Microbiome—Stealth Regulator of Breast Homeostasis and Cancer Metastasis

1. Introduction

External and internal surfaces of animal bodies are entirely covered by microorganisms. For humans, each person contains about 40 trillion microbes, which is as much as the total 30 trillion host cells [1]. These commensals are mostly found in the gut, where their density reaches almost 10 trillion microbes per ml and they weigh about one kilogram per person [2]. It is increasingly evident that these symbionts are not merely passive passengers, but are essential players for fundamental functions of the body, including immunity, metabolism, and energy balance [3]. This notation is well supported by the fact that germ-free (GF) mice manifest serious defects in lymphoid tissue structure and functions [4]. In particular, their gut mucosal immunity is severely compromised due to low numbers of lymphocytes and antibody production [5,6,7].

It is increasingly evident that different physiological conditions between healthy cohorts and cancer patients could be largely attributed to discrete characterization of microbial floras. It is established that not only tumor microbiotas, but also gut microbiotas, of cancer patients are far less diverse than the normal counter-parts—a condition of ‘dysbiosis’. Thus, correcting the microbiome of cancer patients has gained traction as an adjuvant approach. [8,9,10]. For example, normalizing the microbiome of cancer patients through FMTs from healthy cohorts or treatment-responders has been proven for its therapeutic benefits. In fact, treating patients of Trastuzumab-resistant HER2-positive breast cancer with FMT from drug responders improves Trastuzumab-response and tumor immunity [11]. However, the detailed mechanisms of FMT-induced anti-cancer effects have not been fully elucidated.

Along with gut microbiota, breast microbiota is proposed to play critical roles in the breast health and carcinogenesis [8,9,10]. Breast-resident microbes are originated from either the skin/nipple microbiota or those translocated from the guts along with immune cells, such as dendritic cells and macrophages [8,12,13]. Breast microbiota, however, could also be modified by environmental agents, such as aseptic solutions affecting skin microbiota [14]. Gut-breast microbial translocation, termed ‘gut-breast axis’, greatly contributes to the composition of microbiotas of breast tissues and milk [15,16,17,18]. This phenomenon, however, has mostly been conceived in relation to pregnancy and its influence by female sexual hormones [15,16,19,20,21,22]. Thus, it remains unclear whether gut-breast axis exists outside pregnancy on a regular basis and contributes to breast pathophysiology. If this holds true, gut-breast axis may contribute to FMT-mediated anti-breast cancer effects.

2. Breast Tissue Microbiota

The human breast contains a unique microbiota different from those in other parts of the body, playing critical roles in breast health as well as the health of offsprings [23]. Breast tissue microbiota is more diverse (higher α-diversity) than the skin tissue, while each species’ relative abundance (Shannon Index) is similar between them [24]. Sample-to-sample differences of microbiota compositions (β-diversities) are also higher in the breast compared to the skin, owing to major differences found in less abundant microbial species [24]. These features of the breast microbiota are independent of its location within the breast, parity, age, and nationality of the individual [23]. Based on the microbiotas of healthy livers or breast tumors, healthy breast-resident bacteria are expected to mostly reside inside parenchymal cells [25,26]. However, there are clear differences between healthy breast microbiota and breast tumor microbiota [24]. For example, the most abundant bacterial phyla in the healthy breast tissues of women older than 18 years old are Proteobacteria and Firmicutes, whereas these bacteria are under-represented in tumors (Table 1) [27]. Many of these bacteria abundant in healthy breast tissues produce beneficial biomaterials that confer anti-tumor and pro-immunogenic activities to protect the healthy tissue microenvironment.

3. Breast Milk Microbiota

Breast milk microbiota is proposed to be linked to breast tissue microbiota, although there has been no study to confirm their direct relationships. Breast milk microbiota is detectable from the third trimester of pregnancy through lactation. Breast milk, in particular, colostrum (first milk after giving birth), is the primary source of commensals to the newborn [57], whereas maternal-neonatal microbial transfers during pregnancy are conducted through the placenta and amniotic fluid [58,59]. This bacterial transfer through breast milk greatly contributes to the bacterial composition of infants’ guts which is similar to that of breast milk [60]. Breast milk microbiota plays critical roles in the infant’s immune development and his/her health of early and later life. Thus, dysbiosis of the breast milk microbiota would greatly influence infant development [61]. Typically, a baby ingests 1 × 10⁵ to 1 × 10⁷ bacteria a day while consuming approximately 800 mL/day of breast milk [62]. These breast milk bacteria include the genera Lactobacillus, Staphylococcus, Enterococcus, and Bifidobacterium (Table 1) [57]. Breast milk contains olisaccharides which are indigestible by the host, but are digested by enzymes produced by specific gut bacteria, such as bifidobacterial and lactobacilli, which utilize the metabolites for their expansion [63]. In addition, breast milk contains bacterial species that produce short-chain fatty acids (SCFAs), such as butylate, acetate, and formic acid (e.g., Coprococcus, Faecalibacterium, and Roseburia spp). These SCFA-producing bacteria would repopulate the neonatal guts and play a beneficial role in weight gain and adiposity [59,64,65,66].

