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IY35CH08-Trinchieri ARI 18 January 2017 13:30
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Microbes and Cancer∗
Amiran Dzutsev,1,2 Jonathan H. Badger,1
Ernesto Perez-Chanona,1Soumen Roy,1
Rosalba Salcedo,1Carolyne K. Smith,1
and Giorgio Trinchieri1
1Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892, email: trinchig@mail.nih.gov
2Leidos Biomedical Research, Frederick, Maryland 21702
Annu. Rev. Immunol. 2017. 35:199–228
The Annual Review of Immunology is online at
immunol.annualreviews.org
This article’s doi:
10.1146/annurev-immunol-051116-052133
∗This is a work of the U.S. Government and is not
subject to copyright protection in the United
States.
Keywords
microbiota, cancer, carcinogenesis, metabolism and cancer, cachexia,
chemotherapy, immunotherapy
Abstract
Commensal microorganisms (the microbiota) live on all the surface barriers
of our body and are particularly abundant and diverse in the distal gut. The
microbiota and its larger host represent a metaorganism in which the cross
talk between microbes and host cells is necessary for health, survival, and
regulation of physiological functions locally, at the barrier level, and sys-
temically. The ancestral molecular and cellular mechanisms stemming from
the earliest interactions between prokaryotes and eukaryotes have evolved to
mediate microbe-dependent host physiology and tissue homeostasis, includ-
ing innate and adaptive resistance to infections and tissue damage. Mostly
because of its effects on metabolism, cellular proliferation, inflammation,
and immunity, the microbiota regulates cancer at the level of predisposing
conditions, initiation, genetic instability, susceptibility to host immune re-
sponse, progression, comorbidity, and response to therapy. Here, we review
the mechanisms underlying the interaction of the microbiota with cancer and
the evidence suggesting that the microbiota could be targeted to improve
therapy while attenuating adverse reactions.
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INTRODUCTION
Microbial communities have been living on the surface of the Earth for three-fourths of its his-
tory. They have developed a diversity of specialized lineages to adapt to different habitats and have
been instrumental in shaping the evolution of modern life (1). When eukaryotes and multicellular
organisms appeared over a billion years ago, their close interaction with microbes necessitated
their coevolution, establishing persistent relationships including mutualism, commensalism, par-
asitism and a dependency on each other for survival and control of homeostasis; this history is
clearly written in the genomes of eukaryotic hosts and associated microbiota. The phagocytic ca-
pability of eukaryotic cells was preserved from the most primitive organisms to the highest forms
of animal life and was required for both nutrition and defense against pathogens. Phagocytosis
was probably the key trait that eventually gave rise to eukaryotes, by enabling the acquisition
of bacterial endosymbionts that evolved into mitochondria (2, 3). Eventually, mobile phagocytic
cells in higher organisms developed as specialized cell types of the myeloid lineage (granulocytes,
monocytes, macrophages, dendritic cells) that play a central role in wound repair, innate resistance
to infections, and promotion of adaptive immunity (4, 5). Commensal microorganisms, including
eubacteria, archaea, protists, fungi, and viruses, inhabit all the epithelial barrier surfaces of our
body, where bacteria, in particular, are as numerous as human cells (6–8). The unique microbial
genes in our body outnumber human genes by a factor of at least 100, although many microbial
genes are functionally redundant (6). Thus, we are symbionts or metaorganisms composed of host
and microbial cells (9–11). The microbial genome is an integral part of the metaorganism genetic
framework (metagenome), and the wealth of metabolic processes and products (metabolome) it en-
codes profoundly influence all the physiological functions of the body and alter its pathology. Both
microbial and human cells act as sensors for chemical, physical, and biological environmental clues.
Through reciprocal communications they detect homeostatic changes and modify their compo-
sition and/or function accordingly (12). The signaling pathways by which human and microbial
cells in the metaorganism communicate involve small molecule metabolites, nucleic acids, and
physical protein-protein interactions. This cross talk includes molecular and cellular mechanisms
that are also key to innate resistance mediated by myeloid/phagocytic cell (5). Both commensal
microbiota and many pathogens produce “danger molecules” recognized by the immune system
to elicit a reaction. However, tolerance to microbiota is maintained, despite a substantial translo-
cation of microbial products and cells in a steady state, until pathogenic microorganisms breach
the epithelial barriers and then an immune response is mounted (13, 14).
METHODS OF MICROBIAL COMPOSITION IDENTIFICATION
AND FUNCTIONAL ANALYSIS
Elucidation of the interaction between the host and commensal microbiota has been hampered,
until recently, by the fact that only a small fraction of microbial cells could be isolated, cultured in
vitro, and analyzed (15). Although the leading laboratories have made great progress in culturing
previously uncultivable bacterial species (16), the real revolution in microbiota studies over the last
few years has been due to the widespread availability of next-generation sequencing technology.
This has allowed many investigators to analyze the composition of the commensal microbiota in
an unbiased way by sequencing all or part of their genome. The standard method of identifying
bacterial and archaeal taxa in the microbiota is to sequence one or more variable regions of
the gene encoding 16S rRNA (17), although the optimal region may vary by body site. Fungal
composition analysis relies on sequencing of the internal transcribed spacer (ITS) region between
the 18S and 28S rRNAs (18). Analysis of the protist fraction of the microbiota is in its early
stages, but 18S rRNA (which also has variable regions, as in 16S) appears to be the preferred
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method (19). Alternatively, the microbial metagenome can be sequenced using standard shotgun
sequencing protocols. A further degree of information is available with metatranscriptomic data,
where not only the functional potential of the metagenome is revealed, but also the genes that are
actively expressed. Both metatranscriptomic and metagenomic information could be used to infer
relationships with host transcriptomes or proteomes by building trans-kingdom networks (20).
MICROBIAL CONTRIBUTION TO HOST HOMEOSTASIS
In the metaorganism, cross talk between the commensal microbes and the host is essential for main-
tenance of physiological homeostasis, response to environmental changes and survival. Evidence
accumulating in the last few years suggests that the composition of the microbiota at the epithelial
barrier affects systemic functions, including metabolism, energy balance, central nervous system
physiology including cognitive functions, cardiovascular functions, nutrition, circadian rhythm,
inflammation, innate resistance, and adaptive immunity (21–23). Microbes reside along the gas-
trointestinal tract, with the largest microbial population in the colon, ranging between 1013 to
1014 bacteria (24). Approximately 1012 bacteria live on the skin in various communities depending
on factors such as sebum or dryness of the environment (25). The composition of the microbiota
at various anatomical sites is controlled by host genetics, particularly by the polymorphisms in
immune-related genes, as well as by environmental factors, such as lifestyle and nutrition (26).
The initial composition of a newborn’s microbiota largely resembles either the vaginal microbiota
or, if the method of delivery was cesarean section, the skin and environmental microbiota (27).
The gut microbiota matures during the first three years of life. Concurrently, large changes in the
expression of genes related to vitamin biosynthesis and metabolism are also observed. Through-
out adulthood, the microbiota is relatively stable, although disease onset, use of antibiotics, and
changes in alimentation affect its composition (28). In individuals older than 60–70 years, the com-
position of the microbiota gradually changes; a striking negative association is observed between
frailty in elderly individuals and microbial diversity (29, 30).
Many of the effects of the microbiota occur at local barrier sites. For example, in the gut,
the microbiota modulates nutrient absorption, synthesis of vitamins, metabolism of bile and hor-
mones, and fermentation of carbohydrates. In addition, it contributes to barrier fortification and
the establishment of mucosal immunity (22, 31, 32). However, the microbiota also exerts systemic
effects, including modulation of metabolism, inflammation, and immunity (32). The microbiota
trains myeloid and lymphoid cells by providing instructive signals to regulate epigenetic mod-
ifications that calibrate the immune response to inflammatory stimuli, infections, vaccines, and
autoimmunity (33–36). In addition to the gut mucosa, commensals at other epithelial barriers
such as the skin also control local immune homeostasis, protective responses, and tissue pathology
(32). A compartmentalized control of immunity by the skin microbiota has been clearly defined
(37). Although many of the cellular and molecular mechanisms mediated by vicinal microbiota
on barrier homeostasis have been identified, the exact method of systemic influence is yet to be
determined. Traditionally the epithelial barriers and their immune system have been considered
to prevent in healthy organisms the translocation of bacteria into internal tissues. However, it is
now becoming evident that bacteria can penetrate the gut mucosa and skin, diffuse to the draining
lymph nodes, and disseminate systemically with protective rather than pathogenic effects (13, 38,
39). This translocation is amplified in conditions of immune deficiency or during diseases that
compromise the integrity of the epithelium such as colitis or atopic dermatitis (38, 39). Other
mechanisms also contribute to the systemic effects conferred by the microbiota: diffusion of bac-
terial products, host growth factors, cytokines, chemokine-induced migration of barrier immune
cells, and release of vesicles or exosomes from bacteria or immune cells.
