Nihms 1768792

HHS Public Access
Author manuscript
Science. Author manuscript; available in PMC 2022 March 26.
Author Manuscript
Published in final edited form as:

Science. 2021 March 26; 371(6536): . doi:10.1126/science.abc4552.
The microbiome and human cancer

Gregory D. Sepich-Poore1, Laurence Zitvogel2,3,4,5, Ravid Straussman6, Jeff Hasty1,7,8,
Jennifer A. Wargo9,10, Rob Knight1,11,12,*
1Department of Bioengineering, University of California San Diego, La Jolla, CA, USA.
2Gustave Roussy Cancer Campus (GRCC), Equipe Labellisée–Ligue Nationale contre le Cancer,
Villejuif, France.
Author Manuscript
3Institut National de la Santé et de la Recherche Medicale (INSERM) U1015, Villejuif, France.

4Université Paris-Sud, Université Paris-Saclay, Gustave Roussy, Villejuif, France.
5Center of Clinical Investigations in Biotherapies of Cancer (CICBT) 1428, Villejuif, France.
6Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
7BioCircuits Institute, University of California San Diego, La Jolla, CA, USA.
8Molecular Biology Section, Division of Biological Science, University of California San Diego, La
Jolla, CA, USA.
9Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center,
Houston, TX, USA.
Author Manuscript
10Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center,

Houston, TX, USA.
11Department of Pediatrics, University of California San Diego, La Jolla, CA, USA.
12Department of Computer Science and Engineering, University of California San Diego, La Jolla,
CA, USA.
Structured Abstract
BACKGROUND: Historical accounts linking cancer and microbes date as early as four millennia
ago. Post establishment of the germ theory of infectious diseases, clinical research of microbial
influences on cancer began in 1868, when William Busch reported spontaneous tumor regressions
in patients with Streptococcus pyogenes infections. Over the next century, the role of bacteria
Author Manuscript
in carcinogenesis and cancer therapy was discounted due to poor reproducibility, erroneous
microbiological claims, and severe toxicity in patients. However, these provided some of the
first crude demonstrations of cancer immunotherapy. Contemporaneously, the viral theory of
cancer began to flourish, spurred by the 1911 discovery of Rous Sarcoma Virus (RSV), which
transformed benign tissue into malignant tumors in domestic fowl. The subsequent decades-
long search to find a virus behind every human cancer ultimately failed, and many cancers
have been fundamentally linked to somatic mutations. Now the field is encountering intriguing
*
Corresponding author: [email protected].
Authors’ contributions: All authors participated in the writing, editing, and final approval of this article.
Sepich-Poore et al. Page 2
claims of the importance of microbes, including bacteria and fungi, in cancer and cancer
Author Manuscript
therapy. This Review critically evaluates the evidence for these claims in light of modern
cancer biology and immunology, and delineates the roles of microbes in cancer by examining
recent advances in proposed mechanisms, diagnostics, endogenous modulation approaches, and
exogenous therapeutic strategies.
ADVANCES: Few microbes directly cause cancer, but many seem complicit in its growth, often
acting through the host’s immune system; conversely, several have immunostimulatory properties.
Mechanistic analyses of gut microbiota-immune system interactions have demonstrated powerful
effects on innate and adaptive immunity by modulating primary and secondary lymphoid tissue
activities against cancer and tumor immunosurveillance. Many of these pathways invoke Toll-like
receptor (TLR)-initiated cytokine signaling, but microbial metabolic effects in dietary energy
harvest and short-chain fatty acid production, and antigenic mimicry with cancer cells, are also
important. In preclinical models, microbial metabolites also regulate phenotypes of tumor somatic
Author Manuscript
mutations and modulate immune checkpoint inhibitor efficacy.
Emerging evidence also suggests the existence and functional activity of intratumoral
bacteria, with overlapping immunohistochemistry, immunofluorescence, electron microscopy, and
sequencing data on them in ~10 cancer types. Preliminary studies also suggest that fungi and
bacteriophages contribute to gastrointestinal cancers. However, the estimated cellular abundances
of intratumoral microbes is low relative to cancer cells, and knowledge of their functional
repertoire and potency remains limited. Further validation of their prevalence and impact is needed
in diverse cohorts and therapeutic contexts.
The immunomodulatory effects of host microbiota have reinvigorated efforts to change their
composition as a form of immunotherapy. Despite extensive preclinical evidence, translation of
microbiota modulation approaches into humans has yet to broadly materialize into commercialized
Author Manuscript
therapies. Synthetic biology approaches are also gaining traction, however, with engineered
bacterial cancer therapies in preclinical and clinical trial settings.
OUTLOOK: A better understanding of the roles of microbes in cancer has the opportunity
to improve each stage of the cancer care cycle, but major challenges must be surmounted.
Concerted efforts to characterize cancer-associated microbiota among tumor, stool, and blood
samples with gold-standard contamination controls would tremendously aid this progress. This
would be analogous to The Cancer Genome Atlas (TCGA)’s and International Cancer Genome
Consortium (ICGC)’s roles in characterizing the cancer somatic mutation landscape. Large-scale
clinical trials are currently testing the efficacy of microbiota modulation approaches, ranging
from dietary modifications to intratumorally-injected, engineered bacteria. These bacterial cancer
therapies, if safe and effective, could tremendously expand the cancer therapy armamentarium.
Altogether, integrating the host-centric and microbial viewpoints of cancer may improve patient
Author Manuscript
outcomes while providing a nuanced understanding of cancer-host-microbial evolution.

Graphical Abstract

Author Manuscript
Author Manuscript
Opportunities for microbes to impact cancer care. Diagnosis: Cancer-specific, blood-borne

microbial DNA may complement cell-free tumor DNA (ctDNA). Prognosis: Gut and intratumoral
microbiota may stratify patient outcomes; (N)R=(non)responder; TME=tumor microenvironment.
Therapy: Intratumor injection of CD47 nanobody (CD47nb)-producing E. coli may create
systemic antitumor immunity by enhancing dendritic cell (DC) phagocytosis, lymph node (LN)
antigen (Ag) presentation, and cytotoxic T lymphocyte (CTL) activity.
Abstract
Author Manuscript
Microbial roles in cancer formation, diagnosis, prognosis, and treatment have been disputed
for centuries. Recent studies have provocatively claimed that bacteria, viruses, and/or fungi
are pervasive among cancers, key actors in cancer immunotherapy, and engineerable to treat
metastases. Despite these findings, the number of microbes known to directly cause carcinogenesis
remains small. Critically evaluating and building frameworks for such evidence in light of modern
cancer biology is an important task. In this Review, we delineate between causal and complicit
roles of microbes in cancer and trace common themes of their influence through the host’s
immune system, herein defined as the immuno-oncology-microbiome (IOM) axis. We further
review evidence for intratumoral microbes and approaches that manipulate the host’s gut or tumor
microbiome while projecting the next phase of experimental discovery.
The histories of cancer and human microbiota are intimately interwoven. Writings as
Author Manuscript
early as 1550 BCE in the Ebers Papyrus, attributed to the Egyptian physician Imhotep
(c 2600 BCE), suggest a crude treatment for tumors (swellings) involving application of
a poultice to the site followed by an incision, causing an infection (1, 2). In the 13th
century, Peregrine Laziosi described spontaneous regression of his septic, ulcerative tibial
bone malignancy that would have required amputation (2), for which he was canonized in
1726. After establishment of the germ theory of infectious disease, Wilhelm Busch and
Friedrich Fehleisen independently reported in the late 1800s that Streptococcus pyogenes

infections were associated with spontaneous tumor regressions in several patients (3,
Author Manuscript
4). Shortly thereafter, William Coley started testing a highly contentious and sometimes
lethal vaccine of live or heat-killed Streptococcus and Serratia species on terminal
cancer patients, which was only later shown to yield >10-year disease-free survival
in ~30% of them (60 of 210 total), representing the first intentional demonstration of
immunotherapy (5). Contemporaneously, Thomas Glover and Virginia Livingston-Wheeler
claimed, controversially, that bacteria were cultivable from tumors and that bacterial
vaccines were effective against tumors, and suggested a universal bacterial origin of cancer
(6, 7). These early treatment approaches and theories were fraught with error: Livingston-
Wheeler’s bacterial “cause” of cancer, Progenitor cryptocides, turned out to be the skin
commensal Staphylococcus epidermidis (a frequent contaminant), and Glover’s findings
were not reproducible by researchers at the National Institutes of Health (7). With no
mechanistic evidence, irreproducible results, and hazardous therapies, the bacterial theory of
Author Manuscript
cancer was dismissed.
The viral theory of cancer gained traction after Peyton Rous’s 1911 discovery of a
transmissible oncogenic virus in chickens (8). The subsequent decades-long search to find
a virus behind every cancer linked Epstein-Barr, human papilloma, and hepatitis viruses to
carcinogenesis (9) but failed to find a viral cause for most human cancers, and the theory
was overtaken by the somatic mutation hypothesis.
Now, after decades of research thoughtfully characterizing the hallmarks of human cancer
through somatic mutations and other host-centric perspectives (10, 11), the field is
encountering nuanced claims that microbes may play a broad role in cancer diagnosis,
pathogenesis, and treatment (12–26). This reappraisal stems from greater appreciation of
the number of microbes that inhabit the human body (roughly equal to the number of
Author Manuscript
human cells), their gene count that exceeds the human genome’s gene count by ~100-fold
and enables diverse metabolic programming, and their effects on host immune system
development and activity, including antitumor immunosurveillance (27–31). Although most
proposed cancer-microbe relationships focus on gut microbiota (30, 32, 33), recent studies
also contentiously suggest the existence, metabolic activity, and functional importance
of intratumoral microbiota using a combination of imaging, sequencing, and cultivation
techniques, and genetically-engineered and germ-free mouse models (12–14, 18–20, 23, 34).
These studies raise many questions about microbes and cancer. How should microbes be
viewed in light of known host-centric cancer characteristics? To what extent are microbes
causal agents, complicit actors, or passive bystanders? If intratumoral microbes exist, do
they have therapeutic implications? What role do microbes play in patient management?
With these questions in mind, this Review aims to critically evaluate the known roles of
Author Manuscript
microbes in cancer, and to outline the next steps for evaluating their clinical utility.
Overview of the cancer microbiome

Of the estimated ~1012 distinct microbial species on earth (35), just 11 are labeled human
carcinogens, or “oncomicrobes,” by the International Association for Cancer Registries
(IACR) (36). These oncomicrobes cause an estimated 2.2 million cases per year (~13% of
global cancer cases), and their epidemiology, molecular mechanisms, and clinical studies

have been extensively reviewed (36). Strong experimental evidence suggests that additional
Author Manuscript
microbes initiate cancer through genotoxin-mediated mutagenesis; in particular, colibactin (a

DNA alkylator), cytolethal distending toxin (CDT; direct DNAse activity), and Bacteroides
fragilis toxin (Bft; ROS producer) cause mutational signatures found in colorectal, head
and neck, and urinary tract cancers (22, 37–41). Experimental evidence also implicates
several microbes with virulence factors that amplify tumorigenesis via E-cadherin/Wnt/β-
Catenin signaling, including FadA from Fusobacterium nucleatum and AvrA from several
Salmonella strains (42, 43). A few dozen microbial species can thus directly cause cancer,
based on current epidemiological and experimental evidence.
Increasing evidence suggests an important additional category of “complicit” microbes

and microbial functions that promote carcinogenesis but are insufficient to cause cancer
(18, 20, 25, 38, 44–47). This category encapsulates many immunomodulatory functions
of microbiota and their bioactive metabolites in tumor development, and may be linked
Author Manuscript
to the immune system’s role in solid tumorigenesis; the immune system rarely initiates
the incipient lesion but can facilitate progression through tumor-stroma feedback loops,
inflammation, or dysfunctional immunosurveillance (11). One example is that common
p53 mutations are only carcinogenic in the presence of microbially-produced gallic
acid and protective otherwise in the gut, both in vivo and in organoids, suggesting
a microbiome-functional genomic interaction (44). A second is microbially-produced
secondary bile acids, which reduce hepatic sinusoidal CXCL16 expression (the sole ligand
for CXCR6) and prevent CXCR6+ natural killer T (NKT) cell aggregation and liver cancer
immunosurveillance — this carcinogenic effect is eliminated by vancomycin treatment (45).
A third comes from the inability of Kras mutation and p53 loss to produce lung cancer
in germ-free or antibiotic-treated mice: commensal lung microbiota promote expansion and
activation of γδ T cells, which drives tumor-promoting inflammation via local IL-17 and
Author Manuscript
IL-23 release (18). These examples illustrate how microbes or microbial functions can be
complicit in cancer rather than directly causal.
In contrast to the few bona fide oncomicrobes, the many “complicit” microbes and their
functions are broad and under-studied. Complicit microbes require mediators to promote
tumor development, but modulate tumor progression and therapeutic efficacy locally or
from a distance. Complicit microbes are also least understood, requiring comprehension
and integration of host and microbial biology, so we emphasize them in this Review.
Together with known causal mechanisms, the diversity of these “complicit” mechanisms and
their relationships to host-centric cancer hallmarks (10, 11) are notable (Fig. 1), but they
will require more rigorous experimentation and cross-cohort validation to establish clinical
prevalence and utility.
Author Manuscript
Understanding the distribution of microbes across the body is important for understanding
their relationships to cancer. Approximately 4×1013 microbial cells spanning ~3×103 species
inhabit the human body: ~97% of these cells are bacteria in the colon, ~2–3% extra-colonic
bacteria (proximal gut, skin, lungs, etc.), and ~0.1–1% archaea and eukarya (including
fungi) (27, 48). Human-infecting virus and phage counts and diversity may be greater (49).
The high density of colonic bacteria is thought to drive the majority of known microbial
immunomodulatory effects in the mammalian intestinal tract, the largest immune organ in

the body (50), but organ-specific commensals may exert their own overriding influence
Author Manuscript
(18, 46). Intratumorally, Nejman et al. used quantitative PCR (qPCR) of 16S rRNA to
estimate the number of bacteria relative to 40 nanograms of DNA in melanoma, lung,
ovarian, glioblastoma, pancreatic, bone, and breast cancer tissue sections (12). Assuming
tissue homogeneity and 8 picograms of DNA per cancer cell (based on 2.36 average tumor
ploidy from the Pan-Cancer Analysis of Whole Genomes project) (51), the Nejman et
al. data suggest an average pan-cancer percent bacteria composition at 0.68% bacterial
(bootstrapped 95% CI of mean:[0.52%, 0.87%], 1000 iterations), with individual tumors
ranging from no bacteria to nearly 70% bacterial by cell count (12). Applying this percent
bacterial composition to three dimensional and planar contexts equates to ~105-106 bacteria
per palpable 1 cm3 tumor (52) or ~34 bacteria/mm2 (assuming 5000 cells/mm2 (53)), the
latter of which is comparable to the average PD1+ T lymphocyte tumor core density of ~21
cells/mm2 from a recent pan-cancer cohort (54). Importantly, these bacterial composition
Author Manuscript
estimates remain to be confirmed in other cohorts and cancer types and validated with
orthogonal methods. Furthermore, which of these microbial taxa and functions can affect the
host despite their low abundances remains unknown, as does the proportion that are merely
passengers in a nutrient-rich and immunosuppressed space.
Mechanisms and interactions between the gut and tumor microbiome