4. Breast Tumor Microbiota

Microbes within tumors are mostly localized within tumor parenchyma as well as immune cells [26,53]. Since normal tissue microbes presumably reside within parenchymal cells as well, they are proposed to be a major source of intratumoral microbes [67,68]. Nevertheless, breast tumor microbiota is greatly different from healthy breast microbiota, indicating substantial influences of bacterial transfer from other parts of the body during tumorigenesis (Table 1). Breast tumor microbiotas are in general abundant in Fusobacterium, Atopobium, Gluconacterobacter, Hydrogenophaga, and Lactobacillus [24], unlike normal breast microbiotas abundant in Proteobacteria and Firmicutes [27]. Breast tumor microbiota is associated with dysregulation of cell proliferation, metabolic pathways, and immunological responses, contributing to tumor growth and progression (Table 1) [40,45,46,69,70]. Conversely, normal breast microbiota is associated with increased cysteine and methionine metabolism, glycosyltransferases, and fatty acid biosynthesis, promoting immunological responses [24,71,72]. Furthermore, breast tumor microbiota is enriched for Enterobacteriaceae and Staphylococcus, compared to healthy breast microbiota [27]. Both bacteria are known to produce genotoxins that induce DNA damage to help induce malignant progression of host cells [38,39]. In addition, lactic acid producing Lactobacilli, also abundant in breast tumor microbiota, could lower pH and induce metabolic rewiring of the tumor microenvironment (TME), leading to chemotherapy and radiation resistance of tumors [73]. These three taxa of breast tumor-associated bacteria are also found to promote tumor metastasis and colonization, while being transported along with tumor cells to the metastatic site [74]. For colorectal cancer, on the contrary, a different bacterial taxon Fusobacterium is transported along with colon cancer cells to the metastatic site [75], suggesting the roles of different bacteria in metastasis of different types of cancers. The involvement of breast microbiota in tumor metastasis will be further discussed below.

4.1. Breast Cancer Subtype-Specific Microbiota

Different tumor types have distinct microbial compositions, indicating the impacts of different tissue/TME (Table 2) [26]. Furthermore, even among breast tumors, different tumor subtypes (Luminal A, Luminal B, Her2+, and triple negative (TN) types) have distinct microbial compositions (Table 3). This indicates that the heterogeneity of molecular and metabolic profiles and cells of origin among different breast tumor subtypes impacts the fitness of different microbial communities [56,76,77]. For example, luminal subtypes (Luminal A and B) are abundant for the phyla Tenericutes, Proteobacteria, and Planctomycetes. Above all, the most abundant genus in Luminal A tumors is Xanthomonadales (phylum Proteobacteria), while that for Luminal B tumors is Clostridium (phylum Firmicutes) [78]. Conversely, HER2+ breast tumors are abundant in Akkermansia (phylum Verrucomicrobia), Thermi, Firmicutes (Filibacter, Anaerostipes, and Granulicatella_US31), Bacteroidetes (Cloacibacterium, Alloprevotella, and Dyadobacter), and Proteobacteria (Burkholderiales, Helicobacter pylori, PRD01a011B, Stakelama, and Blastomonas) [26,78,79,80,81]. In contrast, TN breast tumors are enriched for Euryarchaeota, Cyanobacteria, Firmicutes, Prevotella, Arcanobacterium, and Brevundimonas [79,82]. In particular, the presence of Listeria fleischmannii (Firmicutes) in TN tumors is shown to be strongly associated with activation of epithelial-to-mesenchymal transition pathway, while the presence of Haemophilus influenza (Pseudomonadota) is correlated with tumor growth and cell cycle progression [83].

4.2. Race/Ethnicity-Specific Breast Cancer Microbiota

In addition to differences in microbiotas among different breast tumor subtypes, races and ethnicities of individuals greatly impact the composition of breast tumor microbiota. This is at least in part attributed to distinct cellular and immunological patterns of breast tumors of Asian, Black and white women (e.g., Asian: high Th1 cells (IFNγ), and megakaryocytes; Black: high dendritic cells, B cells, mesenchymal stem cells, and CXCL9 expression; White: high adipocytes, hematopoietic stem cells, and endothelial cells) [108]. Smith et al. showed that Xanthomonadaceae was the most abundant member in breast tumors from Non-Hispanic White women, whereas genus Ralstonia was most abundant in breast tumors from Non-Hispanic Black women. They also showed that tumors from Non-Hispanic white women were richer in Phylum Bacteroidetes compared to Non-Hispanic Black women [78]. Similarly, Thyagarajan et al. reported that Phylum Bacteroidetes was significantly over-represented in TN breast tumors from white women. Conversely, in TN breast tumors from Black women, phyla Actinobacteria and Thermi and genus Bradyrhizobiaceae were underrepresented. TN tumors from Black women showed reduction of Shannon diversity than adjacent normal tissue, while the trend was reversed for white women [109].