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The effect of the microbiota on innate resistance and immunity is, in part, dependent on its effect
on hematopoiesis. Germfree mice are deficient in bone marrow–derived peripheral myeloid pop-
ulations (40). Recolonization of these mice with microbes provides cues from microbe-associated
molecular patterns (MAMPs) and short-chain fatty acids (SCFAs) to promote hematopoietic ho-
meostasis by activating innate immune receptors (40–42). The maturation of the yolk sac–derived
CNS microglia and their ability to respond to infections was also defective in germ-free mice. This
deficiency was similarly reproduced by treating conventionally raised mice with antibiotics, show-
ing that a complex microbiota is required to maintain the functions of tissue-resident myeloid cells
(43). The functional maturation of circulating neutrophils and their ability to form extracellular
traps is also regulated by the microbiota (44). Diurnal fluctuations of circulating inflammatory
monocytes are regulated by synchronous oscillations of gut microbial composition and through
the level of expression of Toll-like receptors (TLRs) on gut epithelial cells, which control the
expression of the circadian gene Bmal1 (45, 46). Alteration of the circadian clock (e.g., due to jet
lag) induces pathological alteration to the microbiota, termed dysbiosis, promoting a metabolic
syndrome that is transferable by fecal transplantation to germfree mice (47).
Bacterial products can induce expression of type II interferon (IFN-γ), which is known to
affect neutrophil survival and activation (48, 49). This suggests that activated barrier myeloid
cells, among others, can reenter circulation and contribute to systemic microbiota effects (50).
Interleukin-12 (IL-12) produced during intestinal Toxoplasma gondii infection affects monocyte
precursor differentiation in the bone marrow by inducing IFN-γproduction by natural killer
(NK) cells (51). Low concentrations of MAMPs, such as lipopolysaccharide, can also change
myeloid cell activation thresholds for an enhanced inflammatory response, also known as priming
(52). These mechanisms likely contribute to the microbiota’s influence on the responsiveness
of peripheral and tumor-infiltrating myeloid cells (34, 53) and to the generalized Shwartzman
reaction, which also depends on the priming of myeloid cells by low doses of lipopolysaccharide
and IL-12 (54).
MICROBIOTA ASSOCIATION WITH CANCER
Although tumors grow locally before invading other tissues or metastasizing to other parts of the
organism, cancer should be considered a systemic disease affected by and altering the homeostatic
mechanisms that control the physiology of the metaorganism. The growth characteristics of the
tumor cells are dependent on the genetic alterations resulting in the activation of oncogenes, the
silencing of tumor suppressor genes, and other classical hallmarks of cancer (55). However, it is
becoming evident that transformed cells cannot effectively grow without a suitable microenviron-
ment (56). The microbiota, with its ability to modulate the metaorganism’s physiology, contributes
to the establishment of a pro- or antitumor inflammatory milieu (Figure 1). Two findings exem-
plify the requirement of a suitable microenvironment for tumor growth. Rous sarcoma virus, the
first-discovered oncovirus, induces tumors in adult birds at the site of injection or injury, but not
in sterile embryos. This is true even if the infected cells in the embryo express the v-Src oncogene
and show a transformed phenotype in vitro (57). The other example is the finding that more than
one-quarter of the skin cells in aged, sun-exposed eyelid carry clonally expressed cancer-causing
driver mutations similar to those found in squamous cell carcinoma, yet they maintain the physi-
ological differentiation and functions of normal skin without evolving into cancer (58). Although
evidence for the impact of the skin microbiota on the malignant progression of keratinocyte car-
cinomas is still lacking, normal tissue homeostasis and architecture are known to restrain cancer
so that changes in the microenvironment are required for tumor progression (59). Indeed, the
microbiota affects tissue metabolism as well as the differentiation and function of immune cells
202 Dzutsev et al.
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Comorbidity
Cachexia Toxic side eects
of therapy
Antibiotics
Xenobiotics
Probiotics,
prebiotics,
and symbiotics
Lifestyle
Host genetics
Fecal transplantation
Microbiota
Dysbiosis
Diet
Cancer
initiation and progression
Immunosuppression
Inammation
Genotoxicity
Anticancer therapy
Chemotherapy
Immunotherapy
Cell-based
therapy
Radiation
therapy
Lifetime
exposure to
commensals
Metabolic
disorder
Dened microbiota
transfer
Myeloid cells,
innate lymphocytes,
T cells, and B cells
Myeloid cells,
innate lymphocytes,
T cells, and B cells
Myeloid cells,
innate lymphocytes,
T cells, and B cells
Figure 1
Microbiota-dependent regulation of cancer development, progression and treatment. The microbiota plays a
dynamic role, from maintenance of healthy host physiology to disease development. Physiological factors
(blue) such as lifetime exposure to commensals, diet, lifestyle, and host genetics shape the composition of the
microbiota in each individual, which affects response to disease and therapy. Under pathogenic conditions,
changes in microbiota composition (dysbiosis; yellow) may contribute to disease. The microbiota regulates
cancer initiation and progression, comorbidity, and anticancer therapy in part by priming myeloid cells and
(directly or indirectly) lymphoid cells that mediate innate resistance and adaptive immunity. Treatments
targeting microbiota composition ( green), such as probiotics, prebiotics, symbiotics, antibiotics, xenobiotics,
transplantation of fecal or defined microbiota, have potential to modulate cancer progression, cancer-
associated comorbidity, response to therapy, and adverse reactions.
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in the tumor microenvironment, which may foster skin carcinogenesis. In turn, host genetics and
environmental factors may also affect the tumor microenvironment by modifying the composition
and diversity of the microbiota (12, 26). The incidence of keratinocyte cancer and cancer at other
barrier surfaces exposed to the microbiota is elevated in immunosuppressed recipients of organ
transplants (60), likely owing to changes in the microbiota composition at these sites and defective
tumor immunosurveillance.
Epidemiological cancer studies based on the analysis of oral, fecal, and tissue samples to evaluate
the role of the microbiota and dysbiosis have mostly been limited to gastrointestinal and lung
carcinoma (61). These studies have confirmed the role in stomach cancer of Helicobacter pylori,
the only bacterial species recognized as a group 1 human carcinogen by the International Agency
for Research on Cancer (IARC) (62, 63). These studies also identified other emerging candidate
oncogenic bacteria, such as Fusobacterium, enterotoxigenic Bacteroides fragilis (ETBF), and pks+
strains of Escherichia coli (64–67). Traditional epidemiological data showing an association of the
presence of certain species with cancer incidence provide important clues but are in many cases
difficult to interpret because evidence of causality is often lacking. The presence of dysbiosis or
shifts in the abundance of specific microbes may, in fact, be a consequence rather than a cause of
cancer. The microorganisms responsible for cancer initiation may no longer be present when the
patients are analyzed, and dysbiosis may cause homeostatic alterations or epigenetic effects that
contribute to cell transformation and tumor promotion (68, 69). Definitive evidence for the role of
particular species in cancer pathogenesis will come from more molecular-based epidemiological
studies as well as identification of the mechanisms involved in both clinical studies and experimental
animal studies.
ROLE OF THE MICROBIOTA IN DIET- AND OBESITY-ASSOCIATED
CANCER AND CANCER COMORBIDITY
High body mass index (BMI) is a risk factor at the population level for diabetes, cardiovascular
disease, and many common cancers, including colon, liver, gallbladder, postmenopausal breast,
uterus, and kidney cancers (70). High BMI is associated with the pathological inflammation respon-
sible for insulin resistance and altered energy metabolism (71). Colon cancer and gut dysbiosis,
too, are linked to obesity. High-fat diet (HFD) increases tumorigenicity of intestinal cell pre-
cursors by activating peroxisome proliferator-activated receptor delta (72). In hepatocarcinoma,
carcinogenesis is associated with Clostridium cluster XI– and cluster XIVa–induced production of
the secondary bile acid product deoxycholic acid, which induces DNA damage and senescence
in hepatic stellate cells (73, 74). Although it has been estimated for over 30 years that one-third
of all cancers and the majority of gastrointestinal cancers may be due to dietary factors (75),
the epidemiological evidence associating HFD with cancer is modest, unlike the experimental
data demonstrating that HFD causes cardiovascular disease and diabetes (74, 76). Conversely,
high-fiber diets increase the microbe-driven metabolism of SCFAs, such as butyrate, which have
anti-inflammatory properties. In addition, they may protect against gastrointestinal cancers and
lymphoma through the modulation of epigenetic changes by inhibiting histone deacetylases (74,
77). Exposure of female mice to HFD during pregnancy induced obesogenic and diabetogenic
traits in two generations of offspring and was associated with the development of lung and liver
cancers (69, 78). This phenotype was linked to epigenetic changes in genes encoding adiponectin
and leptin, and administration of a normal diet abolished it in three generations. Cancer suscep-
tibility was also induced in recipients of the microbiota from HFD-fed females but was reversed
by treatment with the beneficial probiotic species Lactobacillus reuteri (69, 78). The use of antibi-
otics can persistently reduce bacterial diversity, deplete protective niches that otherwise prevent
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overgrowth of pathogens and pathobionts, and induce gut dysbiosis that may increase the risk of
disease (79, 80). The repeated use of antibiotics in young children resulted in an increased risk of
childhood obesity (81) and up to sevenfold increase in the risk of developing inflammatory bowel
disease (82); thus, it may also increase the risk of colitis-associated cancer. In breast cancer, a
meta-analysis of five case-control studies showed that antibiotic use was associated with a slightly
elevated risk of breast cancer, though this association is controversial (83). Mammary tissue is not
sterile and as the bacterial composition changes in the presence of cancer, the local microbiota
modulates antitumor immunity in the tumor microenvironment (84). The long-term effects of
antibiotics and diets such as HFD and high fiber diet, however, alter the microbial composition
and confound the identification of specific cancer-causing microbes.
