Content uploaded by Emilie Viennois
Author content
All content in this area was uploaded by Emilie Viennois on Jan 23, 2017
Content may be subject to copyright.
Microenvironment and Immunology
Dietary Emulsifier–Induced Low-Grade
Inflammation Promotes Colon Carcinogenesis
Emilie Viennois
1
, Didier Merlin
1,2
, Andrew T. Gewirtz
1
, and Benoit Chassaing
1
Abstract
The increased risks conferred by inflammatory bowel dis-
ease (IBD) to the development of colorectal cancer gave rise to
the term "colitis-associated cancer" and the concept that
inflammation promotes colon tumorigenesis. A condition
more common than IBD is low-grade inflammation, which
correlates with altered gut microbiota composition and met-
abolic syndrome, both present in many cases of colorectal
cancer. Recent findingssuggestthatlow-gradeinflammation
in the intestine is promoted by consumption of dietary
emulsifiers, a ubiquitous component of processed foods,
which alter the composition of gut microbiota. Here, we
demonstrate in a preclinical model of colitis-induced colorec-
tal cancer that regular consumption of dietary emulsifiers,
carboxymethylcellulose or polysorbate-80, exacerbated tumor
development. Enhanced tumor development was associated
with an altered microbiota metagenome characterized by
elevated levels of lipopolysaccharide and flagellin. We found
that emulsifier-induced alterations in the microbiome were
necessary and sufficient to drive alterations in major prolif-
eration and apoptosis signaling pathways thought to govern
tumor development. Overall, our findings support the concept
that perturbations in host–microbiota interactions that cause
low-grade gut inflammation can promote colon carcinogenesis.
Cancer Res; 77(1); 27–40. 2016 AACR.
Introduction
Colorectal cancer is among the most common human malig-
nancies (1) and has been firmly linked to chronic intestinal
inflammation, giving rise to the term "colitis-associated cancer"
(2, 3). The development of colitis-associated cancer in patients
suffering from inflammatory bowel disease (IBD) is one of the
best characterized examples of an association between intestinal
inflammation and carcinogenesis (4–7). Among patients with
ulcerative colitis, the risk of colon cancer has been found to be as
high as 2% at 10 years, 8% at 20 years, and 18% at 30 years after
initial diagnosis (4). In contrast, the lifetime risk of sporadic
colorectal cancer in the United States is only 5% (8).
Gut microbiota is the collective term for the large diverse
community of microorganisms that inhabits the intestine. Gut
microbiota play an important role in health, particularly in
promoting immune system development and aiding metabo-
lism. Alterations in microbiota composition, often referred to
as dysbiosis, are thought to play a central role in the patho-
genesis of numerous intestinal disorders including inflamma-
tory bowel disease (IBD; ref. 9), and are associated with
colorectal cancer (10). However, whether microbial dysbiosis
observed in patients with colorectal cancer is a consequence of
the pathology or is causal remains unclear. An altered micro-
biotacanplayaroleinpromotingcolitis-associated cancer,
not only through induction of inflammation, but also through
the production of toxins that create a favorable niche for tumor
cells (11). Indeed, commensal organisms can have an enor-
mous impact on tumorigenesis through the production of
tumor-promoting genotoxins that can induce chromosomal
instability (12). For example, certain strains of Escherichia coli
harboring the Pks island, involved in the synthesis of the
colibactin toxin, are frequently associated with human colo-
rectal tumors. These strains inhibit DNA mismatch repair
proteins, and were also reported to have carcinogenic effects
in mice (11, 13–16). Besides, treatment of mice with antibiotics
confers some degree of protection against colitis-associated can-
cer, supporting the pivotal role of the gut microbiota during
tumorigenesis (17). Moreover, azoxymethane (AOM)-treated
germ-free IL10
/
mice failed to develop colitis and colorectal
tumors, indicating that the presence of colitogenic bacteria is
essential for the development of colitis-associated cancer (18).
While gut inflammation is classically defined histopathologi-
cally, specifically by the presence of immune cell infiltrates, it is
now appreciated that a much more common form of inflamma-
tion is "low-grade intestinal inflammation," which is defined by
elevated systemic expression of proinflammatory cytokines in the
absence of the classical aggregates of immune cell infiltrates.
Alterations in host–microbiota relationship have been associated
with and can promote low-grade intestinal inflammation (19).
Moreover, it is increasingly appreciated that low-grade chronic
inflammation in the gut can promote metabolic disorders such as
type II diabetes, atherosclerosis, and obesity, which is itself
associated with increased incidence of colon cancer (20, 21).
Emulsifiers are detergent-like molecules that are incorporat-
ed into most processed foods to improve texture and stability,
and we recently demonstrated that emulsifiers disrupted
1
Center for Inflammation, Immunity and Infection, Institute for Biomedical
Sciences, Georgia State University, Atlanta, Georgia.
2
Veterans Affairs Medical
Center, Decatur, Georgia.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Author: Benoit Chassaing, Center for Inflammation, Immunity,
and Infection, Institute for Biomedical Sciences, Georgia State University,
Atlanta GA 30303. Phone: 404-413-3589; Fax: 404-413-3580; E-mail:
bchassaing@gsu.edu
doi: 10.1158/0008-5472.CAN-16-1359
2016 American Association for Cancer Research.
Cancer
Research
www.aacrjournals.org 27
mucus–bacterial interactions, inducing intestinal inflammation
(22). In this recent study, we investigated the effect of two
commonly used emulsifiers, namely carboxymethylcellulose
(CMC) and polysorbate-80 (P80), on the host's intestine. CMC
was previously described to promote overgrowth and inflam-
mation of small intestine in genetically susceptible mice (23),
and P80 is able to increase bacterial translocation across epi-
thelia in vitro (24, 25).
These two emulsifiers are indigestible and mainly excreted in
the feces (26–29), and we recently found that both CMC and P80
promoted microbiota encroachment and increased levels of
proinflammatory flagellin and lipopolysaccharide (LPS), which
correlated with a change in microbiota composition and intesti-
nal inflammation. Such alterations promoted colitis in mice
genetically predisposed to this disorder, and induced low-grade
inflammation and metabolic syndrome in WT mice. Importantly,
such effects were dependent upon the presence of the microbiota
(no phenotype observed in germ-free mice), and fecal microbiota
transplant from emulsifier-treated mice to germ-free mice, trans-
ferred some features of intestinal inflammation and metabolic
syndrome (22).