Furthermore, Parida et al. reported racially distinct bacterial biomarkers for breast tumors. For Asian, Pseudomonas, Terrabacter, Clostridiodes, Aestuariibacter, Succinimonas, Catellicoccus, Leucobacter, Rhizobium, Rhodococcus, Methylobacter and Planctopirus are elevated; for Black, Xanthomonas, Amycolatopsis, Aphanizomenon, Anaerovorax, Aminiphilus, Trichormus, Chlorobium, and Sulfurovum are elevated; and for White: Halonatronum, Salinarchaeum and Amorphus are elevated. Such racial differences in microbial components of breast tumors are proposed to be linked to differential metastasis predictors among races [108].

5. Origin of Breast Tissue Microbiota

The origins of breast tissues and milk microbiotas are currently unclear; however, they are proposed to be derived from the breast skin, the oral cavity of the suckling infant, and the maternal gut through gut-breast axis (Figure 1) [58].

5.1. Microbial Transfer from Breast Skin

Mechanisms of microbial transfer from the skin to breast have not yet been clearly determined, although there are several possible scenarios based on the transfer of pathogenic bacteria from the skin to mammary glands. Abnormal microbiota of the breast skin could contribute to the pathogenicity of breast tissue, attesting to microbial transfer from the skin to the breast tissue [14]. Skin microbes such as Pseudomonas aeruginosa possess fatty-acid metabolising capabilities and could become pathogenic in the breast tissue [110]. Additionally, Staphylococcus aureus enriched in the skin of atopic dermatitis could lead to the formation of breast abscess also colonized by S. aureus [111]. In fact, Staphylococcus is among the most abundant genera in breast tumors and strongly linked to breast cancer metastasis, attesting to the role of skin bacteria in breast tumors [40,47,109,112]. In particular, bacteraemia and colonization of S. aureus in certain tissues could promote the incidence of primary tumors [113,114]. Other bacterial taxa linked to increased breast cancer risk include Bacillus, Bacteroidetes, Brevundimonas, Comamonadaceae, Enterobacteriaceae, and Methylobacterium which are also found in the skin microbiota, supporting the possibility of their transfer from the skin [47,106,109,110]. Furthermore, increased numbers of Corynebacterium and Pseudomonas, usually only found in normal skin flora, could break the skin barrier and penetrate deep into the breast tissue to induce granular lobular mastitis [111,112].

Iatrogenic breakdown of the skin barrier during medical procedures could also result in contamination of the underlying breast tissue by skin commensals [113]. For example, breast skin microbes, such as Staphylococcus epidermidis, play roles in the pathogenesis of breast implant complications including anaplastic large cell lymphoma [113,114,115,116,117]. On the contrary, mechanisms of microbial transfer from the skin to the breast tissue without damaging the skin barrier are more uncertain. Proposed scenarios include retrograde transfer through the nipple and ducts (see the details below) [57] and contamination during nipple aspirate fluid procedure [106].

5.2. Microbial Transfer from Nipple

The nipple of mammary gland contains about ten orifices of milk ducts [123]. It was initially proposed that these ductal openings facilitate bacterial transfer from mother’s skin into the breast milk [124]. However, this possibility was ruled out by the finding that microbial compositions of the nipple skin and nipple aspirate fluid are significantly different [125]. Especially, there are strictly anaerobic species, such as Lactobacilli and bifidobacteria, enriched in the breast milk which are unlikely to have originated from skin microbiota [29]. As a likely mechanism of bacterial transfer through nipples, there are some degrees of retrograde flow of milk back into the mammary glands from the infant’s mouth during suckling [57,126]. Such oscillating milk flows allow mothers to respond to pathogens afflicting infants, build antibodies for them, and transfer these antibodies back to the infants so that they can fight against illnesses. Nevertheless, such retrograde bacterial transfer from infants only serves to influence, but not act as, the original source of the maternal breast microbiota. In fact. certain anerobic bacteria, such as Lactobacillus vini and paracasei, are more abundant in the breast of nulliparous or never-breastfed women than breast-fed women than [35], suggesting that these bacteria are potentially derived from the maternal guts.

5.3. Microbial Transfer via Gut-Breast Axis or Oro-Breast Axis

Translocation of gut bacteria to external tissues is commonly associated with disease conditions that impair the intestinal epithelial tight junctions allowing luminal bacteria to move across the epithelial barrier and get into the bloodstream [127]. However, such bacterial translocation also takes place, although to a lesser extent, in healthy individuals, involving beneficial gut bacteria such as Lactobacilli and Bifidobacteria [128,129,130]. Such non-disease-related bacterial translocation appears to involve select species and be associated with immunotaining and immunomodulation of the host [131,132,133].