Exposure to microbes during natural birth may help the newborn establish a proinflammatory
environment that favors tumor immunosurveillance (85, 86). Data from murine studies have
provided insight into the balance of microbial exposure and immune cell activity. One study
showed that acute intestinal infection induced intolerance to commensals because of dysbiosis-
driven alterations in mucosal integrity and immunity (14, 87). However, this resulted in the
accumulation of Th1-biased, microbe-specific memory T cells that also facilitated protective
responses to subsequent infections and tumor growth (14).
Whereas obesity may initiate certain cancers, anorexia-cachexia syndrome is a cancer-associated
comorbidity observed in a large majority of patients with late-stage, advanced cancer. This wast-
ing of muscle and adipose tissue is also triggered by infection, kidney and intestinal damage,
and chemotherapy (88). It dramatically affects quality of life, decreases the efficacy and tolerabil-
ity of anticancer treatments, and is the predominant cause of cancer-associated comorbidity and
mortality (88). Cancer-associated cachexia is linked to systemic inflammation and characterized
by elevated levels of chemokines and cytokines such as IL-1βand IL-6 (89). Cancer cells in-
duce cachexia through the secretion of proinflammatory cytokines and factors such as parathyroid
hormone–related protein (90). Different microbial species dominate in patients with metabolic
dysfunctions: the archaeon Methanobrevibacter smithii in patients with anorexia nervosa and Lacto-
bacillus spp. in the obese patients (91). Indeed, mice treated with the probiotic L. reuteri, prebiotics,
or both combined (symbiotics) were protected against cancer-associated cachexia, likely because
of enhanced anti-inflammatory, mucosa-protective effects (92, 93). In experimental models of in-
testinal damage or of infection-induced cachexia, E. coli O21:H+, present in the gut, protected
against cachexia (94). E. coli colonized white adipose tissue, where it activated the NLRC4 inflam-
masome and induced production of insulin-like growth factor 1, a hormone that prevents muscle
degradation (94). Changes in the microbial composition of the gut may be a cause or consequence
of cancer cachexia. Regardless, multimodal strategies, including the use of beneficial microbes,
aimed at fortifying barrier function may help reduce the incidence of cachexia (95). Therefore,
further investigation using experimental models may shed light on the role of the gut microbiota
in cancer anorexia/cachexia.
HUMAN CARCINOGENIC MICROORGANISMS
Infection-induced cancer accounts for approximately 16% of the global burden of all human can-
cers, corresponding to approximately 2 million new cases per year (96). The frequency varies by
region, with lower percentages, on average, in more developed countries (7.4%) compared to less
developed countries (22.9%) (96). The IARC classifies 10 microbial agents (7 viruses, 2 parasites,
and 1 bacterium) as group 1 human carcinogens; i.e., their status as carcinogens is based on either
strong evidence in humans, or limited evidence in humans supported by strong evidence in ani-
mals (97). H. pylori, hepatitis B and C viruses (HBV, HCV), and human papillomaviruses (HPV)
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together are responsible for over 90% of all infection-attributed cancers (96). The herpesvirus
Epstein-Barr virus (EBV) is the causative agent of Burkitt lymphoma, nasopharyngeal carcinoma,
and a subset of gastric carcinomas, whereas the Kaposi sarcoma–associated herpesvirus (HSV8)
causes Kaposi sarcoma and other pathologies in immunosuppressed or elderly individuals (98).
HBV and HCV are associated with hepatocellular carcinoma (HCC) (99). High-risk oncogenic
strains of HPV (HPV16, HPV18, and 11 others) are associated with anogenital cancers, a subset
of head and neck cancers, and skin cancers (100, 101). Human T cell lymphotropic virus type 1
is associated with the T cell lymphomas prevalent in certain geographical regions (102). Merkel
cell polyomavirus is the first human virus discovered by metagenomic sequencing and is associ-
ated with the majority of cases of Merkel cell carcinoma, an aggressive skin cancer observed in
immunosuppressed individuals (103).
With the exception of HCV, all human oncogenic viruses encode at least one oncogene and
may directly induce cell transformation (104). However, factors such as infection-associated geni-
tal tract inflammation and vaginal dysbiosis likely play a role in cancer progression even for viruses
such as HPV that have a potent transforming ability (105). HBV and HCV establish chronic liver
infection and account for over 80% of HCCs (106). In some patients, the immune response to
chronic viral infection progresses to fibrosis and cirrhosis and ultimately HCC. Whereas the initial
innate response to HBV infection is modest, HCV actively evades innate responses by inhibiting
both the production and the signaling of type I and type III interferon (106, 107). Unlike HCV,
HBV may directly transform hepatocytes. However, for both viruses, the pathogenesis of HCC
is dependent on immune-related inflammation (106). A higher degree of chronic HBV and liver
pathology correlates with greater gastrointestinal richness of Candida spp. and Saccharomyces cere-
visiae and with a less abundance and diversity of Bifidobacterium spp. (108, 109). The role of the
gut microbiota in regulating liver pathology and progression to HCC in mice has been clearly
documented (110). Interestingly, young mice, similarly to neonates or young children, fail to
clear HBV infection in a hydrodynamic transfection model until an adult-like gut microbiota is
established (111). Oncogene-carrying viruses (HPVs and HBV) require inflammation to promote
tumorigenesis, but this may indeed apply to all oncogenic viruses (112), including the previously
mentioned Rous sarcoma virus (57). As the limited data hitherto published suggest that the com-
mensal microbiota may be involved in regulating the response to infection and progression to
cancer, these represent possible targets for preventive and therapeutic interventions for cancer.
Gastric infection with H. pylori is strongly associated with noncardiac gastric carcinoma and
lymphoma (62). Yet, approximately half of the world population is infected with H. pylori,and
in most cases this infection only develops into well-tolerated gastritis. Only rarely does H. pylori
infection progress to serious lesions such as atrophy, metaplasia, and cancer (62). The virulence and
carcinogenicity of various strains of H. pylori have been associated with the variable expression of
two cytotoxin-encoding genes: cytotoxin-associated gene A (cagA) and vacuolating cytotoxin gene
A(vacA) (113). H. pylori infection, before any damage to the gastric mucosa occurs, cooperates with
the gut microbiota to control energy homeostasis by affecting circulating metabolic gut hormones
(114). H. pylori infection has profound effects on the host immune response. It activates the TLR4
and TLR2 receptors as well as the NLRP3 inflammasome, thus inducing the secretion of both
IL-1βand IL-18. This promotes the activation of both Th1 cell and regulatory T cell (Treg)
responses to protect against asthma, chronic inflammatory diseases, and tuberculosis (115, 116).
Whereas H. pylori directly affects gastric epithelial cells and can compromise genetic integrity by
inducing DNA damage, gastric carcinogenesis requires exposure to the bacterium over multiple
decades, with an initial inflammatory response, epithelium injury and atrophy, reduction in acid
secretory functions, and intestinal metaplasia (117, 118). Often, H. pylori is no longer detectable in
the stomach of seropositive individuals with atrophic body gastritis, suggesting that deterioration
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of the gastric niche and the lowering of acidity due to long-term H. pylori infection cause gastric
dysbiosis dominated by the presence of cancer-provoking species of oropharyngeal or intestinal
origin (119). In developed countries, the incidence of H. pylori infection is decreasing owing to
frequent use of antibiotics, improved hygiene, and eradication protocols using broad-spectrum
antibiotics and proton pump inhibitors (120). Although this decrease correlates with a lower
incidence of gastric inflammation and carcinogenesis, the eradication protocols cause perturbation
of the gut microbiota with possible side effects (120). Also, the absence of H. pylori may remove some
of the beneficial effects of the infection, thereby increasing susceptibility to asthma and obesity
as well as gastroesophageal reflux with increased risk of esophageal and gastric cardia carcinoma
(63). This hypothesis, however, has been challenged by the observation that the incidence of
these pathologies is not increased in certain ethnic Malaysian populations that have a low natural
incidence of H. pylori infection and generally poor sanitation (85). Thus, these findings may suggest
that the relationship between the absence of H. pylori infection and the increased incidence of these
pathologies is not universal and that, in some populations, H. pylori infection may be a marker of
poor hygiene that has a protective effect on asthma, obesity, and esophageal carcinoma (85).
MICROBIOTA AND CANCER AT THE EPITHELIAL BARRIERS
At the epithelial barrier surfaces, the primary contact point between host commensals, the com-
position of the microbiota or the abundance of particular species affects both inflammation and
immunity, as well as the homeostasis of epithelial and stromal cells (22, 121). Although this cross
talk occurs at all barriers (32), it is particularly evident at the level of the lower gastrointesti-
nal tract, where the most bacteria are present (24) (Figure 2). Direct interactions of bacterial
structural components and their metabolites, for example, hydrogen sulfide and p-cresol, with
epithelial, stromal, and hematopoietic cells may have direct genotoxic effects and promote cancer
progression (122–126).