Gut microbiota can regulate many functions of the tumor-bearing meta-organism, typically
through immunomodulation, and putative intratumoral microbes may also be important (31,
55). Known microbial mechanisms can manipulate non-hematopoietic and hematopoietic
components of the gut epithelial barrier, modulate primary and secondary lymphoid organ
activities, and regulate immune tone of the tumor microenvironment (TME). We define
these immune-mediated interactions and collective feedback loops as the immuno-oncology-
Author Manuscript
microbiome (IOM) axis (Fig. 2). Gut-TME crosstalk, especially in non-gastrointestinal

cancers, remains a key area of discovery.
The effects of gut microbiota on primary lymphoid organs—Following allogeneic

hematopoietic stem cell transplantation (HSCT), robust immune reconstitution governs
both relapse and transplant-related patient mortality (56, 57). A recent multi-center,
multinational clinical trial demonstrated that higher diversity of intestinal microbiota is
significantly associated with lower patient mortality following allogeneic HSCT (58).
Moreover, in an allogeneic HSCT trial that co-analyzed daily changes in patient differential
blood counts with >10,000 longitudinal fecal samples, immune-reconstitution dynamics
were closely linked to gut microbiota composition (59). Links between gut microbiota,
nutrition, post-transplant bone marrow (BM) and thymic cellularity, and lympho- and
myelopoiesis, have also been demonstrated in mouse models (57). Gut microbe depletion
Author Manuscript
impairs systemic infection clearance after BM transplant, and sensitizes mice to semi-lethal
doses of radiation. Microbiota-derived compounds can protect against irradiation-induced
hematopoietic injury (60–62) through producing propionate and tryptophan metabolites
(63) or by releasing MAMPs known to maintain BM-derived myeloid cells and neutrophil
function (64, 65). This effect may be explained in part by the delivery of endogenous ligands
for RIG-I (such as 3pRNA and RNA derived from viruses, phages, or bacteria) that can
induce protective type I interferon (IFN-I) signaling in enterocytes and intestinal barrier

repair (66). Post-transplant lymphopoiesis also depends on energy harvest from the diet and
Author Manuscript
potentially on genera whose genomic repertoires encode carbohydrate-active enzymes (57).
The effects of gut microbiota on adaptive immunity—The gut microbiota has broad
effects contributing to host immune tone at steady state and during tumorigenesis (30,
67). Anti-cancer therapies have demonstrated strong links between distinct commensals and
protective antitumor T cell responses: (i) Cyclophosphamide enables Enterococcus hirae
to translocate and stimulate pathogenic TH17 responses and IFN-producing CD8+ T cell
effectors that check tumor growth in sarcoma and lung adenocarcinoma models (68, 69);
(ii) in some patients with melanoma, CTLA-4 blockade allows fecal relative enrichment
of B. thetaiotaomicron and B. fragilis that mediates TLR4- and IL-12-dependent TH1
responses and therapeutic efficacy (70); (iii) PD-(L)1 inhibition leads to T cell priming
against melanoma and is more effective when hosts harbor Bifidobacteria species in their
microbiome (21, 71); (iv) adoptive T cell transfer efficacy against melanoma after total body
Author Manuscript
irradiation depends upon the composition of the microbiota, the translocation of gut bacteria,
and host TLR4 signaling (72–74); (v) oxaliplatin-induced cell death of ileal enterocytes
inversely governs the immunogenic Erysipelotrichaceae and tolerogenic Fusobacteriaceae
proportions in the ileum, dictating the balance between antitumor follicular T helper cells
(TFH) and deleterious TH17 responses in colon cancer (75).
In most of these models, dendritic cells (DC) from the gut-associated lymphoid
tissue (GALT), spleen, or tumor draining lymph node (LN) sense various commensals
(Bifidobacterium spp., B. fragilis, A. muciniphila, B. rodentum, Bacteroidales S24–7),
catalyzing immune responses via IFN-I and IL-12-mediated pathways (17, 70–72, 75–77).
Apart from providing DC adjuvants, the gut microbiota represent an antigen source that
can elicit commensal-specific T cell responses systemically (55, 78). In the context of
Author Manuscript
homologous self-antigens, these commensal-specific immune responses can be deleterious

or protective for the host, depending on the involved peptide(s). For instance, Gil-
Cruz and colleagues demonstrated how homology between B. thetaiotaomicron-derived
β-galactosidase and host cardiac myosin heavy chain 6 could drive lethal autoimmune
inflammatory cardiomyopathy (79); conversely, Nanjundappa and others reported how
cross-reactivity between Bacteroides species-derived integrase and host islet-specific
glucose-6-phosphatase catalytic subunit-related protein (IGRP) could hijack autoreactive
CD8+ T cells to instead suppress colitis (80). Recent studies have further expanded this
cross-reactive homolog list to include exogenous dietary antigens, notably between gliadin
epitopes and gut Pseudomonas fluorescens-derived succinyl-glutamate desuccinylase in the
context of HLA-DQ2.5-mediated celiac disease (81). Molecular mimicry between cancer
and microbial antigens has also been hinted (82) and recently studied in-depth (83). H-2Kb-
Author Manuscript
restricted T cell immune responses against a phage that infects distinct strains of enterococci
(E. hirae) cross-reacted with an oncogenic driver (PSMB4). Oral administration of E. hirae
strains containing this phage then boosted phage-specific T cell responses effective against
extra-intestinal tumors overexpressing PSMB4 during therapy with cyclophosphamide or
anti-PD1 antibodies (83). Similarly, T cells targeting an epitope, SVYRYYGL (SVY),
expressed in the commensal bacterium Bifidobacterium breve, cross-reacted with a model
neoantigen, SIYRYYGL (SIY), expressed by mouse melanoma B16-SIY (73). Moreover,

some human T cells specific for naturally processed melanoma epitopes were found to
Author Manuscript
recognize microbial peptides (83), suggesting clinical significance. However, mechanisms

outside of molecular mimicry that boost antitumor immunity must also exist. For example,
Tanoue and colleagues identified an 11-bacteria cocktail that increased tumor antigen-
specific CD8+ IFN-γ+ T cells in the context of immune checkpoint blockade that were
not cross-reactive with microbial antigens and did not originate from the colon (84).
Gut-derived metabolites can also modulate immune responses. Radiotherapy of

tumor lesions was more effective when vancomycin eliminated Clostridiales-derived
immunosuppressive metabolites (butyrate and propionate), putatively by increasing DC
antigen presentation and concomitant CD8+ T cell priming (72); conversely, gut microbial-
derived propionate and tryptophan pathway metabolites (1H-indole-3-carboxaldehyde,
kynurenic acid) were shown to provide long-term radioprotection in vivo (63). High blood
butyrate and propionate levels were also associated with resistance to CTLA-4 blockade
Author Manuscript
in mice and melanoma patients, with concomitantly increased Treg proportions, reduced
DC and effector T cell activation, and lower responses to IL-2 (85), although they were
also found to be associated with longer progression-free survival during anti-PD-1 treatment
(86). Moreover, prebiotic mucin increasing ex vivo outgrowth of A. muciniphila decreased
growth kinetics of aggressive melanoma in a gut microbiota and T cell-dependent manner,
reducing serum levels of pro-inflammatory and immunosuppressive IL-6, IL-1α, IL-10,
IL-17A, IL-23 cytokines (87); notably, prebiotic inulin operated through a different mode of
action, facilitating the dominance of Bifidobacteria species in the intestines, boosting splenic
cytotoxic T lymphocyte functions, and overcoming melanoma resistance to MEK inhibitors
(87).
The effects of gut microbiota on the tumor microenvironment—The intestinal

Author Manuscript
ecosystem can influence both local and distant neoplasia by impacting their immune context,
influx of myeloid and lymphoid cells, and inflammatory and metabolic patterns. Secretory
components of gut microbiota can be important: for example, outer membrane vesicles
(OMVs) can reprogram the tumor microenvironment (TME) towards a pro-TH1 pattern
(CXCL10, IFNγ) (88), or metabolites including butyrate and niacin can mediate Gpr109a-
dependent induction of IL-18 in colonic epithelium and suppress colitis and colon cancer
(89).
Tumor-associated, NOX2-mediated, myeloid cell ROS production is reduced by antibiotic

administration or germ-free status, reducing oxaliplatin’s capacity to mediate early tumor
genotoxicity (90). Similarly, commensal microbiota primed tumor-associated innate myeloid
cells for TNFα (IL-1β, IL-12 and Cxcl10) production in response to anti-IL-10R/CpG-ODN
Author Manuscript
treatment, and antibiotics, germ-free, or TLR4−/− status attenuated this response and the
TNF-dependent early tumor necrosis (90). Supporting the adjuvant role of commensals
against developing cancers, pasteurized A. muciniphila or its pili-like TLR2 agonist blunted
azoxymethane-induced colitis and colon carcinogenesis by inducing TNFα-producing
cytotoxic T lymphocytes in mesenteric lymph nodes (mLNs) that eventually reached
the colonic mucosa (91). Spontaneous gut bacterial translocation in Tet2−/− mice also
drove pre-leukemic myeloproliferation (PMP), which leads to leukemia if unchecked, in
an IL-6-dependent manner (47). PMP was reversible with antibiotics and abolished in

germ-free mice, suggesting new clinical management opportunities. However, an intact gut
Author Manuscript
microbiome was later shown to be necessary to prevent leukemia progression in genetically

predisposed mice (92).
Non-hematopoietic components of the intestinal mucosa are also linked to the TME (77).
Gene deficient mice and BM chimeras identified a role for RNF5, an E3 ubiquitin ligase,
in immunosurveillance of severe melanoma. Rnf5−/− mice exhibited decreased secretion
of antimicrobial peptides and increased cell death in the ileal crypts, causing changes in
intestinal microbiota community composition. This bowel injury amplified mobilization of
CCR7-expressing DCs to Peyer’s patches, mLNs and melanoma-draining LNs, increasing
IFNγ-producing T lymphocyte tumor infiltration. Confirming a Rnf5−/−-specific microbial
effect, co-housing Rnf5−/− mice with wild type mice, or administering antibiotics, restored
tumor aggressiveness while oral gavage with 11 species overrepresented in Rnf5−/− animals
(Bacteroides and Parabacteroides spp.) into germ-free wild type mice recapitulated tumor
Author Manuscript
immunosurveillance (77). In another study, oxaliplatin-induced caspase 3/7-dependent

ileal apoptosis of crypts coincided with immunogenic bacteria dominance in the ileal
mucosa (75). These commensals regulated TFH cell priming in mLNs, culminating in
B cell activation, Ig production and infiltration of colon cancers with tumor-infiltrating
lymphocytes (TILs) in mice and patients. Anti-CTLA-4-induced gut barrier dysfunction
was also critical for systemic translocation of Bifidobacterium-derived inosine, in turn
promoting TH1 activation and antitumor immunity by agonizing T cell-specific A2AR
signaling in the context of DC costimulation (93). These examples illustrate that barrier
injury is accompanied by a deviation of the local microbiome or translocation of microbial
metabolites, that, in turn, mobilizes DCs to and outside the GALT and contributes to tumor
bed infiltration by activated helper or cytotoxic T cells.
Author Manuscript
The TME comprises not only stromal, tumor, endothelial cells, and hematopoietic
progenitor-derived immune components, but also a dense network of adrenergic nerve
fibres that influence oncogenesis in brain and non-brain cancers (94–97). Interestingly,
enteric nervous system neurons are both affected by the gut microbiota and functionally
tuned according to their location in the gut. A subset of microbiota-responsive neurons
could influence metabolic control independent of the central nervous system (98). These
findings suggest intimate relationships between mucosal or tumoral commensals and tumor
innervation that need further study.
Gut microbiota-mediated effects on anti-cancer drugs—Gut microbes are

intimately involved in biotransformation of xenobiotics, including cancer drugs, with
unintended consequences for clinical cancer control (99). For example, in prostate cancer,
Author Manuscript
abiraterone acetate (AA) was used as an energy source by A. muciniphila and inhibited
Corynebacterium species relying on AA-inhibited androgens for growth (100). Because A.
muciniphila is anti-inflammatory and Corynebacterium species are pro-inflammatory, this
change in their relative abundances increased pharmacologic efficacy of AA therapy. A.
muciniphila’s immunomodulatory effects (78), including association with responders during
PD-1 blockade (17), has prompted speculation that increased A. muciniphila may explain
the efficacy of AA in androgen-independent prostate cancer (100), although this remains

to be tested in large patient cohorts. These types of bidirectional drug-microbiota feedback

Author Manuscript
loops warrant further study.
Intratumor microbiota effects on the tumor microenvironment—Mechanistic

studies of live microbiota within diverse tumor types have been limited, particularly outside
the aerodigestive tract, but many of their effects on the TME appear to suppress local
antitumor immunity (15, 23, 34, 46, 75, 101, 102). Additionally, intratumor microbes
have been reported to have cancer-specific effects on (i) gastrointestinal and urinary tract
mutagenesis via secreted genotoxins, most notably pks+ E. coli-derived colibactin and
B. fragilis-derived toxin (22, 37, 38, 40, 41); (ii) CagA-mediated or IL-17-producing
γδ T cell-mediated inflammation in stomach and lung cancers, respectively (18, 103);
(iii) chemoresistance via direct microbial metabolism (cytidine deaminase degradation of
gemcitabine) in pancreatic cancer or indirect amplification of cancer cell autophagy in
colorectal cancer (14, 104); (iv) tumor proliferation via fungal activation of the host’s C3
Author Manuscript
complement cascade in pancreatic cancer (20); and (v) metastasis through upregulating
tumor matrix metalloproteinases in breast cancer or reducing tumor immunosurveillance
in lung cancer (34, 46). Immunologically, intratumor microbes often create tolerogenic
programming through PRR ligation with lower proportions of TILs, including CD8+ T cells,
and occasionally more CD4+CD25+FoxP3+ Tregs, as observed in colorectal, pancreatic,
breast, and lung cancers (18, 23, 34, 46, 75, 101, 102). However, in certain cases, injection
of intratumoral bacteria or their antigens may conversely provide immunostimulatory
effects, as demonstrated by Coley’s toxins and recent developments in bacterial cancer
therapy (5, 105, 106). In breast cancer, experiments comparing SCID-beige and C57BL/6
mice with intratumor Fusobacterium suggested lymphoid-lineage cells as key mediators
of intratumor microbiota-derived effects on tumor immunosurveillance (34). There are
also associations between intratumor microbiota and immunogenicity, including differential
Author Manuscript
melanoma immunotherapy response and triple-negative breast cancer associations, but their
underlying mechanisms remain uncharacterized (12).
Extra-intestinal barriers and cancer microbiota—Given that the intestinal barrier

offers the largest host-microbial interface and greatest microbial diversity, investigations on
the potential impact of the microbiota in oncogenesis or cancer prognosis have primarily
focused on this barrier (107). These studies could unveil cause-effect relationships between
gut microbial composition changes and compromised tumor immunosurveillance, even in
extraintestinal malignancies. However, it is noteworthy that extra-intestinal cancers can
develop within tissues that harbor their own microbiome and may play a role in the
exacerbation of neoplasia (12, 18, 25, 46).
Author Manuscript
For instance, the lungs’ surface approximates 1 m2/kg of body weight and is not sterile
(108). Experimental evidence in oncogene-driven autochthonous lung cancer models in
mice unveiled that local commensals may be perturbed by carcinogenesis, triggering an
inflammatory cross-talk between alveolar macrophages and IL-17 producing lung resident
γδ T cells contributing to tumor progression (18). The clinical significance of this
observation has been recently brought up in 83 lung cancer patients (25). Tsay et al.
highlighted that microaspiration of supraglottic commensals in lung cancer patients can

affect response to therapies and overall survival, due to a TH17-mediated exacerbated

Author Manuscript
inflammation corollary to immune checkpoint inhibition (25).
Skin is also recognized as our largest and outermost organ, maintaining host homeostasis
through tight interconnections between its resident microbes, keratinocytes, and skin
immune components through metabolic, innate, and cognate immune responses (109).
Compositional shifts in the skin microbiota appear to influence non-melanoma skin
carcinogenesis (110). Similarly, cervical cancer caused by persistent high-risk human
papillomavirus infection is often associated with a deviated cervical microflora (111,
112). The intertwined/interkingdom relationships between commensals and virus-associated
cancers, and their synergistic effects on tumorigenesis need further study, and exploration of
cancer-microbe interactions at other extra-intestinal barriers is warranted.
Cancer microbiome diagnostics

Author Manuscript
Variation in human microbiome composition among body sites (113) contrasts with stable
human genetics exhibiting only minor variation resulting from somatic mosaicism and
clonal hematopoiesis (114). Because both host tissues and microbiota are affected by
carcinogenesis, the genetic heterogeneity of microbes may provide an opportunity to
diagnose and locate disease. For example, a blood-derived TP53 mutation can indicate host
cancer status but implicates >25 cancer types (115); conversely, Streptococcus gallolyticus
(formerly S. bovis) bacteremia can reflect host cancer status and type (colon cancer)
based on its gastrointestinal origin (116, 117). Many challenges exist for microbial-based
diagnostics, including low biomass relative to host and confounding from reagent or
environmental contaminants. Many questions about their uniqueness, prevalence, stability
during cancer treatment, or utility during antibiotic administration remain to be answered
Author Manuscript
and must be addressed before clinical deployment.
Nearly all microbial-based cancer diagnostics are sequencing-based and have focused on
tumors within the aerodigestive tract (31), such as colorectal (118–121), pancreatic (122,
123), and lung cancer (124–126). It was only recently suggested that cancer types outside
of the aerodigestive tract, such as breast or brain cancer, may also harbor microbiota
with unique compositions. Nejman et al. (12) and Poore et al. (13) suggest distinct
intratumoral microbiomes among >30 cancer types (Fig. 3), proposing their applicability
to blood-based diagnostics and providing imaging evidence of these microbes’ intratumoral
spatial distribution and intracellular localization in seven cancer types, although imaging
evidence remains lacking for most cancer types.
Combining multi-region 16S rRNA amplicon sequencing, qPCR, immunohistochemistry