During pregnancy and lactation, maternal gut bacteria translocate to mammary glands so that they could be transferred to offsprings for colonizing their guts. Especially, during late pregnancy, there are synchronous changes in maternal mammary glands and guts. Mammary glands undergo structural and functional remodeling to become specialized organs that produce and transmit nutrients and other components necessary for neonatal growth [134]. Lactating mammary glands are also an effector site of the mucosal-associated lymphoid tissue system, playing essential roles in infants’ immunity [135]. Along with soluble immune factors, breast milk, especially, colostrum, contains select types of leukocytes, such as neutrophils, macrophages, and lymphocytes [136], facilitated by looser tight junctions of breast epithelia after giving a birth [137]. In particular, leukocytes exposed to antigens in the guts may migrate to mammary glands and be transferred to infants through breast milk for their defense and immune training [138,139]. Synchronously, in maternal guts epithelial permeability increases to facilitate bacterial transmigration [140]. Furthermore, gut microbiota undergoes metabolic adaptation to elevated glucose levels, which would further modify their ability to translocate across the intestinal epithelium and reach mammary glands [141].

Over decades, it has been known that breast milk, maternal feces and infant feces share same bacterial species, attesting to their physical connections [16]. Then, Perez et al. reported that a group of gut bacteria appeared to be physically translocated to mesenteric lymph nodes and then to mammary glands during late pregnancy and lactation. Bacteria translocated to the mammary glands would then enter breast milk and be transferred to the infants to establish the microflora of the neonatal guts. Furthermore, the same study showed that viable gut bacteria found in milk-producing breast cells were also detected in peripheral blood mononuclear cells (PBMC), indicating that PBMC helped translocation of these gut bacteria to the breast [131].

The theory of gut-breast bacterial translocation was confirmed by studies demonstrating that orally administered Lactobacilli strains reached the breast milk of mothers [142,143]. Such studies also support another theory of an oro-mammary bacterial translocation brought up by a finding that maternal oral bacteria and milk microbiota partially overlap [144]. Here, the majority of oral bacteria are expected to travel through the gastrointestinal (GI) tract to reach the guts and then be transported to the breast via gut-breast axis, whereas a small fraction of them. In contrast, a small fraction of oral bacteria could directly enter oral and maxillofacial blood circulation to spread to distant tissues/organs [145]. Oro-mammary translocation is in particular important as a cause for the abundant oral bacteria (e.g., Fusobacteria and Streptococci) in breast tumors [145].

6. Mechanisms of Bacterial Translocation

Although the pathway and mechanisms that some bacteria could enter the breast tissue has not been elucidated yet, some works have offered a plausible scientific basis. So far, there are two major mechanisms proposed: internalization/transcytosis by gut epithelia; and direct sampling by phagocytic cells (Figure 2).

6.1. Internalization into Epithelial Cells

Luminal bacteria could be internalized into gut epithelial cells and subsequently taken by DCs or macrophages in the mucosal environment. There are several potential mechanisms for internalization of non-invasive bacteria into gut epithelial cells. First, intestinal epithelia harbor specialized microfold (M) cells that transcytose luminal bacteria to make them available to mucosal immune cells [146]. Alternatively, upon activation of TLR4, non-specialized enterocytes or kidney epithelial cells were found to transcytose gram-negative gut bacteria [147]. Second, metabolic and oxidative stress, including hypoxia, low dose of nitric oxide, and uncoupling of mitochondrial oxidative phosphorylation, could damage tight junction of gut epithelia and induce transcytotic bacterial transfer to epithelia [148,149,150,151]. Third, low concentrations of IFNγ could cause the influx of noninvasive E. coli bacteria into human colon epithelia without affecting cell viability and tight junctions [152]. Such IFNγ-mediated transcytotic bacterial transfer was shown to depend on extracellular signal-regulated kinase (ERK) 1/2 and ADP-ribosylation factor (ARF)-6 [153]. Fourth, infection of intestines with the parasites Giardia lamblia or Campylobacter jejuni could damage gut epithelial barriers and tight junctions and induce penetration of luminal bacteria to the epithelia [154,155]. Fifth, viable non-pathogenic bacteria could enter host cells through endocytic pathways associated with lipid rafts and caveolin-1. Caveolin-1 or cholesterol was in fact found colocalized with bacteria-containing endosomes in epithelial cells [155,156]. These methods exploited by non-invasive bacteria are different from those of invasive pathogens using specialized needle-like systems to inject effector proteins into epithelial cells and manipulate the host cytoskeletons for anchorage and entry [157].