Mice deficient in genes controlling host-microbe cross talk have been extensively used for
studying the mechanisms by which the microbiota affects cancer. In particular, mice deficient in
immunologically relevant genes, such as Tlr5,Il10,Tbx1,andRag2, are not only susceptible to
colitis and colon carcinogenesis, but are also characterized by gut dysbiosis. This susceptibility to
cancer can be transferred to healthy mice by cohousing, fostering, or fecal transplant (65, 127,
128). These findings are relevant to humans because polymorphisms in immunologically relevant
genes affect human microbiota composition and cancer predisposition (26).
Clinical and epidemiological investigations as well as experimental studies in animal models
have already identified bacterial species putatively involved in carcinogenesis on the basis of either
their physical association with the neoplastic lesions or a positive correlation of their abundance
with cancer risk. Mechanisms of bacteria-mediated carcinogenesis have been well characterized
in mice using several microbial species, including Enterococcus faecalis,Streptococcus gallolyticus,en-
teropathogenic E. coli,ETBF,Helicobacter hepaticus,Salmonella enterica,andFusobacterium nucleatum
(64, 65, 129). The bacterial genera Odoribacter and Akkermansia are reportedly enriched in colon
cancer–bearing mice (130), and archaea, such as the order Methanobacteriales, have been found
in the fecal microbiota of patients with colorectal cancer (131).
Fusobacterium spp. are anaerobic, gram-negative bacteria that usually reside in the oropharynx,
where they are involved in oral pathology and participate in the formation of dental biofilms
(132). However, they are also found in the inflamed colon and are particularly enriched in human
colonic adenomas and adenocarcinomas (64, 129). The latter observation may be explained by
the binding of the F. nucleatum protein Fap2 to the carbohydrate moiety Gal-GalNac that is
overexpressed on colonic tumor cells (133). F. nucleatum is immunosuppressive, owing to its
www.annualreviews.org •Microbes and Cancer 207
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ability to recruit tumor-promoting myeloid cells to the intestine of APCMin/+mice and to inhibit
human NK and T cell activity via binding of its Fap2 protein to the TIGIT inhibitory receptor
(64, 134). It also activates β-catenin/Wnt signaling in epithelial cells by the association of its FadA
adhesin to E-cadherin (129). The level of expression of the gene encoding FadA is much higher in
colon tissues of patients with colon neoplasia compared to those of healthy individuals, suggesting
FadA as a potential diagnostic and therapeutic target (129). Fusobacteria have also been identified
IL-22
K
+
K
+
FadA
Fap2
GPR43
GPR109a
Il6, Tnf
pro-IL-18
IL-18
Macrophage
IL-17
Th17 ILC3
CTL
TIGIT
NK
IL-12
TNF IL-23
IL-22BP
SCFAs
VacA
CagA
Colibactin
ROS
IL-6
IL-11
RTK
PI3K/ AKT
MAPK/ ERK
E2F TCF
FadA
FadA
Ca
++
Il18
Treg
Chemokines
NF-ΚB
pSTAT 3
NF-ΚB
pSTAT 3
B-Cat
DCs
Neutrophils
H. pylori
S. enterica
E. coli
H. hepaticus
B. fragilis
Fusobacterium
NLRP6
NLRP3
Recruitment
of myeloid
suppressor cells
E-cadherin
E-cadherin
Xenobiotics
p-cresol
Deoxycholic acid
Clostridium cluster XIVa
Clostridium cluster XIVa
MAMPs
PRRs
MAMPs
PRRs
SCFAs
p53
MSH2
MLH1
ZRANB3
β-Catenin
β-Catenin
CDTH2S
Cag A
AvrA
BFT
208 Dzutsev et al.
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in the biofilms observed in carcinomas of the ascending colon and the tumor-free mucosa of
the same patients (135). The biofilms show upregulation of the polyamine metabolite N1,N12-
diacetylspermine, which is likely responsible for the observed enhanced epithelial proliferation,
diminished E-cadherin, and activation of STAT3 and IL-6 (135, 136).
Patients with colitis and colon cancer also have an increased abundance of E. coli (67). A direct
role in mouse carcinogenesis was demonstrated for strains of E. coli expressing the pks pathogenic-
ity island, which encodes the genotoxin colibactin (67). Both induction of inflammation and a
direct effect of the pks+E. coli are needed for carcinogenesis (67). Colibactin induces alterations
in p53 SUMOylation, inducing cellular senescence associated with the production of growth fac-
tors leading to tumor-promoting effects (137). E. coli along with several other microbial species,
including Campylobacter jejuni,Aggregatibacter actinomycetemcomitans,Haemophilus ducreyi,Shigella
dysenteriae,Helicobacter hepaticus,andS. enterica, also has another type of genotoxic compound
that belongs to the family of cytolethal distending toxins (CDTs). CDT consists of three sub-
units: CdtA, CdtB, and CdtC. Its CdtB subunit has similarities with DNAse-I-like nucleases and
demonstrates potent DNAse activity that causes extensive DNA lesions and apoptosis in target
cells (138). Attaching and effacing E. coli has also been shown, similarly to attaching and effacing
H. pylori, to downregulate the key DNA mismatch proteins MSH2 and MLH1, which are also
mutated in hereditary nonpolyposis colorectal cancer (139, 140). In addition, enteropathogenic
E. coli utilizes another pathway to inhibit DNA repair via the secretory cysteine methyltransferase
NleE, which blocks the function of DNA annealing helicase and endonuclease ZRANB3 (141).
ETBF is a subclass of the human commensal B. fragilis. It is present in the intestine at relatively
low abundance; however, it can act as a pathobiont by causing diarrhea and has been associated
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 2
The role of prokaryotic microbes in cancer initiation and progression. (Far left) Microbial metabolites with direct and indirect
genotoxic activity include products of protein (e.g., H2S, p-cresol) and bile (e.g., deoxycholic acid) degradation as well as products of the
breakdown of liver-detoxified xenobiotics (e.g., sulfation and glucuronidation conjugates). A number of bacterial species can also
produce toxins that are injected into host cells via the type III secretion system. These induce cell toxicity via apoptosis and repression
of proton pump expression. Among the toxins that induce DNA damage are colibactin, produced by some strains of E. coli;and
cytolethal distending toxins (CDTs), produced by Escherichia coli,Campylobacter jejuni,Helicobacter hepaticus,andSalmonella enterica.In
addition to directly damaging DNA, some bacteria, including H. pylori and E. coli, downregulate genome stability–related and repair-
related genes (e.g., TP53, MSH2, MLH1, and ZRANB3). Bacterial species that translocate through the epithelial barrier induce
recruitment of myeloid cells, including reactive oxygen species (ROS)–producing neutrophils that contribute to DNA damage in the
host cell. (Center left) FadA protein from Fusobacterium nucleatum, CagA toxin from H. pylori,Bacteroides fragilis toxin (BFT), and
avirulence protein A (AvrA) from S. enterica Typhi can activate the β-catenin pathway by promoting detachment of β-catenin from
E-cadherin. Some of the same bacterial species, such as H. pylori and S. enterica, also activate the PI3K/AKT and MAPK/ERK pathways
via receptor tyrosine kinases. (Center and far right) Cytokines, such as IL-22, IL-11, and IL-6, promote development of colon cancer via
activation of STAT3. The intestinal microbiota and transmucosally translocated bacterial species, following mucosal damage or tumor
growth, regulate the production of many cytokines—such as IL-6, IL-11, IL-12, IL-18, IL-23, and TNF production by macrophages,
dendritic cells (DCs), and epithelial and mesenchymal cells and IL-22 and IL-17 production by T cells and innate lymphocytes. The
IL-18/IL-22 axis is particularly important in maintaining mucosal homeostasis, and it is tightly regulated. Microbial products induce
epithelial cell production of pro-IL-18, which is cleaved into active IL-18 by inflammasomes. These inflammasomes (NLRP3 and/or
NLRP6) are activated by bacteria-derived small-chain fatty acids (SCFAs) via GPR43 and GPR109a receptors and by commensal
protists. IL-18 then blocks macrophage production of the soluble IL-22 antagonist IL-22BP, and it is required for IL-22 production by
T cells and innate lymphoid cells, thus increasing production and bioavailability of IL-22. IL-22 also induces STAT3 phosphorylation
in epithelial cells, promoting proliferation and secretion of antibacterial peptides, and, in a positive feedback loop, enhances production
of IL-18. Bacteria also activate immunosuppressive mechanisms: F. nucleatum promotes accumulation of immunosuppressive myeloid
cells in colonic tumors and produces the Fap2 protein, which activates the inhibitory receptor TIGIT on natural killer (NK) and T
cells; SCFAs induce regulatory T cells (Tregs), which inhibit local immune response. Abbreviations: CTL, cytotoxic T lymphocyte;
MAMP, microbe-associated molecular pattern.
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with inflammatory bowel disease and colorectal cancer (142). B. fragilis toxin stimulates intestinal
epithelial cell proliferation, which depends in part on E-cadherin degradation and β-catenin
activation. ETBF induces mucosa permeabilization and STAT3 activation in both inflammatory
and epithelial cells, leading to colon carcinogenesis in APCMin/+mice, which is dependent on
simultaneous expansion of both Tregs and Th17 cells (143, 144).
