Exploring the Role of the Gut and Intratumoral Microbiomes in Tumor Progression and Metastasis

Cancer cell dissemination involves invasion, migration, resistance to stressors in the circulation, extravasation, colonization, and other functions responsible for macroscopic metastases. By enhancing invasiveness, motility, and intravasation, the epithelial-to-mesenchymal transition (EMT) process promotes the generation of circulating tumor cells and their collective migration. Preclinical and clinical studies have documented intensive crosstalk between the gut microbiome, host organism, and immune system. According to the findings, polymorphic microbes might play diverse roles in tumorigenesis, cancer progression, and therapy response. Microbial imbalances and changes in the levels of bacterial metabolites and toxins promote cancer progression via EMT and angiogenesis. In contrast, a favorable microbial composition, together with microbiota-derived metabolites, such as short-chain fatty acids (SCFAs), can attenuate the processes of tumor initiation, disease progression, and the formation of distant metastases. In this review, we highlight the role of the intratumoral and gut microbiomes in cancer cell invasion, migration, and metastatic ability and outline the potential options for microbiota modulation. As shown in murine models, probiotics inhibited tumor development, reduced tumor volume, and suppressed angiogenesis and metastasis. Moreover, modulation of an unfavorable microbiome might improve efficacy and reduce treatment-related toxicities, bringing clinical benefit to patients with metastatic cancer.


Introduction
The emerging trend of microbiome research in oncology results from studies uncovering the role of microorganisms in the etiology of several malignancies.Preclinical and clinical studies have also revealed a significant impact of the gut and tumor microbiomes on the efficacy of antitumor therapy and treatment-induced toxicity [1].Moreover, mounting research focuses on the analysis of the microbiome composition in metastatic disease [2].The significant role of the microbiome in oncogenesis and treatment underlines the fact that polymorphic microbiomes, including intestinal, oral, skin, tumor, lung, and vaginal microbiomes, were added to the extended comprehensive concept termed "The Hallmarks of Cancer", which summarizes the key characteristics of tumors.The microbiome directly interacts positively or negatively with other hallmarks of malignancies, such as inflammation, immune impairment, genomic instability, and resistance to antitumor therapy [3].
The study of metastasis biology at the cellular, molecular, biochemical, and physical levels has undergone dramatic growth over the last 20 years.While the precise pathways are still under investigation, recent research has indicated new roles of cancer cells, which involve promoting genes with metastasis-driving mutations, cancer stem cells, circulating tumor cells (CTCs), epithelial-to-mesenchymal transition (EMT), and the metastatic dormancy and dynamic plasticity of cancer cells [4,5].Various studies also demonstrated that the following drive metastatic spread: systemic inflammation; immune system modulation; specific interactions between cancer cells, immune cells, and cells in the tumor microenvironment; the avoidance of anoikis; immune checkpoint regulation; self-seeding, and other mechanisms.Mounting research highlights the role of the intratumoral and mucosal microbiomes in the progression of metastatic processes.
The mechanisms by which the intratumoral microbiota is implicated in metastatic spread are still unclear [6].Recently, a newly developed single-cell RNA-sequencing method, known as INVADEseq, aimed to identify cell-associated bacteria in patient tumor samples and describe the pathways involved in metastatic processes [7].According to the findings, the highly organized distribution of bacteria within tumors affects immune and epithelial cell functions, leading to disease progression [7].Studies reported that specific microorganisms might affect metastatic processes, including EMT, resistance to fluid shear stress, immune system modulation, and matrix metalloproteinase (MMP) regulation [8].EMT-related pathways might be regulated by specific microbial pathogens [9,10].Moreover, the microbiome can change the actin cytoskeleton, contributing to tumor cells' resistance to fluid shear stress [11].As previously shown, Helicobacter pylori supports the remodulation of actin filaments, which results in EMT onset [12,13].In addition, specific microbes within the microbiome contribute to tumor cell adaptation to specific biochemical factors in the tumor microenvironment during metastatic processes [8].
In this review, we aim to summarize the current knowledge about the emerging role of the gut and intratumoral microbiomes in metastasis and cancer progression.We analyze studies focusing on the association between the composition of microbial communities and metastatic disease in patients with different types of malignancies.Moreover, we outline the mechanisms of microbiome involvement in cancer progression and metastasis-related processes, including EMT and angiogenesis.Microbiota modulation represents an evolving phenomenon in cancer patient care, and mounting evidence supports the clinical utility of a microbiome-based approach in cancer initiation, progression, and treatment.

The Mechanisms of Tumor Progression and Metastasis
Tumor progression and metastasis represent multi-step processes, resulting in cancer cell changes that enable them to grow, spread, and establish secondary tumors at distant body sites (Figure 1).seeding".Self-seeding in metastasis is the recruitment of cancer cells and the re-seeding of primary tumors and existing metastases by aggressive cancer cell clones [20,21].