In the current study, we hypothesized that emulsifiers could be
involved in colorectal cancer development through the promo-
tion of low-grade intestinal inflammation and alterations of the
intestinal microbiota. To test this hypothesis, we used the well-
established murine model of colitis-associated cancer using the
carcinogen AOM, followed by two cycles of dextran sulfate
sodium (DSS) in mice subjected to chronic exposures of two
commonly used emulsifiers, CMC and P80. We herein report that
dietary emulsifying agents created and maintained a proinflam-
matory environment in the colon, associated with alterations of
the proliferation/apoptosis balance that resulted in exacerbated
carcinogenesis. These changes were associated with, and depen-
dent upon, alterations in microbiota composition and diversity
that created a favorable niche for tumorigeneisis. These findings
support the concept that a perturbed host–microbiota interaction
resulting in alterations of the intestinal homeostasis can promote
colonic carcinogenesis.
Materials and Methods
Materials
Sodium carboxymethylcellulose (CMC, average M
W
250,000,
degree of substitution ¼0.7) and polysorbate-80 (P80) were
purchased from Sigma.
Mice
Four-week-old male C57BL/6 WT mice were used in this study.
All mice were bred and housed at Georgia State University,
(Atlanta, GA) under institutionally approved protocols (IACUC
# A14033). Mice were housed in specific pathogen-free conditions
and fed ad libitum with regular chow diet. Animals used in Figs.
1–6 and Supplementary Fig. S1–S10 were housed in Helicobacter
positive room, and animals used in Fig. 7 and Supplementary
Fig. S11 were housed in Helicobacter negative room.
Emulsifier agent treatment
Mice were exposed to CMC or P80 diluted in the drinking water
(1.0%). The same water (reverse-osmosis treated Atlanta city
water) was used for the water-treated (control) group. These
solutions were changed every week. Body weights were measured
every week and expressed as percentages of the initial body weight
(day 0 defined as 100%) to study emulsifier effect on body weight
gain. Fresh feces were collected every week for downstream
analysis.
Colitis-associated cancer model
Colitis-associated cancer was induced as previously described
with some modifications (30). As schematized in Supplementary
Fig. S1, after 13 weeks (91 days) of emulsifier administration,
mice were intraperitoneally injected with AOM (10 mg/kg body
weight; Sigma-Aldrich) diluted in PBS and maintained on regular
chow diet and water or emulsifier-supplemented water for 5 days.
Mice were then subjected to two cycles of DSS treatment (MP
Biomedicals), in which each cycle consisted of 2.5% DSS for 7
days followed by a 14-day recovery period with regular water or
emulsifier-supplemented water. Body weights were measured
every week and expressed as percentages of the initial body weight
(day 91 ¼post emulsifier defined as 100%) to study AOM-DSS
protocol effect on body weight gain. After treatment, mice were
fasted for 5 hours, at which time blood was collected by retro-
bulbar intraorbital capillary plexus. Hemolysis-free serum was
generated by centrifugation of blood using serum separator tubes
(Becton Dickinson). After colitis-associated cancer protocol, mice
were euthanized, and colon length, colon weight, spleen weight,
and adipose weight were measured. Organs and blood were
collected for downstream analysis. Colonic tumors were counted
and surface measured using a dissecting microscope. The total
area of tumors for each colon was determined.
As schematized in Supplementary Fig. S11, emulsifier-treated
animals were also weekly treated with AOM (10 mg/kg body
weight) diluted in PBS. At the end of the experiment, mice were
fasted for 15 hours, colonoscopy procedure was performed (Karl
Storz Endoskope) and mice were euthanized and organs collected
as previously described.
Germ-free experiments
Germ-free Swiss Webster mice were kept under germfree con-
ditions in a Park Bioservices isolator in our germ-free facility. CMC
and P80 were diluted to 1% in water and then autoclaved for
germ-free purpose. The same water was used for the water-treated
(control) group. Conventional age-matched and sex-matched
Swiss Webster mice were used in parallel. After 3 months of
emulsifier agent treatment, terminal analyses were performed.
Microbiota transplantation
Cecal contents from Swiss Webster detergent-treated mice were
suspended in 30% glycerol diluted in PBS (1.0 mL) and stocked at
80C until analysis. Germ-free Swiss Webster mice (4 weeks old)
were removed from the isolator and were orally administered
200 mL of fecalsuspension made usingglycerol stocks.Transplanted
mice were monitored for 3 months before terminal analysis.
Colonic myeloperoxidase assay, quantification of fecal Lcn-2
and serum CXCL-1 and IL6 by ELISA
For details, see Supplementary Methods section.
Fecal flagellin and LPS load quantification
We quantified flagellin and LPS as previously described (31)
using human embryonic kidney (HEK)-Blue-mTLR5 and HEK-
BluemTLR4 cells, respectively (Invivogen). We resuspended
Viennois et al.
Cancer Res; 77(1) January 1, 2017 Cancer Research28
fecalmaterialinPBStoafinal concentration of 100 mg/mL
and homogenized using a Mini-Beadbeater-24 without the
addition of beads to avoid bacteria disruption. Supernatants
were serially diluted and applied to mammalian cells. Purified
E. coli flagellin and LPS (Sigma) were used for standard curve
determination. After 24 hours of stimulation, we applied cell
culture supernatant to QUANTI-Blue medium (Invivogen) and
measured alkaline phosphatase activity at 620 nm after 30
minutes.
RNA extraction, real-time RT-PCR, and bacterial quantification
by qPCR
For details, see Supplementary Methods section and Supple-
mentary Table S1.
Fecal microbiota analysis by 16S rRNA gene sequencing using
Illumina technology and metagenome prediction
16S rRNA gene sequencing was performed as previously
described (22), with data deposited in the European Nucleotide
Archive under accession number PRJEB8035. For details, please
see Supplementary Methods.
Ki67 immunohistochemistry
Mouse proximal colon devoid of tumor were fixed in 10%
buffered formalin for 24 hours at room temperature and subse-
quently embedded in paraffin. Tissues were sectioned at 5-mm
thickness and deparaffinized. Sections were incubated in sodium
citrate buffer and cooked in a pressure cooker for 10 minutes for
antigen retrieval. Sections were then blocked with 5% goat serum
in TBS followed by one hour incubation with anti-Ki67 (1:100,
Vector Laboratories) at 37C. After washing with TBS, sections
were treated with biotinylated secondary antibodies for 30 min-
utes at 37C, and color development was performed using the
Vectastain ABC kit (Vector Laboratories). Sections were then
counterstained with hematoxylin, dehydrated, and coverslipped.
Ki67-positive cells were counted per crypt.