In mouse colon carcinogenesis, the polyp-containing epithelium is more permeable than the
contiguous healthy tissue and allows transmucosal bacterial translocation (145). The translocated
bacteria induce the production of proinflammatory IL-6, IL-11, IL-23, IL-17, and IL-22, which
are required for cancer progression (145, 146).
Another pro-inflammatory cytokine, IL-18, is a potent inducer of IFN-γproduction and, when
combined with IL-12, results in type 1 polarization of both innate and adaptive lymphocytes. How-
ever, paradoxically, results in IL-18-deficient mice suggest that in the large intestine this cytokine
has anti-inflammatory properties and mediates mucosal protective mechanisms (147). Mice defi-
cient in IL-18, IL-18R, and MyD88 as well as those deficient for inflammasome-related genes,
which are unable to process pro-IL-18, are more susceptible to dextran sulfate sodium (DSS)-
induced colitis; azoxymethane (AOM)/DSS-induced, colitis-associated cancer; and diet-induced,
nonalcoholic steatohepatitis (148). These mice also have gut dysbiosis characterized by increased
abundance of the phyla Bacteroidetes (Prevotellaceae) and TM7 and greater susceptibility to colon
carcinogenesis that can be transferred to wild-type mice by cohousing or by fecal transplant (128).
Commensal bacteria and protists induce production of IL-18 in intestinal epithelial cells by tran-
scriptionally inducing the production of pro-IL-18, which is then cleaved into biologically active
IL-18 by inflammasomes (149, 150). Bacteria-induced SCFAs activate the epithelial cell inflam-
masome via the metabolite-sensing receptors GPR43 and GPR109A (149). SCFAs also act on
macrophages, dendritic cells, and T cells, in particular, causing the expansion of IL-10-producing
Tregs that limit colonic inflammation and carcinogenesis (151, 152). Whether NLRP3 and/or
NLRP6 are involved in IL-18 induction remains controversial and diametric results have been
published by different laboratories (153). The discordant data could be explained by variations in
the composition of the microbiota in different animal facilities and by the differential distribution
of the two inflammasomes in epithelial and hematopoietic cells.
The ability of IL-18 to protect the mucosa depends on its regulation of IL-22 production and
availability (154–156). IL-22 is produced by T cells and innate lymphoid cells in the intestinal
lamina propria and, through activation of STAT3, induces epithelial cell proliferation and produc-
tion of antibacterial peptides (155). Three regulatory mechanisms have been proposed: (a)IL-18
inhibits IL-22 binding protein (IL-22BP) production by macrophages; (b) IL-18 is required for
IL-22 production by T lymphocytes and innate lymphoid cells; and (c) in a positive feedback
loop, IL-22 induces pro-IL-18 but not its processing by inflammasomes in intestinal epithelial
cells (154–156). In different experimental models of colon carcinogenesis, IL-22, which promotes
mucosa repair, has been observed to be pro- or anticarcinogenic depending on the extent of mu-
cosal damage in the carcinogenic model (154). The paradoxical ability of the proinflammatory
cytokine IL-18 to protect from colitis and colon carcinogenesis (154) may be explained by a re-
cent report showing that IL-18 protects against mucosal inflammation by maintaining a protective
microbiota (157). This article reports that IL-18 signaling in intestinal epithelial cells prevented
mucus-forming goblet cells from maturing, and mice that were deficient for IL-18 or IL-18R in
intestinal epithelial cells and that were cohoused with wild-type mice to equalize the microbiota
composition were resistant to DSS-induced colitis (157).
In several experimental models, SCFAs acting through the GPR109A receptors are anti-
inflammatory and decrease incidence of colon and mammary cancer (152, 158). Some bacterial
species contribute more than others to SCFAs levels, such as the butyrate-producing members of
210 Dzutsev et al.
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Clostridium cluster XIVa (159). SCFAs, particularly butyrate and propionate, are also inhibitors
of histone deacetylases and downregulate expression of proinflammatory genes such as IL-6
and tumor necrosis factor (TNF) (160). However, reducing the production of SCFAs using
either antibiotics or a carbohydrate-poor diet decreases incidence of colonic polyps in ApcMin/+/
Msh2 −/−mice (161). This procarcinogenic effect of SCFA-producing bacteria was not dependent
on inflammation or DNA damage but on induction of hyperproliferation and aberrant β-catenin
signaling in Msh2 −/−cells (161).
Although the microbiota is present on all barrier surfaces, scarce and mostly correlative data
are available for the role of the local microbiota in cancer development outside the gastrointestinal
tract. More studies deciphering the contribution of the microbiota to cancers of the skin, oropha-
ryngeal cavity, lung, and urogenital tract are needed. Also, relatively little attention has been
devoted to microorganisms other than bacteria and viruses, although production of carcinogenic
acetaldehyde by fungi has been proposed to play a role in oral cancer in patients with a mutation
in the AIRE gene and with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(162). Additionally, other members of our microbiota could also contribute to cancer, including
protists and helminths, which have been shown to orchestrate gut immunity (150, 163).
The studies described here have allowed investigators to identify many bacterial species with
carcinogenic activity; however, with the exception of H. pylori, none of them have been formally
proven to be a human carcinogen, for example, by disease prevention upon their elimination from
the host (65).
EFFECTS OF THE GUT MICROBIOTA ON TUMORS
IN DISTANT ORGANS
The microbiota regulates cancer not only at the epithelial barriers that it inhabits but also systemi-
cally at distant sterile sites. One example is the regulation by the gut microbiota of metabolism and
energy balance, which contributes to cancer-predisposing conditions such as obesity and metabolic
disease. Bacteria may also modulate distant malignancies such as endometrial and breast cancer
through a noninflammatory pathway by expressing β-glucuronidases and β-glucuronides that
participate in estrogen metabolism. Thus, the use of antibiotics and dysbiosis may alter estrogen
metabolism and affect the progression of these tumors (119).
The microbiota also metabolizes xenobiotics by transforming heterocyclic aromatic amines
from burned meat or environmental pollutants into genotoxic and procarcinogenic metabo-
lites (164). The colonic procarcinogen AOM is processed by P450 enzymes in the liver and
in the small intestine into carcinogenic methylazoxymethanol (MAM) that also gets transformed
in the liver by UDP-glucuronosyltransferase into inactive MAM-glucuronide. Accumulation of
carcinogenic AOM products in the colon and formation of DNA adducts require gut micro-
bial β-glucuronidase (165) that converts inactive MAM-glucuronide back into active methyla-
zoxymethanol. Colon carcinogenesis induced by AOM is prevented by prebiotics that reduce the
number of β-glucuronidase-expressing bacteria, or by inhibitors of the enzyme (166, 167). Sys-
temic effects of the microbiota can also be inflammatory. In mice where gut dysbiosis was induced
by fungal Candida sp. and by T. gondii infection, the lung macrophages and bone marrow mono-
cyte precursors, respectively, were polarized into cells with antiallergic and anti-inflammatory
characteristics (51, 168).
There are many examples of systemic regulation of carcinogenesis by intestinal microbes in
experimental mouse cancer models. Colonic infection with Helicobacter hepaticus has contrasting
effects on carcinogenesis in the intestine and at distant organs. Colonic H. hepaticus infection
increases ileal and colonic carcinogenesis in APCmin/+mice and in AOM-treated Rag2−/−mice by
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inducing IL-22 production by innate lymphoid cells (155, 169). It also promotes mammary and
prostate carcinoma in APCmin/+/Rag2−/−mice (124, 170) and enhances chemical and viral liver
carcinogenesis (124, 171). However, in Rag2-sufficient animals H. hepaticus infection protects
against intestinal and mammary carcinogenesis by inducing IL-10-producing Tregs (172). In a
different model of mammary carcinogenesis based on expression of SV40 T antigen under the
control of sex steroid hormones, infection with H. hepaticus increases tumor multiplicity, affecting
migration of neutrophils into the mammary gland (173).
Variation in intestinal microbes between different animal facilities or as a consequence of
experimental perturbations profoundly affects incidence of lymphoma and survival of Atm (ataxia
telangiectasia mutated)-deficient mice (174). The gut microbiota contributes to the incidence of
thymic lymphoma in these animals by modulating the TNF-regulated inflammatory tone and by
inducing oxidative stress as well as genotoxicity in leukocytes and epithelial cells (170, 171, 174,
175). In mice TLR5-mediated recognition of commensal microbiota induces production of IL-6
and recruitment of myeloid suppressor cells, enhancing progression of malignant tumors at distal
extramucosal locations (176).