Author Manuscript
(lipopolysaccharide [LPS], lipoteichoic acid [LTA]), immunofluorescence (16S rRNA),

cultivation, and electron microscopy, Nejman et al. (12) surveyed 1010 tumors for bacteria
across melanoma, lung, ovarian, glioblastoma, pancreas, bone, and breast cancers. They
included 811 experimental controls, covering DNA extraction, PCR amplification, and
paraffin embedding, removing 94.3% of bacteria as contaminants. Examining the residual
528 bacterial species revealed significant differences in composition, diversity, and inferred
metabolic functionality between cancer types. Histologic imaging revealed heterogeneous

microbial spatial distributions (Fig. 3B) and their frequent intracellular localization in
Author Manuscript
cancer and immune cells (Fig. 3C). As described above in the Overview of the cancer
microbiome section, qPCR estimated the number of bacteria per tissue section, which we
have graphically depicted as percent bacterial composition per cancer type assuming tissue
homogeneity and 8 picograms of DNA per cancer cell (Fig. 3D). Applying their pipeline to
a melanoma immunotherapy cohort suggested microbiome differences between responders
and non-responders, but not yet a mechanism. Because bacteria were cultured from only
five human breast tumors, the widespread viability of intratumoral bacteria from this study
was unclear, particularly in cancers with reportedly fewer bacteria. However, other studies
have indeed shown cultivable bacteria in breast (127–129), lung (18), prostate (130, 131),
pancreas (14, 15), and colon cancers (19, 132), suggesting broad microbial viability. Still,
basic questions remain about the functional impacts of these intratumoral microbiota and
whether they are parasitic, symbiotic, or passive passengers, and a biopsy specimen is
Author Manuscript
required for analysis, limiting its diagnostic utility.
Poore et al. (13) took a different approach by harvesting all treatment-naive whole genome
and transcriptome studies from The Cancer Genome Atlas (TCGA) (n=18,116 samples; 33
cancer types) to study bacterial, viral, and archaeal nucleic acids. Because no experimental
controls were available, they filtered out historically-known reagent contaminants and
inferred other contaminants using per-sample DNA and RNA concentrations; these
steps removed up to 91.3% of microbial taxa. Machine learning revealed intratumor,
cancer-specific microbial signatures. Because colon cancer is epidemiologically linked
to clinical bacteremia (116, 117), they explored TCGA blood-derived normal samples
(n=1,866 samples) for cancer-specific microbial DNA and reported highly-accurate cancer
discrimination. They validated this blood-based diagnostic approach by comparing plasma-
derived cell-free microbial DNA from 100 patients with lung, prostate, or melanoma cancers
Author Manuscript
to those from 69 HIV-negative, healthy patients while implementing necessary experimental

contamination controls. Although closer to a practical diagnostic approach, the absence of
experimental controls in TCGA, sole reliance on deep sequencing data without orthogonal
approaches, and current lack of explainable mechanism(s) by which microbial DNA enters
into and survives circulation limits these findings. We speculate that the intracellular bacteria
in cancer and immune cells identified by Nejman et al. (12) may provide one source,
though this remains to be demonstrated. A rigorous evaluation alongside blood samples from
patients with non-lethal bodily infections, septic patients, and patients receiving antibiotics
during cancer care are necessary preconditions to broad clinical utility.
Modulation of the cancer microbiome

Author Manuscript
The associations between certain gastrointestinal microbiota and the activity of systemic
lymphoid tissues have stimulated interest in microbial modulation as a powerful
immunotherapeutic modality. If intratumoral microbiota are eventually verified to be
prevalent and immunologically active across most patients, as preliminary data suggest
(12, 13), such interventions must account for microbial niches and their crosstalk (Fig.
4). These dynamics sometimes appear related; for example, modulation of gut microbiota
influences the composition of the intratumoral microbiome in pancreatic cancer, presumably
via pancreatic duct communication (15, 20, 23). However, in other cases, these changes

are incongruent; for instance, antibiotics appear to abrogate immunotherapy response

Author Manuscript
by inhibiting the gut microbiome (133), but paradoxically they improve immunotherapy
efficacy by upregulating PD-1 expression when eliminating the pancreatic intratumoral
microbiome (23). These complexities necessitate more in-depth mechanistic studies of
modulation approaches and better clinical understanding before applying prebiotics,
probiotics, postbiotics, and antibiotics in the setting of cancer.
Antibiotics and the cancer microbiome

The use of antimicrobial therapy in cancer is limited to addressing or preventing known
microbial carcinogens. This includes treating H. pylori-derived gastric lymphomas with
triple or quadruple antibiotic therapy, administering direct-acting antivirals against active
Hepatitis C virus, and vaccinating against major human papillomavirus serotypes and
hepatitis B virus to prevent urogenital, cervical, head and neck, and liver cancers (36, 134,
Author Manuscript
135). Excluding antibiotic-derived chemotherapies (e.g. doxorubicin), there is circumstantial

and conflicting evidence for the use of antibiotics in solid tumors. Several studies in
lung, colon, and pancreatic cancer suggest that eliminating intratumoral microbiota can
check tumor-promoting inflammatory processes, reduce cellular proliferation, or convert a
tolerogenic TME to an immunogenic one (18, 19, 23, 46). However, increasing clinical
evidence suggests that systemic antibiotics abolish immune checkpoint blockade efficacy
and decrease patient survival (133, 136, 137). In hematologic malignancies, preclinical
evidence suggests a careful balance, where either antibiotics or gut bacterial translocation
can trigger leukemic progression in genetically-predisposed hosts (47, 92).
Prebiotics, postbiotics, and dietary interventions to modify the microbiome are also
promising. Dietary effects on cancer were recently reviewed in detail, with many
epidemiological associations but few causal mechanisms (138). Difficulties in dietary
Author Manuscript
data collection have impeded strong conclusions, but metabolomic data that can reveal
dietary intake and concomitant small molecule effectors may help in the future.
Prebiotics (molecules that promote growth of beneficial microbes) such as resistant starch,
inulin, and mucin are promising in preclinical models, improving antitumor immunity
and therapy response in melanoma and colon cancer (87), and are in clinical trials
(e.g. NCT03870607, NCT03950635). Experimental evidence of postbiotic compounds
(microbial-derived molecules) is limited in cancer, but they may provide advantages through
defined composition and manufacturing reproducibility (139).
Gut microbiota can also be modulated in cancer through fecal microbiota transplantation
(FMT), administration of defined microbial consortia, and commercial probiotics. FMT
treats Clostridium difficile (now Clostridioides difficile) colitis effectively (140), with
Author Manuscript
some efficacy in the treatment of immunotherapy-associated colitis (141). The long-term

efficacy and stability of FMT remain unknown (142). Targeting gut microbes clinically is
complicated by factors such as antibiotic pre-conditioning, administration route, frequency
of modulation, and dietary recommendations (142). Ongoing clinical trials suggest that FMT
from donors responsive to immunotherapy may enhance antitumor immune and potentially
clinical responses (NCT03353402, (143)). Additional clinical trials are evaluating the impact
of transferring microbial consortia, ranging in complexity from monoclonal bacterial strains

to multiplexed consortia. Few commercially available probiotic formulations have been

Author Manuscript
tested for impact on antitumor and systemic immunity, with certain formulations actually
increasing tumorigenesis (144). In critically ill patients, commercial probiotic use may even
cause bacteremia (145). Therefore, indiscriminate administration of commercially available
probiotics in cancer patients should be discouraged.
Cancer therapy using exogenous microbiota

Major strides have been made towards engineering exogenous bacterial and viral agents
for cancer therapy, particularly as powerful immunotherapy options or neoadjuvants. Two
such agents have FDA approval: oncolytic viral therapy for advanced melanoma using
talimogene laherparepvec (T-VEC) (146), and bacterial cancer therapy for high-risk,
non-muscle invasive bladder cancer using live-attenuated Mycobacterium bovis (BCG
vaccine) (147). Because oncolytic viruses are non-commensals and have been reviewed
Author Manuscript
elsewhere in detail (148–150), we focus our attention on bacterial cancer therapies

(BCTs). Though historically contentious, BCT is re-gaining attention through synthetic
biology techniques that programmatically limit systemic toxicities while enhancing regional
antitumor immunity (105, 106). Regulatory challenges for BCT agents are considerable
(Fig. 5A), and despite ongoing clinical trials (e.g. NCT04167137), they have yet to be
commercially surmounted (151).
Engineered microbes as cancer drugs

Natural bacterial mechanisms for tumor tropism are numerous (Fig. 5B), with intratumoral
or intravenous injection often leading to ~10,000-fold accumulation in tumors relative to
matched liver, spleen, and lung tissues (152, 153). This affinity for tumor tissue provides
a creative drug chassis and natural bridge from synthetic biology to cancer therapies,
Author Manuscript
whereby cytotoxic payloads can be encoded for programmed delivery by tumor-homing

bacteria (Figs. 5C–G). Thus far, genetically attenuated, auxotrophic, and inducible versions
of Escherichia, Bifidobacterium, Listeria, Shigella, Clostridium, Lactococcus, Vibrio, and
Salmonella species have been engineered and shown antitumor efficacy in preclinical
models with intravenous, intratumor, and oral delivery routes (147). While some approaches
are based on intracellular delivery of drugs via phagocytic uptake of bacteria, others
program bacteria to act as “intratumoral bioreactors” that continuously produce and release
payloads extracellularly as part of colonization. An interesting general approach implements
engineered bacterial lysis, which enables antitumor protein production or release only when
a predefined population density of bacteria is reached (154–157). This dramatically reduces
bacterial colony size and prevents systemic toxicities. Din and colleagues were the first to
demonstrate how non-pathogenic E. coli and Salmonella could be engineered to lyse at a
Author Manuscript
threshold population density, releasing a chemokine, hemolysin, or pro-apoptotic protein,

or all three, into the TME at desired periodic intervals (155). The drugs are delivered
cyclically as the bacterial population is programmed to generate growth-death-regrowth
cycles. Chowdhury et al. then used this design to produce and release an antibody-fragment
nanobody against CD47, which tumors can overexpress to inhibit DC phagocytosis (156).
Intriguingly, this stimulated a tumor-antigen-specific CD8+ T cell response that prevented
metastasis and mediated an abscopal effect, which regressed distal non-injected tumors as

well. This approach further precluded host anemias and thrombocytopenias usually seen
Author Manuscript
with systemic CD47 antagonism, suggesting a clinical opportunity. If intratumoral bacteria

prove to be prevalent across various cancer types, lysis circuit designs may also provide an
opportunity to flexibly engineer patient-specific, tumor-specific commensal strains (Fig. 5E)
or several strains in feedback with each other (Fig. 5F) to regulate payload release. Given
the many encodable cytotoxic payloads (Figs. 5G), a clear demonstration of BCT clinical
efficacy with minimal systemic toxicities could considerably increase the cancer therapy
armamentarium.
Outlook for the cancer microbiome

The last 15 years of microbiome research provide intriguing, though still controversial,
evidence of the relationships between microbes and cancer and the nuances of these
relationships. Few microbes directly cause cancer, but many more seem complicit, and,
Author Manuscript
perhaps counterintuitively, several promote host antitumor immunity. This complexity may
reflect shared evolutionary dynamics between the host’s immune system, its commensal
microbiota, and tumorigenic processes that we are just beginning to uncover (158–160).
A substantial literature gap still separates clinical observations and clinical interventions
targeted at microbiota in cancer. Although gut microbiota modulation in murine
immunotherapy models provides tantalizing results, they have not yet translated to
commercial therapeutic interventions in humans. Moreover, observations in humans and
mice of gut microbes that stratify therapy response, particularly immunotherapy (16, 17,
21, 26), have not uncommonly shown varying taxonomic differences that persist despite
uniform bioinformatic re-analyses, although there is greater concordance when examining
functional profiles (161, 162). Thus, many of the key problems that plagued researchers
Author Manuscript
in the early 20th century — contamination, irreproducibility, patient toxicities — remain

challenges today for microbially-based cancer diagnostics, prognostics, and exogenous
microbial therapeutics. Additional cohorts with carefully curated samples to limit and
mitigate potential contamination are needed to help characterize and understand the impact
of intratumoral microbes on carcinogenesis, cancer progression, and therapy response. Other
efforts are needed to examine non-bacterial relationships with cancer, gastrointestinally
and intratumorally, and their functions, particularly in relationship with known bacterial
functions. Further consortium-level efforts are necessary to assess the quantitative impact
of technical variables (e.g. DNA extraction, sample handling, bioinformatic protocols) on
cancer microbiome data and guide the selection of “gold-standard” pipelines, analogous to
the Microbiome Quality Control consortium’s analysis of fecal amplicon sequencing among
15 laboratories and nine bioinformatic protocols (163).
Author Manuscript
Many of these challenges would be aided by a multi-center, longitudinal, concerted effort to

study microbiota in cancer, analogous to TCGA’s role in elucidating the somatic mutation
landscape, with joint tumor, blood, and stool collection, multi-omic data generation,
and incorporation of experimental contamination controls (Fig. 6) (164). Concurrent meta-
analyses of existent cancer datasets with uniform in silico host depletion, decontamination,
taxonomy calling, and functional profiling may be able to identify global microbial drivers
in cancer pathogenesis and treatment despite technical variation between individual studies

(13, 118, 119, 165–167). Completion of microbiota modulation trials are additionally crucial
Author Manuscript
for guiding clinical applications and increasing the cancer therapy armamentarium (142),
with new evidence demonstrating that modulation of the gut microbiota using FMT in
immunotherapy-refractory melanoma patients is associated with clinical responses and
changes in the gut and tumor immune microenvironment (143) (Davar et al., In press).
In-depth functional analyses at community and per-microbe scales are likely necessary
to elucidate microbial-immune-cancer cell mechanistic interactions and emerging spatial
multi-omic tools may prove invaluable here (168, 169). Engineered organoids with immune
and microbiota niches or metabolites may further help validate or refute microbial causality
or complicity in carcinogenesis, as recently demonstrated by colibactin mutagenesis studies
(22, 170). In sum, although many challenges remain, building a better understanding of the
roles of microbes in cancer may enable a powerful new toolkit for improving patient care.
Author Manuscript
ACKNOWLEDGEMENTS
We thank C. Sepich-Poore (University of Chicago), R. Sullivan (Massachusetts General Hospital), and J. Mesirov
(University of California San Diego) for providing critical review and feedback on the manuscript and figures.
Figures were created with BioRender.com. The authors also wish to acknowledge the patients and their families
who have helped contribute towards a better understanding of this field.
Funding:
G.D.S-P. is supported by a fellowship from the US National Institutes of Health, National Cancer Institute (F30
CA243480). J.H. and R.K. are supported by the National Cancer Institute of the National Institutes of Health under
award number R01CA255206. The L.Z. laboratory is supported by RHU Torino Lumière (ANR-16-RHUS-0008),
the ONCOBIOME project (European Union Horizon 2020 programme), the Seerave Foundation, the French
Agence Nationale de la Recherche (Ileobiome), the French Ligue Contre le Cancer (Équipe Labelisées programme),
the French Association pour la Recherche sur le Cancer, Cancéropôle Ile-de-France, the French Fondation pour
la Recherche Médicale, a donation by Elior and Dassault Systems, the European Research Council, Fondation
Carrefour, the French Institut National du Cancer INSERM (HTE programme), LabEx Immuno-Oncology, SIRIC
Stratified Oncology Cell DNA Repair and Tumour Immune Elimination (SOCRATE) and the CARE network
Author Manuscript
(directed by X. Mariette, Assistance Publique — Hôpitaux de Paris, Kremlin-Bicêtre).
Competing interests:
G.D.S-P. and R.K. are inventors on a US patent application (PCT/US2019/059647) submitted by The Regents of
the University of California and licensed by Micronoma that covers methods of diagnosing and treating cancer
using microbial biomarkers in blood and cancer tissues. G.D.S-P. and R.K. are founders of and report stock
interest in Micronoma. R.K. additionally is a member of the scientific advisory board for GenCirq, holds an equity
interest in GenCirq, and can receive reimbursements for expenses up to US$5,000 per year. R.S. received a grant
from Merck EMD Serono, is a member of the scientific advisory board for Micronoma, and is a paid adviser to
Biomica and BiomX. R.S. is also a co-inventor on a U.S. provisional patent application (63/005,540) submitted
by Yeda Research and Development, the Weizmann Institute of Science, that covers methods of diagnosing and
treating cancer using microbial biomarkers in cancer tissues. J.H. is a founder of and has a financial interest in
GenCirq, which focuses on cancer therapeutics. J.W. is an inventor on a US patent application (PCT/US17/53.717)
submitted by the University of Texas MD Anderson Cancer Center that covers methods to enhance immune
checkpoint blockade responses by modulating the microbiome. J.W. further reports compensation and honoraria
from Imedex, Dava Oncology, Omniprex, Ilumina, Gilead, PeerView, Physician Education Resource, MedImmune
Author Manuscript
and Bristol-Myers Squibb. J.W. serves as a consultant advisory board member for Roche/Genentech, Novartis,
AstraZeneca, GlaxoSmithKline, Bristol-Myers Squibb, Merck, Biothera Pharmaceuticals and Microbiome DX. J.W.
also receives research support from GlaxoSmithKline, Roche/Genentech, Bristol-Myers Squibb and Novartis. J.W.
is an adviser and has stock options for Ella Therapeutics. L.Z. is a founder of everImmune, a biotechnology
company that develops anticancer probiotics and diagnostic tools to define intestinal dysbiosis in cancer. L.Z. also
has an active scientific collaboration (research contract) with Kaleido, Innovate Pharma, and Bioaster, which are
companies involved in the microbiome space.