6.2. Sampling and Transportation by Immune Cells

It has been known that certain type of immune cells, especially, those denoted as CD18+ cells such as dendritic cells (DCs) and macrophages, could penetrate the gut epithelial barrier and directly take up non-pathogenic commensal bacteria from the lumen [158,159]. Especially, DCs are capable of opening tight junctions of intestinal epithelia and sampling lumina bacteria without destroying the epithelial integrity because of their ability to repair damage. This allows non-invasive gut bacteria to spread to extra-intestinal organs [158]. Similarly, macrophages could promote the extra-intestinal dissemination of non-invasive gut bacteria [159]. It was shown that induction of DCs with viable commensal bacteria, but not with dead bacteria, stimulates DC maturation, indicated by the increase in the class II major histocompatibility complex and the B7.2 protein on the cell surface, and their translocation across colon epithelium [160,161,162]. DCs, and possibly macrophages, that have taken up luminal bacteria then migrate to the nearby mesenteric lymph nodes and could stay there up to several days [163]. Such lymphoid tissue resident gut commensals are found to elevate anti-inflammatory signaling to help establish mutualism with host immunity [164]. Alternatively, these lymphoid-resident gut commensal could be taken up by lymphocytes, and transported to distant tissues, such as lactating mammary glands [165,166]. In lactating mammary glands, colonization of immune cells and their bacteria cargos is selective due to regulation by lactogenic hormones and retrograde signaling from suckling infants requesting specific immune cells to fight against their ailments [136,167,168].

7. Functions of Intracellular Microbiota

Increasing evidence demonstrates the existence of different intracellular bacteria in humans and mice [169–172]. The prevalence of intracellular bacteria over extracellular bacteria in tissues is largely attributed to more efficient immunological clearance of the latter than the former [173]. These intracellular microbes are proposed to play direct roles of in the pathophysiology of normal tissues and tumors [174]. This view has been increasingly solidified since the groundbreaking discoveries of the roles of Helicobacter pylori in stomach ulcers and, then, gastric cancer 30 years ago [175]. The recent surge of the next-generation sequencing technologies has allowed investigators to profile microbial compositions of tumor tissues, identify tumor-associated microbes, and study their specific functions. Certain commensal bacteria invade cancer cells and remain inside cells during tumor progression and even metastases [75,174]. This is largely attributed to the fact that intracellular bacteria are better protected than extracellular bacteria in a highly immunologic TME [74].

8. Bacterially Produced Metabolites

As discussed above, microbiotas in human breast milk and breast tissue play essential roles in infants’ development and healthy intestinal microbiota and immunity. In particular, different bacterially-produced metabolites contribute to differences in breast tissue microenvironment and health of offsprings [19]. A group of bacterially produced metabolites are found to exert beneficial effects. For example, short-chain fatty acids (SCFAs), such as butyrate, acetate and formic acid, in the breast milk play promote weight gain and adiposity of infants [176]. Cadaverine, a metabolite produced by bacterial lysine decarboxylase, is found to suppress breast cancer progression and metastasis, although the synthesis is downmodulated in breast cancer patients [177]. Also, indolepropionic acid (IPA) is a bacterial tryptophan metabolite and has cytostatic properties through activation of aryl hydrocarbon and pregnane X receptors. Ectopic application of IPA to breast cancer cells is found to suppress their growth and metastasis [178]. On the other hand, another group of bacteria-derived metabolites exacerbate breast cancer growth. For example, queuine is a nucleobase mostly synthesized by certain pathogenic bacteria, such as Clostridioides difficile and Chlamydia trachomatis, to promote their virulence. Queuine is incorporated into specific transfer RNAs (tRNAs) which drive the expression of genes involved in cell proliferation and migration of breast cancer cells [179,180]. Furthermore, recent studies report anti-tumor effects of bacterial metabolite trimethylamine N-oxide (TMAO) produced by a group of commensal bacteria, such as Clostridia, Bifidobacteria, and Coriobacteria. TMAO could promote tumor cell killing activities of CD8+ T cells and M1-type macrophages. Analysis of clinical tumor samples found that TNBC tumors abundant in Clostridiales are enriched in TMAO and exhibit activated immune microenvironment [181].

9. Breast Tumor-Associated Bacteria

Different levels of bacterial metabolites in normal vs. cancerous breast tissues discussed above are largely attributed to differences in microbial compositions. Decreased ratios of Sphingomonas yanoikuyae to Methylobacterium radiotolerans in the breast tissues are linked to elevated breast cancer risks [106]. Lactobacillus, Staphylococcus, and Enterobacteriaceae are more abundant in tumor-adjacent normal breast tissues compared to healthy breast tissues, indicating their contributions to neoplastic processes [47]. These different tumor-associated bacteria play differential roles in the development of breast cancer. Pro- and ant-tumor roles of select commensal bacteria are discussed below.