BACTERIA AS ANTICANCER TREATMENT
Following the personal and historical observations of tumor regression associated with acute bac-
terial infections, at the end of the nineteenth century, the New York surgeon William Coley suc-
cessfully treated soft tissue sarcoma patients with “Coley’s toxins,” a combination of Streptococcus
pyogenes and gram-negative Bacillus prodigiosus (Serratia marcescens), providing evidence that a severe
localized infection may induce a systemic antitumor immune response (177). Subsequently, several
bacteria or bacterial preparations, such as Corynebacterium parvum and the streptococcal prepa-
ration OK-432, were tested in cancer therapy; local treatment with bacille Calmette Gu´
erin, an
attenuated strain of Mycobacterium bovis, is still a first-line therapy for superficial bladder carcinoma
(178). Many genera of bacteria, including Salmonella,Escherichia,andClostridium, preferentially
accumulate in tumors when delivered systemically, and they have been tested as anticancer agents
(179). A major limitation of cancer therapies is that large tumors contain hypoxic, necrotic, and
quiescent regions in which the tumor cells do not proliferate. These regions are either inaccessible
to drugs or, because of hypoxia, poorly susceptible to DNA damage induced by chemotherapy or
radiation (179). Because obligate anaerobic bacteria such as Clostridium spp. can only proliferate
in hypoxic regions of the tumors, when they are delivered as spores they germinate and prolifer-
ate with antitumor effects only in the hypoxic tissues (180). Although the use of spores could be
considered for large hypoxic tumors, small tumors or metastases are better oxygenated and might
be better treated with facultative anaerobes, such as Salmonella and Escherichia, that are attracted
by small molecules released by tumors (181). The ideal bacterial therapeutic would have the fol-
lowing properties: toxic for tumor cells but not normal cells, selective for the tumor (particularly
regions not targeted by conventional therapies), susceptible to immunological clearance but not
immediately destroyed by the immune response, proliferative, and genetically modifiable (181).
Furthermore, the therapeutic bacteria should be mobile and allow genetic alteration to reduce
systemic toxicity and localization in the tumors (182, 183). For example, deletion in Salmonella of
the Trg gene that encodes a sugar-sensing transmembrane receptor allows the bacteria to deeply
penetrate within poorly vascularized and nutrient-poor regions of the tumor (183). Bacterial
therapeutics can be used for delivery of antitumor molecules such as toxin, cytokines, antigens,
and antibodies, as well as for transfer of genes encoding molecules with antitumor activity (179,
182). The advantages of bacterial cancer therapies include not only their efficacy when other
212 Dzutsev et al.
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therapies fail but also their tumor tropism and ability to proliferate and maintain favorable phar-
macokinetics for an extended period of time (179).
CANCER CHEMOTHERAPY AND THE MICROBIOTA
The microbiota regulates the response to different types of cancer chemotherapy by affecting their
pharmacokinetics, mechanism of action, and toxicity (184, 185) (Figure 3). Intestinal host and
bacterial enzymes control the bioavailability of many oral drugs (186). Exposure to xenobiotics,
including chemotherapy agents, alters the composition, physiology, and gene expression of the
human gut microbiota (187). The gut microbiota also metabolizes injected drugs after biliary excre-
tion and reabsorption (188). For example, tissue carboxylesterase transforms Irinotecan (CPT-11;
an intravenous topoisomerase I inhibitor used for colorectal cancer treatment) into its active form,
SN-38, which is then detoxified in the liver by UDP-glucuronosyltransferases, becoming inactive
SN-38-G before being secreted in the gut (188). When bacterial β-glucuronidase reconverts SN-
38-G in the gut into SN-38, intestinal toxicity and diarrhea are observed (189). β-Glucuronidase
activity in the human gut is mostly associated with abundance of Firmicutes, particularly within
clostridial clusters XIVa and IV (190). Intestinal inflammation induced by CPT-11 therapy can
be successfully treated with antibiotics to decrease the abundance of β-glucuronidase-positive
bacteria or with bacterial β-glucuronidase-specific inhibitors (191).
Platinum-based anticancer drugs inhibit DNA replication by forming intrastrand platinum-
DNA adducts and double-stranded breaks (192). In addition to inducing tumor cell cytotoxicity
and apoptosis, platinum drugs cause severe intestinal toxicity, nephrotoxicity, and peripheral neu-
ropathy that compromise the quality of life (193–195). In germfree mice or in mice depleted of
gut commensals using nonabsorbable broad-spectrum antibiotics, the antitumor efficacy of ox-
aliplatin or cisplatin is dramatically decreased (53). In the absence of commensal microbiota the
drugs still reach the tumor and form platinum-DNA adducts, but DNA damage is severely atten-
uated (53). The microbiota is necessary for training tumor-infiltrating myeloid cells to produce
reactive oxygen species (ROS) via NADPH oxidase 2 (NOX2), which is necessary for platinum
compound–induced DNA damage (53). Mice deficient for the Cybb gene encoding the gp91phox
chain of NOX2 and mice treated with antibodies depleting myeloid cells are poorly responsive
to oxaliplatin (53). Although ROS were known to be involved in the induction of DNA damage
and apoptosis of the tumor cells, the source of ROS was expected to be autocrine (196). However,
observations in microbiota-depleted mice are more compatible with paracrine production of ROS
by NOX2-expressing and ROS-producing tumor infiltrating myeloid cells (53). Administration
of Lactobacillus acidophilus to antibiotics-treated mice restores cisplatin antitumor activity (197).
How precisely the gut commensals and L. acidophilus prime myeloid cells for ROS production in
response to platinum drugs is still unclear. Interestingly, L. acidophilus also attenuates intestinal
toxicity in patients treated with both radiotherapy and cisplatin, suggesting that this probiotic
may enhance the antitumor effect while preventing adverse reactions (197, 198). Acetovanillone,
an inhibitor of NADPH oxidases, protects mice from cisplatin nephrotoxicity by preventing tox-
icity of both the ROS produced by kidney tubular cells soon after treatment and the late ROS
produced by infiltrating myeloid cells (199). Not surprisingly, these observations indicate that the
antitumor effect and the toxicity of platinum compounds are modulated in a similar way by gut
commensals. In devising procedures targeting the microbiota to improve therapeutic efficacy while
limiting toxicity, it will be important to determine how the drugs and the microbiota intersect in
mediating toxicity in tumors and organs and to identify the roles and mechanisms of action of dif-
ferent commensals. Treatment with prebiotics, probiotics, and symbiotics could prevent dysbiosis
www.annualreviews.org •Microbes and Cancer 213
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after chemotherapy and reduce inflammation in the gut and liver, although data from stringent
clinical trials are needed (200).
Antitumor treatment with the alkylating agent cyclophosphamide (CTX) rapidly damages
the mouse gut epithelial barrier by increasing mucosal permeability, resulting in transmucosal
translocation of gram-positive gut bacteria, such as Lactobacillus johnsonii,Lactobacillus murinus,
and Enterococcus hirae, into the mesenteric lymph nodes (201). Within a week, CTX treatment
also alters the composition of commensals in the small intestine of mice, similar to what ob-
served in cancer patients (201, 202). CTX treatment selectively reduces Firmicutes (Clostridium
cluster XIVa,Roseburia,Coprococcus and unclassified Lachnospiraceae) and Spirochaetes (particularly
Untreated CpG-ODN/
Anti-IL-10R Anti-PD-L1
CTX
Anti-CTLA-4 TBI/
Adoptive T cell
transfer
TREATMENTS Cisplatin
Oxaliplatin
Hemorrhagic
necrosis Promoting
antitumor
immunity
Licensing of
T cell–
mediated
killing
Genotoxicity
and cell
death
T cell–
mediated
tumor killing
Immuno-
genic cell
death and
T cell–
mediated
tumor cell
clearance
CELLULAR
RESPONSE TO
ANTICANCER
THERAPY
INTESTINAL
EPITHELIUM
GUT
MICROBIOTA
TUMORS
CTLA-4
T cells
T cells ROS
Transferred
CTLs
PD1/PD-L1
TNF
Monocyte
Dendritic cell
Macrophage
Neutrophils DNA damage
TLR4
TLR4
MyD88
MyD88
Anti-CTLA-4
Anti-CTLA-4
CpG-ODN/
Anti-IL-10R
CpG-ODN/
Anti-IL-10R
Anti-PD-L1
Anti-PD-L1
Total body
irradiation
Total body
irradiation
Cisplatin
Oxaliplatin
Cisplatin
Oxaliplatin
CTX
CTX
No
treatment
No
treatment
L. acidophilus
L. acidophilus
Gram-negative
bacteria
Gram-negative
bacteria
L. johnsonii
L. murinus
E. hirae
B. intestinihominis
L. reuteri
Ruminococcus
A. shahii
L. murinum
L. intestinalis
L. fermentum
Burkholderiales
B. thetaiotaomicron
B. fragilis
Burkholderiales
B. thetaiotaomicron
B. fragilis
B. breve
B. longum
B. adolescentis
TLR9
TLR4
TLR4
THERAPEUTIC
OUTCOME
12
3
4
5
67
pTh17 cells
CTLs
Th1 cells
pTh17 cells
CTLs
Th1 cells
214 Dzutsev et al.
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IY35CH08-Trinchieri ARI 18 January 2017 13:30
Treponema genus) phyla in the small intestinal mucosa while it enhances gram-positive bacteria,
mainly L. johnsonii,L. murinus,E. hirae,andL. reuteri, some of which translocate into mesen-
teric lymph nodes (201). CTX induces immunogenic tumor cell death that, in concert with the
translocated gram-positive bacteria and the accumulation of the gram-negative Barnesiella intes-
tinihominis in the colon, activates pathogenic T helper 17 (pTh17) cells and memory Th1 immune
responses that mediate an antitumor adaptive immune response (201, 203). The pTh17 responses
and the antitumor effect of CTX treatment are reduced in germfree mice and in mice depleted
of gram-positive bacteria by antibiotics (201). The antitumor effect of CTX can be restored in
microbiota-depleted mice by adoptive transfer of pTh17 cells (201).