REFERENCES AND NOTES

Author Manuscript
1. Ebbell B, The Papyrus Ebers: the greatest Egyptian medical document (Levin & Munksgaard,
1937).
2. Hoption Cann SA, van Netten JP, van Netten C, Dr William Coley and tumour regression: a place in
history or in the future. Postgrad. Med. J 79, 672–680 (2003). [PubMed: 14707241]
3. Busch W, Aus der Sitzung der medicinischen Section vom 13 November 1867. Berl Klin
Wochenschr. 5, 137 (1868).
4. Fehleisen F, Ueber die Züchtung der Erysipelkokken auf künstlichem Nährboden und ihre
Übertragbarkeit auf den Menschen. Dtsch. Med. Wochenschr 8, 553–554 (1882).
5. Starnes CO, Coley’s toxins in perspective. Nature. 357, 11–12 (1992). [PubMed: 1574121]
6. Glover TJ, The bacteriology of cancer. Canada Lancet Pract. 75, 92–111 (1930).
7. Livingston-Wheeler therapy. CA Cancer J. Clin 40, 103–108 (1990). [PubMed: 2106368]
8. Rous P, A SARCOMA OF THE FOWL TRANSMISSIBLE BY AN AGENT SEPARABLE FROM
THE TUMOR CELLS. J. Exp. Med 13, 397–411 (1911). [PubMed: 19867421]
Author Manuscript
9. White MK, Pagano JS, Khalili K, Viruses and human cancers: a long road of discovery of molecular
paradigms. Clin. Microbiol. Rev 27, 463–481 (2014). [PubMed: 24982317]
10. Hanahan D, Weinberg RA, The hallmarks of cancer. Cell. 100, 57–70 (2000). [PubMed: 10647931]
11. Hanahan D, Weinberg RA, Hallmarks of cancer: the next generation. Cell. 144, 646–674 (2011).
[PubMed: 21376230]
12. Nejman D, Livyatan I, Fuks G, Gavert N, Zwang Y, Geller LT, Rotter-Maskowitz A, Weiser
R, Mallel G, Gigi E, Meltser A, Douglas GM, Kamer I, Gopalakrishnan V, Dadosh T, Levin-
Zaidman S, Avnet S, Atlan T, Cooper ZA, Arora R, Cogdill AP, Khan MAW, Ologun G, Bussi
Y, Weinberger A, Lotan-Pompan M, Golani O, Perry G, Rokah M, Bahar-Shany K, Rozeman EA,
Blank CU, Ronai A, Shaoul R, Amit A, Dorfman T, Kremer R, Cohen ZR, Harnof S, Siegal T,
Yehuda-Shnaidman E, Gal-Yam EN, Shapira H, Baldini N, Langille MGI, Ben-Nun A, Kaufman
B, Nissan A, Golan T, Dadiani M, Levanon K, Bar J, Yust-Katz S, Barshack I, Peeper DS, Raz
DJ, Segal E, Wargo JA, Sandbank J, Shental N, Straussman R, The human tumor microbiome is
composed of tumor type-specific intracellular bacteria. Science. 368, 973–980 (2020). [PubMed:
32467386]
Author Manuscript
13. Poore GD, Kopylova E, Zhu Q, Carpenter C, Fraraccio S, Wandro S, Kosciolek T, Janssen S,
Metcalf J, Song SJ, Kanbar J, Miller-Montgomery S, Heaton R, Mckay R, Patel SP, Swafford AD,
Knight R, Microbiome analyses of blood and tissues suggest cancer diagnostic approach. Nature.
579, 567–574 (2020). [PubMed: 32214244]
14. Geller LT, Barzily-Rokni M, Danino T, Jonas OH, Shental N, Nejman D, Gavert N, Zwang Y,
Cooper ZA, Shee K, Thaiss CA, Reuben A, Livny J, Avraham R, Frederick DT, Ligorio M,
Chatman K, Johnston SE, Mosher CM, Brandis A, Fuks G, Gurbatri C, Gopalakrishnan V, Kim
M, Hurd MW, Katz M, Fleming J, Maitra A, Smith DA, Skalak M, Bu J, Michaud M, Trauger
SA, Barshack I, Golan T, Sandbank J, Flaherty KT, Mandinova A, Garrett WS, Thayer SP, Ferrone
CR, Huttenhower C, Bhatia SN, Gevers D, Wargo JA, Golub TR, Straussman R, Potential role
of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine.
Science. 357, 1156–1160 (2017). [PubMed: 28912244]
15. Riquelme E, Zhang Y, Zhang L, Montiel M, Zoltan M, Dong W, Quesada P, Sahin I, Chandra V,
San Lucas A, Scheet P, Xu H, Hanash SM, Feng L, Burks JK, Do K-A, Peterson CB, Nejman
D, Tzeng C-WD, Kim MP, Sears CL, Ajami N, Petrosino J, Wood LD, Maitra A, Straussman R,
Author Manuscript
Katz M, White JR, Jenq R, Wargo J, McAllister F, Tumor Microbiome Diversity and Composition
Influence Pancreatic Cancer Outcomes. Cell. 178, 795–806.e12 (2019). [PubMed: 31398337]
16. Gopalakrishnan V, Spencer CN, Nezi L, Reuben A, Andrews MC, Karpinets TV, Prieto PA,
Vicente D, Hoffman K, Wei SC, Cogdill AP, Zhao L, Hudgens CW, Hutchinson DS, Manzo T,
Petaccia de Macedo M, Cotechini T, Kumar T, Chen WS, Reddy SM, Szczepaniak Sloane R,
Galloway-Pena J, Jiang H, Chen PL, Shpall EJ, Rezvani K, Alousi AM, Chemaly RF, Shelburne
S, Vence LM, Okhuysen PC, Jensen VB, Swennes AG, McAllister F, Marcelo Riquelme Sanchez
E, Zhang Y, Le Chatelier E, Zitvogel L, Pons N, Austin-Breneman JL, Haydu LE, Burton EM,
Gardner JM, Sirmans E, Hu J, Lazar AJ, Tsujikawa T, Diab A, Tawbi H, Glitza IC, Hwu WJ,

Patel SP, Woodman SE, Amaria RN, Davies MA, Gershenwald JE, Hwu P, Lee JE, Zhang J,
Coussens LM, Cooper ZA, Futreal PA, Daniel CR, Ajami NJ, Petrosino JF, Tetzlaff MT, Sharma P,
Author Manuscript
Allison JP, Jenq RR, Wargo JA, Gut microbiome modulates response to anti-PD-1 immunotherapy
in melanoma patients. Science. 359, 97–103 (2018). [PubMed: 29097493]
17. Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillère R, Fluckiger A, Messaoudene
M, Rauber C, Roberti MP, Fidelle M, Flament C, Poirier-Colame V, Opolon P, Klein C, Iribarren
K, Mondragón L, Jacquelot N, Qu B, Ferrere G, Clémenson C, Mezquita L, Masip JR, Naltet
C, Brosseau S, Kaderbhai C, Richard C, Rizvi H, Levenez F, Galleron N, Quinquis B, Pons N,
Ryffel B, Minard-Colin V, Gonin P, Soria J-C, Deutsch E, Loriot Y, Ghiringhelli F, Zalcman
G, Goldwasser F, Escudier B, Hellmann MD, Eggermont A, Raoult D, Albiges L, Kroemer G,
Zitvogel L, Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial
tumors. Science. 359, 91–97 (2018). [PubMed: 29097494]
18. Jin C, Lagoudas GK, Zhao C, Bullman S, Bhutkar A, Hu B, Ameh S, Sandel D, Liang XS,
Mazzilli S, Whary MT, Meyerson M, Germain R, Blainey PC, Fox JG, Jacks T, Commensal
Microbiota Promote Lung Cancer Development via γδ T Cells. Cell. 176, 998–1013.e16 (2019).
[PubMed: 30712876]
19. Bullman S, Pedamallu CS, Sicinska E, Clancy TE, Zhang X, Cai D, Neuberg D, Huang K, Guevara
Author Manuscript
F, Nelson T, Chipashvili O, Hagan T, Walker M, Ramachandran A, Diosdado B, Serna G, Mulet N,

Landolfi S, Ramon Y Cajal S, Fasani R, Aguirre AJ, Ng K, Élez E, Ogino S, Tabernero J, Fuchs
CS, Hahn WC, Nuciforo P, Meyerson M, Analysis of Fusobacterium persistence and antibiotic
response in colorectal cancer. Science. 358, 1443–1448 (2017). [PubMed: 29170280]
20. Aykut B, Pushalkar S, Chen R, Li Q, Abengozar R, Kim JI, Shadaloey SA, Wu D, Preiss P, Verma
N, Guo Y, Saxena A, Vardhan M, Diskin B, Wang W, Leinwand J, Kurz E, Kochen Rossi JA,
Hundeyin M, Zambrinis C, Li X, Saxena D, Miller G, The fungal mycobiome promotes pancreatic
oncogenesis via activation of MBL. Nature. 574, 264–267 (2019). [PubMed: 31578522]
21. Matson V, Fessler J, Bao R, Chongsuwat T, Zha Y, Alegre M-L, Luke JJ, Gajewski TF, The
commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients.
Science. 359, 104–108 (2018). [PubMed: 29302014]
22. Pleguezuelos-Manzano C, Puschhof J, Rosendahl Huber A, van Hoeck A, Wood HM, Nomburg J,
Gurjao C, Manders F, Dalmasso G, Stege PB, Paganelli FL, Geurts MH, Beumer J, Mizutani T,
Miao Y, van der Linden R, van der Elst S, Genomics England Research Consortium, Garcia KC,
Author Manuscript
Top J, Willems RJL, Giannakis M, Bonnet R, Quirke P, Meyerson M, Cuppen E, van Boxtel R,
Clevers H, Mutational signature in colorectal cancer caused by genotoxic pks E. coli. Nature. 580,
269–273 (2020). [PubMed: 32106218]
23. Pushalkar S, Hundeyin M, Daley D, Zambirinis CP, Kurz E, Mishra A, Mohan N, Aykut
B, Usyk M, Torres LE, Werba G, Zhang K, Guo Y, Li Q, Akkad N, Lall S, Wadowski B,
Gutierrez J, Kochen Rossi JA, Herzog JW, Diskin B, Torres-Hernandez A, Leinwand J, Wang
W, Taunk PS, Savadkar S, Janal M, Saxena A, Li X, Cohen D, Sartor RB, Saxena D, Miller G,
The Pancreatic Cancer Microbiome Promotes Oncogenesis by Induction of Innate and Adaptive
Immune Suppression. Cancer Discov. 8, 403–416 (2018). [PubMed: 29567829]
24. Fulbright LE, Ellermann M, Arthur JC, The microbiome and the hallmarks of cancer. PLoS
Pathog. 13, e1006480 (2017). [PubMed: 28934351]
25. Tsay J-CJ, Wu BG, Sulaiman I, Gershner K, Schluger R, Li Y, Yie T-A, Meyn P, Olsen E,
Perez L, Franca B, Carpenito J, Iizumi T, El-Ashmawy M, Badri M, Morton JT, Shen N, He
L, Michaud G, Rafeq S, Bessich JL, Smith RL, Sauthoff H, Felner K, Pillai R, Zavitsanou
A-M, Koralov SB, Mezzano V, Loomis CA, Moreira AL, Moore W, Tsirigos A, Heguy A, Rom
Author Manuscript
WN, Sterman DH, Pass HI, Clemente JC, Li H, Bonneau R, Wong K-K, Papagiannakopoulos
T, Segal LN, Lower airway dysbiosis affects lung cancer progression. Cancer Discov. (2020),
doi:10.1158/2159-8290.CD-20-0263.
26. Frankel AE, Coughlin LA, Kim J, Froehlich TW, Xie Y, Frenkel EP, Koh AY, Metagenomic
Shotgun Sequencing and Unbiased Metabolomic Profiling Identify Specific Human Gut
Microbiota and Metabolites Associated with Immune Checkpoint Therapy Efficacy in Melanoma
Patients. Neoplasia. 19, 848–855 (2017). [PubMed: 28923537]
27. Sender R, Fuchs S, Milo R, Revised Estimates for the Number of Human and Bacteria Cells in the
Body. PLoS Biol. 14, e1002533 (2016). [PubMed: 27541692]

28. Belkaid Y, Naik S, Compartmentalized and systemic control of tissue immunity by commensals.
Nat. Immunol 14, 646–653 (2013). [PubMed: 23778791]
Author Manuscript
29. Fung TC, Olson CA, Hsiao EY, Interactions between the microbiota, immune and nervous systems
in health and disease. Nat. Neurosci 20, 145–155 (2017). [PubMed: 28092661]
30. Zitvogel L, Ma Y, Raoult D, Kroemer G, Gajewski TF, The microbiome in cancer immunotherapy:
Diagnostic tools and therapeutic strategies. Science. 359, 1366–1370 (2018). [PubMed: 29567708]
31. Garrett WS, Cancer and the microbiota. Science. 348, 80–86 (2015). [PubMed: 25838377]
32. Gopalakrishnan V, Helmink BA, Spencer CN, Reuben A, Wargo JA, The Influence of the Gut
Microbiome on Cancer, Immunity, and Cancer Immunotherapy. Cancer Cell. 33, 570–580 (2018).
[PubMed: 29634945]
33. Helmink BA, Khan MAW, Hermann A, Gopalakrishnan V, Wargo JA, The microbiome, cancer, and
cancer therapy. Nat. Med 25, 377–388 (2019). [PubMed: 30842679]
34. Parhi L, Alon-Maimon T, Sol A, Nejman D, Shhadeh A, Fainsod-Levi T, Yajuk O, Isaacson B,
Abed J, Maalouf N, Nissan A, Sandbank J, Yehuda-Shnaidman E, Ponath F, Vogel J, Mandelboim
O, Granot Z, Straussman R, Bachrach G, Breast cancer colonization by Fusobacterium nucleatum
accelerates tumor growth and metastatic progression. Nat. Commun 11, 3259 (2020). [PubMed:
Author Manuscript
32591509]
35. Locey KJ, Lennon JT, Scaling laws predict global microbial diversity. Proc. Natl. Acad. Sci. U. S.
A 113, 5970–5975 (2016). [PubMed: 27140646]
36. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, Biological agents.
Volume 100 B. A review of human carcinogens. IARC Monogr. Eval. Carcinog. Risks Hum 100,
1–441 (2012).
37. Wilson MR, Jiang Y, Villalta PW, Stornetta A, Boudreau PD, Carrá A, Brennan CA, Chun E,
Ngo L, Samson LD, Engelward BP, Garrett WS, Balbo S, Balskus EP, The human gut bacterial
genotoxin colibactin alkylates DNA. Science. 363 (2019), doi:10.1126/science.aar7785.
38. Dejea CM, Fathi P, Craig JM, Boleij A, Taddese R, Geis AL, Wu X, DeStefano Shields CE,
Hechenbleikner EM, Huso DL, Anders RA, Giardiello FM, Wick EC, Wang H, Wu S, Pardoll
DM, Housseau F, Sears CL, Patients with familial adenomatous polyposis harbor colonic biofilms
containing tumorigenic bacteria. Science. 359, 592–597 (2018). [PubMed: 29420293]
39. He Z, Gharaibeh RZ, Newsome RC, Pope JL, Dougherty MW, Tomkovich S, Pons B, Mirey
Author Manuscript
G, Vignard J, Hendrixson DR, Jobin C, Campylobacter jejuni promotes colorectal tumorigenesis