Figure 1.
The key processes involved in tumor progression and metastasis.A deep understanding of the crucial events and corresponding mechanisms leading to the formation of distant metastases is essential for developing treatment modalities to target different stages of cancer development and improve patient outcomes.
Cancer cells can induce neutrophils to release neutrophil extracellular traps (NETs), which sequester CTCs and promote the metastatic process [22][23][24][25][26].A certain number of CTCs can be eliminated by anoikis, the programmed apoptosis of cells [27].However, cells can develop an anoikis-resistant state via oncogene activation (e.g., ERBB2 and RAS), an The activation of invasion and metastasis is initiated by epigenetic changes, cellcell interactions, growth factors, cytokines, signals from extracellular matrix components, extracellular matrix mechanical pressures, and the intratumoral microbiota [14].
The metastatic cascade includes the detachment of cancer cells from the primary tumor and the gaining of an invasive phenotype, local invasion into surrounding tissue, intravasation into the circulation, systemic transportation, extravasation, and the formation of colonies at distant sites, with adaptation and proliferation in secondary organs.
CTCs typically arise from epithelial tumor cells that undergo EMT, resulting in the loss of cell-cell adhesion and apical-basal polarity, the reorganization of the cytoskeleton, acquiring properties of tumor stem cells, and resistance to therapy.This process is regulated by transcription factors in tumor cells (Snail 1, Slug, ZEB1, Twist, FOXC2, etc.) and signaling pathways from the tumor microenvironment (WNT, Notch, Hedgehog, TGFβ, FGF, EGF, HGF signaling, etc.).Additionally, the hypoxia and activation of specific signaling pathways, including PI3K, WNT/β-catenin, and MAPK, affect EMT regulation [15,16].Many studies focus not only on CTC detection and enumeration but also on CTC biomarkers, among which EMT markers are of great interest [17][18][19].The most aggressive CTCs are related to the infiltration of the primary tumor or established metastasis in a process of "selfseeding".Self-seeding in metastasis is the recruitment of cancer cells and the re-seeding of primary tumors and existing metastases by aggressive cancer cell clones [20,21].
Before the arrival of tumor cells from primary tumors to the premetastatic niche [30], hematopoietic progenitor cells (VEGFR1-positive) travel from the bone marrow into the circulation and establish themselves in secondary organs, where they adhere to fibronectin, produced by fibroblasts and fibroblast-like cells [31].The adherence is mediated by the integrin VLA-4, expressed by hematopoietic progenitor cells [32].The nidation of tumor cells is primarily influenced by stromal-derived factor 1 (SDF-1), binding to the chemokine receptor CXCR4 [33].CXCR4 receptor expression on breast cancer tumor cells is a typical determinant of bone metastasis [34,35].Its activation results in pseudopodia formation and integrin modulation, followed by the recruitment of endothelial cells (VEGFR2-positive) to the distant site [36].
Cancer cells and the tumor microenvironment produce factors that influence angiogenic processes, with the key drivers being VEGF-A [37,38] binding to VEGFR2 receptor [39].Alterations of protooncogenes (RAS and SRC) and tumor suppressor genes (TP53 and VHL) correlate with VEGF overproduction by tumor cells.Hypoxia is the principal stimulator of VEGF production, and hypoxia-inducible transcription factors (HIF-1α and HIF-2α) play a central role in VEGF regulation.Other angiogenesis inductors, such as FGF1, EGF2, PDGF-B, PDGF-C, and EGF, bind to their respective receptors on blood vessel endothelial cells and induce proliferation and migration [40].Besides the conventional angiogenic mediators, BMP9 signaling and Shh signaling also participate in the process [41].In addition, exosomes released by cancer and immune cells may transport various proangiogenic molecules like VEGF, MMPs, and microRNAs [42].

The Relationship between Microbiome and Cancer Progression-Related Processes
In recent years, the correlation between the microbiome, cancer, and metastatic disease has gained more attention (Figure 2).Many studies confirmed that certain microbes and their metabolites are associated with a better/worse therapy response and patient outcomes.
HIF-2α) play a central role in VEGF regulation.Other angiogenesis inductors, such as FGF1, EGF2, PDGF-B, PDGF-C, and EGF, bind to their respective receptors on blood vessel endothelial cells and induce proliferation and migration [40].Besides the conventional angiogenic mediators, BMP9 signaling and Shh signaling also participate in the process [41].In addition, exosomes released by cancer and immune cells may transport various proangiogenic molecules like VEGF, MMPs, and microRNAs [42].

The Relationship between Microbiome and Cancer Progression-Related Processes
In recent years, the correlation between the microbiome, cancer, and metastatic disease has gained more attention (Figure 2).Many studies confirmed that certain microbes and their metabolites are associated with a better/worse therapy response and patient outcomes.