IMMUNOTHERAPY AND THE GUT MICROBIOTA
Immunotherapy is one of the greatest successes of cancer research (204). Enduring, complete
responses have been obtained in patients with metastatic melanoma and lung cancer even when
previous treatments failed. However, the response in different patients and cell types has varied.
New evidence that the composition of the gut microbiota modulates the response to immunother-
apy opens new possibilities of improving outcomes (53, 205, 206).
An early study showed that in mice preconditioned with total body irradiation (TBI) before
receiving adoptive T cell therapy, the antitumor effect was dependent on the presence of gut
←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−
Figure 3
Gut microbiota in cancer therapy. Gut commensal microorganisms regulate complex cellular networks,
enhancing (red names) or attenuating (blue names) the efficacy of cancer treatments. Some of the mechanisms
by which the microbiota affects the various anticancer therapies (bottom) are depicted here:
() An intact, healthy intestinal epithelial cell layer and mucous layer are an efficient barrier for
luminal commensal bacteria. Together with well-regulated interactions between the intestinal innate
and adaptive immune system, they maintain mucosal homeostasis. Some but not all cancer therapies
damage mucosal integrity and allow transmucosal translocation of commensal bacteria.
() Intratumoral treatment with the immunostimulating TLR9 agonist CpG-ODN combined with in-
hibition of IL-10 signaling (anti-IL10R antibodies) induces rapid tumor hemorrhagic necrosis mediated
by TLR9-induced TNF production. The gut microbiota primes (via a TLR4-dependent mechanism)
tumor-infiltrating myeloid cells to produce TNF in response to CpG-ODN.
() Immunotherapy with anti-CTLA-4 induces mucosal damage and translocation of Burkholderi-
ales and Bacteriodales, which promote anticommensal immunity that acts as an adjuvant for antitumor
immunity and is required for inducing a positive response to therapy.
() The antitumor effect of anti-PD-L1 therapy, which does not damage the gut epithelia, requires
preexisting antitumor immunity that is particularly effective in mice harboring intestinal Bifidobacterium
spp.
() Preconditioning total body irradiation (TBI) enhances the efficacy of adoptive T cell therapy
by inducing mucosal damage, which allows translocation of gram-negative commensals that activate
dendritic cells via TLR4 signaling, augmenting proliferation and cytotoxic functions of the transferred
T cells in the tumor microenvironment.
() Platinum-based drugs, including cisplatin and oxaliplatin, cause DNA damage in tumor cells that
is dependent on both the formation of platinum-DNA adducts and the production of NADPH-oxidase
dependent reactive oxygen species (ROS) by tumor-infiltrating myeloid cells that have been primed in
a MyD88-dependent way by components of the commensal microbiota.
() Cyclophosphamide (CTX) therapy induces immunologic cell death of tumor cells, which elicits
the generation of antitumor pathogenic Th17 (pTh17) cells, Th1 cells, and cytotoxic T lymphocytes
(CTLs), leading to tumor destruction. Optimal generation of this antitumor immune response requires
the activation of tumor-antigen-presenting dendritic cells by components of the intestinal microbiota
that translocate following CTX-induced mucosal damage.
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commensals (207). TBI damaged the intestinal mucosa, inducing translocation of bacteria and
activation of dendritic cells via TLR4 activation. This improved the proliferation and antitumor
effect of the transferred CD8+T cells (207). The beneficial effect of TBI on therapy efficacy was lost
in Tlr4-deficient mice and in antibiotic-treated mice (207). Administration of the TLR4 agonist
lipopolysaccharide to nonirradiated animals enhanced the efficacy of transferred T cells (207).
Intratumoral administration of the TLR9 agonist CpG oligonucleotide (CpG-ODN) along
with IL-10R-blocking antibodies induces a strong inflammatory antitumor response, character-
ized by secretion of proinflammatory cytokines such as TNF and IL-12 that was potentiated by
antagonizing the immunosuppressive role of IL-10 produced by Tregs and myeloid cells (208–
210). CpG-ODN/anti-IL-10R treatment induces rapid hemorrhagic necrosis and repolarization
of tumor-infiltrating dendritic cells and macrophages to a proinflammatory state. These then pro-
mote T cell–mediated antitumor immunity to permanently eliminate the tumors in most mice
(208). In germfree mice or mice whose gut microbiota has been depleted by antibiotic treatment,
tumor-infiltrating myeloid cell production of proinflammatory cytokines in response to CpG-
ODN is poor, preventing the induction of TNF-dependent necrosis and antitumor adaptive im-
munity (53). Tumors of microbiota-depleted mice have only minor alterations in the number and
differentiation of tumor-infiltrating myeloid cells, mostly derived by recruited Ly6C+circulating
inflammatory monocytes. Tumor-infiltrating myeloid cell subsets in microbiota-depleted mice
show modest alterations in their gene expression profile compared with conventional mice. This
observation contrasts with the major gene expression differences between microbiota-depleted and
conventionally raised mice that are observed after CpG-ODN treatment. Myeloid cells from Tlr4-
deficient tumor-bearing mice are also partially unresponsive to CpG-ODN, and administration of
lipopolysaccharide by gavage to microbiota-depleted mice reconstitutes the myeloid cell response
to CpG, suggesting that activation of TLR4 by products of commensal bacteria primes tumor
myeloid cells for responsiveness to TLR9 (53). These results are reminiscent of the reported in-
ability of mononuclear phagocytes in nonmucosal lymphoid organs of germfree mice to respond to
microbial stimulation with transcription of inflammatory genes due to epigenetically closed chro-
matin conformation at those loci (34). Thus, colonizing the mice postnatally introduces chromatin
changes in the myeloid cells, poising them for rapid responses to inflammatory stimuli, similar
to the phenomenon of trained innate resistance recently reported for various innate effector cell
types (34, 211). However, this epigenetic conformation can be reversed by antibiotic-induced de-
pletion of the commensal microbiota for two to three weeks, suggesting either that this chromatin
conformation is not stable and requires continued instructions from the commensal microbiota
or that inflammatory monocytes are newly generated in the bone marrow and require training
(53). The ability of the tumor-infiltrating myeloid cells from animals with altered microbiota to
produce TNF in response to CpG-ODN intratumoral treatment correlated with the abundance of
specific bacterial genera. For example, TNF production was positively correlated with the frequen-
cies of gram-negative Alistipes spp. and gram-positive Ruminococcus spp. in the fecal microbiota.
Frequencies of Lactobacillus species that are considered to be probiotics with anti-inflammatory
properties, including L. murinum,L. intestinalis,andL. fermentum, negatively correlated with TNF
production (53). Alistipes shahii administration by gavage to mice previously exposed to antibiotics
restored TNF production, whereas L. fermentum impaired TNF production in conventionally
raised mice (53). Thus, the training of myeloid cells for response to CpG-ODN is reversed when
the commensal microbiota is depleted, and individual bacterial strains can either increase or at-
tenuate priming for TNF production. Selective antibiotics, prebiotics, or probiotics that modify
microbiota composition could be considered for optimizing anticancer immunotherapy.
Immunity plays a dual role in cancer. As part of immunosurveillance, it may prevent or de-
lay the growth of the tumor by destroying tumor cells or creating a hostile microenvironment.
216 Dzutsev et al.
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On the other hand, it can also promote tumor initiation and progression by activating an anti-
inflammatory microenvironment or selecting cells that are able to evade the antitumor immune
response (212). In patients with progressive tumors, anticancer immunity is dormant or sup-
pressed, unable to prevent tumor growth. However, in many patients, it can be reactivated by
releasing the immunological brakes responsible for tumor escape (204, 213). Immune checkpoint
inhibitors include antibodies against cytotoxic T lymphocyte–associated protein 4 (CLTA-4), ex-
pressed on activated T effector cells and Tregs, and programmed cell death protein 1 (PD1) or its
ligand PDL-1. These inhibitors have had enduring clinical efficacy in many cancer patients (214).
Anti-CTLA-4 antibodies enhance T cell immune responses and proliferation by preventing the
T cell–suppressive interaction of CTLA-4 with its CD80 and CD86 ligands (214). Anti-PD1 and
anti-PD-L1 antibodies avoid T cell exhaustion and maintain T cell effector functions by prevent-
ing the binding of PD1 on activated T cells with PD-L1 that is expressed on the tumor cells and
other stromal cells (214). As with many effective anticancer therapies, checkpoint inhibitors may
have immune-related adverse effects. Onset of colitis and hypophysitis is mostly observed in re-
sponse to anti-CTLA-4 antibodies, whereas blocking PD1/PD-L1 interactions can lead to thyroid
dysfunctions and pneumonitis (215). Variability in patient response and susceptibility to adverse
reactions has been primarily attributed to the genetic characteristics of the tumors (including the
number of mutations and possibly the number of neoantigens); however, the immune status of the
patient and the possible effect of the microbiota in modulating it are additional important factors
(213, 214).