Terminal deoxynucleotidyl transferase deoxyuridine
triphosphate nick-end labeling assay
To quantitate the number of apoptotic cells in colonic
epithelial cells, mouse proximal colon devoid of tumor were
fixed in 10% buffered formalin for 24 hours at room temper-
ature, embedded in paraffin, sectioned at 5-mm thickness,
deparaffinized, and stained for apoptotic nuclei according to
the manufacturer's instructions using the In Situ Cell Death
Detection Kit (Roche Diagnostics). Terminal deoxynucleotidyl
transferase deoxyuridine triphosphate nick-end labeling
(TUNEL)-positive cells overlapping with DAPI nuclear staining
were counted per crypt.
Statistical analysis
Data are presented as means SEM. Significance was deter-
mined using ttests, with each treatment group compared with
the control group. Two-way group ANOVA corrected for mul-
tiple comparisons using Bonferroni posttest was used for body
weight over time and alpha diversity analysis (GraphPad Prism
software, version 6.01). and # indicate statistically significant
differences.
Results
Dietary emulsifying agents induce low-grade intestinal
inflammation associated with metabolic syndrome
Multiple mouse litters were equally split at weaning into three
groups that received either water, CMC, or P80 in drinking water
(1.0% w/v or v/v, respectively) for 13 weeks, as reported previ-
ously (Supplementary Fig. S1; ref. 22). In accord with our previous
work, emulsifier consumption resulted in features of chronic low-
grade intestinal inflammation, including shortened colons and
splenomegaly (Supplementary Fig. S2). Fecal lipocalin-2 (Lcn2),
which is a sensitive and broadly dynamic marker of intestinal
inflammation in mice (32), was used to quantify the intestinal
inflammation, and showed that emulsifier-treated mice exhibited
elevated fecal Lcn2 levels after 9 weeks of dietary emulsifier
consumption (day 63; Supplementary Fig. S2A–S2C), confirming
the induction of low-grade inflammation. As expected, both CMC
and P80 induced a modest but statistically significant increase in
body mass (Supplementary Fig. S2D). Emulsifier treatment also
impaired glycemic control as assessed by fasting blood glucose
concentration (Supplementary Fig. S2E), and was associated with
increased food consumption (Supplementary Fig. S2F), confirm-
ing our previous observation that emulsifiers induce low-grade
intestinal inflammation and impair glucose metabolism (22).
Emulsifier consumption exacerbates carcinogenesis in a colitis-
associated cancer model
In an effort to investigate whether low-grade intestinal inflam-
mation induced by emulsifier consumption might predispose to
the development of colitis-associated carcinogenesis, mice that
had consumed emulsifiers for 90 days were subsequently admin-
istered AOM and DSS while maintaining emulsifier consumption
except during DSS administration (Supplementary Fig. S1). All
groups of AOM/DSS-treated mice displayed acute weight loss
during DSS treatment and, when euthanized, exhibited gross
features of inflammation, including increased colon and spleen
weights (Fig. 1A–E). While emulsifier treatment, by itself, induced
some indicators of inflammation such as colon shortening and
mild splenomegaly, the extent of inflammatory changes induced
by AOM/DSS treatment (i.e., fold change induced by AOM/DSS
treatment) was not greater in mice that had consumed emulsifiers.
However, emulsifier consumption increased tumor development
in response to AOM/DSS compared with AOM/DSS control
animals, as assessed by number and size of tumors (Fig.
1F–H). Histologic examination revealed the presence of larger
adenomas and increased areas of inflammatory cell infiltration in
emulsifier/AOM/DSS-treated animals compared with the water/
AOM/DSS group (Supplementary Fig. S3). Histologically, no
gross difference between water-treated and emulsifier-treated
mice was observed in the absence of AOM/DSS (Supplementary
Fig. S3). We next examined the expression of proinflammatory
cytokines by qRT-PCR using mRNA extracted from whole distal
colon devoid of tumor. mRNAs were thus isolated from a mixture
of epithelial cells, immune cells, and other colonic cells, allowing
the analysis of global molecular alterations of the colon. Emul-
sifier consumption by itself resulted in increased expression of the
proinflammatory cytokine CXCL1, confirming that emulsifiers
induce the development of low-grade intestinal inflammation
(Fig. 2A–E). AOM/DSS significantly increased the expression
levels of all the tested cytokines/chemokines (IL6, CXCL1, CXCL2,
IL22, and TNFa) compared with the corresponding control
Emulsifying Agents Promote Colonic Carcinogenesis
www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 29
Viennois et al.
Cancer Res; 77(1) January 1, 2017 Cancer Research30
groups (Fig. 2A–E), as were the levels of circulating CXCL1 (Fig.
2F). After AOM/DSS-induced carcinogenesis, both CXCL1 and
CXCL2 were significantly increased in emulsifier-treated group
compared with water-treated group (Fig. 2B and C), supporting
the previous observation that AOM/DSS-induced tumors were
infiltrated by inflammatory cells. Importantly, we noted that mice
receiving P80 exhibited the highest tumor and a greater increase of
the CXCL1 and CXCL2 compared with CMC (Fig. 2B and C). As a
further readout of the inflammatory state of the intestine, we next
examined colonic myeloperoxidase (MPO) activity and fecal Lcn2
level following AOM/DSS treatment (Fig. 3A and B). These
analyses confirmed the observation that emulsifier's consump-
tion per se was sufficient to drive intestinal inflammation, as
revealed by a moderate but nonetheless significant increase in
MPO in emulsifier-only treated groups compared with water-only
treated group (Fig. 3A). Furthermore, emulsifiers led to an exacer-
bation of the intestinal inflammation observed after AOM/DSS, as
revealed by both MPO and Lcn2 measurements at day 141 (Fig.
3A and B).
Dietary emulsifying agents alter the intestinal microbiota
composition, leading to a proinflammatory intestinal
environment
We next considered the possibility that emulsifier-induced
alterations of the gut microbiota might underlie its tumor-pro-
moting effects. Microbiota composition analysis were previously
performed on CMC- and P80-treated animals and revealed a
strong clustering following treatment irrespective of cage group-
ing, thus confirming our observation that emulsifiers alter micro-
biota composition (22). On the basis of previous reports that
some pathovars of Escherichia coli can produce the procarcino-
genic toxin colibactin (11, 14, 33), we aimed to elucidate whether
such pathovars were playing a role in the observed emulsifier-
induced exacerbation of tumorigenesis. The quantification of
g-Proteobacteria, Enterobacteriaceae, Escherichia coli, or ClbB
encoding gene (colibactin polyketide synthesis system) revealed
that none of these phylotypes were significantly altered following
emulsifier consumption (day 0, day 21, and day 63) or after
tumorigenesis induction (day 141), suggesting that colibactin-
producing bacteria were not involved in the aforementioned
emulsifier-induced exacerbation of tumorigenesis (Supplemen-
tary Fig. S4). We next performed a more in-depth analysis of the
intestinal microbiota after emulsifier treatment, and found that
both CMC and P80 led to a significant reduction of microbiota
diversity after 9 weeks (day 63) of treatment (Supplementary Fig.
