Microorganisms 09 01021

microorganisms
Review
Role of Gut Microbiota and Probiotics in Colorectal Cancer:
Onset and Progression
Edgar Torres-Maravilla 1 , Anne-Sophie Boucard 1 , Amir Hossein Mohseni 2 , Sedigheh Taghinezhad-S 2 ,
Naima G. Cortes-Perez 3 and Luis G. Bermúdez-Humarán 1, *
1 Micalis Institute, Université Paris-Saclay, INRAE, AgroParisTech, 78350 Jouy-en-Josas, France;

edgar.torres-maravilla@inrae.fr (E.T.-M.); Anne-Sophie.Boucard@inrae.fr (A.-S.B.)
2 Department of Microbiology, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University,
Tehran 1477893855, Iran; amho.mohseni@gmail.com (A.H.M.); taghinezhad.m@gmail.com (S.T.-S.)
3 Université Paris-Saclay, INRAE, AgroParisTech, UMR 0496, 78350 Jouy-en-Josas, France;
naima.cortes-perez@inrae.fr
* Correspondence: luis.bermudez@inrae.fr
Abstract: The gut microbiota plays an important role in maintaining homeostasis in the human
body, and the disruption of these communities can lead to compromised host health and the onset of
disease. Current research on probiotics is quite promising and, in particular, these microorganisms
have demonstrated their potential for use as adjuvants for the treatment of colorectal cancer. This re-
view addresses the possible applications of probiotics, postbiotics, synbiotics, and next-generation
probiotics in colorectal cancer research.

Keywords: microbiota; probiotics; lactic acid bacteria; cancer; colorectal cancer
Citation: Torres-Maravilla, E.;
Boucard, A.-S.; Mohseni, A.H.;
Taghinezhad-S, S.; Cortes-Perez, N.G.;
Bermúdez-Humarán, L.G. Role of
1. Introduction
Gut Microbiota and Probiotics in
Colorectal Cancer: Onset and Currently, colorectal cancer (CRC) is the third most common cancer worldwide,
Progression. Microorganisms 2021, 9, with more than one million new cases and 600,000 deaths each year [1]. There are two
1021. https://doi.org/10.3390/ types of CRC: colitis-associated, caused by the presence of a mutation in the TP53 gene,
microorganisms9051021 and sporadic, caused by a mutation in the adenomatous polyposis coli (APC) gene. How-
ever, genetic factors play a relatively minor role in cancer development (<10% to 30%);
Academic Editor: Gisèle LaPointe instead, cancer risk is greatly influenced by extrinsic (e.g., environmental) factors such as
infectious agents, antibiotic administration, high-fat diets, red meat consumption, and a
Received: 18 April 2021 deficiency in fiber intake [2,3]. All of these components are known to alter gut microbiota
Accepted: 7 May 2021 and induce dysbiosis [4], defined as perturbations in commensal communities that can lead
Published: 10 May 2021 to the deficient education of the host immune system and the subsequent development
of immune-mediated diseases. Dysbiosis can be categorized into three types: (i) loss of
Publisher’s Note: MDPI stays neutral beneficial species, (ii) expansion of pathobionts or potentially harmful species, and (iii)
with regard to jurisdictional claims in
loss of overall microbial diversity [5]. All three types of dysbiosis have been observed in
published maps and institutional affil-
CRC patients.
iations.
One of the means by which healthy gut microbiota may exert their anticancer ef-
fects is through the beneficial metabolites they produce, which can have antioxidant and
anti-inflammatory properties, regulate bowel barrier function, act as vitamins, and rep-
resent a source of energy. Instead, the gut microbiota of CRC patients can have direct
Copyright: © 2021 by the authors. pro-tumorigenic effects; for example, a gavage of fecal samples from CRC patients was
Licensee MDPI, Basel, Switzerland. observed to promote intestinal carcinogenesis in both germ-free and conventional mice [6].
This article is an open access article
Recent research into probiotics, and into microbiota more generally, has yielded promising
distributed under the terms and
outcomes and has demonstrated the serious potential of these assemblages as co-adjuvants
conditions of the Creative Commons
in colon cancer therapies. This review addresses the current state of research on the
Attribution (CC BY) license (https://
role of the gut microbiota, as well as the efficacy of probiotics, postbiotics, synbiotics,
creativecommons.org/licenses/by/
and next-generation probiotics (NGPs), in CRC treatment and prevention.
4.0/).
Microorganisms 2021, 9, 1021. https://doi.org/10.3390/microorganisms9051021 https://www.mdpi.com/journal/microorganisms

Microorganisms 2021, 9, 1021 2 of 24
2. Gut Microbiota
The microbiota, also referred to as the microflora, is defined as the entire population
of microbes present within the human body, which principally includes bacteria, archaea,
and eukarya [7], as well as viruses [8]. The quantity of these microorganisms is staggering.
The human gastrointestinal tract alone can host nearly 100 trillion (1014 ) microorganisms [9],
a number nearly three times greater than the total number of cells in the entire human
body (recently recalculated as 3.7 × 1013 ) [10]. From a physiological point of view, the mi-
crobiota makes up about 2% of an adult’s body mass, almost equivalent to the size of the
human brain or liver [11], which has led some researchers to refer to the microbiota as
the forgotten human organ [12,13]. Many essential body processes require the presence
of these diverse and numerous microorganisms, as they provide the host with nutrients,
metabolize indigestible compounds, and can help in the defense against colonization by
opportunistic pathogens, as well as possess immune-modulatory properties [14].
With the use of next-generation DNA sequencing technologies and metagenomic anal-
ysis, it has been shown that the gut microbiota of vertebrates is composed of approximately
500–1000 different bacterial species, of which 98% are represented by two dominant phyla,
Bacteroidetes and Firmicutes [15–17]. One of the most surprising discoveries has been the
fact that the number of genes in the gut microbiota is approximately 100 times larger than
the human genome and appears to represent a co-evolutionary relationship [17]. Due to the
numerous interactions among different microbial species, human host cells, and the exter-
nal environment, the microbiota can also be conceptualized as being a dynamic ecological
community [18]. In this sense, a dynamic equilibrium of the microbiota in the human body
is necessary for health, which can be disrupted by environmental factors and external stim-
uli such as the use of antibiotics, illness, stress, aging, bad dietary habits, and lifestyle [19].
These alterations frequently result in microbial imbalances—dysbiosis—with direct links
to multiple pathological conditions [20].
For example, between 2018 and 2021, a search for “microbiota dysbiosis” and “dis-
eases” in PubMed returned 5,617 published articles describing links between dysbiosis
of the gut microbiota and diseases such as obesity [21], autism spectrum disorders [22],
cardiovascular diseases [23], diarrhea [24], alcoholic liver disease [25], acute-on-chronic
liver failure [26], arthritis [27], lung diseases [28], autoimmune diseases [29], lupus erythe-
matosus [30], coeliac disease [31], intestinal inflammatory diseases such as Crohn’s disease,
colitis and irritable bowel syndrome (IBD) [32], and colorectal cancer [33] (these account
for 531 citations by themselves).
3. CRC and Gut Microbiota