Figure 2.
The involvement of the gut and intratumoral microbiome in metastatic processes.Not only cancer development but also the type of anti-cancer therapy affects the diversity of microbial composition in the gastrointestinal tract and alters microbial-associated metabolites.The deregulated thickness of the gut mucosal layer might be responsible for bacterial translocation and development of bloodstream infections.Gastrointestinal dysbiosis results in the inflammation that promotes cancer cell spread due to changed immune responses.Microbes within the tumor microenvironment affect the progression of cancer via modulated immunity and changed inflammatory signaling pathways.Moreover, studies observed the relationship between intratumoral bacteria and metastasis via increased resistance to mechanical stress.Abbreviations: EMT, epithelial-to-mesenchymal transition.
Understanding the mechanisms by which unfavorable microbes have an impact on tumor progression is an active area of recent research.Therefore, intensive research in numerous ongoing clinical trials might shed more light on prognostic microbial markers for treatment outcomes in metastatic disease (Table 1).The identification of microbial biomarkers will help to understand how the microbiome is implicated in cancer progression.Understanding the mechanisms by which unfavorable microbes have an impact on tumor progression is an active area of recent research.Therefore, intensive research in numerous ongoing clinical trials might shed more light on prognostic microbial markers for treatment outcomes in metastatic disease (Table 1).The identification of microbial biomarkers will help to understand how the microbiome is implicated in cancer progression.

Microbiome in Epithelial-to-Mesenchymal Transition
The association between specific microbiome compositions, the inflammatory response, and therapy resistance related to EMT is still under debate [43].Alterations in the gut microbiome might support EMT via the TGFβ, WNT, and Notch signaling pathways, and Slug, SNAIL, Twist, ZEB1, and ZEB2 transcription factors resulted in invasive and metastatic cancer processes [44][45][46].A pathological microbiome in pancreatic ductal adenocarcinoma (PDAC) patients promoted EMT via the activation of transcription factors, including TGFβ and TNFα [47].Antibiotic-induced gut dysbiosis supported macrophage activation and the production of inflammatory cytokines, subsequently promoting EMT in colorectal cancer (CRC) [48].Fusobacterium nucleatum, Enterococcus faecalis, Bacteroides fragilis, Escherichia coli, and Salmonella enterica are known microbes involved in CRC progression, producing virulence factors that contribute to EMT and cancer progression [49].F. nucleatum-infected CRC cells supported the EMT cell phenotype and elevated cancer cell migration, as well as tumorsphere formation [50].Similarly, EMT-related morphological changes and the upregulation of mesenchymal marker ZEB1 were documented in cag pathogenicity island+ H. pylori-infected gastric epithelial cells [51].Marques et al. noted that the H. pylori infection of gastric cells reduced Afadin and increased ZEB1, vimentin, Slug, Snail, and N-cadherin.Since Afadin regulates adherens and tight junctions, its loss due to bacterial infection might play a role in aggressive gastric cancer phenotypes [52].
The tumor-associated butyrate-producing bacterium SM4/1, E. coli str.K-12 substrate MG1655, Saccharomonospora viridis DSM 43,017, and E. coli O157:H7 str.EC4115 correlated with the expression of EMT-related genes (TGFB, RhoA, vimentin-associated genes, SNAI2, SNAI3, and TWIST1) in muscle-invasive bladder cancer.In addition, the level of both E. coli O157:H7 str.EC4115 and Oscillatoria sp.CCAP 1459/13 was negatively associated with E-cadherin expression [53].In the T24 cell line, infection with E. coli significantly upregulated vimentin, reactive oxygen species levels, and stemness markers (CD44, NANOG, SOX2, and OCT4), while it downregulated CK19.The authors observed that E. coli-infected bladder cancer cells had a specific elongated morphology with a shortage of contact between cells, while non-infected cells had an adhesive ability with epithelial morphology [54].Gingival squamous cell carcinoma tissue samples showed the presence of Porphyromonas gingivalis [55].Porphyromonas supports the invasion and metastasis of oral cancer cells via EMT processes.The exposure of cancer cells to P. gingivalis for a long period promoted migration, invasiveness, and resistance to chemotherapeutics [56,57].The infection of gingival epithelial cells with P. gingivalis increased ZEB1 expression, leading to EMT regulation.Conversely, bacterial strains lacking FimA did not promote the expression of ZEB1 [58].
Serum levels of bacterial lipopolysaccharide (LPS) were found to be elevated in individuals diagnosed with esophageal cancer.An in vitro study by Peng et al. revealed an increase in N-cadherin and vimentin in EC109 cells after treatment with LPS, accompanied by reduced E-cadherin levels.The achieved results suggest the potential role of LPS in promoting migration, invasion, and EMT initiation [59].Microbiota might control cancer stem cells via miRNA and circRNA regulation [60].Broad-spectrum antibiotic treatment upregulated the level of mmu_circ_0000730, showing its association with cancer cell stemness and EMT via an upregulated SOX9 expression level.As reported, siRNA targeted mmu_circ_0000730-inhibited invasive/migrative processes and reduced metastasis development [61].Yan et al. confirmed the involvement of F. nucleatum in cancer stem cells and EMT crosstalk in cancer progression.The expression levels of EMT markers (E-cadherin and N-cadherin) and a cancer stem cell marker (Nanog) correlated with the abundance of F. nucleatum in CRC tissue samples [62].Previous data observed that infection with Fusobacterium upregulated miR-21 levels, and the upregulation of miR-21 correlated with distant metastases and cancer stage [63].In breast cancer, bacterial metabolites, such as short-chain fatty acids (SCFAs), lithocholic acid, cadaverine, and indole derivatives, exert an influence on the EMT process [64,65].Ujlaki et al. screened the antineoplastic properties of bacterial-associated metabolites.The application of 3-hydroxyphenyl acetic acid, 4-hydroxybenzoic acid, and vanillic acid led to a hyperproliferative impact on breast cancer cells, while the application of butyric acid, glycolic acid, d-mannitol, 2,3-butanediol, and trans-ferulic acid had cytostatic effects.Anti-EMT properties were documented in the case of applied 3-hydroxyphenyl acetic acid, 4-hydroxybenzoic acid, 2,3-butanediol, and hydrocinnamic acid [65].The treatment of breast cancer cells with indolepropionic acid, a microbiota-derived tryptophan metabolite, reverted EMT [66].

