [145]. Cancer is a systemic disease: inflammatory immune cells, cytokines and chemokines affect tumor growth, metastasis, and spread [144].

Inflammation is involved in almost all stages of tumorigenesis [145]. It promotes tumorigenesis by altering host physiology. Through high-throughput sequencing, Arthur et al. [46] found that colitis promotes tumorigenesis by altering the composition of the intestinal flora in mice, IL-10 deficient mice were highly susceptible to colitis and that colitis induces the expansion of genotoxic microorganisms. Chronic inflammation is involved in carcinogenesis through mutations, genomic instability and epigenetic modifications [37]. Inflammation stimulates pluripotent stem cells or progenitor cells in various tissues to produce cancer stem cells [37]. In addition, inflammatory bowel diseases have been shown to cause mutations in cancer-related genes, such as TP53, to accumulate in intestinal epithelial cells [146]. Inflammatory cells, such as neutrophils and $\gamma$$\delta$T cells, can further aid in tumor adhesion and metastasis [147].

Anti-microbial antibiotics can inhibit infection and inflammation [148] and play a regulatory role in intestinal flora. Inflammation is the body’s defense response to exogenous injury stimuli. Viral and bacterial infections can directly or indirectly (e.g., through chronic inflammation) induce abnormal DNA methylation, leading to oncogenic mutations in host cells [149]. In cases of inflammation, antibiotics do not have direct inhibitory roles. They play a preventive role by eliminating the pathogens responsible for inflammation. For instance, for inflammatory bowel disease caused by virulent E. colistrains, Bacteroides spp, and Mycobacterium avium subspecies paratuberculosis, the use of ciprofloxacin, metronidazole, rifaximin, clarithromycin and other antibiotics can improve the clinical symptoms [150]. For Helicobacter pylori infection that may lead to gastric muco-associated lymphoid tissue lymphoma and early gastric cancer, a combination of PPI, clarithromycin, and nitroimidazole is available as first-line treatment, with simultaneous or sequential administration ranging from 3 to 14 days [151].

Chemotherapeutic agents are associated with barrier damage and critical function deteriorations of tissues and organs [152]. Anti-tumor antibiotics cause tissue cell destruction and epithelial barrier damage [64, 116], which may trigger inflammation. However, antibiotics are ineffective against this type of inflammation. Intestinal flora can play an important role in clearing inflammation and inhibit the formation of a micro-environment that is conducive for tumor growth.

Anti-microbials antibiotics play a certain role in regulating intestinal flora. A healthy and stable intestinal environment is of great significance in improving the inflammatory environment and in maintaining the homeostasis of the body’s internal environment. Intestinal flora and their metabolites have a major role in this process. However, some intestinal flora have the potential to induce the occurrence and metastasis of inflammation-associated cancer [4, 111, 112]. Therefore, selection of appropriate intestinal microbiota is a major challenge in microbiome therapy. Symbiotic flora exerts different effects on the type of inflammation in different treatment regimens, which affects tumor treatment outcomes, highlighting the need to manipulate the human gut microbiota to promote cancer treatment [65, 153]. After metronidazole treatment of fecal microbiota transplantation donor mice, their microbiota still retained the ability to control inflammation, accompanied by enrichment of Lactobacillus and iNKT cells [153]. Thus, antibiotics have the ability to maintain and promote normal defense functions.

Elucidation of the joint modes of action can provide new ideas for controlling inflammation. Normal and steady states of the intestinal microbiome are closely associated with chronic inflammation and tumor growth. Elucidation of the relationship between antibiotics, intestinal flora and inflammation, correct use of anti-microbial antibiotics for early prevention and maintenance of healthy bacterial homeostasis to achieve the synergistic effects of the two in inhibiting inflammation and tumor emergence is a direction for further studies.

The combination of broad-spectrum antibiotics (ampicillin & colistin & streptomycin) and imipenem was associated with intestinal microecological disorders and weakened anti-tumor effects of anti-CTLA-4 [9]. Treatment of natal K/BxN mice with vancomycin or ampicillin strongly inhibited rheumatoid arthritis development [154]. However, targeted inhibitory effects of antibiotic therapy on microbiota, such as inhibition of Gram-negative bacteria by vancomycin, anaerobic bacteria and gram-negative bacteria by metronidazole and neomycin sulfate, and gram-positive bacteria by ampicillin, all reduced Th17 cell numbers by varying degrees. Recovery of Th17 cells was achieved after the recovery of microbiota [60]. Thus, intestinal microbiota can alleviate antibiotic-induced damage to immune functions. Proper regulation of intestinal flora can also alleviate and counter the effects of incidences as well as severity of intestinal lesions caused by antibiotic therapy to enhance drug efficacy. A recent study [155] evaluated the relationship between pATB, concurrent ATB and OS, progression-free survival, and objective response rates in 302 stage IV NSCLC patients. Contrary to previous reports that antibiotics weaken the efficacy of ICIs and affects prognosis, it was found that antibiotic exposures do not affect clinical outcomes of first-line immunization combined with chemotherapy in NSCLC patients. There are positive correlations between antibiotics and tumor immunity. Pingyangmycin, a bleomycin-type antineoplastic antibiotic with good clinical therapeutic effects enhances the therapeutic outcomes of anti-PD-1 antibodies associated with tumor-infiltrating CD8${}^{+}$ T cell enlargement [156].

Therefore, antibiotics can play a beneficial role in tumor immunotherapy, regulating bacterial balance by increasing the presence of anti-tumor beneficial microorganisms and inhibiting the abundance of tumor-associated flora.