Compared to healthy individuals, CRC patients harbor a distinct mucosa-associated
microbiota. For instance, the effect of CRC on the microbiota is generally characterized by
an increase in microbial diversity that seems to progress with cancer development—late
CRC samples (stage III and stage IV) generally display higher richness levels than early
CRC samples do (stage I and stage II) [34]. At the phylum level, increasing numbers of
Bacteroidetes, Firmicutes, and Fusobacteria and decreasing numbers of Proteobacteria in
mucosa-associated microbiota are observed. At the genus level, CRC progression tends
to be associated with the proliferation of Fusobacterium, Peptostreptococcus, Streptococcus,
and Ruminococcus and a decline in Lactobacillus and Granulicatella [34,35] (Figure 1). Dif-
ferences in mucosa-associated microbiota can also be observed before the appearance of a
cancerous state. For example, Flemer et al. [36] detected significant differences between
the mucosa-associated microbiota from subjects with polyps and from healthy controls,
suggesting that the gut microbiota is involved in cancer development from a very early
stage. Another large-cohort multi-omics dataset indicated that shifts in the microbiome
and metabolome occur from the very early stages of the development of colorectal cancer,
which could be of possible etiological and diagnostic importance [37]. Accordingly, it has
been reported that a long-term exposure (≥2 months) to antibiotics in early to middle
adulthood is associated with an increased risk for colorectal adenoma at the age of 60 [38].
Microorganisms 2021, 9, x FOR PEER REVIEW 3 of 25
Microorganisms 2021, 9, 1021 metabolome occur from the very early stages of the development of colorectal cancer, 3 of 24
which could be of possible etiological and diagnostic importance [37]. Accordingly, it has
been reported that a long-term exposure (≥2 months) to antibiotics in early to middle
adulthood is associated with an increased risk for colorectal adenoma at the age of 60 [38].
Interestingly, the microbiota alterations observed in CRC patients are not restricted to
Interestingly, the microbiota alterations observed in CRC patients are not restricted to the
the tumor site; they can also be seen in the surrounding healthy tissue. Indeed, in multi-
tumor site; they can also be seen in the surrounding healthy tissue. Indeed, in multiple
ple cohorts, the mucosa-associated microbiota from paired samples of tumor tissue and
cohorts, the mucosa-associated microbiota from paired samples of tumor tissue and
nearby nontumor mucosa was similar with regard to both individual taxa and the overall
nearby nontumor mucosa was similar with regard to both individual taxa and the overall
microbiota composition [34,35,39,40].
microbiota composition [34,35,39,40].
Figure 1. Schematic of the roles of probiotic and harmful bacteria in CRC context. Disruption of the gut microbiota balance
Figure 1. Schematic of the roles of probiotic and harmful bacteria in CRC context. Disruption of the gut microbiota balance
is associated with CRC development, and regulation of probiotic bacteria is associated with CRC remission. (The figure
is associated with CRC development, and regulation of probiotic bacteria is associated with CRC remission. (The figure was
was created with Biorender.com).
created with Biorender.com).
A comparative study of coupled fecal and mucosal samples demonstrated that, alt-
A comparative study of coupled fecal and mucosal samples demonstrated that, al-
hough fecal microbiota only partially reflects the community at the mucus layer, differ-
though fecal microbiota only partially reflects the community at the mucus layer, differences
ences
due todue CRC to are
CRC areevident
still still evident insamples
in fecal fecal samples [36]. Thus,
[36]. Thus, this noninvasive
this noninvasive approach
approach is the
is the most commonly used sampling method in gut microbiome
most commonly used sampling method in gut microbiome studies. Fecal samples studies. Fecal samples
from
from CRC patients
CRC patients differ differ significantly
significantly fromofthose
from those of healthy
healthy subjects,subjects, both in microbial
both in microbial richness
richness and community
and community composition.composition.
In fecal In fecal samples,
samples, CRC development
CRC development is usually
is usually as-
associated
sociated with an in
with an increase increase in pro-inflammatory
pro-inflammatory or pathogenic
or pathogenic species belonging
species belonging to phyla
to phyla Proteobac-
Proteobacteria and Fusobacteria and a decrease in beneficial species of
teria and Fusobacteria and a decrease in beneficial species of phylum Firmicutes [41]. phylum Firmicutes
[41]. As observed
As observed withwith the mucosa-associated
the mucosa-associated microbiota,
microbiota, the fecal
the fecal microbiota
microbiota of CRCof CRC pa-
patients
tients is dynamic, with characteristic changes during cancer progression.
is dynamic, with characteristic changes during cancer progression. In a Chinese cohort, In a Chinese co-
hort, fecal samples from healthy individuals were dominated by Bacteroidetes
fecal samples from healthy individuals were dominated by Bacteroidetes and Firmicutes, and Fir-
micutes, the abundance
the abundance of which of which decreased
decreased with progression
with progression along the along the polyp–adenoma–
polyp–adenoma–carcinoma
carcinoma
sequence. In sequence.
contrast,Inthecontrast, the abundance
abundance of Proteobacteria
of Proteobacteria was noted towas noted to
increase increase
with colon
cancer development [42]. Such shifts have even been seen in the relative abundance of in-
dividual bacterial taxa. In particular, Firmicutes, Actinobacteria, Lachnospiraceae, and the
genus Desulfovibrio have been shown to be specific to early-stage CRC, while the genera
with colon cancer development [42]. Such shifts have even been seen in the relative abun-
dance of individual bacterial taxa. In particular, Firmicutes, Actinobacteria, Lachnospi-
raceae, and the genus Desulfovibrio have been shown to be specific to early-stage CRC,
Solobacterium, Peptostreptococcus, Corynebacterium, Parvimonas, Neisseria, Porphyromonas,
while the genera Solobacterium, Peptostreptococcus, Corynebacterium, Parvimonas, Neisseria,
Gemella, and the families Alcaligenaceae and Enterobacteriaceae appear to be associated
Porphyromonas, Gemella, and the families Alcaligenaceae and Enterobacteriaceae appear to
with malignancy [37,43–45] (Figure 2).
be associated with malignancy [37,43–45] (Figure 2).
Figure
Figure 2. 2. Overviewofofthe
Overview theimplications
implicationsof
ofgut
gut bacteria
bacteria in
in the
thedevelopment
developmentand
andprogression
progressionofof
colorectal cancer.
colorectal Shift
cancer. from
Shift from
healthy intestine to CRC intestine: (a) normal epithelium, (b) poly P (small benign growth), (c) early adenoma, (d) late
healthy intestine to CRC intestine: (a) normal epithelium, (b) poly P (small benign growth), (c) early adenoma, (d) late
adenoma, and (e) carcinoma. (The figure was created with Biorender.com).
adenoma, and (e) carcinoma. (The figure was created with Biorender.com).
3.1. Inflammation and CRC
3.1. Inflammation and CRC
Chronic inflammation is an established risk factor for CRC, as patients with inflam-
Chronic
matory bowelinflammation
diseases (IBD)isconsistently
an establishedhaverisk factorrisk
a higher for than
CRC,the asgeneral
patientspopulation
with inflam-
matory bowel diseases (IBD) consistently have a higher risk
of developing CRC [46,47]. Correspondingly, an increase in pro-inflammatory species than the general population
has
ofbeen
developing
repeatedly reported in CRC patients (Figure 3). The most prevalent and mostspecies
CRC [46,47]. Correspondingly, an increase in pro-inflammatory de-
has beenbacterium
scribed repeatedly in reported
CRC fecalin andCRC patients (Figuremicrobiota
mucosa-associated 3). The most prevalent and
is Fusobacterium most
nucle-
atum [45], which, in murine models, increases the proliferation of CRC cells and colonic nu-
described bacterium in CRC fecal and mucosa-associated microbiota is Fusobacterium
cleatum [45], which,
tumorigenesis in murine
by activating models,
TLR4 increases
signaling the proliferation
to NF-κB, thus promoting of CRC the cells and colonic
infiltration of
tumorigenesis by activating
specific pro-inflammatory TLR4 cell
myeloid signaling
subsetsto NF-κB,
into tumors thus promoting
[48–50]. the infiltration
Interestingly, a recent of
specific
study foundpro-inflammatory
that more thanmyeloid
40% of CRCcell patients
subsets into tumors
exhibited [48–50].
identical Interestingly,
strains a recent
of F. nucleatum
study
in bothfound
tumor thatandmore
salivathan 40% ofsuggesting
samples, CRC patients exhibited
that F. nucleatum identical
in CRC strains of F.
originates nucleatum
from the
inoral
both tumor
cavity [51].and saliva
In this, F. samples,
nucleatumsuggesting that F.tonucleatum
does not appear in CRC originates
be alone; meta-analyses from
of geo-
the oral cavity
graphically and[51].
technically F. nucleatum
In this,diverse cohortsdoes
havenot appear several
identified to be alone; meta-analyses
oral commensal and of
geographically
pathogenic bacteria and technically diverse cohorts
that are significantly enriched have identified
in CRC samples, several oral commensal
including members ofand
pathogenic
the generabacteria that are significantly
Fusobacterium, Porphyromonas, enriched in CRC samples,
Parvimonas, including members
Peptostreptococcus, Gemella, of
Prevotella,
the genera and Solobacterium.
Fusobacterium, Taken together,
Porphyromonas, these results
Parvimonas, reinforce the hypothesis
Peptostreptococcus, of an
Gemella, Prevotella,
oral–gut
and translocation
Solobacterium. Taken route that is these
together, associated
resultswith inflammation
reinforce and CRCof[39,52–55].
the hypothesis an oral–gut
Other examples
translocation of well-known
route pro-inflammatory
that is associated species with
with inflammation and links
CRC to CRC are colibactin-
[39,52–55]. Other exam-
producing
ples Escherichia
of well-known coli, which enhances
pro-inflammatory inflammation
species with links and to CRC the production of reactive
are colibactin-producing
oxygen species
Escherichia (ROS)enhances
coli, which in tumorsinflammation
in early-stageand CRC the[56], and enterotoxigenic
production Bactoroides
of reactive oxygen species
fragilis,inwhich
(ROS) tumors mediates inflammation
in early-stage through
CRC [56], andthe Th17 responseBactoroides
enterotoxigenic and NF-κBfragilis,
activation,
which
thus inducing
mediates myeloid cell-dependent
inflammation through the Th17 distalresponse
colon tumorigenesis
and NF-κB[57]. The increase
activation, thus in pro-
inducing
inflammatory
myeloid species observed
cell-dependent in CRC
distal colon correlates with
tumorigenesis [57].a reduction
The increase in anti-inflammatory
in pro-inflammatory
species observed in CRC correlates with a reduction in anti-inflammatory species belonging
to the beneficial genera Ruminococcus, Bifidobacterium, Lachnospira, Oribacterium, Desulfovib-
rio, Clostridiales, and Lactobacillus [44,55,58]. Furthermore, the alterations observed in CRC
with respect to microbiota composition may also translate into changes in metabolite

