cancers

Review
Gut Microbiota and Cancer: From Pathogenesis
to Therapy
Silvia Vivarelli 1 , Rossella Salemi 1 , Saverio Candido 1 , Luca Falzone 1 , Maria Santagati 2 ,
Stefania Stefani 2 , Francesco Torino 3 , Giuseppe Luigi Banna 4 , Giuseppe Tonini 5 and
Massimo Libra 1,6, *
1 Department of Biomedical and Biotechnological Sciences, Oncologic, Clinic and General Pathology Section,
University of Catania, 95123 Catania, Italy; [email protected] (S.V.); [email protected] (R.S.);
[email protected] (S.C.); [email protected] (L.F.)
2 Department of Biomedical and Biotechnological Sciences, Section of Microbiology, University of Catania,
95123 Catania, Italy; [email protected] (M.S.); [email protected] (S.S.)
3 Department of Systems Medicine, Medical Oncology, Tor Vergata University of Rome, 00133 Rome, Italy;
[email protected]
4 Division of Medical Oncology, Cannizzaro Hospital, 95126 Catania, Italy; [email protected]
5 Department of Medical Oncology, University Campus Bio-Medico of Rome, 00128 Rome, Italy;
[email protected]
6 Research Center for Prevention, Diagnosis and Treatment of Cancer, University of Catania,
95123 Catania, Italy
* Correspondence: [email protected]; Tel.: +39-095-478-1271




Received: 14 December 2018; Accepted: 27 December 2018; Published: 3 January 2019


Abstract: Cancer is a multifactorial pathology and it represents the second leading cause of death
worldwide. In the recent years, numerous studies highlighted the dual role of the gut microbiota
in preserving host’s health. Gut resident bacteria are able to produce a number of metabolites
and bioproducts necessary to protect host’s and gut’s homeostasis. Conversely, several microbiota
subpopulations may expand during pathological dysbiosis and therefore produce high levels of
toxins capable, in turn, to trigger both inflammation and tumorigenesis. Importantly, gut microbiota
can interact with the host either modulating directly the gut epithelium or the immune system.
Numerous gut populating bacteria, called probiotics, have been identified as protective against the
genesis of tumors. Given their capability of preserving gut homeostasis, probiotics are currently
tested to help to fight dysbiosis in cancer patients subjected to chemotherapy and radiotherapy.
Most recently, three independent studies show that specific gut resident species may potentiate the
positive outcome of anti-cancer immunotherapy. The highly significant studies, uncovering the tight
association between gut microbiota and tumorigenesis, as well as gut microbiota and anti-cancer
therapy, are here described. The role of the Lactobacillus rhamnosus GG (LGG), as the most studied
probiotic model in cancer, is also reported. Overall, according to the findings here summarized,
novel strategies integrating probiotics, such as LGG, with conventional anti-cancer therapies are
strongly encouraged.

Keywords: microbiota; microbiome; cancer; anti-cancer therapy; integrated therapy; inflammasomes;

probiotics; Lactobacillus rhamnosus GG

1. Introduction
Cancer is the second leading cause of death worldwide [1,2]. Cancer formation is the result of the
stochastic intracellular accumulation of spontaneous mutations during DNA replication, combined
with the environment exposure and lifestyle habits, both able to significantly influence cancer risk [3,4].