Fusobacterium nucleatum is a common bacterial species in CRC tissues. Treatment of colon cancer xenografted mice with metronidazole reduced the Fusobacterium load in the tumor micro-environment and slowed down tumor cell proliferation [66]. Similarly, C4/butyrate produced by gut bacteria such as Clostridia impairs APC activities while reducing vancomycin-enhanced RT anti-tumor activities. Interestingly, vancomycin treatment targeted Gram-positive bacterial populations, including Clostridia and reduced SCFA and C4 concentrations in stool and tissue samples [66]. These results inform on antibiotic-targeted interventions in cancer-associated microbiota, providing a basis for potential treatment of cancer patients.

Antibiotic intervention of intestinal flora has a positive role in tumor immunity. Intestinal flora of some patients with pancreatic cancer promotes immunosuppression and tumor immune evasion by inducing Foxp3${}^{+}$ Treg cell proliferations [61]. Inhibition of relevant microbial growth by using appropriate antibiotics can control precancerous lesions and cancer progression. Butyrate induces T cell changes via epigenetic changes of the Foxp3 gene [84], implying that antibiotics act on intestinal bacteria to regulate the effects of their metabolites. Antibiotic mixture (ABX) ablation of the microbiome can inhibit pancreatic duct adenocarcinoma (PDA) invasion [157]. Therefore, oral antibiotic induced immunogenic reprogramming of the tumor micro-environment after ablation, including increased differentiation of M1 macrophages, enhanced TH1 differentiation of CD4${}^{+}$ T cells and activation of CD8${}^{+}$ T cells, significantly up-regulates PD-1 expressions on effector T cells, and promotes checkpoint targeted immunotherapeutic outcomes. Thus, modulation of the microbiome by oral antibiotic orientation has great prospects for enhancing immunotherapy.

The use of antibiotics and probiotics to rebuild healthy intestinal micro-environments has a certain reference value. Refilling of mice fecal microbiome reversed intratumor immunogenicity changes attributed to antibiotic ablation bacteria, so that genes associated with T cell proliferation and immune activation were up-regulated in tumors of antibiotic-treated mice. These findings imply that the reconstructed microbiome better regulated the immunogenicity of PDA. In addition, antibiotics are beneficial for preventing the transfer of intestinal micro-organisms and maintaining a stable microbial distribution in vivo. Disruption of intestinal vascular barrier leads to systemic transmissions of intestinal bacteria and CRC liver colonization [34], while ABX treatment effectively reduces bacterial metastasis to the liver and intestinal lumen.

Therefore, interactions between antibiotics and intestinal flora can effectively alleviate immune function damage caused by antibiotic treatment, while targeting tumor or immune related intestinal flora components. Studies have evaluated the synergistic effects of intestinal microbiota and anti-tumor drugs in stimulating anti-tumor adaptive immune functions [17], although there are few studies on antibiotic drugs. Combination immunotherapy regimens are a key direction for future cancer treatment. The combination of antibiotics and ICIs is a potential approach for experimental treatment of cancer. Interactions between antibiotic therapy, intestinal microbial diversity, and anti-tumor immunotherapy are another scientific pathway that is worth of explorating.

Common anti-tumor antibiotics, such as DOXO, are highly toxic with various side effects in patients. Studies have developed a lipid delivery system co-loaded with curcumin and DOXO, which makes use of their synergistic anti-tumor effects. It can initially achieve therapeutic effects of attenuation and efficiency enhancement [62]. Thus, antibiotics can synergistically work with other drugs to achieve better anti-tumor outcomes.

Double-hit lymphoma, a tumor with a higher degree of malignancy, involves the activation of both MYC and B-cell lymphoma-2 oncogenes. Experimentally, the combination of tigecycline, which is effective against MYC-driven lymphomas, with the B-cell lymphoma-2 inhibitor (Venetoclax), showed significant anti-tumor effects. Tigecycline has good prospects for combination with Rituximab, the current first-line treatment option for lymphoma [158].

Anti-tumor effects of antibiotic combinations have a certain relationship with intestinal flora. Intestinal homeostasis was shown to contribute to normal anti-tumor immune functions of mice [63, 159]. A combination of antibiotics are conducive for regulating the effects of microflora imbalance caused by a single antibiotic. Intestinal tumors are often accompanied by dysregulated intestinal flora. Polysaccharides regulate the ratio of beneficial to harmful bacteria in the intestinal flora to maintain a healthy intestinal microbiota balance [160]. Jiyan Su et al. [161] performed 16S rRNA sequencing analysis on mice 4T1-breast cancer models and found that the combined action of paclitaxel (PTX) and Ganoderma spore powder polysaccharide (SGP) improved the PTX-induced intestinal microbiome dysregulation.

The combination of anti-bacterial agents and radiotherapy has improved therapeutic outcomes. Vancomycin combined with RT exhibited better anti-tumor effects than the use of a model drug alone [66]. Vancomycin in combination with RT can reshape the tumor micro-environment, but also be IFN-$\gamma$ and CD8 dependent, enhancing local antigen presentation accompanied by cytotoxic T cell infiltrations in the tumor.

With regards to single use of antibiotics for anti-tumor treatment, efficacies should be improved while side effects should be minimized. Synergistic effects from the combination of antibiotics with other drugs can significantly improve intestinal flora abundance and species diversity. Enhanced anti-tumor effects of antibiotics directly inhibits tumor development. Regulating the occurrence and development of tumors that are caused by antibiotic-mediated dysbiosis can also indirectly inhibit tumor proliferation. Drug combinations can also improve tumor attenuation efficiencies of antibiotics while maintaining normal bacterial homeostasis. Thus, combinations of antibiotics with other anti-tumor drugs have better tumor control outcomes.