S5A and S5B), as well as profound alteration of the bacterial
community at the phylum, class, and order levels (Supplementary
Fig. S5C–S5E). Such alterations were characterized by an increase
in Bacteroidales and a decrease in Clostridiales orders upon CMC
or P80 consumption (Supplementary Fig. S5C–S5E). LEfSe (LDA
effect size), used to identify the most differentially abundant
taxons and OTUs between water and emulsifier-treated groups,
revealed a decrease of numerous Firmicutes members, such as
Lactobacillus, upon emulsifier consumption, together with an
increase of Bacteroidetes members (Supplementary Fig. S6).
We next wanted to investigate whether such alterations of the
microbiota were associated with any modification of its inherent
ability to induce proinflammatory gene expression in the host.
Hence, we measured the capacity of feces from control and
emulsifier-treated mice to activate proinflammatory gene expres-
sion via the LPS and flagellin receptors Toll-like receptor 4 (TLR4)
and TLR5, respectively. As previously reported, at day 63, exposure
to emulsifiers increased levels of bioactive fecal LPS and flagellin
(Fig. 3C–H). Elevated fecal flagellin and LPS were not associated
with an elevation in the total fecal bacterial load (Supplementary
Fig. S7), indicating that this increase in microbiota proinflamma-
tory potential was independent of bacterial load and, rather likely
a consequence of altered species composition. The induction of
tumorigenesis by AOM/DSS was found to further increase the
proinflammatory potential of the microbiota, in both water and
emulsifier-treated groups (Fig. 3E and H). The analysis of the
predicted metagenome indicated the presence of an altered meta-
genome in emulsifier-treated animals compared with water-only
treated animals (Fig. 4; Supplementary Figs. S8 and S9; ref. 34).
Using principal coordinate analysis, a strong and distinct cluster-
ing (P¼0.01) was observed between metagenomes of water and
CMC or P80-treated groups at day 63 (Fig. 4C and D), while
predicted metagenomes of all groups were similar at day 0 (Fig. 4A
and B). This was further confirmed using Volcano plots of Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathways abun-
dance, demonstrating a drastic alteration in microbiota metagen-
omes following emulsifier consumption at day 63 (Fig. 4E). Such
alterations were characterized, in emulsifier-treated animals, by a
decrease in the richness of signaling pathways, in accord with the
observed decrease in bacterial community richness (Supple-
mentary Fig. S5). Moreover, the analysis of significantly altered
metabolic pathways revealed that emulsifier consumption led
to an increased proportion of bacterial genes involved in LPS
biosynthesis, bacterial motility, and secretion systems (Fig. 4F
and G; Supplementary Figs. S8 and S9), correlating with the
observationofanincreasedproinflammatory microbiota under
emulsifier consumption. Altogether, these data demonstrate
that emulsifier consumption drastically altered the intestinal
microbiota composition, resulting in a basal low-grade proin-
flammatory environment in the intestine that predisposed to
subsequent tumorigenesis.
Dietary emulsifying agents disturb the proliferation/apoptosis
balance of epithelial cells
The increased tumor burden observed in emulsifier-treated
animals suggested the possibility of increased cell proliferation
in those animals. Hence, proliferation of colonic epithelial cells
was analyzed by Ki67 staining, which revealed that the consump-
tion of emulsifiers by itself (i.e., no AOM/DSS) resulted in
increased cell proliferation relative to the water-treated control
group (Fig. 5). AOM/DSS treatment increased the number of
Figure 1.
Dietary emulsifiers promote colitis-associated cancer. WT mice were exposed to drinking water containing CMC or P80 (1.0%) for 13 weeks. Mice were then injected
intraperitoneally with AOM (10 mg/kg body weight), maintained for 7 days, and then subjected to a two-cycle DSS treatment (each cycle consisted of 7 days
of 2.5% DSS and 14 days of H2O). Colon weights (A), colon lengths (B), spleen weights (C), fat-pad mass (D), body weight over time (E), representative colon samples
from each experimental group at the end of the AOM/DSS protocol (F), number of tumor per mouse (G), total tumor surface determined using a dissecting
microscope fitted with an ocular micrometer (H). Data are the means SEM (n¼5–8). Significance was determined using ttest (,P<0.05) or two-way group
ANOVA corrected for multiple comparisons with a Bonferroni test (#, statistical significance).
Emulsifying Agents Promote Colonic Carcinogenesis
www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 31
Figure 2.
Dietary emulsifiers promote intestinal inflammation and carcinogenesis. Analysis of IL6 (A), CXCL1 (B), CXCL2 (C), IL22 (D), and TN FamRNA expression (E) by qRT-
PCR in the colon following emulsifier treatment and following the induction of colonic neoplasia. F, Analysis of serum CXCL1 level by ELISA following emulsifier
treatment and following the induction of colonic neoplasia. Data are the means SEM (n¼5–8). Significance was determined using ttest (,P<0.05).
Viennois et al.
Cancer Res; 77(1) January 1, 2017 Cancer Research32
Figure 3.
Dietary emulsifiers favor a
proinflammatory microbiota. Colonic
MPO levels (A), fecal Lcn2 concentration at
day 141 (B), and bioactive levels of fecal
flagellin (FliC; C–E) and LPS (F–H) assayed
with TLR5 and TLR4 reporter cells,
respectively, at day 0 (C, F), day 63 (D, G),
and day 126 (E, H). Data are the means
SEM (n¼5–8). Significance was
determined using ttest (,P<0.05).
Emulsifying Agents Promote Colonic Carcinogenesis
www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 33
Viennois et al.
Cancer Res; 77(1) January 1, 2017 Cancer Research34
Ki67-positive cells in all groups of mice, but the proliferation level
remained significantly higher in AOM/DSS-treated mice that had
consumed emulsifiers, in accord with the notion that emulsifier
promotion of tumorigeneis involves increased cell proliferation
(Fig. 5). To further address the role of cell turnover in this process,
we performed TUNEL-based quantification of apoptosis in colon-
ic sections from emulsifier-treated mice. Analogous to our results
for cell proliferation, we observed that consumption of emulsi-
fiers, alone, increased the basal level of TUNEL
þ
cells. Moreover,
this difference between the water and emulsifier-consuming
groups was further increased in response to AOM/DSS treatment.
For both proliferative and apoptotic cells counts, similar results
were obtained when subdividing the crypts (top/middle/bottom,
data not shown). Together, these results indicate that emulsifier
consumption upregulates both apoptosis/proliferation in the
intestinal epithelium, resulting in an increased cell turnover, and
creating an opportune milieu for the tumorigenesis.