species belonging to the beneficial genera Ruminococcus, Bifidobacterium, Lachnospira, Ori-
bacterium, Desulfovibrio, Clostridiales, and Lactobacillus [44,55,58]. Furthermore, the altera-
tions observed in CRC with respect to microbiota composition may also translate into
concentrations. Specifically, a metabolomic study detected a direct association between
changes in metabolite concentrations. Specifically, a metabolomic study detected a direct
significantly lower abundances of Clostridia and Lachnospiraceae in CRC and reduced
association between significantly lower abundances of Clostridia and Lachnospiraceae in
quantities of the metabolites p-aminobenzoate and conjugated linoleic acid, which are
CRC and reduced quantities of the metabolites p-aminobenzoate and conjugated linoleic
known to exhibit anti-inflammatory and anti-cancerogenic properties [59].
acid, which are known to exhibit anti-inflammatory and anti-cancerogenic properties [59].
Figure
Figure 3.
3. Intestinal
Intestinal inflammation
inflammation caused
caused by
by gut
gut microbiota
microbiota contributes
contributes to
to the
the onset
onset of
of CRC.
CRC. Dysbiotic
Dysbiotic bacteria
bacteria can
can elicit
elicit
immune imbalances and facilitate the translocation of gut microbiota and/or its metabolites due to a leaky gut to the tissues
immune imbalances and facilitate the translocation of gut microbiota and/or its metabolites due to a leaky gut to the
and systemic circulation. These events may lead to the stimulation of an inflammatory state and ultimately to the devel-
tissues and systemic circulation. These events may lead to the stimulation of an inflammatory state and ultimately to the
opment of CRC. Thus, the production of IL-6 and IL-23, in turn, trigger the expression of IL-17A and contribute to the
development of
development ofCRC
CRC.through
Thus, the production
STAT3 of IL-6
activation. and IL-23,
In addition, in turn,
TNF-α and trigger the expression
IL-1 promote of IL-17Aand
pro-inflammatory andpro-tumor-
contribute
to the development
igenic of CRC
activities of COX-2 through
that STAT3
stimulate activation.
growth In addition,and
and angiogenesis TNF-α and
inhibit IL-1 promote
apoptosis pro-inflammatory
in CRC. (The figure wasand pro-
created
tumorigenic
with activities of COX-2 that stimulate growth and angiogenesis and inhibit apoptosis in CRC. (The figure was
Biorender.com).
created with Biorender.com).
3.2. DNA Damage and CRC
3.2. DNA Damage and CRC
Certain members of the CRC-enriched microbiota are able to directly induce DNA
damage Certain members
to colonic of thecells.
epithelial CRC-enriched
For example, microbiota are in
some strains ablethetoCRC-associated
directly inducefamily
DNA
damage to colonic epithelial cells. For example, some strains in the CRC-associated
Enterobacteriaceae produce ROS and colibactin, a toxin responsible for oncogenic muta- family
Enterobacteriaceae
tions in host colonicproduce ROS
epithelial and[60,61].
cells colibactin,
The apivotal
toxin responsible
role of thisfor oncogenic
toxin mutations
in carcinogenesis
in host colonic epithelial cells [60,61]. The pivotal role of this toxin
was confirmed by the finding that colibactin-producing E. coli promote tumorigenesis in in carcinogenesis
wasMin/+
Apc confirmed
; IL10−/−−by theinfinding
mice that colibactin-producing
a colibactin-dependent E. coliApromote
manner [62]. tumorigenesis
recent study identified in
a
Apc Min/+ ; IL10 / − mice in a colibactin-dependent manner [62]. A recent study identified
distinct mutational signature of genotoxic pks+ E. coli in human intestinal organoids, and
a distinct
this mutationalwas
same signature signature of genotoxic
also detected pks+ CRC
in human E. coligenomes.
in human intestinal
This suggestsorganoids,
that the
and this same signature was also detected in human CRC genomes.
underlying mutational process may be the direct result of past exposure to bacteria This suggests thatthat
the
underlying mutational process may be the direct result of past exposure to bacteria that
carried the colibactin-producing pks pathogenicity island [60]. However, E. coli is far from
carried the colibactin-producing pks pathogenicity island [60]. However, E. coli is far from
the only species with this capability. Molecular studies of F. nucleatum have discovered
the only species with this capability. Molecular studies of F. nucleatum have discovered the
the virulence protein FadA and its involvement in the transformation of epithelial cells
virulence protein FadA and its involvement in the transformation of epithelial cells and
the promotion of colon tumorigenesis [63]. A meta-analysis of CRC fecal metagenomes
confirmed the significant enrichment in both the colibactin-producing gene cluster pks and
the F. nucleatum adhesin fadA [53]. In vitro, enterotoxins produced by B. fragilis have been
associated with DNA damage and genomic instability [64], while, in mice, Peptostreptococcus
anaerobius, another CRC-enriched species, was shown to interact with TLR2/4 receptors
on host cells to induce ROS production, increasing cholesterol biosynthesis and activating
pro-oncogenic factors and CRC-promoting pathways [65]. CRC patients present increased
amounts of sulphate-reducing bacteria belonging to genus Desulfovibrio [37,44], which
explains the elevated levels in late-stage CRC of dissimilatory sulfate reductase subunit
A, a gene responsible for the production of genotoxic hydrogen sulfide [37]. Multiple
metabolomic analyses have reported increases in polyamines, such as putrescine and
cadaverine, in CRC fecal samples compared to healthy controls [41,64]; in particular,
the polyamine spermidine is known to promote colibactin-associated genotoxicity [66].
Finally, evidence from whole metagenomics analysis has linked the relative abundance
of some members of the CRC microbiota with the methylation or demethylation of host
genes, indicating that epigenome dysregulation may be another means by which CRC-
associated dysbiosis promotes colon carcinogenesis [58]. For example, the B. fragilis toxin
is able to induce epigenetic changes in vitro in HT-29 colon epithelial cells. The toxin alters
the expression of specific genes, the accessibility of certain transcription factor binding
sites, and the coordination between regions with different degrees of methylation, which,
together, increases the risk of colon tumorigenesis [67].
3.3. Short-Chain Fatty Acids and CRC

Short-chain fatty acids (SCFAs) are the primary end-products of the fermentation
of polysaccharides and nondigestible carbohydrates that remain available to the gut mi-
crobiota. Butyrate, acetate, and propionate are the most abundant SCFAs. Butyrate,
in particular, has a remarkable array of colonic health-promoting and antineoplastic prop-
erties; along with being the preferred energy source for colonocytes, it maintains mucosal
integrity, reduces pro-inflammatory cytokines, and induces apoptosis in CRC cell lines [68].
Compared to healthy controls, the fecal microbiota of patients with CRC and advanced
colorectal adenoma demonstrates significant reductions in the abundance of butyrate-
producing bacteria [55,69], and these reductions are dependent on CRC progression. No-
tably, the abundance of Oscillospira declines in the transition from advanced adenoma
to stage 0 CRC, whereas levels of Haemophilus decrease in the transition from stage 0 to
early-stage CRC [45]. A meta-analysis of fecal metagenomes confirmed a significant de-
crease in the carbohydrate-degradation genes responsible for SCFA production in CRC [53].
These changes in the microbiome and metagenome coincide with a decrease in butyrate
concentration in CRC patients [41,69].
3.4. Bile Acid Metabolism and CRC

Primary bile acids are synthesized in the liver, conjugated to taurine or glycine,
and released in the gut. Upon reaching the colon, bile acids are deconjugated by bile
salt hydrolases of the gut microbiota and are subsequently transformed into dangerous
secondary bile acids by 7α-dehydroxylating bacteria [70]. Alterations in this process have
been associated with CRC. Metabolomic profiling confirmed the presence of elevated
levels of secondary bile acids, including deoxycholic acid (DCA), in adenomas and/or
intramucosal carcinomas [37,71]. In mice, DCA has been found to induce alterations in the
gut microbiota that are accompanied by impairments in the intestinal barrier, low-grade
inflammation, and colonic tumors [72,73]. DCA-induced dysbiosis is characterized by an
increased abundance of pathogens and a decreased abundance of probiotics, and this shift
in microbial community structure can be sufficient by itself to cause disease independent
of DCA treatment [73]. Instead, the secondary ursodeoxycholic acid (UDCA), known for
its anti-carcinogenic properties, is less abundant in CRC patients [69,74].
Generally speaking, a high-fat and low-fiber diet has long been known to represent a
risk factor for CRC; specifically, this diet correlates with lower levels of colonic SCFAs and
higher levels of colonic secondary bile acids and mucosal proliferative biomarkers of cancer
risk [75]. Interestingly, directed dietary changes (switch from high-fat/low-fiber to low-
fat/high-fiber diet and vice versa) resulted in remarkable reciprocal changes in mucosal
biomarkers of cancer risk, with an increased saccharolytic fermentation and butyrogenesis
and a suppressed secondary bile acid synthesis in the low-fat/high-fiber diet group [75].
4. Microbiota Biomarkers for CRC Diagnosis