Microbiome and Angiogenesis
Tumor angiogenesis is involved not only in tumor growth but also in tumor progression and metastasis.According to findings, microbiota plays a role in angiogenesis via VEGF and inflammatory cells [67].Studies showed that microbiota-derived SCFAs may have an impact on angiogenesis.Butyrate, a major member of SCFAs, plays a role in many cellular processes.Decreased levels of sodium butyrate supported angiogenesis [68].Lithocholic acid is a bacterial metabolite, and it plays a role as a tumor promoter in human CRC cells [69].Enterotoxin produced by enterotoxigenic E. coli might activate a cGMP-dependent signaling pathway that decreases VEGF and VCAM-1, both associated with angiogenesis and tumor metastasis [70].Trimethylamine N-oxide (TMAO), as a microbiome-derived metabolite, increased CRC cell proliferation in vitro.Moreover, in vivo experiments confirmed higher levels of TMAO in the circulation, an increased tumor volume, and promoted angiogenesis in choline-fed CRC mice [71].Changes in the gut microbiome participate in the transformation of adenomas into carcinomas.These alterations resulted from reduced butyrate levels and the secretion of mutagenic metabolites, including spermidine and trimethylamine.Therefore, the CRC microbiome might promote inflammation, angiogenesis, and apoptosis via increased/decreased histamine, glutamine, and pyruvate levels [72].The crosstalk between the microbiome, macrophages, and cancer cells is still unrevealed.CRC-derived exosomes can interplay with macrophages within the tumor microenvironment and participate in angiogenesis.Moreover, macrophages activated by microbiome-derived exosomes might induce inflammation [73].Feces transferred from CRC patients into a model of germ-free and azoxymethane-treated mice upregulated genes associated with angiogenesis, invasion, metastasis, and proliferation [74].Four quorumsensing peptides from Bacillus subtilis (PhrG), Streptococcus mitis (CSP), and E. coli (EDF) with its tripeptide analog NWN promoted angiogenesis and breast cancer cell invasion [75].Similarly, the bacteria-produced quorum-sensing peptides such as EntF-metabolite from Enterococcus faecium, Phr0662 from Bacillus spp., and EDF derived from E. coli stimulated colon cancer cell invasion and angiogenesis [76].

The Studies of Microbiome Composition in Metastatic Disease
Recently, Hilmi et al. studied samples obtained from the lymph nodes, lungs, and livers of patients suffering from different cancer types, such as breast, lung, and colorectal malignancies.A higher presence of F. nucleatum was specific to lung metastases.The microbial load in lymph node metastases was lower than in liver and lung metastases.However, the authors did not observe a relationship between the type of primary tumor and the microbial composition in metastases [77].The level of Eubacterium halli in stool samples is negatively associated with fatigue in patients with advanced, metastatic, unresectable colon, ovarian, cervical, and non-small-cell lung cancers [78].In vivo experiments confirmed that gut microbial depletion via a broad-spectrum antibiotic cocktail reduced the incidence of metastases in melanoma, pancreatic, or colon cancer murine models [79].Spakowicz et al. performed a retrospective analysis of 690 patients treated with immunotherapy for metastatic melanoma or non-small-cell lung cancer.The results showed that antibiotics and corticosteroids reduced overall survival (OS), but no direct microbiome measurements were performed [80].