Two recent studies have identified the important role of the microbiota in modulating the
efficacy of anti-CTLA-4 and anti-PD-L1 therapy (205, 206). The anti-CTLA-4 treatment was
found to be ineffective in antibiotic-treated mice and in germfree mice (205). Treatment of mice
with anti-CTLA-4 antibodies consistently induces T cell–mediated mucosal damage in the ileum
and colon and alters the proportion of bacterial species both in the intestine and in the feces (205).
Bacteroides thetaiotaomicron or B. fragilis, when orally administered to microbiota-depleted mice,
corrected the deficient response to anti-CTLA-4 by activating intratumoral dendritic cells and
inducing a Th1 response in the tumor-draining lymph nodes (205). Of particular interest, feeding
with B. fragilis and Burkholderia cepacia combined not only enhanced the anticancer response but
also prevented the intestinal inflammation and colitis induced by anti-CTLA-4 (205). The correla-
tion between adverse effects and microbiota composition was confirmed by clinical trials in which
the increased frequency of the Bacteroidetes phylum was found to be correlated with resistance to
colitis, whereas underrepresented genetic pathways involved in polyamine transport and B vitamin
biosynthesis were associated with increased risk (216). In mice, selective antibiotics alter the com-
position of the gut microbiota affecting the anti-CTLA-4 response: e.g., vancomycin prevents loss
of Bacteroidales and Burkholderiales and enhances the efficacy of anti-CTLA-4 therapy (205). The
melanoma patients treated with anti-CTLA-4 clustered into three fecal microbiota enterotypes:
Cluster A was dominated by Prevotella spp. and clusters B and C by Bacteroides spp. After treatment,
some of the patients in cluster B acquired the cluster C enterotype. When germfree mice were
colonized with the patients’ fecal microbiota, the mice colonized with cluster C enterotype showed
expansion of B. thetaiotaomicron or B. fragilis and an ability to respond to anti-CTLA-4 treatment
(205). Thus, anti-CTLA-4 treatment may, in some cases, alter the composition of the gut micro-
biota in a direction that favors its antitumor activity. Anti-CTLA-4 elicits, both in mice and in the
humans, a Th1 response specific for either B. thetaiotaomicron or B. fragilis. Microbiota-depleted
mice recover their ability to respond to anti-CTLA-4 therapy when B. fragilis–specific T cells are
adoptively transferred (205). A similar observation was reported for mucosal damage induced by
infection that also induces a persistent memory Th1 cell response against commensal bacteria that,
upon reinfection at the same anatomical site, prime for a polarized Th1 response (14). However,
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in the case of the anti-CTLA-4 treatment, the mucosal response to commensals and the antitumor
response are at different anatomical sites and the mechanism regulating migration and tropism of
the T cells involved is not yet understood.
Unlike anti-CTLA4, anti-PD-L1 treatment in mice does not induce intestinal damage, and
its anticancer effect does not have an absolute requirement for the presence of gut commensals
(206). However, B16 melanoma grew faster in C57BL/6 mice purchased from Taconic (TAC)
than in those purchased from Jackson Laboratory ( JAX) (206). Anti-PD-L1 almost completely
arrested growth of the smoldering tumors of JAX mice, whereas it only slowed the progression
of the faster-growing tumors of TAC mice (206). More CD8+T cells infiltrated within tumors
in JAX mice compared to TAC mice, suggesting that the slower tumor growth and better re-
sponsiveness to anti-PD-L1 in JAX mice was due to a more robust anticancer immune response
(206). TAC mice cohoused with JAX mice acquired the same rate of tumor growth, antitumor
resistance, and responsiveness to anti-PD-L1 observed in JAX mice (206). The responsiveness of
JAX mice to anti-PD-L1 correlated with the fecal abundancy of the Bifidobacterium species, in-
cluding B. breve,B. longum,andB. adolescentis (206). A commercially available probiotic cocktail of
Bifidobacterium species (including B. breve and B. longum), administered alone or with anti-PD-L1,
to TAC mice enhanced CD8+T cell–induced antitumor activity (206). Treatment of TAC mice
with Bifidobacterium spp. or anti-PD-L1 showed equivalent therapeutic efficacy (206). Thus, unlike
anti-CTL-4, anti-PD-L1 treatment does not require an inflammatory immune activation that is
supported by the presence of the commensal microbiota. However, the presence of Bifidobacterium
spp. in the gut microbiota fosters a more robust antitumor immune response that, when enhanced
by anti-PD-L1, prevents tumor progression.
CONCLUSIONS
Recent technological advances have revolutionized our understanding of human metaorganism
physiology. This has opened new possibilities for basic and clinical science, including precision
medicine. Although novel sequencing technologies have contributed most to the success of mi-
crobiology studies, progress in other areas has been critical as well: improved bacterial culture
methodology, gnotobiotic technology, bioinformatics, computational science (including an expo-
nentially growing number of reference data sets), microscopy, and systems biology. Indeed, we
might safely predict that microbiota research will benefit from both experimental and computa-
tional advances. For example, there is a need for software that interprets larger metagenomic data
sets and presents hierarchically organized functional data such as novel microorganism-centered
annotated pathways and other gene function interpretations. Metagenomic and metatranscrip-
tomic analyses will be improved by new reference genomes from human and animal microbiota.
This will be aided by new single-molecule sequencing methods that generate reads tens of kilobases
long, easing genome assembly of even uncultivable microbes. Single-cell sequencing technologies
will also allow us to sequence microbial genomes and transcriptomes without the need of culturing
the microorganisms. New computational methods will be developed to better understand the ki-
netics of microbial communities and to identify keystone species that have greater influence than
their abundance may imply. Advances in bacterial culture methods and artificial gut development
[e.g., “robogut” technology (217) and “gut on a chip” (218)] will serve to validate computational
predictions and identify novel microbe-microbe and host-microbe interactions. Finally, the def-
inition of microbiota will be broadened to include not just prokaryotes, unicellular eukaryotes,
and viruses but also small multicellular animals residing in the gut (e.g., helminths) and in the skin
(e.g., Demodex mites). Our internal ecosystem is much more diverse than previously realized.
218 Dzutsev et al.
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The studies reviewed here have led us to conclude that many of the mechanisms of microbe-
dependent host physiology and tissue homeostasis are derived from ancestral mechanisms stem-
ming from the earliest interactions between prokaryotes and eukaryotes. Phagocytosis began as a
method of obtaining nutrients and probably led to the genesis of mitochondria. Phagocytes and
myeloid cells, in particular, communicate with commensal microbiota to dictate responses in-
volving immune defense, tissue homeostasis, repair, cancer development, and response to cancer
therapy (219). As phagocytes act as bridges between all the leukocyte subsets involved in resistance
to infection and tissue damage, the cross talk between the host and microbiota transverses both
innate and adaptive immunity.
The composition and functional status of different members of the microbial community can
modulate or even control cancer initiation and progression, comorbidity, and response to ther-
apy. We are at a critical point when the accumulated knowledge of host-microbe interactions
can be used to design new therapeutic strategies. The commensal microbiota is a target for in-
terventions to prevent cancer, control its progression, induce its destruction via genetically en-
gineered bacteria, and prevent cancer-associated comorbidities such as cachexia that can affect
treatment success and quality of life. Indeed, recent studies demonstrating that microbes can
modulate anticancer therapies offer the prospect that targeting the gut microbiota may improve
therapeutic efficacy and attenuate toxicity and adverse reactions. Currently, however, few ther-
apeutic approaches target microbes; clinical applications are still from the dark ages. Probiotics
and prebiotics might be a more precise tool for manipulating the gut microbiota than antibi-
otics. However, until now, there has been little consistent or sufficient evidence of their clinical
efficacy in many microbiota-related diseases. An exception is Clostridium difficile infection, where
crude community-altering therapies, such as fecal transplantation, have been successful (220).
Progress in bacterial ecology and in understanding the role of bile acids in controlling C. dif-
ficile infection has identified bacterial species that for treating C. difficile infection (221) as an
alternative to fecal transplantation. Therapeutic use of bacterial species presents the challenge of
formulation that will achieve efficient colonization in the intestine after oral delivery. Whereas
preparations of facultative anaerobic bacteria may be relatively easy to formulate and adminis-
ter, obligate anaerobes may present more obstacles. Fortunately, many obligate anaerobes, like
Clostridium spp., form spores that germinate only in hypoxic environments. Spores are durable
and survive dry, oxygen-rich environments and therefore could be used as a vector for micro-
bial delivery. These characteristics of the spores also make them attractive for use in anticancer
treatments, since they would germinate in the hypoxic environment of large tumors. Looking
at the road ahead there is hope that we will overcome the many challenges of using microbes
as anticancer therapeutics. In addition to identifying therapeutic microbes, we have to address
the temporal and geographical variability of the microbiota in human populations. Through the
accumulation of clinical data, we need to identify key metabolic processes of the healthy micro-
biota and microbial taxa that promote resistance to disease or improve therapeutic outcomes in
different clinical situations. As we identify beneficial microbes and metabolic processes that pro-
mote resistance to disease or improve efficacy of available treatments, microbiology will likely
become an important component of precision and personalized medicine for cancer and other
diseases.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
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