through the action of cytolethal distending toxin. Gut. 68, 289–300 (2019). [PubMed: 30377189]
40. Boot A, Ng AWT, Chong FT, Ho S-C, Yu W, Tan DSW, Iyer NG, Rozen SG, Characterization of
colibactin-associated mutational signature in an Asian oral squamous cell carcinoma and in other
mucosal tumor types. Genome Res. 30, 803–813 (2020). [PubMed: 32661091]
41. Barrett M, Hand CK, Shanahan F, Murphy T, O’Toole PW, Mutagenesis by Microbe: the Role of
the Microbiota in Shaping the Cancer Genome. Trends Cancer Res. 6, 277–287 (2020).
42. Silva-García O, Valdez-Alarcón JJ, Baizabal-Aguirre VM, Wnt/β-Catenin Signaling as a Molecular
Target by Pathogenic Bacteria. Front. Immunol 10, 2135 (2019). [PubMed: 31611869]
43. Rubinstein MR, Wang X, Liu W, Hao Y, Cai G, Han YW, Fusobacterium nucleatum promotes
colorectal carcinogenesis by modulating E-cadherin/β-catenin signaling via its FadA adhesin. Cell
Host Microbe. 14, 195–206 (2013). [PubMed: 23954158]
44. Kadosh E, Snir-Alkalay I, Venkatachalam A, May S, Lasry A, Elyada E, Zinger A, Shaham M,
Vaalani G, Mernberger M, Stiewe T, Pikarsky E, Oren M, Ben-Neriah Y, The gut microbiome
switches mutant p53 from tumour-suppressive to oncogenic. Nature. 586, 133–138 (2020).
Author Manuscript
[PubMed: 32728212]
45. Ma C, Han M, Heinrich B, Fu Q, Zhang Q, Sandhu M, Agdashian D, Terabe M, Berzofsky
JA, Fako V, Ritz T, Longerich T, Theriot CM, McCulloch JA, Roy S, Yuan W, Thovarai V, Sen
SK, Ruchirawat M, Korangy F, Wang XW, Trinchieri G, Greten TF, Gut microbiome-mediated
bile acid metabolism regulates liver cancer via NKT cells. Science. 360 (2018), doi:10.1126/
science.aan5931.
46. Le Noci V, Guglielmetti S, Arioli S, Camisaschi C, Bianchi F, Sommariva M, Storti C, Triulzi
T, Castelli C, Balsari A, Tagliabue E, Sfondrini L, Modulation of Pulmonary Microbiota by

Antibiotic or Probiotic Aerosol Therapy: A Strategy to Promote Immunosurveillance against Lung

Metastases. Cell Rep. 24, 3528–3538 (2018). [PubMed: 30257213]
Author Manuscript
47. Meisel M, Hinterleitner R, Pacis A, Chen L, Earley ZM, Mayassi T, Pierre JF, Ernest JD, Galipeau
HJ, Thuille N, Bouziat R, Buscarlet M, Ringus DL, Wang Y, Li Y, Dinh V, Kim SM, McDonald
BD, Zurenski MA, Musch MW, Furtado GC, Lira SA, Baier G, Chang EB, Eren AM, Weber CR,
Busque L, Godley LA, Verdú EF, Barreiro LB, Jabri B, Microbial signals drive pre-leukaemic
myeloproliferation in a Tet2-deficient host. Nature. 557, 580–584 (2018). [PubMed: 29769727]
48. Almeida A, Mitchell AL, Boland M, Forster SC, Gloor GB, Tarkowska A, Lawley TD, Finn RD,
A new genomic blueprint of the human gut microbiota. Nature. 568, 499–504 (2019). [PubMed:
30745586]
49. Dion MB, Oechslin F, Moineau S, Phage diversity, genomics and phylogeny. Nat. Rev. Microbiol
18, 125–138 (2020). [PubMed: 32015529]
50. Chassaing B, Kumar M, Baker MT, Singh V, Vijay-Kumar M, Mammalian gut immunity. Biomed.
J 37, 246–258 (2014). [PubMed: 25163502]
51. The ICGC/TCGA Pan-Cancer Analysis of Whole Genomes Consortium, Pan-cancer analysis of
whole genomes. Nature. 578 (2020), pp. 82–93. [PubMed: 32025007]
Author Manuscript
52. Del Monte U, Does the cell number 10(9) still really fit one gram of tumor tissue? Cell Cycle. 8,
505–506 (2009). [PubMed: 19176997]
53. Erdag G, Schaefer JT, Smolkin ME, Deacon DH, Shea SM, Dengel LT, Patterson JW, Slingluff
CL Jr, Immunotype and immunohistologic characteristics of tumor-infiltrating immune cells are
associated with clinical outcome in metastatic melanoma. Cancer Res. 72, 1070–1080 (2012).
[PubMed: 22266112]
54. Kather JN, Suarez-Carmona M, Charoentong P, Weis C-A, Hirsch D, Bankhead P, Horning M,
Ferber D, Kel I, Herpel E, Schott S, Zörnig I, Utikal J, Marx A, Gaiser T, Brenner H, Chang-
Claude J, Hoffmeister M, Jäger D, Halama N, Topography of cancer-associated immune cells in
human solid tumors. Elife. 7 (2018), doi:10.7554/eLife.36967.
55. Zitvogel L, Ayyoub M, Routy B, Kroemer G, Microbiome and Anticancer Immunosurveillance.
Cell. 165, 276–287 (2016). [PubMed: 27058662]
56. Michelis FV, Messner HA, Loach D, Uhm J, Gupta V, Lipton JH, Seftel MD, Kuruvilla J, Kim
DD, Early lymphocyte recovery at 28 d post-transplant is predictive of reduced risk of relapse in
patients with acute myeloid leukemia transplanted with peripheral blood stem cell grafts. Eur. J.
Author Manuscript
Haematol 93, 273–280 (2014). [PubMed: 24725056]

57. Staffas A, Burgos da Silva M, Slingerland AE, Lazrak A, Bare CJ, Holman CD, Docampo MD,
Shono Y, Durham B, Pickard AJ, Cross JR, Stein-Thoeringer C, Velardi E, Tsai JJ, Jahn L, Jay
H, Lieberman S, Smith OM, Pamer EG, Peled JU, Cohen DE, Jenq RR, van den Brink MRM,
Nutritional Support from the Intestinal Microbiota Improves Hematopoietic Reconstitution after
Bone Marrow Transplantation in Mice. Cell Host Microbe. 23, 447–457.e4 (2018). [PubMed:
29576480]
58. Peled JU, Gomes ALC, Devlin SM, Littmann ER, Taur Y, Sung AD, Weber D, Hashimoto D,
Slingerland AE, Slingerland JB, Maloy M, Clurman AG, Stein-Thoeringer CK, Markey KA,
Docampo MD, Burgos da Silva M, Khan N, Gessner A, Messina JA, Romero K, Lew MV,
Bush A, Bohannon L, Brereton DG, Fontana E, Amoretti LA, Wright RJ, Armijo GK, Shono
Y, Sanchez-Escamilla M, Castillo Flores N, Alarcon Tomas A, Lin RJ, Yáñez San Segundo L,
Shah GL, Cho C, Scordo M, Politikos I, Hayasaka K, Hasegawa Y, Gyurkocza B, Ponce DM,
Barker JN, Perales M-A, Giralt SA, Jenq RR, Teshima T, Chao NJ, Holler E, Xavier JB, Pamer
EG, van den Brink MRM, Microbiota as Predictor of Mortality in Allogeneic Hematopoietic-Cell
Author Manuscript
Transplantation. N. Engl. J. Med 382, 822–834 (2020). [PubMed: 32101664]

59. Schluter J, Peled JU, Taylor BP, Markey KA, Smith M, Taur Y, Niehus R, Staffas A, Dai A,
Fontana E, Amoretti LA, Wright RJ, Morjaria S, Fenelus M, Pessin MS, Chao NJ, Lew M,
Bohannon L, Bush A, Sung AD, Hohl TM, Perales M-A, van den Brink MRM, Xavier JB, The
gut microbiota is associated with immune cell dynamics in humans. Nature (2020), doi:10.1038/
s41586-020-2971-8.
60. Brook I, Ledney GD, Effect of antimicrobial therapy on the gastrointestinal bacterial flora,
infection and mortality in mice exposed to different doses of irradiation. J. Antimicrob. Chemother
33, 63–72 (1994).

61. Burdelya LG, Krivokrysenko VI, Tallant TC, Strom E, Gleiberman AS, Gupta D, Kurnasov OV,
Fort FL, Osterman AL, Didonato JA, Feinstein E, Gudkov AV, An agonist of toll-like receptor
Author Manuscript
5 has radioprotective activity in mouse and primate models. Science. 320, 226–230 (2008).
[PubMed: 18403709]
62. Liu W, Chen Q, Wu S, Xia X, Wu A, Cui F, Gu Y-P, Zhang X, Cao J, Radioprotector WR-2721
and mitigating peptidoglycan synergistically promote mouse survival through the amelioration of
intestinal and bone marrow damage. J. Radiat. Res 56, 278–286 (2015). [PubMed: 25617317]
63. Guo H, Chou W-C, Lai Y, Liang K, Tam JW, Brickey WJ, Chen L, Montgomery ND, Li X,
Bohannon LM, Sung AD, Chao NJ, Peled JU, Gomes ALC, van den Brink MRM, French
MJ, Macintyre AN, Sempowski GD, Tan X, Sartor RB, Lu K, Ting JPY, Multi-omics analyses
of radiation survivors identify radioprotective microbes and metabolites. Science. 370 (2020),
doi:10.1126/science.aay9097.
64. Khosravi A, Yáñez A, Price JG, Chow A, Merad M, Goodridge HS, Mazmanian SK, Gut
microbiota promote hematopoiesis to control bacterial infection. Cell Host Microbe. 15, 374–381
(2014). [PubMed: 24629343]
65. Clarke TB, Davis KM, Lysenko ES, Zhou AY, Yu Y, Weiser JN, Recognition of peptidoglycan
Author Manuscript
from the microbiota by Nod1 enhances systemic innate immunity. Nat. Med 16, 228–231 (2010).
[PubMed: 20081863]
66. Fischer JC, Bscheider M, Eisenkolb G, Lin C-C, Wintges A, Otten V, Lindemans CA, Heidegger S,
Rudelius M, Monette S, Porosnicu Rodriguez KA, Calafiore M, Liebermann S, Liu C, Lienenklaus
S, Weiss S, Kalinke U, Ruland J, Peschel C, Shono Y, Docampo M, Velardi E, Jenq RR, Hanash
AM, Dudakov JA, Haas T, van den Brink MRM, Poeck H, RIG-I/MAVS and STING signaling
promote gut integrity during irradiation- and immune-mediated tissue injury. Sci. Transl. Med 9
(2017), doi:10.1126/scitranslmed.aag2513.
67. Honda K, Littman DR, The microbiota in adaptive immune homeostasis and disease. Nature. 535,
75–84 (2016). [PubMed: 27383982]
68. Viaud S, Saccheri F, Mignot G, Yamazaki T, Daillère R, Hannani D, Enot DP, Pfirschke C,
Engblom C, Pittet MJ, Schlitzer A, Ginhoux F, Apetoh L, Chachaty E, Woerther P-L, Eberl
G, Bérard M, Ecobichon C, Clermont D, Bizet C, Gaboriau-Routhiau V, Cerf-Bensussan N,
Opolon P, Yessaad N, Vivier E, Ryffel B, Elson CO, Doré J, Kroemer G, Lepage P, Boneca IG,
Ghiringhelli F, Zitvogel L, The intestinal microbiota modulates the anticancer immune effects of
Author Manuscript
cyclophosphamide. Science. 342, 971–976 (2013). [PubMed: 24264990]

69. Daillère R, Vétizou M, Waldschmitt N, Yamazaki T, Isnard C, Poirier-Colame V, Duong CPM,
Flament C, Lepage P, Roberti MP, Routy B, Jacquelot N, Apetoh L, Becharef S, Rusakiewicz S,
Langella P, Sokol H, Kroemer G, Enot D, Roux A, Eggermont A, Tartour E, Johannes L, Woerther
P-L, Chachaty E, Soria J-C, Golden E, Formenti S, Plebanski M, Madondo M, Rosenstiel P,
Raoult D, Cattoir V, Boneca IG, Chamaillard M, Zitvogel L, Enterococcus hirae and Barnesiella
intestinihominis Facilitate Cyclophosphamide-Induced Therapeutic Immunomodulatory Effects.
Immunity. 45, 931–943 (2016). [PubMed: 27717798]
70. Vétizou M, Pitt JM, Daillère R, Lepage P, Waldschmitt N, Flament C, Rusakiewicz S, Routy B,
Roberti MP, Duong CPM, Poirier-Colame V, Roux A, Becharef S, Formenti S, Golden E, Cording
S, Eberl G, Schlitzer A, Ginhoux F, Mani S, Yamazaki T, Jacquelot N, Enot DP, Bérard M, Nigou
J, Opolon P, Eggermont A, Woerther P-L, Chachaty E, Chaput N, Robert C, Mateus C, Kroemer
G, Raoult D, Boneca IG, Carbonnel F, Chamaillard M, Zitvogel L, Anticancer immunotherapy
by CTLA-4 blockade relies on the gut microbiota. Science. 350, 1079–1084 (2015). [PubMed:
26541610]
Author Manuscript
71. Sivan A, Corrales L, Hubert N, Williams JB, Aquino-Michaels K, Earley ZM, Benyamin
FW, Lei YM, Jabri B, Alegre M-L, Chang EB, Gajewski TF, Commensal Bifidobacterium
promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science. 350, 1084–1089
(2015). [PubMed: 26541606]
72. Uribe-Herranz M, Rafail S, Beghi S, Gil-de-Gómez L, Verginadis I, Bittinger K, Pustylnikov
S, Pierini S, Perales-Linares R, Blair IA, Mesaros CA, Snyder NW, Bushman F, Koumenis
C, Facciabene A, Gut microbiota modulate dendritic cell antigen presentation and radiotherapy-
induced antitumor immune response. J. Clin. Invest 130, 466–479 (2020). [PubMed: 31815742]