Gastrointestinal Malignancies
Gut dysbiosis and intestinal inflammation represent critical factors in CRC progression [81].Many studies documented differences in the gut microbiome composition between early and definitive CRC stages [82,83].Higher levels of F. nucleatum in fecal and tumor samples correlated with advanced-stage CRC in patients.This microorganism activated tumor-derived CCL20 expression, increased macrophage infiltration, induced M2 polarization, and enhanced metastasis [84].Similarly, F. nucleatum was more abundant in feces from patients with positive lymph node metastases.In vitro experiments showed that bacterial infection increased the migration of HCT-116 and LoVo cells.However, the heatkilled bacterium could not support cell migration.Accordingly, an in vivo analysis showed that an injection of Fusobacterium-treated cells induced lung metastatic nodules in nude mice [85].Fusobacterium might contribute to CRC metastases via declined m 6 A modifications in CRC cells and patient-derived xenografts, which resulted in more aggressive cancer cell phenotypes [86].The presence of Fusobacterium in primary colon adenocarcinoma correlated with Bacteroides, Selenomonas, and Prevotella in distant metastases.Viable Fusobacterium was detected in murine xenografts, and metronidazole administration significantly reduced intratumoral Fusobacterium load and tumor growth [87].
Wang et al. assessed the effect of combined regorafenib plus toripalimab in patients with refractory metastatic CRC.Patients not responding to combined treatment were reported to have an increased amount of F. nucleatum.A lower response rate was documented in patients with liver metastases [88].However, supplementation with Faecalibacterium prausnitznii could reverse CRC progression via several mechanisms and reduce inflammation [89].In mice, a colonic bacterial biofilm composed of E. coli and B. fragilis was responsible for faster CRC onset, disease progression, and mortality than colonization only with one bacterial strain alone [90].The B. fragilis toxin is responsible for lost cell adhesion resulting in EMT [91].Changes in the EMT-associated genes SNAI1 and VIM were documented after the co-cultivation of Caco-2 cells with bacteria from CRC biopsies.The downregulation of SNAI1 was observed in all affected cells.However, VIM expression changes depended on co-cultivation with specific bacteria.Proteus vulgaris increased VIM expression, but Proteus mirabilis and Pseudomonas aeruginosa were associated with reduced expression [92].Sun et al. assessed the effect of the Quxie capsule (QX) as herbal medicine on the gut microbiome in patients with unresectable metastatic CRC.A decreased amount of Actinobacteria became elevated after QX treatment.An LEfSe analysis showed that the microbiome before QX treatment was composed of c_Gammaproteobacteria and g_Lachnospiraceae UCG 008, while f_Ruminococcaceae and g_Peptostreptococcus dominated after QX treatment.Moreover, high levels of CD4 and CD8 cells were observed in the QX-treated group [93].
The poor prognosis of pancreatic cancer reflects the necessity of novel non-invasive markers in the management of patients suffering from metastatic or locally advanced pancreatic cancer.Therefore, a fecal microbiome analysis might help to determine potential microbiome-related biomarkers in the early diagnosis of patients [94].Jeong et al. investigated the microbiome in tumor and normal adjacent tissue samples from patients with PDAC.A higher number of metastases in lymph nodes correlated with a higher abundance of Comamonas and Turicibacter in tumor tissues, while there was a lower amount of Tepidimonas, Enhydrobacter, Turicibacter, and Wautersiella in normal tissues.Alpha diversity was higher in patients with cancer recurrence with low levels of Streptococcus and Akkermansia in tumor samples [95].
The administration of a nanoparticle conjugation of ginsenoside Rg3 (NpRg3) reduced Firmicutes and increased Bacteroidetes and Verrucomicrobia in the dimethylnitrosamine-induced hepatocellular carcinoma murine model.The results confirmed that NpRg3 administration suppressed the development of lung metastases.Although circulating tumor cells from primary tumors are the main factor affecting cancer metastasis, NpRg3 might inhibit metastatic spreading via the reduced mobilization of endothelial progenitor cells in the circulation [96].