Unlike the well-described causal role of Helicobacter pylori in gastric ulceration and
cancer, a specific and universal microorganism that triggers CRC has not been identified.
Instead, the evidence points to a shift in microbial composition, accompanied by changes in
microbial gene abundance and microbe-associated metabolites, which all tend to be propor-
tionate with the degree of malignancy. It is not clear whether these species and metabolites
directly cause tumorigenesis; if not, the culprit may be the microenvironment created
by these structural shifts, which then promotes inflammation, proliferation, and cancer
progression [37,54]. Regardless, the identification of reproducible microbial biomarkers for
CRC may enable the design of noninvasive tools for CRC diagnosis. For example, CRC pa-
tients demonstrate enrichments in pro-inflammatory F. nucleatum and reduced levels of
beneficial Faecalibacterium prausnitzii and Bifidobacterium. In vitro, F. nucleatum displays
strong bacteriostatic activities against these probiotic bacteria. Thus, the ratio of F. nu-
cleatum/F. prausnitzii and F. nucleatum/Bifidobacterium has been proposed as a biomarker
for screening early CRC [76]. Going further, a multi-cohort analysis of gut metagenomes
identified seven CRC-enriched bacteria—B. fragilis, F. nucleatum, Porphyromonas asaccha-
rolytica, Parvimonas micra, Prevotella intermedia, Alistipes finegoldii, and Thermanaerovibrio
acidaminovorans—which performed well in distinguishing CRC samples from controls
across different populations and may, thus, have potential for universal use for noninvasive
CRC diagnosis [52]. The association between CRC and Streptococcus gallolyticus subsp.
gallolyticus (formerly known as S. bovis type I) has also been reported [77–79]. A meta-
analysis performed by Boleij et al. [78] reported an association between the infection from
S. gallolyticus subsp. gallolyticus and CRC in 65% of cases. The strong association between
S. bovis infection and colonic adenomas and carcinomas has led to the speculation about
the possible oncogenic (driver or passenger) role of this bacterium. Indeed, the oral admin-
istration of S. gallolyticus in a mouse model of azoxymethane (AOM)-induced CRC led to a
higher number of tumors, higher level of dysplasia, and increased cell proliferation and
β-catenin levels in colon crypts as compared to control mice treated with L. lactis strain [79].
In any case, every infection by any of these subspecies should lead to a colonoscopy diag-
nosis and should not be underestimated, and a complete intestinal examination is highly
recommended for patients presenting a S. bovis bacteremia, especially when S. gallolyticus
subsp. gallolyticus is involved (biotype I) [80].
A metabolomic analysis of a Chinese cohort identified 20 gene markers that signifi-
cantly differentiated CRC-associated and control samples. Four of these markers—butyryl-
CoA dehydrogenase from F. nucleatum, two transposases from P. anaerobius, and RNA
polymerase subunit β (rpoB) from P. micra—were found to also be present in Danish,
French, and Austrian cohorts. This suggests that even though human populations may
differ with respect to the structure of gut microbial communities, there may be certain
universal signatures of CRC-associated microbial dysbiosis [54]. Furthermore, levels of gut
microbiota-derived metabolites, such as SCFAs, bile acids, and protein-derived metabo-
lites, have been repeatedly associated with CRC progression and have been proposed as
complementary biomarkers for the early screening of CRC [81]. In addition to providing
diagnostic markers, analysis of the gut microbiota might also help in CRC prognosis.
For example, F. nucleatum enrichment in cancer tissue is associated with a shorter survival
and may, therefore, act as a potential prognostic marker [82]. Another potential method for
the noninvasive detection/diagnosis of CRC could rely on the correlation of metabolomic
signatures from fecal samples. Recently, analyses of stools have led to the identification of
several relevant fecal metabolites. In addition to SCFAs (acetate and butyrate), metabolites
such as xenobiotics, heme, peptides/amino acids (proline and cysteine), vitamins, and co-
factors all demonstrated alterations in CRC samples [37,83]. In addition, CRC metagenome
analyses have highlighted enrichments in protein and mucin catabolism genes, and de-
pletions in carbohydrate degradation genes and bile acid genes, which could be used as
signatures for CRC diagnostics. For example, intestinal Clostridium species are known to
contribute to the conversion of primary to secondary bile acids via the 7α-dehydroxylation
pathway encoded in the bai operon. Bai was found to be highly enriched in stools from
CRC patients and may represent a possible CRC biomarker. However, bai enrichment is
also a consequence of a diet rich in meat and fat; therefore, more studies are necessary to
elucidate any potential biomarker function [53]. In general, further research is needed to
identify microbes that are universally associated with CRC for use in early noninvasive
diagnosis and prognosis.
5. Other Microbiota Microorganisms