Dietary emulsifiers alter epithelial cell proliferation and
apoptosis in a microbiota-dependent manner
To further investigate how emulsifier consumption impacted
proliferation/apoptosis, we next analyzed by qRT-PCR the expres-
sion levels of genes that control proliferation (cyclin D1, D2,
Ki67), apoptosis (BCL2 and BAD), and angiogenesis (VEGFA). As
shown in Fig. 7, we observed that, following dietary emulsifier
consumption, the expression of cyclin D1, cyclin D2, and Ki67-
encoding genes were significantly deregulated (Fig. 7A–C), while
the b-catenin pathway were found unaltered (Supplementary Fig.
S10). In the AOM/DSS-treated groups, the expression levels of
these genes were further deregulated, without any difference
observed between water- and emulsifier-treated groups (data not
shown). The anti- and proapoptotic encoding genes Bcl2 and Bad
(Fig. 7D and E) and the marker of angiogenesis VEGFA (Fig. 7F)
remained unaltered under emulsifier's consumption.
We next sought to investigate whether the unbalanced prolif-
eration/apoptosis and the molecular alterations observed in
emulsifier-treated mice were driven by alterations in gut micro-
biota. We therefore analyzed the expression of the same genes in
germ-free animals treated with dietary emulsifiers, and impor-
tantly found that none of those genes have an altered expression
under germ-free conditions (Fig. 7A–F), thus indicating that the
presence of an altered microbiota is a prerequisite for subsequent
perturbations in proliferation and apoptosis processes. Finally,
we investigated whether the altered microbiota driven by emul-
sifier consumption was sufficient to alter intestinal expression of
genes involved in the proliferative/apoptosis balance. As pre-
sented in Fig. 7G–L, we found that transfer of microbiota from
emulsifier-consuming mice to germ-free mice, that were not fed
emulsifiers, recapitulated perturbations of cyclin D1 and D2
expression, thus suggesting that emulsifier-induced alterations of
intestinal microbiota composition plays a central and direct role
in the promotion of carcinogenesis.
Dietary emulsifiers induce carcinogenesis in AOM-treated
animals
We next sought to investigate whether emulsifier consumption
may be sufficient to drive colonic carcinogenesis in animals
treated with AOM but without exogenously induced severe intes-
tinal inflammation. For this purpose, WT mice were exposed to
drinking water containing CMC or P80 (1.0%) for 12 weeks, and
were injected intraperitoneally with AOM (10 mg/kg body
weight) weekly, for a total of seven injections. The combination
of AOM injections and emulsifier consumption leads to substan-
tial intestinal inflammation, as characterized by increase in colon
and spleen weights and by increased proinflammatory cytokine
expressions and fecal Lcn2 levels (Fig. 7A–L). Importantly, CMC-
induced intestinal inflammation was sufficient to induce carci-
nogenesis in a subset of animals, that was associated with altera-
tions of proliferative pathways (Fig. 7M–R), suggesting that, at
least in presence of some mutagens, emulsifier-induced low grade
inflammation and/or alteration of proliferation pathways is suf-
ficient to drive colon carcinogenesis.
Discussion
Mounting evidence implicates alteration of the gut microbiota,
that is, dysbiosis, as an important determinant of colon cancer. A
major tenet in this indictment is that the microbial dysbiosis is a
major driver of gut inflammation, which, when occurring chron-
ically, is strongly associated with an increased incidence of colon
cancer. More recently, it has been shown that the microbiota
composition can influence tumor development beyond merely
driving inflammation (11, 14, 33, 35). Herein, we add to this body
of knowledge in several ways thus providing new insights into
factors that may drive microbial dysbiosis in the first place and,
moreover, elucidate how an altered microbiota can result in
increased tumor development. Specifically, we report that, in
mice, consumption of dietary emulsifiers resulted in an altered
gut microbiota composition associated with increased levels of
flagellin and LPS, hence creating a low-grade proinflammatory
environment. The latter was associated with altered rates of
proliferation/apoptosis that predisposed animals to exacerbated
tumor development when subjected to a chemical-induced model
of colitis-associated cancer. In addition, emulsifier consumption
Figure 4.
Profound metagenome alteration following CMC and P80 consumption. WT mice were exposed to drinking water containing CMC or P80 (1.0%) for 13 weeks.
PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to predict the metagenomes, subsequently
analyzed by principal coordinates analysis of the beta diversity using binary Jaccard method at day 0 (Aand B) and day 63 (Cand D). E, KEGG pathways were
visualized on a volcano plot. From left to right, water-treated versus CMC-treated at day 0; water-treated versus CMC-treated at day 63; water-treated
versus P80-treated at day 0; water-treated versus P80-treated at day 63. For each KEGG identifier, the difference in abundance between the two groups is
indicated in log
2
fold change on x-axis (with positive values corresponding to an increase in emulsifier-treated group compared with water-treated group, and
negative values corresponding to a decrease in emulsifier-treated group compared with water-treated group), and significance between the two groups is
indicated by log
10
Pvalue on the y-axis. Red dots correspond to KEGG identifiers with a Pvalue <0.05 between emulsifier-treated and water-treated groups. Orange
dots correspond to KEGG identifiers with at least a 2-fold decreased or increased abundance in emulsifier-treated group compared with water-treated group.
Green dots correspond to KEGG identifiers with at least a 2-fold decreased or increased abundance in emulsifier-treated group compared with water-treated
group and with a Pvalue <0.05. Fand G, Predicted metagenomes were categorized at level 3 of the KEGG pathways, and pathways involved in LPS biosynthesis,
secretion system synthesis, and motility were graphed. Data are the means SEM (n¼5–8). Significance was determined using ttest (,P<0.05).
Emulsifying Agents Promote Colonic Carcinogenesis
www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 35
Figure 5.
Dietary emulsifiers alter epithelial cell proliferation and apoptosis during colitis-associated cancer development. Aand B, Epithelial cell proliferation was analyzed by
IHC using the proliferation marker Ki67 in colonic tissue sections. A, Representative images of Ki67 staining. Scale bar, 200 mm. B, Ki67
þ
cells were counted
and averaged per crypt. Cand D, Epithelial cell apoptosis was analyzed by TUNEL assay. C, Representative confocal images of TUNEL assay. TUNEL, green;
DNA, blue. Scale bar, 25 mm. D, TUNEL
þ
DAPI
þ
cells were counted and averaged per crypt. Data are the means SEM (n¼5–8). Significance was determined using
ttest (,P<0.05).
Viennois et al.
Cancer Res; 77(1) January 1, 2017 Cancer Research36
was sufficient to drive development of colonic tumors in some
animals treated with AOM (without DSS) and was associated with
altered proliferation. The level of exposure used herein in mice is
intended to approximately model the case of humans who eat
large amounts of processed foods, many of which contain CMC,
P80, and/or other emulsifiers exceeding 2% of the product by
weight (23).