Lung Cancer
Huang et al. observed that bronchial washing fluid might characterize the microbial composition of lung tumor tissues.Higher amounts of Veillonella, Megasphaera, Actinomyces, and Arthrobacter genera were documented in a group of lung adenocarcinoma patients with-out distant metastases than in squamous cell lung carcinoma patients without metastases.However, lower levels of Capnocytophaga and Rothia were found in lung adenocarcinoma with distant metastases than in squamous cell lung carcinoma with distant metastases.Specifically, the Streptococcus genus was more reduced in lung adenocarcinoma with metastases than in lung adenocarcinoma without metastases.The results confirmed that the microbial composition differed in histologic types of lung cancer with or without metastases [97].Another microbiome study showed higher Legionella in lung tissues from patients who developed metastases, while Thermus was more abundant in tissue samples from patients with advanced-stage (IIIB and IV) lung cancer [98].A respiratory tract infected with Streptococcus pneumoniae promoted the development of liver metastases in the murine model via TLR2 activation [99].Higher exposure to cigarette smoke and non-typeable Haemophilus influenzae supported lung metastasis growth in the lung cancer model.The authors noted that cigarette smoke caused the disruption of pulmonary barrier integrity, allowing for the translocation of bacterial factors into tumors [100].

Breast Cancer
Some studies highlighted the differences in the microbiome composition between samples from breast tumors and non-malignant breast tissues [101].For the first time, Buchta Rosean and a research team documented that dysbiotic changes in the gut microbiome increased the dissemination process in the HR+ breast cancer murine model.Antibiotic pretreatment caused changes at the phylum and genus levels.The amounts of Alistipes, Blautia, Parabacteroides, and Escherichia/Shigella genera were elevated in mice with antibiotic-induced commensal dysbiosis.Antibiotic-induced dysbiosis enhanced tumor cell dissemination in peripheral blood, lungs, and axillary lymph nodes [102].An analysis of fecal samples revealed that Acinetobacter, Bacilli, Campylobacter, Collinsella, Lactobacillales, Veillonella, Streptococcus, Epsilonproteobacteria, Pseudomonadales, and Moraxellaceae were more abundant in the gut microbiome of breast cancer patients with bone metastases than in control samples.However, the analysis revealed significantly decreased amounts of Megamonas, Clostridia, Akkermansia, Gemmiger, and Paraprevotella in the fecal samples of metastatic patients.Therefore, the authors suggested that lower levels of Megamonas and Akkermansia might correlate with the presence of bone metastases [103].The tumor microbiome depletion induced by antibiotic treatment inhibited lung metastasis development in the murine spontaneous breast-tumor model.As detected, the intratumoral administration of specific bacteria, such as Staphylococcus xylosus, Ligilactobacillus animalis, and Spilopsyllus cuniculi, elevated the number of lung metastases without affecting primary tumors in mice [11].
Parhi et al. confirmed the pro-metastatic effect of F. nucleatum in the murine model.A tail vein microbe injection supported the progression of AT3 mammary tumors and the development of lung metastases.Mammary tumors were colonized by Fusobacterium via bacterial attachment to display Gal-GalNAc on tumor cells [104].Zhu et al. observed that the administration of broad-spectrum antibiotics promoted cancer metastases and decreased the survival rate, showing low Bifidobacterium in antibiotic-treated mice.However, fecal microbiota transplantation (FMT) from SPF mice caused the restoration of the gut microbiome composition [61].Neoadjuvant chemotherapy decreased the intratumoral level of Streptococcus while elevating the amount of Pseudomonas.A higher abundance of Brevundimonas and Staphylococcus was reported in primary breast tumors from women with distant metastases.In vitro experiments showed that breast cancer cells treated with 10% P. aeruginosa-conditioned media decreased doxorubicin-mediated cleaved caspase 7 in breast cancer cells [105].

Head and Neck Cancers
Eun et al. documented statistically significant differences in the oral microbiome between patients with and without metastases in oral squamous cell carcinoma.An analysis of the oral microbiome in saliva samples from patients with tumors in the oral cavity showed that Tannerella and Fusobacterium genera were more abundant in participants without metastases, while Prevotella, Stomatobaculum, Bifidobacterium, Peptostreptococcaceae, Shuttleworthia, and Finegoldia had a higher presence in metastatic patients [106].
A study with 802 nasopharyngeal cancer patients revealed that a higher bacterial load within tumors correlated with worse metastasis-free survival and patient OS.The predominant bacterial taxa in the tumor samples were Corynebacterium and Staphylococcus [107].Yuan et al. revealed specific intratumoral bacterial taxa, including Pseudomonas, Rhodococcus, Sphingomonas, Streptococcus, Granulicatella, Haemophilus, and Coriobacteriales, which predicted papillary thyroid cancer invasiveness.A higher alpha diversity was documented in tumor samples from patients with advanced stages than in patients with lower disease stages.The interaction between the tumor microbiome and thyroid hormones might contribute to cancer progressiveness, but more research is needed in this area [108].