73. Bessell CA, Isser A, Havel JJ, Lee S, Bell DR, Hickey JW, Chaisawangwong W, Glick
Bieler J, Srivastava R, Kuo F, Purohit T, Zhou R, Chan TA, Schneck JP, Commensal bacteria
Author Manuscript
stimulate antitumor responses via T cell cross-reactivity. JCI Insight. 5 (2020), doi:10.1172/
jci.insight.135597.
74. Paulos CM, Wrzesinski C, Kaiser A, Hinrichs CS, Chieppa M, Cassard L, Palmer DC, Boni A,
Muranski P, Yu Z, Gattinoni L, Antony PA, Rosenberg SA, Restifo NP, Microbial translocation
augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4
signaling. J. Clin. Invest 117, 2197–2204 (2007). [PubMed: 17657310]
75. Roberti MP, Yonekura S, Duong CPM, Picard M, Ferrere G, Tidjani Alou M, Rauber C, Iebba
V, Lehmann CHK, Amon L, Dudziak D, Derosa L, Routy B, Flament C, Richard C, Daillère R,
Fluckiger A, Van Seuningen I, Chamaillard M, Vincent A, Kourula S, Opolon P, Ly P, Pizzato
E, Becharef S, Paillet J, Klein C, Marliot F, Pietrantonio F, Benoist S, Scoazec J-Y, Dartigues
P, Hollebecque A, Malka D, Pagès F, Galon J, Gomperts Boneca I, Lepage P, Ryffel B, Raoult
D, Eggermont A, Vanden Berghe T, Ghiringhelli F, Vandenabeele P, Kroemer G, Zitvogel L,
Chemotherapy-induced ileal crypt apoptosis and the ileal microbiome shape immunosurveillance
and prognosis of proximal colon cancer. Nat. Med (2020), doi:10.1038/s41591-020-0882-8.
76. Shi Y, Zheng W, Yang K, Harris KG, Ni K, Xue L, Lin W, Chang EB, Weichselbaum RR, Fu Y-X,
Author Manuscript
Intratumoral accumulation of gut microbiota facilitates CD47-based immunotherapy via STING

signaling. J. Exp. Med 217 (2020), doi:10.1084/jem.20192282.
77. Li Y, Tinoco R, Elmén L, Segota I, Xian Y, Fujita Y, Sahu A, Zarecki R, Marie K, Feng Y, Khateb
A, Frederick DT, Ashkenazi SK, Kim H, Perez EG, Day C-P, Segura Muñoz RS, Schmaltz R,
Yooseph S, Tam MA, Zhang T, Avitan-Hersh E, Tzur L, Roizman S, Boyango I, Bar-Sela G,
Orian A, Kaufman RJ, Bosenberg M, Goding CR, Baaten B, Levesque MP, Dummer R, Brown K,
Merlino G, Ruppin E, Flaherty K, Ramer-Tait A, Long T, Peterson SN, Bradley LM, Ronai ZA,
Gut microbiota dependent anti-tumor immunity restricts melanoma growth in Rnf5−/− mice. Nat.
Commun 10, 1492 (2019). [PubMed: 30940817]
78. Ansaldo E, Slayden LC, Ching KL, Koch MA, Wolf NK, Plichta DR, Brown EM, Graham DB,
Xavier RJ, Moon JJ, Barton GM, Akkermansia muciniphila induces intestinal adaptive immune
responses during homeostasis. Science. 364, 1179–1184 (2019). [PubMed: 31221858]
79. Gil-Cruz C, Perez-Shibayama C, De Martin A, Ronchi F, van der Borght K, Niederer R, Onder
L, Lütge M, Novkovic M, Nindl V, Ramos G, Arnoldini M, Slack EMC, Boivin-Jahns V, Jahns
Author Manuscript
R, Wyss M, Mooser C, Lambrecht BN, Maeder MT, Rickli H, Flatz L, Eriksson U, Geuking
MB, McCoy KD, Ludewig B, Microbiota-derived peptide mimics drive lethal inflammatory
cardiomyopathy. Science. 366, 881–886 (2019). [PubMed: 31727837]
80. Hebbandi Nanjundappa R, Ronchi F, Wang J, Clemente-Casares X, Yamanouchi J, Sokke
Umeshappa C, Yang Y, Blanco J, Bassolas-Molina H, Salas A, Khan H, Slattery RM, Wyss
M, Mooser C, Macpherson AJ, Sycuro LK, Serra P, McKay DM, McCoy KD, Santamaria P, A
Gut Microbial Mimic that Hijacks Diabetogenic Autoreactivity to Suppress Colitis. Cell. 171,
655–667.e17 (2017). [PubMed: 29053971]
81. Petersen J, Ciacchi L, Tran MT, Loh KL, Kooy-Winkelaar Y, Croft NP, Hardy MY, Chen Z,
McCluskey J, Anderson RP, Purcell AW, Tye-Din JA, Koning F, Reid HH, Rossjohn J, T cell
receptor cross-reactivity between gliadin and bacterial peptides in celiac disease. Nat. Struct. Mol.
Biol 27, 49–61 (2020). [PubMed: 31873306]
82. Snyder A, Makarov V, Merghoub T, Yuan J, Zaretsky JM, Desrichard A, Walsh LA, Postow MA,
Wong P, Ho TS, Hollmann TJ, Bruggeman C, Kannan K, Li Y, Elipenahli C, Liu C, Harbison CT,
Wang L, Ribas A, Wolchok JD, Chan TA, Genetic basis for clinical response to CTLA-4 blockade
Author Manuscript
in melanoma. N. Engl. J. Med 371, 2189–2199 (2014). [PubMed: 25409260]

83. Fluckiger A, Daillère R, Sassi M, Sixt BS, Liu P, Loos F, Richard C, Rabu C, Alou MT, Goubet
A-G, Lemaitre F, Ferrere G, Derosa L, Duong CPM, Messaoudene M, Gagné A, Joubert P, De
Sordi L, Debarbieux L, Simon S, Scarlata C-M, Ayyoub M, Palermo B, Facciolo F, Boidot R,
Wheeler R, Boneca IG, Sztupinszki Z, Papp K, Csabai I, Pasolli E, Segata N, Lopez-Otin C,
Szallasi Z, Andre F, Iebba V, Quiniou V, Klatzmann D, Boukhalil J, Khelaifia S, Raoult D, Albiges
L, Escudier B, Eggermont A, Mami-Chouaib F, Nistico P, Ghiringhelli F, Routy B, Labarrière N,
Cattoir V, Kroemer G, Zitvogel L, Cross-reactivity between tumor MHC class I-restricted antigens
and an enterococcal bacteriophage. Science. 369, 936–942 (2020). [PubMed: 32820119]

84. Tanoue T, Morita S, Plichta DR, Skelly AN, Suda W, Sugiura Y, Narushima S, Vlamakis H, Motoo
I, Sugita K, Shiota A, Takeshita K, Yasuma-Mitobe K, Riethmacher D, Kaisho T, Norman JM,
Author Manuscript
Mucida D, Suematsu M, Yaguchi T, Bucci V, Inoue T, Kawakami Y, Olle B, Roberts B, Hattori

M, Xavier RJ, Atarashi K, Honda K, A defined commensal consortium elicits CD8 T cells and
anti-cancer immunity. Nature. 565, 600–605 (2019). [PubMed: 30675064]
85. Coutzac C, Jouniaux J-M, Paci A, Schmidt J, Mallardo D, Seck A, Asvatourian V, Cassard L,
Saulnier P, Lacroix L, Woerther P-L, Vozy A, Naigeon M, Nebot-Bral L, Desbois M, Simeone
E, Mateus C, Boselli L, Grivel J, Soularue E, Lepage P, Carbonnel F, Ascierto PA, Robert C,
Chaput N, Systemic short chain fatty acids limit antitumor effect of CTLA-4 blockade in hosts
with cancer. Nat. Commun 11, 2168 (2020). [PubMed: 32358520]
86. Nomura M, Nagatomo R, Doi K, Shimizu J, Baba K, Saito T, Matsumoto S, Inoue K, Muto
M, Association of Short-Chain Fatty Acids in the Gut Microbiome With Clinical Response to
Treatment With Nivolumab or Pembrolizumab in Patients With Solid Cancer Tumors. JAMA
Network Open. 3 (2020), p. e202895. [PubMed: 32297948]
87. Li Y, Elmén L, Segota I, Xian Y, Tinoco R, Feng Y, Fujita Y, Segura Muñoz RR, Schmaltz
R, Bradley LM, Ramer-Tait A, Zarecki R, Long T, Peterson SN, Ronai ZA, Prebiotic-Induced
Anti-tumor Immunity Attenuates Tumor Growth. Cell Rep. 30, 1753–1766.e6 (2020). [PubMed:
Author Manuscript
32049008]
88. Kim OY, Park HT, Dinh NTH, Choi SJ, Lee J, Kim JH, Lee S-W, Gho YS, Bacterial outer
membrane vesicles suppress tumor by interferon-γ-mediated antitumor response. Nat. Commun 8,
626 (2017). [PubMed: 28931823]
89. Singh N, Gurav A, Sivaprakasam S, Brady E, Padia R, Shi H, Thangaraju M, Prasad PD,
Manicassamy S, Munn DH, Lee JR, Offermanns S, Ganapathy V, Activation of Gpr109a,
receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and
carcinogenesis. Immunity. 40, 128–139 (2014). [PubMed: 24412617]
90. Iida N, Dzutsev A, Stewart CA, Smith L, Bouladoux N, Weingarten RA, Molina DA, Salcedo
R, Back T, Cramer S, Dai R-M, Kiu H, Cardone M, Naik S, Patri AK, Wang E, Marincola FM,
Frank KM, Belkaid Y, Trinchieri G, Goldszmid RS, Commensal bacteria control cancer response
to therapy by modulating the tumor microenvironment. Science. 342, 967–970 (2013). [PubMed:
24264989]
91. Wang L, Tang L, Feng Y, Zhao S, Han M, Zhang C, Yuan G, Zhu J, Cao S, Wu Q, Li L, Zhang
Author Manuscript
Z, A purified membrane protein from Akkermansia muciniphila or the pasteurised bacterium

blunts colitis associated tumourigenesis by modulation of CD8+ T cells in mice. Gut (2020),
doi:10.1136/gutjnl-2019-320105.
92. Vicente Dueñas C, Janssen S, Oldenburg M, Auer F, González Herrero I, Casado-García A, Isidro-
Hernández M, Raboso-Gallego J, Westhoff P, Pandyra AA, Hein D, Gössling KL, Alonso-López
D, De Las Rivas J, Bhatia S, Garcia-Criado FJ, García Cenador MB, Weber APM, Köhrer K,
Hauer J, Fischer U, Sanchez-Garcia I, Borkhardt A, An intact gut microbiome protects genetically
predisposed mice against leukemia. Blood (2020), doi:10.1182/blood.2019004381.
93. Mager LF, Burkhard R, Pett N, Cooke NCA, Brown K, Ramay H, Paik S, Stagg J, Groves RA,
Gallo M, Lewis IA, Geuking MB, McCoy KD, Microbiome-derived inosine modulates response to
checkpoint inhibitor immunotherapy. Science. 369, 1481–1489 (2020). [PubMed: 32792462]
94. Amit M, Takahashi H, Dragomir MP, Lindemann A, Gleber-Netto FO, Pickering CR, Anfossi S,
Osman AA, Cai Y, Wang R, Knutsen E, Shimizu M, Ivan C, Rao X, Wang J, Silverman DA, Tam
S, Zhao M, Caulin C, Zinger A, Tasciotti E, Dougherty PM, El-Naggar A, Calin GA, Myers JN,
Loss of p53 drives neuron reprogramming in head and neck cancer. Nature. 578, 449–454 (2020).
Author Manuscript
[PubMed: 32051587]
95. Venkataramani V, Tanev DI, Strahle C, Studier-Fischer A, Fankhauser L, Kessler T, Körber C,
Kardorff M, Ratliff M, Xie R, Horstmann H, Messer M, Paik SP, Knabbe J, Sahm F, Kurz FT,
Acikgöz AA, Herrmannsdörfer F, Agarwal A, Bergles DE, Chalmers A, Miletic H, Turcan S,
Mawrin C, Hänggi D, Liu H-K, Wick W, Winkler F, Kuner T, Glutamatergic synaptic input to
glioma cells drives brain tumour progression. Nature. 573, 532–538 (2019). [PubMed: 31534219]
96. Zeng Q, Michael IP, Zhang P, Saghafinia S, Knott G, Jiao W, McCabe BD, Galván JA, Robinson
HPC, Zlobec I, Ciriello G, Hanahan D, Synaptic proximity enables NMDAR signalling to promote
brain metastasis. Nature. 573, 526–531 (2019). [PubMed: 31534217]

97. Venkatesh HS, Morishita W, Geraghty AC, Silverbush D, Gillespie SM, Arzt M, Tam LT, Espenel
C, Ponnuswami A, Ni L, Woo PJ, Taylor KR, Agarwal A, Regev A, Brang D, Vogel H, Hervey-
Author Manuscript
Jumper S, Bergles DE, Suvà ML, Malenka RC, Monje M, Electrical and synaptic integration of
glioma into neural circuits. Nature. 573, 539–545 (2019). [PubMed: 31534222]
98. Muller PA, Matheis F, Schneeberger M, Kerner Z, Jové V, Mucida D, Microbiota-modulated CART
enteric neurons autonomously regulate blood glucose. Science. 370, 314–321 (2020). [PubMed:
32855216]
99. Spanogiannopoulos P, Bess EN, Carmody RN, Turnbaugh PJ, The microbial pharmacists within
us: a metagenomic view of xenobiotic metabolism. Nature Reviews Microbiology. 14 (2016), pp.
273–287. [PubMed: 26972811]
100. Daisley BA, Chanyi RM, Abdur-Rashid K, Al KF, Gibbons S, Chmiel JA, Wilcox H, Reid G,
Anderson A, Dewar M, Nair SM, Chin J, Burton JP, Abiraterone acetate preferentially enriches
for the gut commensal Akkermansia muciniphila in castrate-resistant prostate cancer patients.
Nat. Commun 11, 4822 (2020). [PubMed: 32973149]
101. Mima K, Sukawa Y, Nishihara R, Qian ZR, Yamauchi M, Inamura K, Kim SA, Masuda A,
Nowak JA, Nosho K, Kostic AD, Giannakis M, Watanabe H, Bullman S, Milner DA, Harris
Author Manuscript
CC, Giovannucci E, Garraway LA, Freeman GJ, Dranoff G, Chan AT, Garrett WS, Huttenhower
C, Fuchs CS, Ogino S, Fusobacterium nucleatum and T Cells in Colorectal Carcinoma. JAMA
Oncol. 1, 653–661 (2015). [PubMed: 26181352]
102. Hamada T, Zhang X, Mima K, Bullman S, Sukawa Y, Nowak JA, Kosumi K, Masugi Y, Twombly
TS, Cao Y, Song M, Liu L, da Silva A, Shi Y, Gu M, Li W, Koh H, Nosho K, Inamura K, Keum
N, Wu K, Meyerhardt JA, Kostic AD, Huttenhower C, Garrett WS, Meyerson M, Giovannucci
EL, Chan AT, Fuchs CS, Nishihara R, Giannakis M, Ogino S, Fusobacterium nucleatum in
Colorectal Cancer Relates to Immune Response Differentially by Tumor Microsatellite Instability
Status. Cancer Immunol Res. 6, 1327–1336 (2018). [PubMed: 30228205]
103. Suzuki M, Mimuro H, Kiga K, Fukumatsu M, Ishijima N, Morikawa H, Nagai S, Koyasu
S, Gilman RH, Kersulyte D, Berg DE, Sasakawa C, Helicobacter pylori CagA phosphorylation-
independent function in epithelial proliferation and inflammation. Cell Host Microbe. 5, 23–34
(2009). [PubMed: 19154985]
104. Yu T, Guo F, Yu Y, Sun T, Ma D, Han J, Qian Y, Kryczek I, Sun D, Nagarsheth N, Chen Y, Chen
H, Hong J, Zou W, Fang J-Y, Fusobacterium nucleatum Promotes Chemoresistance to Colorectal
Author Manuscript
Cancer by Modulating Autophagy. Cell. 170, 548–563.e16 (2017). [PubMed: 28753429]

105. Forbes NS, Engineering the perfect (bacterial) cancer therapy. Nat. Rev. Cancer 10, 785–794
(2010). [PubMed: 20944664]
106. Zhou S, Gravekamp C, Bermudes D, Liu K, Tumour-targeting bacteria engineered to fight cancer.
Nat. Rev. Cancer 18, 727–743 (2018). [PubMed: 30405213]
107. Garrett WS, Gordon JI, Glimcher LH, Homeostasis and inflammation in the intestine. Cell. 140,
859–870 (2010). [PubMed: 20303876]
108. O’Dwyer DN, Dickson RP, Moore BB, The Lung Microbiome, Immunity, and the Pathogenesis
of Chronic Lung Disease. J. Immunol 196, 4839–4847 (2016). [PubMed: 27260767]
109. Stacy A, Belkaid Y, Microbial guardians of skin health. Science. 363, 227–228 (2019). [PubMed:
30655428]
110. Squarzanti DF, Zavattaro E, Pizzimenti S, Amoruso A, Savoia P, Azzimonti B, Non-Melanoma
Skin Cancer: news from microbiota research. Crit. Rev. Microbiol 46, 433–449 (2020). [PubMed:
32692305]
Author Manuscript
111. Liu J, Luo M, Zhang Y, Cao G, Wang S, Association of high-risk human papillomavirus infection
duration and cervical lesions with vaginal microbiota composition. Ann Transl Med. 8, 1161
(2020). [PubMed: 33241010]
112. So KA, Yang EJ, Kim NR, Hong SR, Lee J-H, Hwang C-S, Shim S-H, Lee SJ, Kim TJ, Changes
of vaginal microbiota during cervical carcinogenesis in women with human papillomavirus
infection. PLoS One. 15, e0238705 (2020). [PubMed: 32941440]
113. Human Microbiome Project Consortium, Structure, function and diversity of the healthy human
microbiome. Nature. 486, 207–214 (2012). [PubMed: 22699609]

114. Campbell IM, Shaw CA, Stankiewicz P, Lupski JR, Somatic mosaicism: implications for disease
and transmission genetics. Trends Genet. 31, 382–392 (2015). [PubMed: 25910407]
Author Manuscript
115. Bailey MH, Tokheim C, Porta-Pardo E, Sengupta S, Bertrand D, Weerasinghe A, Colaprico A,