While human studies of microbial dysbiosis, inflammation,
and cancer are primarily limited to associations, that germ-free
mice are protected against colonic carcinogenesis supports the
notion that dysbiosis is not purely a consequence of disease but,
rather, also plays a role in driving the development of colorectal
cancer (9, 36–38). Moreover, in this study, we demonstrated that
microbiota dysbiosis that precedes the initiation of colonic
tumorigenesis was sufficient to promote cancer development.
Importantly, the induction of colonic tumorigenesis similarly
induced intestinal inflammation in emulsifier- and water-treated
groups, showing that the observed phenotypes were not only due
to an exacerbation of inflammation during DSS, but that the low-
grade intestinal inflammation that precedes the initiation of
carcinogenesis was playing a central role. While, in mice, emul-
sifier consumption promotes increased food consumption and
adiposity and, in humans, obesity itself is associated with colon
cancer, we speculate that the link between emulsifiers and cancer
is not obesity per se but rather low-grade inflammation. Accord-
ingly, emulsifiers increased food consumption in AOM/DSS-
treated mice without a corresponding increase in weight, likely
reflecting increased energy demand, suggest that inflammation
and associated increases in cell turnover are more germane.
However, it is possible that emulsifier-induced alterations in
microbiota can cause increased energy intake that might promote
carcinogenesis irrespective of adiposity.
One mechanism by which microbiota was previously reported
to promote colorectal cancer is via a transient contact between
colibactin-producing E. coli and epithelial cells that subsequently
became malignant (11, 14, 33, 35). We thus hypothesized that
colibactin-producing E. coli could be involved in the emulsifier-
induced exacerbation of tumorigenesis. However, the quantifica-
tion of Proteobacteria, Enterobacteriaceae, Escherichia coli, and
ClbB-encoding gene negated this possibility. Although emulsi-
fiers did not drive substantial changes in abundance of such
pathovars, inflammation, alteration of microbiota and its capacity
to generate proinflammatory signals were critical for tumor devel-
opment. Indeed, upon emulsifier's exposure, the diversity of
microbiota was decreased and the abundance of some phyla,
class, and orders was altered (decrease of Firmicutes, notably
Clostridiales class and Lactobacillus member, and increase of
Bacteroidetes). Lactobacilli have been associated with protection
against colonic carcinogenesis via antioxidant, antiproliferation
properties and immunomodulatory and antitumor effects
(39–41). We observed a greater abundance of bacteroidetes in
both CMC- and P80-treated mice, correlating with the observa-
tion that intestinal mucosal surface of patients with adenoma
displayed increased abundance of Bacteroidetes (42). However,
this same study reported higher abundance of Firmicutes in
adenoma patients while we observed a decrease of this phylum
upon emulsifier's consumption (42). While little is known about
how emulsifiers impact microbiota, the findings of our current
study, in conjunction with previous reports, suggest that the
consumption of emulsifiers induce a shift of the gut microbial
population creating a favorable environment for colonic
Figure 6.
Dietary emulsifiers alter epithelial cell proliferation and apoptosis in a
microbiota-dependent manner. Conventional and germfree Swiss-Webster WT
mice were exposed to drinking water containing CMC or P80 (1.0%) for 13 weeks.
Intestinal microbiota from conventional Swiss-Webster WT mice exposed to
drinking water containing CMC or P80 (1.0%) for 13 weeks were transplanted to
germfree Swiss-Webster WT mice. Analysis of cyclin D1 (Aand G), cyclin D2
(Band H), Ki67 (Cand I), BCL2 (Dand J), BAD (Eand K), and VEGFA
mRNA expression (Fand L) by qRT-PCR in the colon following emulsifier
treatment under germ-free conditions (A–F) and following microbiota
transplantation (G–L).
Emulsifying Agents Promote Colonic Carcinogenesis
www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 37
Figure 7.
Dietary emulsifiers promote intestinal inflammation and carcinogenesis in the absence of DSS. WT mice were exposed to drinking water containing CMC or P80 (1.0%)
for 12 weeks. Mice were injected intraperitoneally with AOM (10 mg/kg body weight) weekly for a total of 7 injections. Colon weights (A), colon lengths (B),
spleen weights (C), and fat-pad mass (D). E–I, Analysis of IL6 (E), CXCL1 (F), CXCL2 (G), IL22 (H), and TNFamRNA expression (I) by qRT-PCR in colons of emulsifier-
AOM–treated mice. J–L, Fecal Lcn2 concentration at day 0 (J), 56 (K), and 84 (L). M, Representative colonoscopy from each experimental group at the end
of the protocol. Nand O, Number of tumor per mouse (N) and total tumor surface (O) determined using a dissecting microscope fitted with an ocular micrometer.
P–R, Analysis of cyclin D1 (P), cyclin D2 (Q), and Ki67 mRNA expression (R) by qRT-PCR in colons of emulsifier-AOM–treated mice. Data are the means SEM
(n¼10). Significance was determined using ttest (,P<0.05).
Viennois et al.
Cancer Res; 77(1) January 1, 2017 Cancer Research38
carcinogenesis (22, 23). Gut microbes could indeed act through
various pathways including proliferation, immune system, or
inflammation. We quantified the proinflammatory potential of
the microbiota associated with the consumption of CMC or P80
and showed increased level of bioactive flagellin and LPS. It was
recently reported that in humans, serum anti-flagellin and anti-
LPS antibody concentrations positively correlate with colorectal
cancer risk (43). Importantly, TLR4-deficient mice, unable to
recognize bacterial LPS, are protected from colon carcinogenesis
(44), further highlighting the central role played by increased fecal
flagellin and/or LPS loads in the carcinogenesis initiation and
promotion.
Comparative metagenomes analyses of CMC- or P80-treated
mice and water control mice showed that the shift of bacteria was
accompanied by alterations of several metabolic pathways, with a
decrease in overall metabolic pathway richness and an increased
proportion of bacterial genes involved in LPS biosynthesis, as well
as bacterial motility and secretion systems. These observations
were correlating with the increased proinflammatory ability of
microbiota measured under emulsifier consumption. Important-
ly, in addition of promoting and maintaining low-grade intestinal
inflammation, we observed in the current study that the con-
sumption of dietary emulsifiers also induced alterations of some
major proliferation and apoptosis actors in a microbiota-depen-
dent manner. The observation that such disruptions were effi-
ciently transferred to mice receiving fecal microbiota of emulsifier-
treated donors revealed that emulsifier-induced alteration of
microbiota composition plays a central role in the promotion
of carcinogenesis. The previous observation that genetically engi-
neered animals that develop intestinal inflammation, such as
TLR5KO and NLRP3 mice, are not necessarily predisposed to
colonic carcinogenesis (unpublished data and ref. 45, respective-
ly) highlights the concept that alterations of proliferation and
apoptosis pathways in a microbiota-dependent manner may be
the central mechanism that mediates the emulsifier-dependent
increase of carcinogenesis.