Genitourinary Cancers
A study of the bacterial composition of fecal samples from patients with advanced metastatic castrate-resistant prostate cancer treated with immunotherapy showed differences between responders and non-responders to pembrolizumab.Bacteroides thetaiotaomicron, B. fragilis, and Roseburia unassigned dominated in responder samples.In contrast to other studies showing that the presence of favorable Akkermansia muciniphila correlated with a better anti-cancer treatment response, Peiffer et al. observed its lower amount in responder fecal samples [109].The level of intratumoral microbes, including Methylobacterium radiotolerans JCM 2831, Delftia acidovorans SPH-1, Rhodococcus erythropolis PR4, Stenotrophomonas maltophilia K279a, and Meiothermus silvanus DSM 9946, negatively correlated with the tumor-node-metastasis stage in patients with prostate cancer [110].
A higher fecal microbial diversity and A. muciniphila enrichment were associated with a better clinical response to nivolumab or nivolumab plus ipilimumab in patients with metastatic renal cell carcinoma [111].However, an analysis of fecal samples from patients with metastatic renal cell carcinoma noted that Akkermansia was not responsible for treatment efficacy due to its detection in the gut microbiome of seven responders and six non-responders.Differences in the baseline gut microbiome between responding and non-responding patients were not supposed to predict patient outcomes [112].Probiotic supplementation elevated the level of Bifidobacterium spp. in patients with metastatic renal cell carcinoma.A clinical benefit from targeted therapy using vascular endothelial growth factor-tyrosine kinase inhibitors was observed in patients enriched with Barnesiella intestinihominis and A. muciniphila in the microbiome [113].Antibiotic supplementation targeting Bacteroides spp. in the gut microbiome improved progression-free survival (PFS) in patients with metastatic renal cell carcinoma who received VEGF-tyrosine kinase inhibitors (TKIs) [114].

Metastatic Melanoma
Oral and gut microbial communities were diverse in 112 patients with metastatic melanoma.Significant enrichments of Bacteroidales in the fecal microbiome and Lactobacillales in the oral microbiome were documented in patient samples using 16S rRNA sequencing.In the case of response to programed cell death protein 1 (PD-1) blockade, alpha diversity was higher in the responders' fecal microbiome, but differences in the oral microbiome were not observed between responders and non-responders to therapy [115].Mizuhashi et al. found an association between the level of the genus Corynebacterium in swab samples and melanoma progression.The results showed that Corynebacterium was present in 76.9% and 28.6% of patients at stages III/IV and I/II, respectively [116].A microbiome analysis of fecal samples from 42 metastatic melanoma patients before immunotherapy uncovered an enrichment of Bifidobacterium longum, Collinsella aerofaciens, and E. faecium in the gut microbiome of responding patients, whereas Ruminococcus obeum and Roseburia intestinalis were more abundant in non-responders [117].The gut microbiome of metastatic melanoma patients responding to PD-1 blockade was enriched in Clostridiales.On the contrary, a higher amount of Bacteroidales dominated in non-responding patients [118].Vitali et al. found that Prevotella, Clostridium IV, Holdemania, and Anaerofustis were abundant in melanoma patients' gut microbiome.According to the authors, a richer gut microbiome was shown in patients with stage I/II melanoma than in patients with metastatic disease [119].
The fundamental studies focusing on the microbiome composition in metastatic disease are summarized in Table 2.The authors observed a higher abundance of R. obeum and R. intestinalis in non-responders, while the other 3 species were enriched significantly in the responder gut microbiome. [117] Melanoma Patients

Clostridiales and Bacteroidales
Higher bacterial diversity with the prevalence of Clostridiales was observed in the gut microbiome of responding patients.However, the dominance of Bacteroidales within the gut microbiome characterized non-responders. [118]