Wendl MC, Kim J, Reardon B, Ng PK-S, Jeong KJ, Cao S, Wang Z, Gao J, Gao Q, Wang F,
Liu EM, Mularoni L, Rubio-Perez C, Nagarajan N, Cortés-Ciriano I, Zhou DC, Liang W-W, Hess
JM, Yellapantula VD, Tamborero D, Gonzalez-Perez A, Suphavilai C, Ko JY, Khurana E, Park
PJ, Van Allen EM, Liang H, MC3 Working Group, Cancer Genome Atlas Research Network,
Lawrence MS, Godzik A, Lopez-Bigas N, Stuart J, Wheeler D, Getz G, Chen K, Lazar AJ, Mills
GB, Karchin R, Ding L, Comprehensive Characterization of Cancer Driver Genes and Mutations.
Cell. 173, 371–385.e18 (2018). [PubMed: 29625053]
116. Klein RS, Recco RA, Catalano MT, Edberg SC, Casey JI, Steigbigel NH, Association of
Streptococcus bovis with carcinoma of the colon. N. Engl. J. Med 297, 800–802 (1977).
[PubMed: 408687]
117. Kwong TNY, Wang X, Nakatsu G, Chow TC, Tipoe T, Dai RZW, Tsoi KKK, Wong MCS, Tse
G, Chan MTV, Chan FKL, Ng SC, Wu JCY, Wu WKK, Yu J, Sung JJY, Wong SH, Association
Between Bacteremia From Specific Microbes and Subsequent Diagnosis of Colorectal Cancer.
Gastroenterology. 155, 383–390.e8 (2018). [PubMed: 29729257]
Author Manuscript
118. Wirbel J, Pyl PT, Kartal E, Zych K, Kashani A, Milanese A, Fleck JS, Voigt AY, Palleja A,
Ponnudurai R, Sunagawa S, Coelho LP, Schrotz-King P, Vogtmann E, Habermann N, Niméus
E, Thomas AM, Manghi P, Gandini S, Serrano D, Mizutani S, Shiroma H, Shiba S, Shibata
T, Yachida S, Yamada T, Waldron L, Naccarati A, Segata N, Sinha R, Ulrich CM, Brenner H,
Arumugam M, Bork P, Zeller G, Meta-analysis of fecal metagenomes reveals global microbial
signatures that are specific for colorectal cancer. Nat. Med 25, 679–689 (2019). [PubMed:
30936547]
119. Thomas AM, Manghi P, Asnicar F, Pasolli E, Armanini F, Zolfo M, Beghini F, Manara S, Karcher
N, Pozzi C, Gandini S, Serrano D, Tarallo S, Francavilla A, Gallo G, Trompetto M, Ferrero G,
Mizutani S, Shiroma H, Shiba S, Shibata T, Yachida S, Yamada T, Wirbel J, Schrotz-King P,
Ulrich CM, Brenner H, Arumugam M, Bork P, Zeller G, Cordero F, Dias-Neto E, Setubal JC,
Tett A, Pardini B, Rescigno M, Waldron L, Naccarati A, Segata N, Metagenomic analysis of
colorectal cancer datasets identifies cross-cohort microbial diagnostic signatures and a link with
choline degradation. Nat. Med 25, 667–678 (2019). [PubMed: 30936548]
Author Manuscript
120. Yachida S, Mizutani S, Shiroma H, Shiba S, Nakajima T, Sakamoto T, Watanabe H, Masuda

K, Nishimoto Y, Kubo M, Hosoda F, Rokutan H, Matsumoto M, Takamaru H, Yamada M,
Matsuda T, Iwasaki M, Yamaji T, Yachida T, Soga T, Kurokawa K, Toyoda A, Ogura Y,
Hayashi T, Hatakeyama M, Nakagama H, Saito Y, Fukuda S, Shibata T, Yamada T, Metagenomic
and metabolomic analyses reveal distinct stage-specific phenotypes of the gut microbiota in
colorectal cancer. Nat. Med 25, 968–976 (2019). [PubMed: 31171880]
121. Flemer B, Lynch DB, Brown JMR, Jeffery IB, Ryan FJ, Claesson MJ, O’Riordain M, Shanahan F,
O’Toole PW, Tumour-associated and non-tumour-associated microbiota in colorectal cancer. Gut.
66, 633–643 (2017). [PubMed: 26992426]
122. Farrell JJ, Zhang L, Zhou H, Chia D, Elashoff D, Akin D, Paster BJ, Joshipura K, Wong DTW,
Variations of oral microbiota are associated with pancreatic diseases including pancreatic cancer.
Gut. 61, 582–588 (2012). [PubMed: 21994333]
123. Half E, Keren N, Reshef L, Dorfman T, Lachter I, Kluger Y, Reshef N, Knobler H, Maor Y, Stein
A, Konikoff FM, Gophna U, Fecal microbiome signatures of pancreatic cancer patients. Sci. Rep
9, 16801 (2019). [PubMed: 31727922]
Author Manuscript
124. Yan X, Yang M, Liu J, Gao R, Hu J, Li J, Zhang L, Shi Y, Guo H, Cheng J, Razi M, Pang S,
Yu X, Hu S, Discovery and validation of potential bacterial biomarkers for lung cancer. Am. J.
Cancer Res 5, 3111–3122 (2015). [PubMed: 26693063]
125. Lee SH, Sung JY, Yong D, Chun J, Kim SY, Song JH, Chung KS, Kim EY, Jung JY, Kang YA,
Kim YS, Kim SK, Chang J, Park MS, Characterization of microbiome in bronchoalveolar lavage
fluid of patients with lung cancer comparing with benign mass like lesions. Lung Cancer. 102,
89–95 (2016). [PubMed: 27987594]
126. Greathouse KL, White JR, Vargas AJ, Bliskovsky VV, Beck JA, von Muhlinen N, Polley EC,
Bowman ED, Khan MA, Robles AI, Cooks T, Ryan BM, Padgett N, Dzutsev AH, Trinchieri G,

Pineda MA, Bilke S, Meltzer PS, Hokenstad AN, Stickrod TM, Walther-Antonio MR, Earl JP,
Mell JC, Krol JE, Balashov SV, Bhat AS, Ehrlich GD, Valm A, Deming C, Conlan S, Oh J, Segre
Author Manuscript
JA, Harris CC, Interaction between the microbiome and TP53 in human lung cancer. Genome
Biol. 19, 123 (2018). [PubMed: 30143034]
127. Urbaniak C, Cummins J, Brackstone M, Macklaim JM, Gloor GB, Baban CK, Scott L, O’Hanlon
DM, Burton JP, Francis KP, Tangney M, Reid G, Microbiota of human breast tissue. Appl.
Environ. Microbiol 80, 3007–3014 (2014). [PubMed: 24610844]
128. Urbaniak C, Gloor GB, Brackstone M, Scott L, Tangney M, Reid G, The Microbiota of Breast
Tissue and Its Association with Breast Cancer. Appl. Environ. Microbiol 82, 5039–5048 (2016).
[PubMed: 27342554]
129. Cantwell AR Jr, Kelso DW, Microbial findings in cancers of the breast and in their metastases
to the skin. Implications for etiology. J. Dermatol. Surg. Oncol 7, 483–491 (1981). [PubMed:
6166642]
130. Gorelick JI, Senterfit LB, Vaughan ED Jr, Quantitative bacterial tissue cultures from 209
prostatectomy specimens: findings and implications. J. Urol 139, 57–60 (1988). [PubMed:
3336106]
Author Manuscript
131. Cohen RJ, Shannon BA, McNeal JE, Shannon T, Garrett KL, Propionibacterium acnes associated
with inflammation in radical prostatectomy specimens: a possible link to cancer evolution? J.
Urol 173, 1969–1974 (2005). [PubMed: 15879794]
132. Castellarin M, Warren RL, Freeman JD, Dreolini L, Krzywinski M, Strauss J, Barnes R, Watson
P, Allen-Vercoe E, Moore RA, Holt RA, Fusobacterium nucleatum infection is prevalent in
human colorectal carcinoma. Genome Res. 22, 299–306 (2012). [PubMed: 22009989]
133. Pinato DJ, Howlett S, Ottaviani D, Urus H, Patel A, Mineo T, Brock C, Power D, Hatcher
O, Falconer A, Ingle M, Brown A, Gujral D, Partridge S, Sarwar N, Gonzalez M, Bendle M,
Lewanski C, Newsom-Davis T, Allara E, Bower M, Association of Prior Antibiotic Treatment
With Survival and Response to Immune Checkpoint Inhibitor Therapy in Patients With Cancer.
JAMA Oncol. 5, 1774–1778 (2019). [PubMed: 31513236]
134. Lowy DR, Schiller JT, Preventing Cancer and Other Diseases Caused by Human Papillomavirus
Infection: 2017 Lasker-DeBakey Clinical Research Award. JAMA. 318, 901–902 (2017).
[PubMed: 28876435]
135. Stolte M, Bayerdörffer E, Morgner A, Alpen B, Wündisch T, Thiede C, Neubauer A, Helicobacter
Author Manuscript
and gastric MALT lymphoma. Gut. 50 Suppl 3, III19–24 (2002). [PubMed: 11953328]
136. Huang X-Z, Gao P, Song Y-X, Xu Y, Sun J-X, Chen X-W, Zhao J-H, Wang Z-N, Antibiotic use
and the efficacy of immune checkpoint inhibitors in cancer patients: a pooled analysis of 2740
cancer patients. Oncoimmunology. 8, e1665973 (2019). [PubMed: 31741763]
137. Derosa L, Hellmann MD, Spaziano M, Halpenny D, Fidelle M, Rizvi H, Long N, Plodkowski
AJ, Arbour KC, Chaft JE, Rouche JA, Zitvogel L, Zalcman G, Albiges L, Escudier B, Routy
B, Negative association of antibiotics on clinical activity of immune checkpoint inhibitors in
patients with advanced renal cell and non-small-cell lung cancer. Ann. Oncol 29, 1437–1444
(2018). [PubMed: 29617710]
138. Steck SE, Angela Murphy E, Dietary patterns and cancer risk. Nature Reviews Cancer. 20 (2020),
pp. 125–138. [PubMed: 31848467]
139. Rad AH, Abbasi A, Kafil HS, Ganbarov K, Potential Pharmaceutical and
Food Applications of Postbiotics: A review. Curr. Pharm. Biotechnol (2020),
doi:10.2174/1389201021666200516154833.
Author Manuscript
140. van Nood E, Vrieze A, Nieuwdorp M, Fuentes S, Zoetendal EG, de Vos WM, Visser CE,
Kuijper EJ, Bartelsman JFWM, Tijssen JGP, Speelman P, Dijkgraaf MGW, Keller JJ, Duodenal
infusion of donor feces for recurrent Clostridium difficile. N. Engl. J. Med 368, 407–415 (2013).
[PubMed: 23323867]
141. Wang Y, Wiesnoski DH, Helmink BA, Gopalakrishnan V, Choi K, DuPont HL, Jiang Z-D,
Abu-Sbeih H, Sanchez CA, Chang C-C, Parra ER, Francisco-Cruz A, Raju GS, Stroehlein
JR, Campbell MT, Gao J, Subudhi SK, Maru DM, Blando JM, Lazar AJ, Allison JP, Sharma
P, Tetzlaff MT, Wargo JA, Jenq RR, Fecal microbiota transplantation for refractory immune
checkpoint inhibitor-associated colitis. Nat. Med 24, 1804–1808 (2018). [PubMed: 30420754]

142. McQuade JL, Daniel CR, Helmink BA, Wargo JA, Modulating the microbiome to improve
therapeutic response in cancer. Lancet Oncol 20, e77–e91 (2019). [PubMed: 30712808]
Author Manuscript
143. Baruch EN, Youngster I, Ben-Betzalel G, Ortenberg R, Lahat A, Katz L, Adler K, Dick-Necula D,
Raskin S, Bloch N, Rotin D, Anafi L, Avivi C, Melnichenko J, Steinberg-Silman Y, Mamtani R,
Harati H, Asher N, Shapira-Frommer R, Brosh-Nissimov T, Eshet Y, Ben-Simon S, Ziv O, Khan
MAW, Amit M, Ajami NJ, Barshack I, Schachter J, Wargo JA, Koren O, Markel G, Boursi B,
Fecal microbiota transplant promotes response in immunotherapy-refractory melanoma patients.
Science (2020), doi:10.1126/science.abb5920.
144. Arthur JC, Gharaibeh RZ, Uronis JM, Perez-Chanona E, Sha W, Tomkovich S, Mühlbauer M,
Fodor AA, Jobin C, VSL#3 probiotic modifies mucosal microbial composition but does not
reduce colitis-associated colorectal cancer. Sci. Rep 3, 2868 (2013). [PubMed: 24100376]
145. Yelin I, Flett KB, Merakou C, Mehrotra P, Stam J, Snesrud E, Hinkle M, Lesho E, McGann
P, McAdam AJ, Sandora TJ, Kishony R, Priebe GP, Genomic and epidemiological evidence of
bacterial transmission from probiotic capsule to blood in ICU patients. Nat. Med 25, 1728–1732
(2019). [PubMed: 31700189]
146. Conry RM, Westbrook B, McKee S, Norwood TG, Talimogene laherparepvec: First in class
Author Manuscript
oncolytic virotherapy. Hum. Vaccin. Immunother 14, 839–846 (2018). [PubMed: 29420123]
147. Kramer MG, Masner M, Ferreira FA, Hoffman RM, Bacterial Therapy of Cancer: Promises,
Limitations, and Insights for Future Directions. Front. Microbiol 9, 16 (2018). [PubMed:
29472896]
148. Zheng M, Huang J, Tong A, Yang H, Oncolytic Viruses for Cancer Therapy: Barriers and Recent
Advances. Mol Ther Oncolytics. 15, 234–247 (2019). [PubMed: 31872046]
149. Ylösmäki E, Cerullo V, Design and application of oncolytic viruses for cancer immunotherapy.
Curr. Opin. Biotechnol 65, 25–36 (2019). [PubMed: 31874424]
150. Bommareddy PK, Shettigar M, Kaufman HL, Integrating oncolytic viruses in combination cancer
immunotherapy. Nat. Rev. Immunol 18, 498–513 (2018). [PubMed: 29743717]
151. Charbonneau MR, Isabella VM, Li N, Kurtz CB, Developing a new class of engineered
live bacterial therapeutics to treat human diseases. Nat. Commun 11, 1738 (2020). [PubMed:
32269218]
152. Zheng JH, Nguyen VH, Jiang S-N, Park S-H, Tan W, Hong SH, Shin MG, Chung I-J, Hong Y,
Bom H-S, Choy HE, Lee SE, Rhee JH, Min J-J, Two-step enhanced cancer immunotherapy with
Author Manuscript
engineered Salmonella typhimurium secreting heterologous flagellin. Sci. Transl. Med 9 (2017),
doi:10.1126/scitranslmed.aak9537.
153. Roberts NJ, Zhang L, Janku F, Collins A, Bai R-Y, Staedtke V, Rusk AW, Tung D, Miller M, Roix
J, Khanna KV, Murthy R, Benjamin RS, Helgason T, Szvalb AD, Bird JE, Roy-Chowdhuri S,
Zhang HH, Qiao Y, Karim B, McDaniel J, Elpiner A, Sahora A, Lachowicz J, Phillips B, Turner
A, Klein MK, Post G, Diaz LA Jr, Riggins GJ, Papadopoulos N, Kinzler KW, Vogelstein B,
Bettegowda C, Huso DL, Varterasian M, Saha S, Zhou S, Intratumoral injection of Clostridium
novyi-NT spores induces antitumor responses. Sci. Transl. Med 6, 249ra111 (2014).
154. Swofford CA, Van Dessel N, Forbes NS, Quorum-sensing Salmonella selectively trigger protein
expression within tumors. Proc. Natl. Acad. Sci. U. S. A 112, 3457–3462 (2015). [PubMed:
25737556]
155. Din MO, Danino T, Prindle A, Skalak M, Selimkhanov J, Allen K, Julio E, Atolia E, Tsimring
LS, Bhatia SN, Hasty J, Synchronized cycles of bacterial lysis for in vivo delivery. Nature. 536,
81–85 (2016). [PubMed: 27437587]
Author Manuscript
156. Chowdhury S, Castro S, Coker C, Hinchliffe TE, Arpaia N, Danino T, Programmable bacteria
induce durable tumor regression and systemic antitumor immunity. Nat. Med 25, 1057–1063
(2019). [PubMed: 31270504]
157. Scott SR, Din MO, Bittihn P, Xiong L, Tsimring LS, Hasty J, A stabilized microbial ecosystem
of self-limiting bacteria using synthetic quorum-regulated lysis. Nat Microbiol. 2, 17083 (2017).
[PubMed: 28604679]
158. Davenport ER, Sanders JG, Song SJ, Amato KR, Clark AG, Knight R, The human microbiome in
evolution. BMC Biol. 15, 127 (2017). [PubMed: 29282061]