Cancers 2019, 11, 38; doi:10.3390/cancers11010038 www.mdpi.com/journal/cancers

Cancers 2019, 11, 38 2 of 26

For example, the exposure to infectious agents, UV radiation and toxic substances, individual diet and
lifestyle, strongly influences cancer rise, although the risk mainly depends on the dose, the duration
and the combination of such injuries, coupled with the individual genetic background [5].
In the recent years, numerous evidences pointed towards the central role of commensal bacteria
colonizing body surfaces as key determinants of health or pathologic conditions, including cancer [6].
Among the human symbiotic microbial populations, the gut microbiota is the most extensively studied
and it deeply influences host’s homeostasis [7]. Gut microbiota is the name given to the heterogeneous
population of commensal microorganisms, mainly bacteria, but also fungi, archaea and viruses,
populating the intestinal tract, mostly the large intestine, and it can be considered as one factor to
which we are persistently exposed, at high doses, throughout the entire lifespan [8]. Among a 1000 of
different bacterial species within the gut microbiota, the highly represented ones are the Firmicutes
and Bacteroidetes phyla [9]. The gut microbiota performs a number of vital functions, including
production of vitamins, metabolization of dietary compounds, protection against the expansion and
systemic infiltration of gut pathogens [10–12]. The gut microbial balance has a key role in the correct
fulfilment of all these pivotal metabolic functions. Any imbalance in this delicate equilibrium may
lead to an impaired microbiota, condition called dysbiosis, linked with several human pathologies,
including cancer [13].
The gut microbiome, defined as the whole genome of the host’s gut microbiota, encodes 100-fold
more genes than the human genome [14]. Within the last decade, the advent of metagenomics,
combining next-generation sequencing (NGS) with computational analysis of the 16S rRNA amplicons,
allowed to characterize both diversity and abundance of the gut microbiome. Progresses in
metagenomics studies, together with the advancements in transcriptomics and metabolomics,
allowed to describe the impact of individual bacterial species on host’s health [15,16]. That represents
a step forward, from descriptive microbiome composition analyses, to functional studies, which are
nowadays helping to understand the true impact of the microbiome architecture on human
health [17,18]. However, this research is at its beginning, and all these growing number of associative
and functional studies need to be further corroborated by results obtained from larger clinical
studies [19].
Among all the pathologies linked with the gut microbiome, tumorigenesis is one of the mostly
studied. The link has been found both with local gastro-intestinal cancers, as well as with other distal
tumors [20]. Metabolomics and metagenomics studies highlighted the dual role of the gastro-intestinal
microbiome in cancer prevention, tumorigenesis and anti-cancer therapy [21]. In fact, the gut
microbiome can either be tumor-suppressive or oncogenic [22,23]. Although this link is studied
since long ago, it is only partially characterized. In fact, all the current knowledge emphasizes the
complexity and bidirectionality of the connection existing between microbiome and cancer. As a
consequence, cancer development may alter the microbiome and, in turn, microbiome changes may
affect cancer progression [24].
In this review, the up-to-date studies uncovering the tight link between gut microbiota and
tumorigenesis will be described. Moreover, the importance of probiotics supplementation with
anti-cancer therapy will be also discussed, including the key role and the future applications of a
probiotic model studied in cancer, Lactobacillus rhamnosus GG (LGG), often used to treat cancer patients’
intestinal dysbiosis.

2. Gut Microbiota and Host’s Tumorigenesis

2.1. Gut Microbiota and Host Crosstalk

The gut is bidirectionally connected with the nervous system through the so-called “gut-brain
axis” (GBA), which includes the central nervous system (CNS), the autonomic nervous system
(ANS), the enteric nervous system (ENS), the hypothalamic pituitary adrenal (HPA) axis and the
Cancers 2019, 11, 38 3 of 26

entero-endocrine system (EES). Hormones and neuro-hormones secreted at each of these listed GBA
levels may modulate the gastro-intestinal digestive and metabolic activities, and vice-versa [25].
For that reason, the gut represents a complex interface between the gastro-intestinal resident
microbiota and the human body. There is a bidirectional crosstalk between gut resident microbes
and host’s GBA, in which the gut functions as the communication gatekeeper [26]. In particular,
it is known that host’s hormones and neuro-hormones are able to modify the gut microbiome
composition, as during stress response [27]. Importantly, the gastro-intestinal entero-endocrine cells
secrete over 30 different peptide hormones, involved in several functions, such as gastro-intestinal
motility, digestive functions and neuromodulation [28]. A number of these hormones can be sensed
by the gut bacteria, as in the case of leptin and ghrelin, which, in turn, finely tune the gut microbiota
composition in rodents [29,30].
In the same way, the gut microbial population secrete active molecules which can be sensed by
the gut cells and whose effects are then transduced to the GBA [27]. In fact, it has been widely studied
that the gut microbiota may produce or transform molecules affecting several aspects of human
health, including: host’s metabolism modulation, gut barrier integrity maintenance, xenobiotics
and drug metabolism, protection against gastro-intestinal pathogens and host’s immune system
modulation [31–34].
With regards to the host’s metabolism, it is known that certain commensal bacteria produce
essential micronutrients, such as vitamin K and various components of vitamin B. Also, members of
the Bacterioides family are able to synthetize the anti-diabetic compound linoleic acid, to catabolize
secondary bile acids and to break down phenolic compounds. Moreover, a number of gut commensals
can modify small aminoacids into signaling molecules, as for example histidine to histamine or
glutamate to γ-aminobutyric acid (GABA) [35].
Additionally, it has been reported that gut resident commensals can produce hormone-like
metabolites, such as the short chain fatty acids (SCFAs), as a consequence of the bacterial fermentation
of dietary fibers in the large intestine [36]. The nature of the SCFAs synthetized depends on both diet
and microbiota specific composition. The SCFAs, once produced by the gut bacteria, are transported
through the bloodstream, and used by the liver as the main source of energy. Importantly, SCFAs have
a role in controlling the glucose and the lipidic metabolism, by affecting the intestine hormone peptide
secretion, including Peptide YY (PYY) and Glucagon-like peptide 1 (GLP-1) [37].