An important distinction between this study and others exam-
ining microbiota, inflammation, and cancer, is that emulsifier
consumption does not induce histopathologically evident (i.e.,
classic) inflammation, but rather induces only low-grade inflam-
mation. Such low-grade inflammation is associated with and may
cause obesity and its interrelated metabolic diseases, that is,
metabolic syndrome. Hence, the mechanisms described herein
may be relevant not only to the potential promotion of colon
cancer by one class of food additive, emulsifiers, but may be a
broad mechanism whereby any inducer of low-grade inflamma-
tion, including obesity itself, may increase potential for cancer
development. While the increased risk of cancer development in
obese are modestly less than that of IBD patients (46), given the
very high and increasing prevalence of obesity in all developed
countries, low-grade inflammation may prove to be a major factor
that underlies the increasing incidence of colon cancer. Hence, we
propose that numerous factors that induce low-grade inflamma-
tion, including consumption of dietary emulsifiers, may promote
a hostile environment in the colon by modifying the microbiota
composition, leading to low-grade intestinal inflammation and
alterations in the colonic proliferation/apoptosis balance, there-
fore creating a favorable niche for colonic tumorigenesis.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: E. Viennois, D. Merlin, A.T. Gewirtz, B. Chassaing
Development of methodology: E. Viennois, B. Chassaing
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): E. Viennois, B. Chassaing
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): E. Viennois, B. Chassaing
Writing, review, and/or revision of the manuscript: E. Viennois, D. Merlin,
A.T. Gewirtz, B. Chassaing
Study supervision: B. Chassaing
Grant Support
This work was supported by NIH grants DK099071 and DK083890 (A.T.
Gewirtz). B. Chassaing is a recipient of the Career Development Award from the
Crohn's and Colitis Foundation of America (CCFA). E. Viennois is a recipient of
the Research Fellowship Award from the CCFA. D. Merlin is a recipient of a
Research Career Scientist Award from the Department of Veterans Affairs.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received May 13, 2016; revised October 25, 2016; accepted October 26, 2016;
published OnlineFirst November 7, 2016.
References
1. Weir HK, Thun MJ, Hankey BF, Ries LA, Howe HL, Wingo PA, et al. Annual
report to the nation on the status of cancer, 1975–2000, featuring the uses
of surveillance data for cancer prevention and control. J Natl Cancer Inst
2003;95:1276–99.
2. Merlin D, Si-Tahar M, Sitaraman SV, Eastburn K, Williams I, Liu X, et al.
Colonic epithelial hPepT1 expression occurs in inflammatory bowel dis-
ease: transport of bacterial peptides influences expression of MHC class 1
molecules. Gastroenterology 2001;120:1666–79.
3. Wojtal KA, Eloranta JJ, Hruz P, Gutmann H, Drewe J, Staumann A, et al.
Changes in mRNA expression levels of solute carrier transporters in
inflammatory bowel disease patients. Drug Metab Dispos 2009;37:
1871–7.
4. Eaden JA, Abrams KR, Mayberry JF. The risk of colorectal cancer in ulcerative
colitis: a meta-analysis. Gut 2001;48:526–35.
5. Ahmadi A, Polyak S, Draganov PV. Colorectal cancer surveillance in
inflammatory bowel disease: the search continues. World J Gastroenterol
2009;15:61–6.
6. Ekbom A, Helmick C, Zack M, Adami HO. Increased risk of large-bowel
cancer in Crohn's disease with colonic involvement. Lancet 1990;
336:357–9.
7. Jess T, Gamborg M, Matzen P, Munkholm P, Sorensen TI. Increased risk of
intestinal cancer in Crohn's disease: a meta-analysis of population-based
cohort studies. Am J Gastroenterol 2005;100:2724–9.
8. Thorsteinsdottir S, Gudjonsson T, Nielsen OH, Vainer B, Seidelin JB.
Pathogenesis and biomarkers of carcinogenesis in ulcerative colitis. Nat
Rev Gastroenterol Hepatol 2011;8:395–404.
9. Chassaing B, Darfeuille-Michaud A. The commensal microbiota and enter-
opathogens in the pathogenesis of inflammatory bowel diseases. Gastro-
enterology 2011;140:1720–28.
10. Arthur JC, Jobin C. The complex interplay between inflammation, the
microbiota and colorectal cancer. Gut Microbes 2013;4:253–8.
11. Arthur JC, Perez-Chanona E, Muhlbauer M, Tomkovich S, Uronis JM, Fan
TJ, et al. Intestinal inflammation targets cancer-inducing activity of the
microbiota. Science 2012;338:120–3.
www.aacrjournals.org Cancer Res; 77(1) January 1, 2017 39
Emulsifying Agents Promote Colonic Carcinogenesis
12. Wang X, Huycke MM. Extracellular superoxide production by Enterococcus
faecalis promotes chromosomal instability in mammalian cells. Gastro-
enterology 2007;132:551–61.
13. Maddocks OD, Short AJ, Donnenberg MS, Bader S, Harrison DJ. Attaching
and effacing Escherichia coli downregulate DNA mismatch repair protein
in vitro and are associated with colorectal adenocarcinomas in humans.
PLoS One 2009;4:e5517.
14. Nougayrede JP, Homburg S, Taieb F, Boury M, Brzuszkiewicz E, Gottschalk
G, et al. Escherichia coli induces DNA double-strand breaks in eukaryotic
cells. Science 2006;313:848–51.
15. Buc E, Dubois D, Sauvanet P, Raisch J, Delmas J, Darfeuille-Michaud A,
et al. High prevalence of mucosa-associated E. coli producing cyclomo-
dulin and genotoxin in colon cancer. PLoS One 2013;8:e56964.
16. Prorok-Hamon M, Friswell MK, Alswied A, Roberts CL, Song F, Flanagan
PK, et al. Colonic mucosa-associated diffusely adherent afaCþEscherichia
coli expressing lpfA and pks are increased in inflammatory bowel disease
and colon cancer. Gut 2014;63:761–70.
17. Klimesova K, Kverka M, Zakostelska Z, Hudcovic T, Hrncir T, Stepankova R,
et al. Altered gut microbiota promotes colitis-associated cancer in IL-1
receptor-associated kinase M-deficient mice. Inflamm Bowel Dis 2013;
19:1266–77.