Microbiota Modulation and Cancer Progression
Gut microbiome modulation leading to increased intestinal barrier and anti-inflammatory responses might inhibit pro-tumorigenic processes, including cancer progression, migration, invasion, and angiogenesis [120,121].The intragastric administration of Lactobacillus reuteri FLRE5K1 inhibited the incidence of tumors in BALB/C mice injected with melanoma cells.The results indicated that L. reuteri FLRE5K1 might restrain the development of tumors due to the blockade of the migration and colonization of cancer cells [122].A preclinical study noted that fecal transplants from obese mice to lean mice with B16F10 tumors stimulated melanoma development and supported cancer progression [123].The aim of recent studies is to assess the effect of probiotic supplementation on tumor progression.
Chen et al. showed that probiotics composed of B. longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus bulgaricus, and Streptococcus thermophilus attenuated the development of lung metastases and prolonged survival in melanoma-bearing mice.Probiotic supplementation led to increased levels of Lachnospiraceae, Streptococcus, and Lachnoclostridium [124].Aerosolized probiotic Lactobacillus rhamnosus, which reached lung murine tissue, reduced the number of lung metastases.Moreover, aerosolized L. rhamnosus and Bifidobacterium bifidum increased the effect of conventional chemotherapeutic dacarbazine in melanomabearing mice [125].A Prohep probiotic product composed of L. rhamnosus GG, E. coli Nissle 1917, and VSL#3 (1:1:1) orally administered to mice bearing hepatocellular carcinoma retarded tumor growth and inhibited angiogenesis.Prohep administration altered the gut microbiome toward valerate producer Oscillibacter and propionate producer Prevotella, both known for their ability to reduce Th17 polarization and support Treg/Tr1 cells in the gut.The results showed that probiotics reduced the recruitment of Th17 cells, which secrete pro-inflammatory cytokines to the tumor microenvironment.Additionally, several angiogenic factors, including ANG2, FLT-1, KDR, TEK, and VEGF-A, were downregulated in the probiotic-supplemented group [126].Daily oral administration of CBM588 containing Clostridium butyricum prolonged PFS in patients with metastatic renal cell carcinoma treated with nivolumab and ipilimumab.The level of Bifidobacterium spp. was higher in probiotic-supplemented patients who responded to immunotherapy, while the results showed a decline in Desulfovibrio spp.However, the toxicity rate was not different between the supplemented and control groups [127].A probiotic mixture of eight bacterial strains mitigated the length and severity of chemotherapy-associated diarrhea in CRC animal models with liver metastases.Moreover, probiotics support gastrointestinal regeneration after chemotherapeutic treatment [128].Shang et al. documented that an intragastric probiotic mixture of B. longum, B. bifidum, L. acidophilus, and L. plantarum attenuated cancer cell proliferation and even the development of metastasis in mouse models of CRC [129].Baruch et al. performed FMT from 2 selected donors previously treated with immunotherapy for metastatic melanoma into 10 recipients with confirmed progression on PD-1 blockade.The presence of favorable Lachnospiraceae was observed in both donors.The feces transfer from donors caused a shift in the gut microbiome in metastatic melanoma recipients with abundant favorable Veillonellaceae and a decline in B. bifidum [130].

Conclusions and Future Directions
The gut and intratumoral microbiomes can influence cancer progression and metastatic processes in various ways, including inducing inflammation and immune system modulation, affecting metabolism and providing energy for cancer cell spread, promoting the angiogenesis caused by microbial metabolites, and impacting cancer treatment efficacy to control metastatic disease.Particular attention should be paid to addressing the ability of specific microorganisms and microbiota-derived metabolites to shape the immune system and tumor microenvironment, potentially promoting the growth and spread of cancer cells.Longitudinal multi-center clinical trials targeting the microbiome composition and function in cancer patients before, during, and after treatment would help uncover distinctive microbiome-associated patterns and their effects on recurrence and survival rates.However, the standardization of methods and procedures for microbiome analyses will be crucial to obtain the most relevant results for clinical use.
Mounting evidence reported a significant correlation between the efficacy of immunotherapy and the gut microbiome composition in patients with metastatic disease.Tumor microbiome profiling along with gut microbiome characterization in individual patients could lead to future personalized therapy to increase the efficacy and decrease the toxicity of anti-cancer treatment.Furthermore, the tumor and gut microbiomes could undergo similar evolution, and the microbiome composition could be assessed at several time points.Another important field of research includes therapeutic interventions aimed at modifying the tumor and/or gut microbiome, including different approaches, such as antibiotic therapy; the modification of the tumor microenvironment; treatments affecting immune cells; and the colonization of beneficial bacteria by prebiotics, probiotics, and fecal microbiome transplant.Further research is required to obtain more evidence elucidating the mechanisms and signaling pathways behind the microbiota-host interactions that drive metastasis and malignant progression.A deeper understanding could help integrate microbiome-based interventions into clinical practice for improving cancer patient outcomes.

Figure 1 .
Figure 1.The key processes involved in tumor progression and metastasis.A deep understanding of the crucial events and corresponding mechanisms leading to the formation of distant metastases is essential for developing treatment modalities to target different stages of cancer development and improve patient outcomes.

Figure 2 .
Figure 2. The involvement of the gut and intratumoral microbiome in metastatic processes.Not only cancer development but also the type of anti-cancer therapy affects the diversity of microbial composition in the gastrointestinal tract and alters microbial-associated metabolites.The deregulated thickness of the gut mucosal layer might be responsible for bacterial translocation and development of bloodstream infections.Gastrointestinal dysbiosis results in the inflammation that promotes cancer cell spread due to changed immune responses.Microbes within the tumor microenvironment affect the progression of cancer via modulated immunity and changed inflammatory signaling pathways.Moreover, studies observed the relationship between intratumoral bacteria and metastasis via increased resistance to mechanical stress.Abbreviations: EMT, epithelial-to-mesenchymal transition.

Table 1 .
Exploring the microbial markers associated with treatment outcomes in advanced or metastatic cancer patients (according to https://ClinicalTrials.gov/, accessed on 18 October 2023).

Table 2 .
Detection of specific microorganisms in advanced/metastatic cancer.The table summarizes fundamental preclinical/clinical studies and their major findings.