2.2. Gut Microbiota as a Tumor-Suppressor

Given the above illustrated complex crosstalk occurring between the host and its gut microbiota,
it is not surprising that the gut microbial population may affect pathological processes, such as cancer
genesis and development, either in a positive or in a negative way, depending on its own composition.
Remarkably, a number of microbes-derived molecules show an anti-tumor activity.
In particular, microbial-derived SCFAs may have an anti-cancer effect. For example, gut bacterial
butyrate and propionate are able to inhibit host’s tumor cells histone deacetylases with a general
anti-cancer effect. Such mechanism is the cause of the anti-tumoral in vitro and in vivo effect of butyrate
observed in both colorectal cancer (CRC) and lymphoma [38,39]. Some of the probiotics’ derived
molecules and metabolites are able to modulate host’s immune system, thereby triggering an indirect
immune-mediated response against tumor development. For example, the widely studied bacterial
lipopolysaccharide (LPS), a major component of the outer membrane in gram-negative bacteria,
activates the host’s cell surface receptor toll-like receptor 4 (TLR4), belonging to the family of pattern
recognition receptors (PRRs), thus activating immune T cell-mediated response against cancer cells [40].
In the same way, the monophosphoryl lipid A (MPL) from Salmonella enterica has been currently used
as adjuvant in the vaccine formulation used against anti-cervical carcinoma [41]. Moreover, bacterial
derived pyridoxine, a group B vitamin, can stimulate host’s antitumoral immunosurveillance [42].
Several commensal bacteria play a probiotic role thanks to their capability to confer health benefits,
either protecting against gut dysbiosis or enhancing host’s immune defense mechanisms [22,24].
Cancers 2019, 11, 38 4 of 26

The administration of such probiotics, as for example Mutaflor (Escherichia coli Nissle 1917) combined
Cancers 2019, 11, x 4 of 26
with the intestinal antibiotic rifaximin, demonstrated a clear anti-inflammatory activity, enhancing
the anti-inflammatory effect of rifaximin in a rat model of inflammatory bowel disease [43].
enhancing the anti-inflammatory effect of rifaximin in a rat model of inflammatory bowel disease
Additionally, many probiotics have shown a potential antineoplastic activity. For example, probiotics
[43]. Additionally, many probiotics have shown a potential antineoplastic activity. For example,
or probiotics-derived metabolites administered to mice can, in turn, to inhibit tumor growth. One good
probiotics or probiotics-derived metabolites administered to mice can, in turn, to inhibit tumor
example is ferricrome metabolite secreted from Lactobacillus casei, able to trigger apoptosis in tumor
growth. One good example is ferricrome metabolite secreted from Lactobacillus casei, able to trigger
cells
apoptosis inpathway
via JNK direct
tumor cells viaactivation [44]. Itdirect
JNK pathway has been also reported
activation in several
[44]. It has studies
been also that Lactobacilli
reported in several
may stimulate host’s immune cells such as NK cells or dendritic cells (DC) or TH1
studies that Lactobacilli may stimulate host’s immune cells such as NK cells or dendritic cells (DC) response, which,
or
inTH1
turn, leads to the elimination of cancerous or precancerous cells, although the
response, which, in turn, leads to the elimination of cancerous or precancerous cells, although exact bacterial
bioproduct mediating
the exact bacterial such stimulatory
bioproduct mediatingeffect still needs toeffect
such stimulatory be identified
still needs[45–48]. A summary
to be identified of the
[45–48]. A
gut microbiome anti-cancer functions is illustrated in Figure 1.
summary of the gut microbiome anti-cancer functions is illustrated in Figure 1.