While the role of the bacterial microbiota in CRC has been extensively studied, little
data are available regarding other members of the intestinal community, such as viruses,
fungi, and archaea [84]. With respect to fungi, Richard et al. [40] noted no differences
in mucosal samples from CRC patients compared to healthy controls. In fecal samples,
however, fungal dysbiosis has been observed, with an increased ratio of Basidiomycota to
Ascomycota in CRC patients. Ecological analysis revealed a higher number of co-occurring
correlations among fungi and more co-exclusive correlations between fungi and bacteria in
CRC compared with control samples, indicating possible roles in colorectal carcinogenesis
for synergistic intra-fungal relationships and antagonistic bacterial–fungal associations [85].
However, an indirect protective role of fungi was also recently reported in mice, as fungal
commensals were found to induce IL-18 and thus inhibit colitis-associated CRC [86].
Regarding enteric archaea, alterations in community composition have been observed
during tumorigenesis. In particular, fecal samples from patients with CRC demonstrated
significant enrichments in halophilic archaea and depletions in methanogenic archaea.
Furthermore, CRC-associated halophiles were positively associated with the oncogenic
bacterium B. fragilis and were negatively correlated with butyrate-producing Clostridium
species [87].
Little is known about the viral component of the CRC-associated microbiome. Two re-
cent studies reported an increase in the diversity of the gut bacteriophage community in
patients with CRC. The authors described altered interactions between bacteriophages
and oral bacterial commensals, suggesting that the bacteriophages modulate the bacterial
community and, through those interactions, indirectly influence the bacteria that drive col-
orectal cancer progression [88,89]. A more thorough understanding of the virome, of which
bacteriophages are an important part, is crucial to understand the etiology and progression
of CRC. Indeed, viruses may play a pivotal role from a very early stage; broadly infectious
phages in the colon can lyse, and thereby disrupt the biofilms of, the existing bacterial
communities in the intestinal mucosa. This alteration then enables the growth of oncogenic
bacteria that are able to transform epithelial cells and disrupt tight junctions to infiltrate
the epithelium, thereby provoking an inflammatory immune response [89].
Although extensive research remains to be performed, these findings suggest that
dysbiosis in fecal communities of archaea, fungi, and viruses may contribute, together with
or in addition to bacteria, to colon tumorigenesis.
6. Probiotics in CRC
Currently, a broad search is ongoing for alternatives to help in the treatment of cancer.
As mentioned above, dysbiosis of the microbiota is closely associated with cancer risk and
CRC development. This dysbiosis can be redressed by probiotic strains, which, in effect,
shift the composition of the microbiota toward more favorable species. Probiotics are
defined as “live microorganisms which, when administered in adequate amounts, confer a
health benefit on the host” [90]. Commercial and medicinal probiotics have demonstrated
potentials beyond simply modulating the gut microbiota; they have become attractive and
6. Probiotics in CRC
Currently, a broad search is ongoing for alternatives to help in the treatment of can-
cer. As mentioned above, dysbiosis of the microbiota is closely associated with cancer risk
and CRC development. This dysbiosis can be redressed by probiotic strains, which, in
Microorganisms 2021, 9, 1021
effect, shift the composition of the microbiota toward more favorable species. Probiotics
9 of 24
are defined as “live microorganisms which, when administered in adequate amounts,
confer a health benefit on the host” [90]. Commercial and medicinal probiotics have
demonstrated potentials beyond simply modulating the gut microbiota; they have be-
promising
come agents
attractive of promising
and host–microbiome
agents modulation therapiesmodulation
of host–microbiome for several diseases,
therapiesincluding
for sev-
CRCdiseases,
eral (Figure 4).
including CRC (Figure 4).
Figure 4. Positive effects of microbiota and probiotics in CRC. Some probiotic strains and their metabolites are able to
Figure 4. Positive effects of microbiota and probiotics in CRC. Some probiotic strains and their metabolites are able to
enhance the intestinal immune system by inhibiting the development of cancer cells. Probiotics promote macrophage and
enhance the intestinal immune system by inhibiting the development of cancer cells. Probiotics promote macrophage
dendritic cell differentiation, enhancing the activation of cytotoxic T-cells and NK cells. (The figure was created with Bio-
and dendritic cell differentiation, enhancing the activation of cytotoxic T-cells and NK cells. (The figure was created
render.com).
with Biorender.com).
Several studies have documented both their safety and effectiveness in enhancing the
Several studies have documented both their safety and effectiveness in enhancing
action of chemotherapy in the fight against cancer, as well as attenuating the side-effects
the action of chemotherapy in the fight against cancer, as well as attenuating the side-
of conventional treatments; there is some evidence to support the hypothesis that probi-
effects of conventional treatments; there is some evidence to support the hypothesis that
otics may also be able to minimize the development and progression of CRC by mitigating
probiotics may also be able to minimize the development and progression of CRC by
the aggressiveness of tumors. At present, probiotics have been extensively adopted by
mitigating the aggressiveness of tumors. At present, probiotics have been extensively
adherents of ‘wellness’ lifestyles, who consume foods that, in addition to their nutritional
adopted by adherents of ‘wellness’ lifestyles, who consume foods that, in addition to their
value, offer benefits to improve the overall well-being. To meet this demand, many com-
nutritional value, offer benefits to improve the overall well-being. To meet this demand,
mercial products containing probiotic microorganisms have been developed and made
many commercial products containing probiotic microorganisms have been developed
available throughout the world. Such products typically make use of the genera Lactoba-
and made available throughout the world. Such products typically make use of the genera
cillus, Bifidobacterium,
Lactobacillus, Lactococcus,
Bifidobacterium, Streptococcus,
Lactococcus, and Enterococcus.
Streptococcus, However,
and Enterococcus. other strains,
However, other
such as Bacillus, Saccharomyces, and next-generation probiotics (NGPs), are currently un-
strains, such as Bacillus, Saccharomyces, and next-generation probiotics (NGPs), are currently
der study for use in treating CRC (Table
under study for use in treating CRC (Table 1).1).
Table 1. List of probiotics used in in vitro and in vivo CRC studies.
Microorganism Function Mechanism Year Reference
Reduction in tumor in Activation of immune response by enhancing Th1 helper 2013 [91]
acidophilus
colitis-associated CRC models lymphocytes and M1 macrophages 2019 [92]
Decreasing the expression of the inflammation-associated
L. acidophilus strain genes; reducing the levels of TNF-α, IL-6,
Alleviation of gut inflammation 2018 [93]
MTCC 5401 and malonaldehyde; increasing the levels of superoxide
and catalase
Inhibition of H. pylori adherence through the production
L. acidophilus
of acetic acid and other bactericidal substances.
Protection against H. pylori 2019 [94]
Prevention of TLR4/NF-κB signaling, and production of
L. bulgaricus
the IL-8 pro-inflammatory cytokine
L. acidophilus Inhibition of the incidence of Elevation of IFN-γ and IL-10 serum levels and the
2019 [95]
B. bifidum colonic lesions number of CD4+ and CD8+ cells
Stimulation of immune response, effect on apoptosis,
L. acidophilus Cytotoxic effect on tumor cells 2018 [96]
and inactivation of NF-κB inflammatory pathway
L. acidophilus Prevention of the formation of Inhibition of pre-neoplastic lesions and reduction in the
B. animalis advanced aberrant crypt foci activity of antioxidant enzymes (SOD) and 2019 [97]
subsp. lactis and CRC apoptosis-related proteins (caspase-3 and Bcl-2)
L. acidophilus
Protection against toxic and
CL1285
reactive chemical species and
Stimulation of quinone reductase activity 2020 [98]
L. casei LBC80R inhibition of colon cancer (HT-29)
cell proliferation
L. rhamnosus CLR2
Reduction of enteropathogenic E. Creation of a strong physical barrier against EPEC
L. reuteri 2016 [99]
coli (EPEC) infection infection by binding to the mucus layer
Induction of apoptosis by increasing the expression of
Anticancer effect on colon
Lactobacillus EPSs Caspase 3, Caspase 9, and BAX, and reducing the levels 2019 [100]
cancer cells
of Bcl-2
Regulation of cancer cells proliferation and apoptosis
Protection against
L. casei through modulation of IL-22 and upregulation of 2017 [101]
CRC development
caspase-7, respectively
Restoration of T cell populations and regulation of IFN-γ
L. lactis Prevention of CRC development 2020 [102]
production in the CD4+ T cell population
Inhibition of colitis-associated Suppression of inflammation and apoptosis,
L. plantarum 2015 [103]
carcinogenesis and elevation of IgA secretion
L. plantarum
Prevention of CRC development Upregulation of IL-18 production 2020 [104]
L. salivarius
Reduction in the elevated expression of miR-155 and
onco-miR miR-21a, elevation in the levels of
B. longum Colon cancer treatment 2019 [105]
tumor-suppressing miR-145 and miR-15a,
and downregulation in NF-κb and miR-146a
Enhancement of SCFAs production and reducing the
B. longum (BB536-y) Inhibition of CRC growth 2018 [106]
amount of Bacteroides fragilis enterotoxin
Modulation of Notch- or Wnt/β-catenin signaling
Prevention and treatment of
Lactobacilli cocktail pathway, apoptosis, and downregulation of 2020 [107]
colon cancer
cell proliferation
Inhibition of intestinal epithelial
L. rhamnosus KCTC Regulation of p53-p21-Cdk1/Cyclin B1 signaling pathway
apoptosis and suppression of CRC 2019 [108]
12202BP by downregulating the expression of Cyclin B1 and Cdk1
cell proliferation
Reducing fecal procarcinogenic enzymes, oxidants,
L. rhamnosus MD 14 Anticancer effect and aberrant crypt foci, downregulating numerous 2020 [109]
oncogenes, and upregulating tumor-suppressing p53
L. casei ATCC334 Inhibition of CRC cell growth Induction of apoptosis by upregulation of DDIT3 2021 [110]
Regulation of the intestinal barrier integrity and
Reduction in the size and number
endogenous antioxidant defense system by increasing the
VSL#3 of pre-neoplastic lesions in a 2020 [111]
level of SCFAs and enzymes, and alterations in the
model of colitis-associated cancer
general composition of the intestinal microbiota
Table 1. Cont.
Microorganism Function Mechanism Year Reference