9.1. Origin of Breast-Tumor Resident Bacteria

Breast tumor-resident bacteria are proposed to be derived from the similar origins as those in normal breast tissues, namely, breast skin, the oral cavity of the suckling infant, maternal gut through gut-breast axis, and maternal oral cavity through oro-breast axis [182]. However, how these bacteria have traveled to distant tumors remains largely unknown. Bacterial strains found in tumors are mostly present in the gut microbiome, supporting the possibility of gut-breast axis [183]. Oral microbiota is also one of the potential sources of breast tumor resident bacteria [184]. In particular, Fusobacterium nucleatum, a major human oral bacterium, is commonly found in breast tumor cells [30], while it is rarely found in the intestine and thus is expected to reach tumors through the circulatory system [185]. Furthermore, a study by Nejman, et al. showed that bacteria in tumor-adjacent normal breast tissues had intermediate compositions between those of breast tumors and normal tissues [26]. This indicates that there are bacterial transfers between neighboring tissues that result in heterogeneity within tumors.

9.2. Major Breast-Tumor Resident Bacterial Species

There are several major bacterial species frequently found in breast tumor samples.

9.2.1. Fusobacterium nucleatum

Fusobacterium nucleatum is a common opportunistic bacterium in the oral cavity and is a potential causative agent of periodontitis and oral carcinomas [186]. This bacterium is also elevated in various types of solid tumors, especially in colorectal tumors, compared to the matched healthy tissues [187]. It is also associated with liver metastasis, indicating the broad spread of this oral pathogen [75]. F. nucleatum localizes at tumor sites by attaching to the cell surface galactose-N-acetylgalactosamine (Gal-GalNAc) through its lectin Fap2 [29,174]. In particular, intravascularly injected Fap2-expressing F. nucleatum strain ATCC 23726 specifically colonizes mammary tumors in mice, whereas Fap2-deficient bacteria fail to do so. Furthermore, F. nucleatum secretes an amyloid-like filament FadA which not only helps the attachment and invasion of the bacterium to host cells [188], but also serves as the scaffold of biofilm formation and promotes cancer progression [189]. Within tumor cells F. nucleatum induces proinflammatory signaling through TNFα, NF-kB and IL-6/IL-8 pathways [190,191,192] and promotes tumor growth, epithelial-to-mesenchymal transition (EMT), metastasis, and therapy resistance, while also suppressing NK cell-mediated tumor cell killing and T cell infiltration into tumors [29,94].

9.2.2. Streptococcus

Streptococcus is an oral bacterium found to promote metastasis and colonization of metastasized breast cancer cells [74]. Streptococcus mutans is a gram positive bacterium associated with dental caries (cavities). This bacterium could invade endothelial cells through Toll-like receptor 2 that triggers the production of proinflammatory IL-6/IL-8 and monocyte chemoattractant protein-1 (MCP1). Inflamed endothelial cells elevate permeability of the blood vessel, leading to various systemic conditions. For example, intravenously injected S. mutans is shown to induce lung vascular inflammation (e.g., thrombosis) and promote breast cancer metastasis to the lungs [193,194]. In addition, S. cuniculiIn, originally isolated from the respiratory tract of wild animals [195], is shown to promote metastatic potential of tumor cells by reorganizing actin cytoskeletons to resist sheer stress during invasion [74]. In contrast, another strains of Streptococcus confer beneficial effects on breast cancer treatment. S. salivarius, an abundant probiotic bacterium found in breast milk, is shown to suppress breast cancer growth when applied ectopically. Similarly, S. pneumoniae, the bacterium responsible for pneumonia and lung cancer [196], produces endopeptidase O (PepO) virulence protein. Ectopic administration of PepO to mouse model of triple-negative breast cancer (TNBC) is shown to activate TLR2/4 in tumor-associated macrophages and suppresses breast tumor growth [197].

9.2.3. Staphylococcus, and Enterobacteriaceae

Staphylococcus and Enterobacteriaceae are intestinal bacteria that could induce DNA damage within host cells. Staphylococci produce a toxin alpha phenol-soluble modulins (PSMα) and specific lipoproteins (Lpls). PSMα could incur DNA damage, whereas Lpls dampen DNA damage repair signaling, compromising genomic integrity of the host cell [39]. Furthermore, S. aureus, the bacterium usually found in the upper respiratory tract and skin, lowers the immunogenicity of the TME by suppressing effector T cells and promoting regulatory T cells [198]. In addition, S. xylosus, a skin commensal, promotes metastatic potential of tumor cells by reorganizing actin cytoskeletons to resist fluid sheer stress (FSS) during invasion [74]. Enterobacteriaceae (e.g., E. coli and Salmonella) are mostly intestinal commensals, and systemic infection with these bacteria is a common complication in cancer patients [199]. Similar to Staphylococcus, Enterobacteriaceae, especially those harboring the polyketide synthase (pks) island, produce a genotoxin Colibactin that causes DNA double strand breaks [200]. Enterobacteriaceae also impairs the expression of p53 tumor suppressor upon DNA damage, contributing to genomic instability of the host cell [201]. Furthermore, Enterobacteriaceae-infected host tumor cells produce the bactericidal lysophosphatidylcholines, which is found to be elevated in breast tumors and promote tumor growth and metastasis [202,203].