159. Foster KR, Schluter J, Coyte KZ, Rakoff-Nahoum S, The evolution of the host microbiome as an
ecosystem on a leash. Nature. 548, 43–51 (2017). [PubMed: 28770836]
Author Manuscript
160. Janney A, Powrie F, Mann EH, Host–microbiota maladaptation in colorectal cancer. Nature. 585
(2020), pp. 509–517. [PubMed: 32968260]
161. Gharaibeh RZ, Jobin C, Microbiota and cancer immunotherapy: in search of microbial signals.
Gut. 68, 385–388 (2019). [PubMed: 30530851]
162. Limeta A, Ji B, Levin M, Gatto F, Nielsen J, Meta-analysis of the gut microbiota in predicting
response to cancer immunotherapy in metastatic melanoma. JCI Insight. 5 (2020), doi:10.1172/
jci.insight.140940.
163. Sinha R, Abu-Ali G, Vogtmann E, Fodor AA, Ren B, Amir A, Schwager E, Crabtree J, Ma S,
Microbiome Quality Control Project Consortium, Abnet CC, Knight R, White O, Huttenhower
C, Assessment of variation in microbial community amplicon sequencing by the Microbiome
Quality Control (MBQC) project consortium. Nat. Biotechnol 35, 1077–1086 (2017). [PubMed:
28967885]
164. Eisenhofer R, Minich JJ, Marotz C, Cooper A, Knight R, Weyrich LS, Contamination in Low
Microbial Biomass Microbiome Studies: Issues and Recommendations. Trends Microbiol. 27,
Author Manuscript
105–117 (2019). [PubMed: 30497919]

165. Lozupone CA, Stombaugh J, Gonzalez A, Ackermann G, Wendel D, Vázquez-Baeza Y, Jansson
JK, Gordon JI, Knight R, Meta-analyses of studies of the human microbiota. Genome Res. 23,
1704–1714 (2013). [PubMed: 23861384]
166. Gonzalez A, Navas-Molina JA, Kosciolek T, McDonald D, Vázquez-Baeza Y, Ackermann G,
DeReus J, Janssen S, Swafford AD, Orchanian SB, Sanders JG, Shorenstein J, Holste H, Petrus
S, Robbins-Pianka A, Brislawn CJ, Wang M, Rideout JR, Bolyen E, Dillon M, Caporaso JG,
Dorrestein PC, Knight R, Qiita: rapid, web-enabled microbiome meta-analysis. Nat. Methods 15,
796–798 (2018). [PubMed: 30275573]
167. Davis NM, Proctor DM, Holmes SP, Relman DA, Callahan BJ, Simple statistical identification
and removal of contaminant sequences in marker-gene and metagenomics data. Microbiome. 6,
226 (2018). [PubMed: 30558668]
168. Merritt CR, Ong GT, Church SE, Barker K, Danaher P, Geiss G, Hoang M, Jung J, Liang Y,
McKay-Fleisch J, Nguyen K, Norgaard Z, Sorg K, Sprague I, Warren C, Warren S, Webster PJ,
Zhou Z, Zollinger DR, Dunaway DL, Mills GB, Beechem JM, Multiplex digital spatial profiling
Author Manuscript
of proteins and RNA in fixed tissue. Nat. Biotechnol 38, 586–599 (2020). [PubMed: 32393914]
169. Shi H, Shi Q, Grodner B, Lenz JS, Zipfel WR, Brito IL, De Vlaminck I, Highly multiplexed
spatial mapping of microbial communities. Nature (2020), doi:10.1038/s41586-020-2983-4.
170. Min S, Kim S, Cho S-W, Gastrointestinal tract modeling using organoids engineered with cellular
and microbiota niches. Exp. Mol. Med 52, 227–237 (2020). [PubMed: 32103122]
171. Abed J, Emgård JEM, Zamir G, Faroja M, Almogy G, Grenov A, Sol A, Naor R, Pikarsky
E, Atlan KA, Mellul A, Chaushu S, Manson AL, Earl AM, Ou N, Brennan CA, Garrett WS,
Bachrach G, Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment
by Binding to Tumor-Expressed Gal-GalNAc. Cell Host Microbe. 20, 215–225 (2016). [PubMed:
27512904]
172. Abed J, Maalouf N, Manson AL, Earl AM, Parhi L, Emgård JEM, Klutstein M, Tayeb S, Almogy
G, Atlan KA, Chaushu S, Israeli E, Mandelboim O, Garrett WS, Bachrach G, Colon Cancer-
Associated May Originate From the Oral Cavity and Reach Colon Tumors via the Circulatory
System. Front. Cell. Infect. Microbiol 10, 400 (2020). [PubMed: 32850497]
Author Manuscript
173. Wassenaar TM, Insights from 100 Years of Research with Probiotic. Eur. J. Microbiol. Immunol
6, 147–161 (2016).
174. Gurbatri CR, Lia I, Vincent R, Coker C, Castro S, Treuting PM, Hinchliffe TE, Arpaia N, Danino
T, Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies. Sci. Transl.
Med 12 (2020), doi:10.1126/scitranslmed.aax0876.
175. Tanouchi Y, Smith RP, You L, Engineering microbial systems to explore ecological and
evolutionary dynamics. Curr. Opin. Biotechnol 23, 791–797 (2012). [PubMed: 22310174]
176. Liao MJ, Din MO, Tsimring L, Hasty J, Rock-paper-scissors: Engineered population dynamics
increase genetic stability. Science 365, 1045–1049 (2019). [PubMed: 31488693]

177. Lagier J-C, Dubourg G, Million M, Cadoret F, Bilen M, Fenollar F, Levasseur A, Rolain J-M,
Fournier P-E, Raoult D, Culturing the human microbiota and culturomics. Nat. Rev. Microbiol
Author Manuscript
16, 540–550 (2018). [PubMed: 29937540]

178. Thorsson V, Gibbs DL, Brown SD, Wolf D, Bortone DS, Ou Yang T-H, Porta-Pardo E, Gao
GF, Plaisier CL, Eddy JA, Ziv E, Culhane AC, Paull EO, Sivakumar IKA, Gentles AJ, Malhotra
R, Farshidfar F, Colaprico A, Parker JS, Mose LE, Vo NS, Liu J, Liu Y, Rader J, Dhankani V,
Reynolds SM, Bowlby R, Califano A, Cherniack AD, Anastassiou D, Bedognetti D, Mokrab Y,
Newman AM, Rao A, Chen K, Krasnitz A, Hu H, Malta TM, Noushmehr H, Pedamallu CS,
Bullman S, Ojesina AI, Lamb A, Zhou W, Shen H, Choueiri TK, Weinstein JN, Guinney J, Saltz
J, Holt RA, Rabkin CS, Cancer Genome Atlas Research Network, Lazar AJ, Serody JS, Demicco
EG, Disis ML, Vincent BG, Shmulevich I, The Immune Landscape of Cancer. Immunity. 48,
812–830.e14 (2018). [PubMed: 29628290]
Author Manuscript
Author Manuscript
Author Manuscript

Author Manuscript
Author Manuscript
Author Manuscript
Fig. 1. Examples by which microbial mechanisms intersect with key cancer pathways (10, 11).
Microbiota-derived metabolites, genotoxins, and antigens influence host antitumor
immunity, inflammation, energetics, cellular signaling, and metastasis. Abbreviations:
MMP=matrix metalloproteinases; SCFAs=short-chain fatty acids; mAb=monoclonal
antibody.
Author Manuscript

Author Manuscript
Author Manuscript
Fig. 2. Defining the immuno-oncology-microbiome (IOM) axis.

Gut and TME microbiota regulate host metabolism and immunity, which ultimately
influence antitumor immunity. (A) Gut microbial metabolites and byproducts influence host
lympho- and myelopoiesis, including during allogeneic HSCT and radiotherapy (59, 63).
Author Manuscript
(B) Cyclophosphamide (CTX)-derived gut epithelial damage enables E. hirae translocation

and antitumor immunity (68, 69). (C) Gut translocation of Bifidobacterium species or
its antigens can increase IFN-I signaling and antitumor immunity (73, 76). (D) Microbes
within the tumor microenvironment (TME) can be either immunosuppressive (often PRR-
mediated) or immunogenic, including shaping response to immunotherapy (12, 23). Cancer
(neo)antigens may share epitopes with microbes through molecular mimicry (73, 83).
Microbial hematogenous spread (117, 171, 172) or colonized micrometastases (19) may
complete this feedback loop that originated in the gut. Abbreviations: MAMPs=microbe-
associated molecular pattern; SCFAs=short-chain fatty acids; GALT=gut-associated
lymphoid tissue; mLN=mesenteric lymph node; DC=dendritic cell; OMVs=bacterial outer
membrane vesicles; NK=natural killer cell; PRR=pattern recognition receptor; TIL=tumor-
infiltrating lymphocytes.
Author Manuscript

Author Manuscript
Author Manuscript
Author Manuscript
Fig. 3. Current landscape of the cancer microbiome.

(A) Body diagram of all cancers currently linked to microbiota, where the colored dots
reflect reference numbers and are colored according to the major theme of the referenced
paper (diagnosis, mechanism, prognosis, treatment). Dots are included based on existing
preclinical and clinical data. (B) Representative histology, immunohistochemistry (IHC) for
lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and immunofluorescence (IF) for
Author Manuscript
bacterial 16S rRNA in six cancers. (C) Representative transmission electron microscopy
(TEM) images with overlaid 16S rRNA immunofluorescence of intracellular bacteria
(arrows) in breast cancer. (D) Estimation of tumor percent bacterial composition across
seven cancer types assuming tissue homogeneity and 8 picograms of DNA per cancer cell.
Black lines depict distributional quantiles (25%–50%–75%); white dots reflect averages.
(B-D) Adapted from Nejman et al. (12)

Author Manuscript
Author Manuscript
Author Manuscript
Fig. 4. Considerations when modulating the endogenous cancer microbiome.

Diet, medications, and prebiotics, postbiotics, probiotics, and antibiotics all have the
capacity to modify the gut and tumor microbiomes. Bi-directional influences may exist
between these microbiomes and cancer therapies (chemotherapy and immunotherapy). For
instance, chemotherapy can cause compositional changes in the gut microbiome, which
in turn enhance treatment efficacy (75); in other cases, chemotherapy may be degraded
by microbes (14). Thus, modification of the gut and/or tumor microbiomes may be
advantageous for one modality of therapy while disadvantageous for another. Dotted arrows
denote gaps in the literature.
Author Manuscript

Author Manuscript
Author Manuscript
Author Manuscript
Fig. 5. Synthetic biology for exogenous cancer therapeutics.

(A) Regulatory considerations for engineering bacteria against cancer (151). (B) Diverse
sources of intratumoral bacteria include organ-specific commensals (18, 19, 25, 46, 112),
gut communication (15, 20, 23), hematogenous spread (171, 172), and intra-metastatic
spread (19). (C) Some probiotics, such as E. coli Nissle 1917, possess strong safety records
(173), have been shown to naturally migrate to solid tumors in animal models, and can be
Author Manuscript
programmed to produce and deliver therapies from within solid tumors (155). (D) Complex
population dynamics can be engineered to generate the cyclical delivery of therapeutics
(155, 156, 174). (E) Future efforts will likely center on engineering and testing strains that
are found naturally in patient-specific tumors. (F) Engineered ecologies can be designed to
create tailored, tumor-specific therapeutic cocktails (175, 176). (G) Multiple drug payloads
can be encoded by one or more engineered strains against tumors.

Author Manuscript
Author Manuscript
Fig. 6. Study design for characterizing cancer-associated microbiota and their functional
impacts.
Opportunities exist to perform large-scale identification of the presence and function
of cancer-associated microbiota, beginning with longitudinal cohorts and multi-region
sampling. Existing tools can be used to gather multi-omic information on host immune
cells, cancer cells, microbiota, and metabolites (51, 177, 178). In vitro and in vivo disease
models of a patient’s tumor and intestine can then be used to verify or rebut the predicted
functional impact and mechanism(s) of a given microbe (or its metabolites) and its causality
in carcinogenesis (160, 170).
Author Manuscript
Author Manuscript

Nihms 1768792

Uploaded by

Copyright:

Available Formats

Nihms 1768792

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Nihms 1768792

Uploaded by

Copyright:

Available Formats

HHS Public Access

Published in final edited form as:

The microbiome and human cancer

3Institut National de la Santé et de la Recherche Medicale (INSERM) U1015, Villejuif, France.

10Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center,

mutations and modulate immune checkpoint inhibitor efficacy.

outcomes while providing a nuanced understanding of cancer-host-microbial evolution.

Science. Author manuscript; available in PMC 2022 March 26.

Opportunities for microbes to impact cancer care. Diagnosis: Cancer-specific, blood-borne

Science. Author manuscript; available in PMC 2022 March 26.

cancer was dismissed.

Overview of the cancer microbiome

Science. Author manuscript; available in PMC 2022 March 26.

microbes initiate cancer through genotoxin-mediated mutagenesis; in particular, colibactin (a

Increasing evidence suggests an important additional category of “complicit” microbes

Science. Author manuscript; available in PMC 2022 March 26.

Mechanisms and interactions between the gut and tumor microbiome

microbiome (IOM) axis (Fig. 2). Gut-TME crosstalk, especially in non-gastrointestinal

The effects of gut microbiota on primary lymphoid organs—Following allogeneic

Science. Author manuscript; available in PMC 2022 March 26.

potentially on genera whose genomic repertoires encode carbohydrate-active enzymes (57).

homologous self-antigens, these commensal-specific immune responses can be deleterious

Science. Author manuscript; available in PMC 2022 March 26.

recognize microbial peptides (83), suggesting clinical significance. However, mechanisms

Gut-derived metabolites can also modulate immune responses. Radiotherapy of

The effects of gut microbiota on the tumor microenvironment—The intestinal

Tumor-associated, NOX2-mediated, myeloid cell ROS production is reduced by antibiotic

Science. Author manuscript; available in PMC 2022 March 26.

microbiome was later shown to be necessary to prevent leukemia progression in genetically

immunosurveillance (77). In another study, oxaliplatin-induced caspase 3/7-dependent

Gut microbiota-mediated effects on anti-cancer drugs—Gut microbes are

Science. Author manuscript; available in PMC 2022 March 26.

to be tested in large patient cohorts. These types of bidirectional drug-microbiota feedback

loops warrant further study.

Intratumor microbiota effects on the tumor microenvironment—Mechanistic

Extra-intestinal barriers and cancer microbiota—Given that the intestinal barrier

Science. Author manuscript; available in PMC 2022 March 26.

affect response to therapies and overall survival, due to a TH17-mediated exacerbated

inflammation corollary to immune checkpoint inhibition (25).

Cancer microbiome diagnostics

and must be addressed before clinical deployment.

Combining multi-region 16S rRNA amplicon sequencing, qPCR, immunohistochemistry

(lipopolysaccharide [LPS], lipoteichoic acid [LTA]), immunofluorescence (16S rRNA),

Science. Author manuscript; available in PMC 2022 March 26.

required for analysis, limiting its diagnostic utility.

to those from 69 HIV-negative, healthy patients while implementing necessary experimental

Modulation of the cancer microbiome

Science. Author manuscript; available in PMC 2022 March 26.

are incongruent; for instance, antibiotics appear to abrogate immunotherapy response

Antibiotics and the cancer microbiome

135). Excluding antibiotic-derived chemotherapies (e.g. doxorubicin), there is circumstantial

some efficacy in the treatment of immunotherapy-associated colitis (141). The long-term

Science. Author manuscript; available in PMC 2022 March 26.

to multiplexed consortia. Few commercially available probiotic formulations have been

Cancer therapy using exogenous microbiota

elsewhere in detail (148–150), we focus our attention on bacterial cancer therapies

Engineered microbes as cancer drugs

whereby cytotoxic payloads can be encoded for programmed delivery by tumor-homing

threshold population density, releasing a chemokine, hemolysin, or pro-apoptotic protein,