18. Uronis JM, Muhlbauer M, Herfarth HH, Rubinas TC, Jones GS, Jobin C.
Modulation of the intestinal microbiota alters colitis-associated colorectal
cancer susceptibility. PLoS One 2009;4:e6026.
19. Chassaing B, Gewirtz AT. Gut microbiota, low-grade inflammation, and
metabolic syndrome. Toxicol Pathol 2014;42:49–53.
20. Vijay-Kumar M, Aitken JD, Carvalho FA, Cullender TC, Mwangi S, Srini-
vasan S, et al. Metabolic syndrome and altered gut microbiota in mice
lacking Toll-like receptor 5. Science 2010;328:228–31.
21. Gregor MF, Hotamisligil GS. Inflammatory mechanisms in obesity. Annu
Rev Immunol 2011;29:415–45.
22. Chassaing B, Koren O, Goodrich JK, Poole AC, Srinivasan S, Ley RE, et al.
Dietary emulsifiers impact the mouse gut microbiota promoting colitis and
metabolic syndrome. Nature 2015;519:92–6.
23. Swidsinski A, Ung V, Sydora BC, Loening-Baucke V, Doerffel Y, Verstraelen
H, et al. Bacterial overgrowth and inflammation of small intestine after
carboxymethylcellulose ingestion in genetically susceptible mice. Inflamm
Bowel Dis 2009;15:359–64.
24. Roberts CL, Keita AV, Duncan SH, O'Kennedy N, Soderholm JD, Rhodes
JM, et al. Translocation of Crohn's disease Escherichiacoli across M-cells:
contrasting effects of soluble plant fibres and emulsifiers. Gut 2010;59:
1331–9.
25. Roberts CL, Rushworth SL, Richman E, Rhodes JM. Hypothesis: Increased
consumption of emulsifiers as an explanation for the rising incidence of
Crohn's disease. J Crohns Colitis 2013;7:338–41.
26. Bar A, Van Ommen B, Timonen M. Metabolic disposition in rats of regular
and enzymatically depolymerized sodium carboxymethylcellulose. Food
Chem Toxicol 1995;33:901–7.
27. Food and Agriculture Organization of the United Nations. Food Additives
Series. Safety evaluation of certain food additives; 1999. Geneva, Switzer-
land: World Health Organization. Available from: http://www.inchem.
org/pages/jecfa.html.
28. Food Safety Commission of Japan. Available from: https://www.fsc.go.jp/
english/evaluationreports/foodadditive/polysorbate_report.pdf.
29. Treon J, Gongwer L, Nelson M, Kirschman J. Chemistry, physics, and
application of surface active substances. New York, NY:Gordon and Breach;
1967.
30. Greten FR, Eckmann L, Greten TF, Park JM, Li ZW, Egan LJ, et al. IKKbeta
links inflammation and tumorigenesis in a mouse model of colitis-asso-
ciated cancer. Cell 2004;118:285–96.
31. Chassaing B, Koren O, Carvalho FA, Ley RE, Gewirtz AT. AIEC pathobiont
instigates chronic colitis in susceptible hosts by altering microbiota com-
position. Gut 2014;63:1069–80.
32. Chassaing B, Srinivasan G, Delgado MA, Young AN, Gewirtz AT, Vijay-
Kumar M. Fecal lipocalin 2, a sensitive and broadly dynami c non-invasive
biomarker for intestinal inflammation. PLoS One 2012;7:e44328.
33. Cougnoux A, Dalmasso G, Martinez R, Buc E, Delmas J, Gibold L, et al.
Bacterial genotoxin colibactin promotes colon tumour growth by inducing
a senescence-associated secretory phenotype. Gut 2014;63:1932–42.
34. Langille MG, Zaneveld J, Caporaso JG, McDonald D, Knights D, Reyes JA,
et al. Predictive functional profiling of microbial communities using 16S
rRNA marker gene sequences. Nat Biotechnol 2013;31:814–21.
35. Chassaing B, Gewirtz AT. Pathobiont hypnotises enterocytes to promote
tumour development. Gut 2014;63:1837–8.
36. Sears CL, Garrett WS. Microbes, microbiota, and colon cancer. Cell Host
Microbe 2014;15:317–28.
37. Engle SJ, Ormsby I, Pawlowski S, Boivin GP, Croft J, Balish E, et al.
Elimination of colon cancer in germ-free transforming growth factor beta
1-deficient mice. Cancer Res 2002;62:6362–6.
38. Laqueur GL, Matsumoto H, Yamamoto RS. Comparison of the carci-
nogenicity of methylazoxymethanol-beta-D-glucosiduronic acid in con-
ventional and germfree Sprague-Dawley rats. J Natl Cancer Inst 1981;
67:1053–5.
39. Zhong L, Zhang X, Covasa M. Emerging roles of lactic acid bacteria in
protection against colorectal cancer. World J Gastroenterol 2014;20:
7878–86.
40. Matsuzaki T, Yokokura T, Mutai M. Antitumor effect of intrapleural
administration of Lactobacilluscasei in mice. Cancer Immunol Immunother
1988;26:209–14.
41. Thirabunyanon M, Hongwittayakorn P. Potential probiotic lactic acid
bacteria of human origin induce antiproliferation of colon cancer cells
via synergic actions in adhesion to cancer cells and short-chain fatty acid
bioproduction. Appl Biochem Biotechnol 2013;169:511–25.
42. Sanapareddy N, Legge RM, Jovov B, McCoy A, Burcal L, Araujo-Per ez F, et al.
Increased rectal microbial richness is associated with the presence of
colorectal adenomas in humans. ISME J 2012;6:1858–68.
43. Kong SY, Tran HQ, Gewirtz AT, McKeown-Eyssen G, Fedirko V, Romieu I,
et al. Serum endotoxins and flagellin and risk of colorectal cancer in the
European Prospective Investigation into Cancer and Nutrition (EPIC)
Cohort. Cancer Epidemiol Biomarkers Prev 2016;25:291–301.
44. Fukata M, Chen A, Vamadevan AS, Cohen J, Breglio K, Krishnareddy S, et al.
Toll-like receptor-4 promotes the development of colitis-associated colo-
rectal tumors. Gastroenterology 2007;133:1869–81.
45. Chow MT, Sceneay J, Paget C, Wong CS, Duret H, Tschopp J, et al. NLRP3
suppresses NK cell-mediated responses to carcinogen-induced tumors and
metastases. Cancer Res 2012;72:5721–32.
46. Berger NA. Obesity and cancer pathogenesis. Ann N Y Acad Sci 2014;
1311:57–76.
Cancer Res; 77(1) January 1, 2017 Cancer Research40
Viennois et al.