Figure1.1.Anti-tumoral
Figure Anti-tumoral effects of
ofthe
thegut
gutmicrobiota.
microbiota. Probiotics andand
Probiotics other gut gut
other resident bacteria
resident are able
bacteria are
to secrete molecules, capable, in turn, to fight tumor growth and prevent tumorigenesis
able to secrete molecules, capable, in turn, to fight tumor growth and prevent tumorigenesis through through
severalmechanisms.
several mechanisms.Schematic
Schematic of
of the
the intestinal
intestinal layers,
layers, from
from top to bottom: mucus
mucus and
and microbiota,
microbiota,
gutepithelium.
gut epithelium.Into
Intothe
thegrey
greyboxes
boxesareareillustrated,
illustrated, from
from top
top to
to bottom,
bottom, the microorganism
microorganism species
species
implicatedininthe
implicated theanti-cancer
anti-cancerprocess,
process,thethemolecules
moleculesproduced
produced andand the corresponding effects induced
induced
withinthe
within thehost.
host.Abbreviations:
Abbreviations:MPL,
MPL,monophosphoryl
monophosphoryllipid lipid A;
A; LPS,
LPS, lipopolysaccharide.
lipopolysaccharide.

2.3.
2.3.Gut
GutMicrobiota
Microbiotaasasa aTumor-Promoter
Tumor-Promoter
Gut
Gutdysbiosis
dysbiosisandandthetheconsequent
consequent development
development of of pathogenic
pathogenic populations within the
populations within the gut
gut
microbiota,
microbiota,may contributetotoa awide
may contribute wide variety
variety of pathologies,
of pathologies, even even
in sitesindistant
sites distant
from thefrom the gut,
gut, ranging
ranging from inflammation,
from bowel bowel inflammation, to neurodegenerative
to neurodegenerative diseasesdiseases (including
(including Parkinson’s
Parkinson’s disease)disease) and
and cancer
cancer [43,49,50].
[43,49,50].
Regarding
Regardingcancer,
cancer,within
withinaadysbiotic
dysbioticgut,
gut,certain
certainbacterial
bacterial pathogens
pathogens can negatively
negatively affect
affect either
either
thehost’s
the host’s metabolism
metabolism or orthe
thehost’s gutgut
host’s andand
immune
immunesystem functionalities,
system thereby
functionalities, triggering
thereby tumor
triggering
growth
tumor [51]. Importantly,
growth gastro-intestinal
[51]. Importantly, dysbiosis
gastro-intestinal has been
dysbiosis haslinked
been with
linkedboth
withlocal and
both distant
local and
tumorstumors
distant [52]. Microbial pathogenspathogens
[52]. Microbial are knownareto drive
knowntheto 20% of tumorigenesis
drive and a larger number
the 20% of tumorigenesis and a
of malignancies
larger are associatedare
number of malignancies with microbial
associated commensal
with microbialimbalance,
commensal orimbalance,
dysbiosis [53]. In line with
or dysbiosis [53].
Inthat,
line many preclinical
with that, many studies performed
preclinical studiesusing germ-free
performed using mice models mice
germ-free demonstrated how the gut
models demonstrated
microbiome
how is able to deeply
the gut microbiome is ableaffect canceraffect
to deeply genesis and progression
cancer genesis and through
progressiondifferent
throughmechanisms
different
[33,54,55].
mechanisms [33,54,55].
Helicobacterpylori
Helicobacter pyloriproduced
producedprotein
protein CagA
CagA was
was the
the first
first bacterial
bacterial protein
protein shown
shown to to be
be involved
involved
in human cancer [56]. Although only Helicobacter pylori is included among class
in human cancer [56]. Although only Helicobacter pylori is included among class I carcinogens I carcinogens by the
by
World Health Organization (WHO) [57], several studies performed in cell culture and animal models,
assessed the ability of additional microbiota populations to affect host’s DNA replication and
integrity [58–60]. In fact, during pathogenic infections, when the gut microbiome is affected by
Cancers 2019, 11, 38 5 of 26