9.3. Roles of Intracellular Microbes in Breast Tumor Initiation/Development

According to the International Agency for Research on Cancer (IARC), 18-20% of cancers are caused by biological carcinogens such as oncogenic viruses and bacteria [204]. The roles of these microbes in cancer initiation and development involve six major mechanisms: genome instability/mutation, epigenetic modification, chronic inflammation, immune evasion, metabolic regulation, and metastasis [205].

9.3.1. Genome Instability/Mutation

The induction of genomic instability and mutation is one of the major carcinogenic mechanisms of the microbes. Oncoviruses are one of the major breast tumor-causing agents, including human papilloma virus (HPV), mouse mammary tumor virus (MMTV), Epstein-Barr virus (EBV), and bovine leukemia virus (BLV) [206]. They integrate the viral genome into the host chromosome to induce genetic mutations, while oncoproteins are produced by the integrated viral genome. For example, the HPV E7 oncoprotein directly inhibits cGas-STING pathway involved in the expression of type I interferon and pro-inflammatory factors, leading to immune escape [207,208]. EBV LMP1 oncoprotein upregulates oncogenic signaling pathways, such as the NF-κB pathway, involved in cell proliferation [209], while MMTV oncovirus-infected cells escape apoptosis by activation of Src tyrosine kinase pathway [210].

Likewise, certain carcinogenic bacteria, such as pks+ Escherichia coli and Bacteroides fragilis, secrete carcinogenic toxins that induce DNA damage, which results in elevated tumor onset and mortality [211]. The toxic produced by Bacteroides fragilis also promotes the expression of the enzyme spermine oxidase producing reactive oxygen species (ROS) that causes DNA damage [212]. The oncobacterium Fusobacterium nucleatum secretes FadA, a key adhesin, that activates the E-cadherin/β-catenin pathway to upregulate checkpoint kinase 2 (CHK2), inducing DNA damage [213]. Fusobacterium nucleatum infection also downmodulates Ku70/p53 DNA damage repair pathway, exacerbating DNA double-strand breaks (DSBs) [214]. E. coli and Staphylococcus epidermis isolated from breast tumors could cause DSBs even in cervical cancer cells, demonstrating non-tissue-specific tumorigenicity [47]. Furthermore, H. pylori and E. coli expressing EspF effector protein could suppress DNA mismatch repair mechanisms, augmenting genome instability and tumorigenesis [211,215].

Bacterial metabolites could also induce DNA damage to promote tumor development. Breast tumor tissues contain the elevated levels β-glucuronidase, a carcinogenic enzyme [125,216], that generates reactive intermediates from 2-amino-3-methylimidazo [4,5-f]quinoline to induce DNA damage [217]. Furthermore, Streptococcus anginosus and Porphyromonas gingivalis can convert ethanol to acetaldehyde that could form DNA adducts or inhibit DNA repair enzymes, causing DNA damage [218,219].

9.3.2. Tumor Metastasis

Over the past decade, it has been unveiled that intratumoral bacteria play critical roles in tumor metastasis. The initial study by Bullman et al. reported that primary colorectal tumors and their metastases shared the same viable bacterial components and that these bacteria were able to promote tumor cell growth and survival [75]. Furthermore, recent studies demonstrated that these tumor-resident bacteria are in fact localized in the cytosol of tumor cells and transported to the metastatic sites by tumor cells. During tumor cell metastasis, the intracellular bacteria promote tumor cell survival by allowing them to overcome physical and biochemical hurdles of unfavorable environment through adaptations termed pro-metastatic processes [67,74,220]. During pro-metastatic adaptations, tumor cells acquire capabilities of breaking tissue boundaries, controlling the local environment, conferring immune suppression and resistance to mechanical stress, and remodulating tumor cell intrinsic properties, such as epithelial-to-mesenchymal transition (EMT), stemness, and adhesion (Figure 3) [220]. Heer are some examples of intratumoral bacteria playing role in pro-metastasis and metastasis of tumor cells.

Food bacteria Listeria, in particular, Listeria monocytogenes, is an intracellular pathogen found in decaying food [221]. L. monocytogenes has been long utilized to develop cancer vaccines because it induces potent innate and adaptive immunological responses [222]. However, this bacterium could also reside within breast tumor cells and promote the growth and metastasis of tumor cells, worsening prognosis of patients [223,224]. Intratumoral L. monocytogenes induces cytoskeletal reorganization of tumor cells through its actin nucleation protein ActA and promotes tumor cell survival under FSS in the circulation [224]. Furthermore, such actin nucleation also recruits the ubiquitin conjugating enzyme Ube2N that activates TAK1-p38 MAP kinase signaling that controls tumor cell metastasis [225,226]. Not only the pathogenic strain, but also a non-pathogenic strain L. fleischmannii resides within breast tumors and is strongly associated with elevated expression of EMT-associated genes [83].