Regulation of apoptosis by changing the
intracellular calcium concentrations,
L. lactis Anti-metastatic effects on
and downregulating the expression of CEA, 2018 [112]
subsp. lactis multiple colon cancer cell lines
CEAM6, and matrix metalloproteinases
(MMP2 and MMP9)
Decreasing proliferation, increasing apoptosis,
Inhibition of intestinal
butyricum suppressing the Wnt/β-catenin signaling pathway, 2020 [113]
tumor development
and modulating the composition of gut microbiota
P. pentosaceus FP3
L. salivarius Inhibition of colon cancer
Production of SCFAs (propionic and butyric acid) 2013 [114]
FP35 and FP25 cell proliferation
E. faecium FP51
Improvement of CRC Downregulating pro-inflammatory cytokines and 2020 [115]
L. gasseri 505
anti-apoptotic factors, and upregulating
Prevention of hepatic toxicity anti-inflammatory cytokines and
2020 [116]
induced by CRC pro-apoptotic factors
Cancer immunotherapy
A. muciniphila Improvement of anti-PD-1 blockade efficacy 2018 [117]
treatments
Prevention of necrotic enteritis Reducing pathogen abundance in the cecum
2018 [118]
B. pullicaecorum and CRC and ileum
Anticancer effect and Production of butyrate and upregulation of
2020 [119]
inhibition of CRC cell growth SLC5A8 and GPR43
The beneficial effects of probiotics have been demonstrated both in vitro and in pre-
clinical trials, particularly for species of Lactobacillus, which has been, by far, one of the most
documented genera. Many studies have examined the ways in which probiotic strains
affect or interact with pathogenic microorganisms that contribute to the development of
CRC, such as Helicobacter pylori, Salmonella, B. fragilis, F. nucleatem, and some strains of E.
coli. These pathogens are capable of degrading the gut and releasing highly toxic com-
pounds, thus compromising the balance of intestinal homeostasis. Probiotics fight against
the proliferation of harmful microorganisms through lowering the pH of the environment,
producing bacteriocins, and reducing the level of pro-carcinogenic enzymes [120]. There
has been a particular focus on infection by H. pylori, which can potentially increase the
risk of colorectal cancer. Specifically, many studies have reported a link between H. pylori
infection and increased serum levels of gastrin; hypergastrinemia is associated with rectal
cell proliferation and stimulates the growth of colorectal cancer cells and the development
of colon adenomas [121]. In human gastric epithelial cells, Lactobacillus acidophilus and
Lactobacillus bulgaricus were found to protect against the negative effects of H. pylori in two
ways. First, both strains inhibited H. pylori adherence through the production of acetic acid
and other bactericidal substances. Then, L. bulgaricus was found to prevent TLR4/NF-κB
signaling and, thus, the production of the IL-8 pro-inflammatory cytokine that can lead to
chronic inflammation as a result of H. pylori infection [94]. Based on the results of meta-
analyses, though, it does not seem that probiotic therapies can be considered alternatives
to anti-H. pylori treatment. Rather, the association of probiotics with standard antibiotic
treatment could significantly improve the eradication rates of H. pylori, as well as decrease
the side-effects of current medication therapy [122].
Recent studies have provided strong evidence to support a causative role for col-
ibactin, a genotoxin of unknown structure, in CRC [123]. Colibactin is produced by E. coli,
specifically through the action of genes encoded in the 52-kb polyketide synthase (pks)
pathogenicity island [124]. Two strains of Lactobacillus reuteri, ATCC PTA 6475 and ATCC
53608, were found to reduce infection by enteropathogenic E. coli (EPEC). Although the ex-
act mechanism is still unclear, it could be related to competitive exclusion, i.e., competition
between probiotic and pathogenic strains for binding sites on the epithelial surface. It is
conceivable that, by binding to the mucus layer, L. reuteri could create a stronger physical
barrier against EPEC infection [99].
CRC is frequently associated with impairments to the immune system. TNF-α, IL-6,
IL-1, and chemokines induce tumor growth by promoting angiogenesis and suppressing
immune-mediated tumor elimination, while dendritic cells (DCs) and natural killer (NK)
cells play a critical role in the early defense against cancer [125]. Probiotics can enhance
innate immune functions, including the phagocytic activity of neutrophils and the cytotoxic
activity of NK cells; indeed, such abilities might lie at the root of their anti-infectious or
anticancer effects [126]. For example, strains of lactic acid bacteria (LAB) were reported to
regulate the maturation of myeloid DCs, polarizing the subsequent T-cell activity toward
Th1, Th2, or even T-reg responses. Additionally, in a colitis-associated model of CRC,
oral administration of Lactobacillus casei BL23 protected against tumor development through
the modulation of IL-22, a cytokine that promotes the proliferation of cancer cells, and the
upregulation of caspase-7, a gene involved in apoptosis [101].
Pro-inflammatory cytokines provide critical protection against colorectal tumorigene-
sis, and probiotics have been found to mediate this role. For example, the anti-tumorigenic
cytokine IL-18 promotes protective host immunity through the actions of CD8+ cytotoxic
T cells (Tc), NK cells, and Th1-driven macrophage activation [127]. This cytokine is cru-
cial for the homeostasis, mucosal repair, and proliferation/differentiation of intestinal
epithelial cells, as well as the induction of goblet cell mucus production, the expression
of tight junction proteins, and the secretion of anti-bacterial peptides that are essential
in preventing CRC development. A recent study of aged IL-18-deficient mice found that
Lactococcus lactis subsp. cremoris C60 restored T cell populations in small intestinal lamina
propria, which led to a rebound in IFN-γ production in the CD4+ T cell population [102].
Similarly, the probiotic strains Lactobacillus plantarum and Lactobacillus. salivarius were able
to augment IL-18 production in both in vitro and rat models of CRC [104]. In addition
to LAB species, yeast is also able to immunomodulate IL-18 levels. In a CRC model, Sac-
charomyces cerevisiae was reported to have a pro-apoptotic effect via upregulation of the
expression of IL-18; downregulation of the expression of TNF-α, IL-17, and IL-1β; and
inactivation of the NF-κB and mTOR signaling pathways via downregulation of the target
molecule, which is overactivated in CRC [128]. Probiotic strains are also able to modulate
host immune functions through the production of derived molecules or cell envelope
components [129]. For example, the administration of high-dose lysates of L. acidophilus
significantly reduces the number of visible tumors and average body weight in colitis-
associated CRC models. L. acidophilus lysates act as immunological adjuvants to activate
the immune response; in particular, a significant enhancement was observed in subsets
detected for Th1 helper lymphocytes (CD3+ , CD4+ , and IFN-γ+ ) and M1 macrophages
(CD11b+ , F4/80+ , and CD86+ ) in mesenteric lymph nodes [91,92]. In a mouse model of
AOM-induced CRC, oral consumption of the probiotics L. acidophilus and Bifidobacterum
bifidum increased IFN-γ and IL-10 serum levels and the number of CD4+ and CD8+ cells.
Administration of the probiotics inhibited the incidence of colonic lesions by about 57% for
L. acidophilus and 27% for B. bifidum compared to the AOM-only group [95].
Regulation in Apoptotic Genes in CRC by Probiotics