Another tumor metastasis-associated bacterium is Fusobacterium nucleatum, an opportunistic bacterium usually found in the oral cavity, but also abundant in breast tumors [29]. Intratumoral F. nucleatum promotes tumor cell invasion and suppresses immunological response through several different mechanisms [29]. First, F. nucleatum produces a virulence factor FadA, an amyloid protein that helps binding of the pathogen to host cells [227]. FadA upregulates Mir4435-2HG, which then induces the expression of SNAIL1 triggering EMT of host cells [94]. Second, F. nucleatum elevates the expression of MMP-9 that degrades extracellular matrix to assist tumor cell invasion. Third, F. nucleatum upregulates the expression of an adhesion molecule ICAM1 through the ALPK1/NF-κB axis that promotes tumor cell adhesion to endothelial cells during intravasation [228]. Fourth, F. nucleatum induces the production of extracellular vesicles that promote the expression of TLR4 in neighboring tumor cells to help their growth and metastasis [229]. Fifth, F. nucleatum elevates the expression of immune checkpoint receptors, TIGIT and CEACAM1, that suppress immunological responses. Lastly, F. nucleatum directly invades and kills tumor-infiltrating lymphocytes, including NK cells and T cells [29].

The third tumor metastasis-associated bacterium is Bacteroides fragilis, an abundant commensal bacterium in the colon and breast tumors. B. fragilis produces an enterotoxin, B. fragilis toxin (BFT), a zinc-dependent metalloprotease commonly associated with inflammatory colon diseases. Intratumorally produced BFT could elevate the growth and metastatic potentials of breast tumor cells by inducing the expression of stem cell/EMT-associated genes such as Slug and Twist [108,230,231]. As the mechanisms of this phenomenon, it is shown that BFT induces the cleavage of E-cadherin on the surface of tumor cells, which then triggers nuclear localization of β-catenin and Notch effector NICD. Activation of both the Wnt and Notch signaling pathways greatly promotes stemness and metastasis of tumors [232].

Furthermore, Staphylococcus and Lactibacillus, commensal bacteria abundant in breast tumor cells, have been shown to translocate to the lungs along with metastasizing tumor cells. These intracellular bacteria inhibit RhoA/ROCK-induced contractility of tumor cells while being exposed to FSS, conferring protection against mechanical force-induced apoptosis of tumor cells during metastasis [74]. As a potential mechanism, the same group proposed the possible involvement of ADP-ribosyltransferase C3 exoenzyme produced by these bacteria. This enzyme inhibits Rho GTPases to counteract immune cell activities and is well studied as their virulence factor of select bacterial species, such as Staphylococcus and Bacilus [233,234].

10. Discussion

Cumulative evidence unveils that intratumoral microbes are not only the mere biomarkers for breast cancer phenotype and prognosis, but also the causes of breast cancer initiation and metastasis. Such roles of intratumoral microbes suggest that they could serve as potential targets for breast cancer treatment and prevention. Thus, the efficacy of the use antibiotic(s) in combination with chemotherapy has been tested for breast cancer treatment. However, these antibiotics are reported to show both positive and negative effects, depending on whether they target tumor-resident bacteria or intestinal microbes [235]. For example, a Phase II study of combining Moxifloxacin, a fourth-generation quinolone with broad-spectrum coverage of breast tumor-resident bacteria, with treatment of physician’s choice (TPC: capecitabine, eribulin, gemcitabine, paclitaxel, or nab-paclitaxel) reported a promising efficacy and well-tolerated toxicities in patients with metastatic breast cancer [236]. On the other hand, a retrospective study on 772 women with triple-negative breast cancer treated with antimicrobials along with standard cytotoxic chemotherapy found that these patients had overall poorer survivals than those without antimicrobials [237]. Such deleterious effects of antimicrobial use for breast cancer patients are largely due to intestinal microbiota disorder that impairs immune function and triggers a systemic inflammatory response [235]. One possible solution to such corundum is to repopulate beneficial bacterial flora by supplementing probiotics and prebiotics to cancer patients treated with or without antibiotics. Furthermore, more recent strategies include fecal matter transplant of health cohorts to patients with breast cancer resistant to standard chemotherapy. Therapeutic manipulation of tumor microbiome is an emerging research field which would revolutionize cancer therapy in the near future.

11. Conclusion

In summary, recent studies have unveiled that intratumoral microbes play critical roles in breast cancer development and metastasis and serve as potential targets for breast cancer treatment and prevention.