Another way in which probiotics can affect CRC is through the regulation of genes
implicated in cell proliferation and apoptosis. One rarely studied example involves the
modulation of microRNAs (miRNA), which can act as either oncogenes or tumor sup-
pressors based on the cellular microenvironment where they are expressed. In a model
of CRC induced by AOM/dextran sodium sulfate (DSS) treatment, levels of miR-155
(which induces resistance to chemotherapeutic agents) are dramatically increased [130].
The oral administration of B. longum to CRC mice resulted in a significant decrease in the
elevated expression of miR-155, as well as that of the onco-miR miR-21a; moreover, in both
healthy and CRC mice, treatment with B. longum increased levels of tumor-suppressing
miR-145 and miR-15a. This probiotic treatment resulted in the downregulation of both NF-
κB and miR-146a (which regulates IL-1β and IL-6 expressions) [105]. The probiotic’s effect
on miR-21 is of particular interest given that the expression of miR-21 leads to enhanced
cell proliferation, intravasation, cell migration, and metastasis, as well as declined rates of
apoptosis, which, together, contribute to an enhanced cancer incidence and the diminished
efficacy of drug therapies [131]. Furthermore, miR-21 is frequently upregulated in several
kinds of carcinomas, for instance, colon and gastric cancers [132]. This suggests that drugs
or supplements that can inhibit miR-21 might help in colon cancer treatment, as well as
support the function and efficacy of chemotherapies.
A probiotic strain need not be alive to exert beneficial effects; dead probiotics or
even cell components have been reported to effectively combat cancer. In a 2015 study by
Lee et al. [103], a dead strain of L. plantarum inhibited AOM/DSS-induced colitis-associated
carcinogenesis in mice better than the live bacterium did. This was reportedly due to the
effects of inflammation suppression, apoptosis, and enhanced IgA secretion. AOM/DSS
control animals possessed colon tumors, but administration with dead L. plantarum signifi-
cantly suppressed the development of neoplasia by increasing the levels of secretory IgA.
Specifically, it appeared that dead probiotics were more easily taken up by M cells than
pure live probiotics were, thus generating a stronger secretory immune response.
A recent study on the probiotic L. reuteri demonstrated the anti-metastatic and an-
tiproliferative effects of high-molecular-weight secretory molecules-cell-free supernatant
components from heat-killed sonicated probiotic bacteria [96]. An exopolysaccharide (EPS)
of L. acidophilus 20079 was noted to have a direct cytotoxic effect on tumor cells via mech-
anisms of apoptosis, stimulation of the immune response, and inactivation of the NF-κB
inflammatory pathway. Extracted EPS from this strain may thus represent a promising
therapeutic strategy for cancer [96]. Furthermore, EPSs produced by multiple strains of
probiotic lactic acid bacteria, including Lactobacillus plantarum GD2, Lactobacillus rhamnosus
E9, Lactobacillus brevis LB63 isolated from healthy infant feces, and Lactobacillus delbrueckii
subsp. bulgaricus B3 isolated from yogurt, were reported to have an anticancer effect on
colon cancer cells (HT-29). In this case, Lactobacillus EPSs were found to induce apoptosis
in CRC in vitro through the increased expression of Caspase 3, Caspase 9, and BAX and
decreased levels of Bcl-2, which led to a decline in cancer cell survival [100].
Some probiotics have been implicated in the inhibition of the epidermal growth factor
receptor (EGFR) pathway, which can play an important role in CRC-related signaling.
Some studies suggest that, during CRC, overexpression of the genes EGFR and HER-2 re-
sults in the deregulation of this pathway, leading to increased cell proliferation, prolonged
cell survival, anti-apoptotic effects, and metastasis [133]. For this reason, these two genes
are now potential targets for anticancer therapies such as cetuximab and trastuzumab
(anti-CRC drugs) and anti-EGFR and HER-2 monoclonal antibodies, which are already
available on the market. The related Notch and Wnt/β-catenin pathways have also been
shown to be modulated by probiotics, in this case, by a cocktail of lactobacilli (L. cocktail),
to generate antitumor effects in HT-29 cells in vitro. Specifically, the L. cocktail resulted
in the Notch- or Wnt-induced promotion of apoptosis and the downregulation of cell
proliferation. Therefore, the use of probiotic lactobacilli as nutritional supplements may
both prevent colon cancer and represent a cost-effective and safe means of CRC treat-
ment [107]. Generally, the pathogenesis of CRC is highly correlated with the deregulation
of the Wnt/β-catenin signaling pathway. The major effector of the canonical Wnt signaling
pathway is β-catenin (encoded by the CTNNB1 gene), which has a variety of cellular func-
tions. In more than half of all cancer cases—including colorectal carcinoma, breast cancer,
and liver carcinoma—nuclear localization of β-catenin always induces tumorigenesis and
promotes the proliferation and survival of cancer cells [134].
In summary, some of the suggested mechanisms by which probiotics promote CRC
prevention include the improvement of the host immune response, induction of apoptosis,
and inhibition of tyrosine kinase signaling pathways [135,136].
7. Synbiotics
There has been a great deal of interest in the use of probiotics in combination with
prebiotics, nonviable food components that confer health benefits on the host associated
with modulation of the microbiota [137]. Gibson and Roberfroid [138] introduced the
term “synbiotic” to describe the combination of synergistically acting probiotics and pre-
biotics. With respect to colorectal carcinogenesis, synbiotics have been demonstrated to
have protective effects via multiple different mechanisms, including the modulation of
the intestinal microbiota and immune response, reduction of inflammation, biosynthesis
of compounds with antitumor activity, and improvement in the antioxidant system [139].
For example, the effects of co-administration of the probiotic VSL#3 and the prebiotic
yacon (Smallanthus sonchifolius, a tuberous root rich in phenolic compounds and with a
high soluble fiber content) were tested in a model of colitis-associated carcinogenesis.
The synbiotic demonstrated numerous potential benefits: it supported the integrity of the
intestinal barrier, increased the concentrations of SCFAs, as well as enzymes involved in the
endogenous antioxidant defense system, and led to alterations in the general composition
of the intestinal microbiota [111]. Furthermore, in a model of carcinogenesis induced by
1,2-dimethylhydrazine (DMH), treatment with VSL#3 and yacon reduced the size and
number of pre-neoplastic lesions. In particular, the synbiotic was found to increase the
secretion of IL-2 and IL-4; the former has effects on the regulation of immune cells and
has been inversely correlated with tumor size, while the latter occurs concomitantly with
the expression of TLR4, resulting in the improvement of the innate immune response and
antitumor defense [111]. In a similar study, Lin et al. [97] demonstrated the protective
effect of a combination of germinated brown rice (GBR) and L. acidophilus/Bifidobacterium
animalis subsp. lactis in a DMH/DSS rat model. The synbiotic inhibited preneoplastic
lesions (aberrant crypt foci) and decreased the activity of antioxidant enzymes (SOD) and
apoptosis-related proteins in the colon (caspase-3 and Bcl-2). The authors hypothesized
that, as GBR is a good substrate for certain colonic bacteria—and, thus, promotes fer-
mentation and the production of SCFAs in the colon—the colonic epithelium may use
the increased supply of SCFAs to produce additional mucin. In this way, the synbiotic
may modulate the colonic secretion of mucins and their alterations during colorectal car-
cinogenesis to prevent the formation of more advanced aberrant crypt foci. Similarly,
the consumption of yogurt containing B. longum (BB536-y) and fructo-oligosaccharides
was found to enhance the amounts of SCFAs in fecal samples from healthy individuals.
This combination significantly suppressed the amount of Bacteroides fragilis enterotoxin
detected, and the SCFAs exerted a growth-inhibitory activity in human colon cancer cell
lines [106]. Further evidence of the supportive effects of SCFA production have been found
in studies of multiple probiotic strains. For example, treatment with the butyrate-producing
bacterium Clostridium butyricum ATCC 19398 significantly inhibited intestinal tumor devel-
opment in Apcmin/+ (Min, multiple intestinal neoplasia) mice by decreasing proliferation
and increasing apoptosis. This strain suppressed the Wnt/β-catenin signaling pathway
and modulated the gut microbiota composition, as demonstrated by decreases in some
pathogenic and bile acid-biotransforming bacteria, and increases in some beneficial bacteria,
including those that produce SCFAs [113]. An inhibited proliferation of colon cancer cells
was also noted in response to the production of SCFAs, mostly propionic and butyric acid,
by Pediococcus pentosaceus FP3, L. salivarius FP35, L. salivarius FP25, and Enterococcus faecium
FP51 [114].
Another mechanism by which synbiotics have been documented to restore intestinal
homeostasis in CRC is by improving antioxidant properties. For example, the adminis-
tration of ginger extract, together with L. acidophilus strain MTCC 5401, had a positive
effect on reducing gut inflammation (i.e., decreases in TNF-α and IL-6 levels) and de-
creasing the expression of the inflammation-associated genes Cox-2, iNOS, and c-Myc.
Interestingly, treatment with either ginger extract or LAB alone failed to produce any
effect on the antioxidant properties; together, however, they caused significant declines
in the levels of malonaldehyde (MDA, a mutagen and tumor promoter) and significant
increases in the levels of superoxide (SOD) and catalase (CAT), two important enzymatic
antioxidants [93]. The antioxidant epigallocatechin gallate (EGCG) has demonstrated the
potential for use as a prebiotic with Lactobacillus species because, unlike many bacteria,
they possess the phenol decarboxylase and inducible acid phenol reductase activities that
are necessary to metabolize phenolic acids such as EGCG [140]. Finally, a recent study of
the combination of Cudrania tricuspidata leaf extract with Lactobacillus gasseri 505 reported
that this synbiotic releases bioactive peptides from β-casein and phenolic compounds with
antioxidant activities. In an AOM/DSS model, this treatment ameliorated the effects of
cancer by downregulating pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, and IL-6) and
anti-apoptotic factors (Bcl-2 and Bcl-xL), and upregulating anti-inflammatory cytokines
(IL-4 and IL-10) and pro-apoptotic factors (p53, p21, and Bax). Furthermore, synbiotic
treatment decreased the expression of the inflammation-associated enzymes iNOS and
COX-2 [115]. These effects were corroborated by work in a mouse model that demonstrated
the ability of this combination to prevent the hepatic toxicity induced by CRC [116].
A particularly interesting example may be the combination of Lactobacillus and cranber-
ries. Studies have indicated that phenolic compounds from cranberries act as antimicrobial
substances against food pathogens such as E. coli; however, they do not exert an inhibitory
effect on some Lactobacillus strains and may even act as growth-promoting factors for probi-
otics. Indeed, a combination of concentrated cranberry juice and cell walls extracted from
a probiotic biomass (L. acidophilus CL1285, L. casei LBC80R, and L. rhamnosus CLR2) was
found to exert an increased inhibitory effect against HT-29 cells. Furthermore, the phenolic
compounds and probiotic biomass stimulated the activity of quinone reductase, a phase II
detoxifying enzyme that offers protection against toxic and reactive chemical species [98].
8. Postbiotics
Postbiotics are the complex mixtures of metabolic products secreted by probiotics
in cell-free supernatants—including enzymes, secreted proteins, short-chain fatty acids,
vitamins, secreted biosurfactants, amino acids, peptides, and organic acids—that exert
beneficial effects on the host, directly or indirectly [141]. As postbiotics do not contain
live microorganisms, the risks associated with their intake are minimized. Postbiotics are
conceptually similar to paraprobiotics, which are the inactivated microbial cells of probi-
otics (intact or ruptured, containing cell components such as peptidoglycans, teichoic acids,
and surface proteins) or crude cell extracts (i.e., with complex chemical composition) [142].
A recent example focused on L. rhamnosus (LR) KCTC 12202BP, which is known to inhibit
the cytokine-mediated apoptosis of mouse and human intestinal epithelial cells by regu-
lating signaling pathways. In lysates, An et al. [108] identified an LR-derived therapeutic
protein, p8, that suppressed CRC proliferation. This protein translocated specifically to the
cytosol of DLD-1 (human CRC cell line) cells, where it downregulated the expression of Cy-
clin B1 and Cdk1 (p53-p21-Cdk1/Cyclin B1 signaling pathway), both of which are required
for cell cycle progression. Another tumor-suppressive molecule, ferrichrome, was iden-
tified in conditioned media of the probiotic strain L. casei ATCC334 [143]. Ferrichrome is
known to be a siderophore and a mediator of the bacterial anti-tumor function on colorectal
cancer, inducing apoptosis by the activation of c-jun N-terminal kinase. Subsequently,
the anti-tumor effect of ferrichrome was tested in an AOM-DSS model of carcinogenesis,
where it was found to induce apoptosis via the upregulation of DDIT3 (DNA damage
inducible transcript 3). However, ferrichrome did not demonstrate any anti-inflammatory
activity in a DSS-mouse model, indicating that it inhibits cancer cell growth but not the
advent of a precancerous condition such as inflammation [110].
The term ‘metabiotics’ refers to the functional metabolites secreted by probiotics that
can optimize host-specific physiological functions; these are emerging as potential anti-
cancer agents due to their ability to alter metabolic processes in the gut lumen and reduce
the severity of colon carcinogenesis [144]. For example, a metabiotic extract from L. rham-
nosus MD 14 demonstrated anticancer potential in the DMH rat model by reducing fecal
procarcinogenic enzymes, oxidants, and aberrant crypt foci; downregulating numerous
oncogenes (K-ras, β-catenin, Cox-2, and NF-κB); and upregulating tumor-suppressing p53.
The metabiotic signature of L. rhamnosus MD 14 was characterized by several short-chain
fatty acids (i.e., acetate, butyrate, and propionate), as well as other active compounds
(i.e., acetamide, thiocyanic acid, and oxalic acid; [109]). Lactococcus lactis subsp. lactis
produces a lantibiotic bacteriocin, nisin A, that was recently found to prevent the growth
of cancer cells. Nisin demonstrated anti-metastatic effects on multiple colon cancer cell
lines, including LS180, SW48, HT-29, and Caco-2. It was hypothesized that nisin’s probiotic
effects might be caused by changes in intracellular calcium concentrations, which play an
important role in apoptosis, specifically through downregulating the gene expression of
carcinoembryonic antigen (CEA), carcinoembryonic cell adhesion molecule 6 (CEAM6),
and two matrix metalloproteinases (MMP2 and MMP9) [112].
9. Next-Generation Probiotics
Recent studies have highlighted many potential next-generation probiotics (NGPs).
These include Prevotella copri and Christensenella minuta, which control insulin resistance;
Parabacteroides goldsteinii, Akkermansia muciniphila, and Bacteroides thetaiotaomicron, which
reverse obesity and insulin resistance; F. prausnitzii, which protects mice against intestinal
diseases; and Bacteroides fragilis, which reduces inflammation and exhibits anticancer
effects [145]. In particular, A. muciniphila may have further potential for use in anticancer
immunotherapy, such as treatments that target programmed cell death protein 1 (PD-1).
Intriguing results were seen in an investigation of the differences in the microbiota of
patients who responded to anti-PD1 therapy and those who did not. A. muciniphila was
found to be particularly enriched in the microbiota of responders, and its importance was
confirmed using fecal microbiota transplantation into germ-free mice. A. muciniphila was
able, by itself, to improve the compromised efficacy of the anti-PD-1 blockade in mice
that were given the microbiota from nonresponders. This finding has particular relevance
given the increased popularity of cancer immunotherapy treatments aimed at the PD-
1 protein and its ligand, PD-L1 (programmed death ligand 1), which have shown benefits
in patients with various types of cancer [117]. Another next-generation probiotic currently
under study is Butyricicoccus pullicaecorum, which can prevent necrotic enteritis and reduce
pathogen abundance in the cecum and ileum, and has been reported to be safe in a human
intervention trial [118]. This bacterium has also been linked to CRC, as it was found to be
significantly less abundant in the stools of patients with late-stage CRC. The anticancer
effects of B. pullicaecorum appear to be linked to its high production of butyrate, which
was reported to inhibit CRC cell growth via the upregulation of SLC5A8 and GPR43 in
an animal model of DMH/DSS tumorigenesis [119]. SLC5A8 and GPR43 are known to
serve as tumor suppressors; mice lacking SLC5A8 develop CRC, while the activation of
GPR43 prevents colon inflammation and carcinogenesis [146].
10. Discussion
CRC is one of the most common cancers in the world, affecting approximately 1 mil-
lion people. The occurrence of CRC can have genetic or environmental origins but can also
be due to a previously established disease (such as IBD). Another emerging factor that plays
a part in CRC susceptibility is the composition of the intestinal microbiota. Indeed, the gut
microbiota can affect many physiological functions involved in the control of epithelial
cell proliferation and differentiation, prevention of pathogen growth, and stimulation of
intestinal immunity. Many key discoveries regarding the role of the microbiota in CRC
have originated from the use of axenic mouse models. For example, when conventional
IL-10-deficient mice are administered AOM, they develop colitis and carcinomas in the
colon, while axenic AOM-IL-10−/− mice are tumor-free and without histological damage.
Similarly, axenic AOM-IL-10−/− mice colonized with Bacteroides vulgatus present more
tumors than conventional mice [147]. In general, the microbiota of CRC patients has
been shown to be different from that of healthy individuals, with the genera Fusobacte-
ria, Bacteroides, and Prevotella being more predominant [41,148]. In addition, the mucosa
of these patients presents higher levels of adherent-invasive E. coli [149]. Some strains
are also known to have pro-carcinogenic characteristics and can initiate CRC onset [60].
For example, Enterococcus faecalis produces superoxide ions (O2− ) that can be converted to
hydrogen peroxide (H2 O2 ), which causes DNA damage [150]. Some strains of E. coli pro-
duce the genotoxin colibactin, which induces cuts in DNA [56,61]; similarly, the production
of a toxin by B. fragilis is responsible for the degradation of a tumor suppressor protein,
E-cadherin, leading to cell proliferation and permeability of the intestinal barrier [151].
Altogether, these results demonstrate the strength of the association between alterations in
gut microbiota composition and CRC.
Recent advances in our understanding of the composition of the microbiota have
highlighted the implications of beneficial bacterial “probiotics” for human health. Cur-
rently, most of the anticancer effects of probiotic bacteria have been studied in vitro or
using in vivo animal models. Together, these studies have revealed that the anti-CRC
effects of probiotics arise through various mechanisms, including: (i) alteration of the com-
position of the microbiota, (ii) inactivation of carcinogenic compounds, (iii) competition
with pathogenic or CRC-promoting bacteria, (iv) stimulation of the immune response,
(v) regulation of apoptosis and cell differentiation, (vi) fermentation of undigested nu-
trients, and vii) pH acidification [136]. In humans, one of the best studied groups of
probiotics is the lactic acid bacteria, which may prevent the development of CRC. In-
deed, the consumption of dairy products containing Lactobacillus seems to be related to a
low incidence of CRC [92,96,103,105,106,152]. In addition, epidemiological studies have
shown that, even among individuals with a high-fat diet (which favors CRC development),
the incidence of this cancer was lower in consumers of milk, yogurt, and other dairy
products [153–155]. Furthermore, there is strong evidence that many of the anticancer
effects of probiotic bacteria are mediated through their production of metabolites such
as SCFAs [119]. Indeed, SCFAs (in particular, butyrate) can induce changes in apoptosis,
cell cycle arrest, and cell differentiation; for this reason, there is currently a great deal of
interest in the use of butyrate-producing bacteria, such as F. prausnitzii, to treat CRC [145].
To conclude, the microbial ecosystem of the intestine exerts a considerable influence
on the human physiology through its metabolic and immune functions. A disturbance in
intestinal homeostasis and in the gut microbiota can favor the appearance of certain patholo-
gies, such as CRC. Due to the deep and fundamental links between these pathologies and
the gut microbiota, the modulation of the species composition of these communities repre-
sents an attractive therapeutic alternative. In this context, the beneficial effects of probiotic
bacteria on CRC have been well established, but studies have thus far been limited to only
a few bacterial effectors and host molecular mechanisms. In parallel, a large number of
studies have demonstrated the potential anticancer effects of probiotics in vitro or in vivo,
but the evaluation of their curative effects on CRC has been more complicated. Recent
studies in the technological advancement to analyze the human intestinal microbiota have
established a new paradigm for the development of tools in the early detection of CRC
through biomarkers. The identification of the exacerbation of a specific group of pathogenic
bacteria or their metabolites will define a personalized strategy to counteract intestinal
microbial dysbiosis. One of the strategies could be the use of probiotic strains to counteract
intestinal microbial dysbiosis, one of the etiological agents of CRC and other intestinal
disorders. To develop effective probiotic-based therapies against CRC, it will be essential
to improve the characterization of the crosstalk between the microbiota and the host and to
further elucidate the beneficial mechanisms of probiotics.
Author Contributions: Conceptualization, L.G.B.-H., writing—original draft preparation, E.T.-M.,

A.-S.B., N.G.C.-P., L.G.B.-H., writing—review and editing, E.T.-M., A.-S.B., A.H.M., S.T.-S., N.G.C.-P.,
L.G.B.-H. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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microorganisms

1 Micalis Institute, Université Paris-Saclay, INRAE, AgroParisTech, 78350 Jouy-en-Josas, France;

Microorganisms 2021, 9, 1021. https://doi.org/10.3390/microorganisms9051021 https://www.mdpi.com/journal/microorganisms

3. CRC and Gut Microbiota

Microorganisms 2021, 9, 1021 4 of 24

Microorganisms 2021, 9, 1021 5 of 24

3.3. Short-Chain Fatty Acids and CRC

3.4. Bile Acid Metabolism and CRC

4. Microbiota Biomarkers for CRC Diagnosis

5. Other Microbiota Microorganisms

Table 1. List of probiotics used in in vitro and in vivo CRC studies.

Microorganism Function Mechanism Year Reference

Microorganism Function Mechanism Year Reference

Regulation in Apoptotic Genes in CRC by Probiotics

Author Contributions: Conceptualization, L.G.B.-H., writing—original draft preparation, E.T.-M.,

Informed Consent Statement: Not applicable.

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