the World Health Organization (WHO) [57], several studies performed in cell culture and animal
models, assessed the ability of additional microbiota populations to affect host’s DNA replication
and integrity [58–60]. In fact, during pathogenic infections, when the gut microbiome is affected
by dysbiosis, bacterial pathogens can expand and release a large amount of toxins which, in turn,
induce host’s DNA breaks, thus contributing to genomic instability, tumor initiation and progression
in those predisposed cells [61–63]. This is the case of colibactin and cytolethal distending toxin (CDT)
both produced by Escherichia coli and displaying a DNAse activity. Once released in the proximity
of the gastrointestinal epithelium, the toxins generate DNA double-strand breaks within the host’s
epithelial cells, thus promoting a transient cell cycle arrest, allowing for genomic mutations to arise,
and finally leading to tumor formation [64]. Gut pathogenic bacteria can also interfere with DNA
damage response and repair pathways, as in the case of Shigella flexneri, inducing host’s cells p53
degradation via the secretion of its enzymes inositol phosphate phosphatase D (IpgD) and cysteine
protease-like virulence gene A (VirA), therefore increasing the probability of introducing mutations
during the DNA damage response in infected cells [65]. In the same way, the product of the cytotoxin
associated gene A (CagA) from Helicobacter pylori, induces the proteasome-mediated degradation of
p53 in gastric epithelial cells, by interfering with the host’s AKT pathway, thus promoting the rise of
gastric cancer [66].
Moreover, gut bacteria can modulate several host’s cellular proliferative and pro-survival
pathways, therefore contributing to cancer. For example, Helicobacter pylori derived CagA protein,
Fusobacterium nucleatum effector adhesin A (FadA) and Bacteroides fragilis metalloproteinase toxin
(MP toxin) are all capable to interact (directly or indirectly) with the host’s epithelial E-cadherin,
thus disrupting the intercellular junctions and activating β-catenin signaling. This, in turn, triggers
cell proliferation and the potential cancerogenic transformation of those affected host’s cells [67–69].
In the same direction, the Salmonella enterica effector avirulence protein A (AvrA) is able to translocate
into host’s cells and activate β-catenin via its intrinsic de-ubiquitinase activity [70].
As for the β-catenin signaling, other virulence factors released in the extracellular gut
milieu during a pathogenic infection, can potentially induce cancer transformation when infecting
pre-transformed cells, through the activation of other pro-survival intrinsic host’s cellular pathways,
such as MAPK and AKT, as for CagA from Helicobacter pylori, controlling the hosts MAPK pathway
or AvrA from Salmonella enterica, triggering both MAPK and AKT pathways [71,72]. In particular,
CagA from Helicobacter pylori can bind many host’s proteins intracellularly, including the protein
tyrosine phosphatase SHP-2. CagA-SHP-2 complex formation deregulates the phosphatase activity of
SHP-2, which, in turn, promotes Ras/MAPK signaling activation [73].
Additionally, pathogenic bacteria may indirectly affect host’s tumorigenesis.
Different mechanisms can mediate this effect. One is the generation of oxidative stress, leading
to cell autonomous genomic mutations [74,75]. Another one consists either in the enhancement
of the inflammation or the inhibition of the host’s immune response, thus helping the tumor
immune-escape [76]. For example, Helicobacter pylori or Bacteroides fragilis are both able to activate the
host’s spermine oxidase, which, in turn, generates hydrogen peroxide and reactive oxygen species
(ROS)-induced accumulation of DNA damage [77,78]. Enterococcus faecalis produces extracellular
superoxide and derivative oxygen species is capable to diffuse into host’s cells. In turn, the increase in
the oxidative milieu enhances the possibility of host’s cellular DNA mutations [79].
Moreover, relevant bacteria can stimulate cancer formation by blocking immune-effectors that
normally inhibit tumorigenesis. For example, Fusobacterium nucleatum inhibits for its own advantage
host’s Natural Killer (NK) cells, in order to recruit at the site of the infection myeloid suppressor cells,
therefore indirectly helping cancer genesis. Such mechanism is mediated by the bacterial virulence
factor Fap2, able to bind and block the NK inhibitory receptor TGIT, thus arresting the NK-mediated
tumor cell attack [80].
Finally, certain microbiota species may interfere with host’s hormones metabolism. In fact, it has
been widely studied the link between bacterial secretion of the β-glucuronidase enzymes and the
Cancers 2019, 11, 38 6 of 26

increased bioavailability of the host’s estrogen hormones (both originating from hepatic catabolism
and phytoestrogens). When gut dysbiosis is coupled with an increase in the β-glucuronidase-secreting
bacteria, such as Clostridium leptum and Clostridium coccoides, the enzyme deconjugates liver-catabolized
and plant-derived estrogens, enabling them to bind and activate the estrogen receptors expressed
byCancers
target2019, 11, [81].
cells x Estrogen receptors activation promotes cell proliferation in tissues responding 6 of 26
to
estrogens, as breast and endometrium [82]. Accordingly, this augmented intake of estrogen hormones is
responding to estrogens, as breast and endometrium [82]. Accordingly, this augmented intake of
linked with an increased risk of developing breast cancer, supporting the finding that the gut microbiota
estrogen hormones is linked with an increased risk of developing breast cancer, supporting the
composition of women with breast cancer differs from that from healthy controls, and suggesting that
finding that the gut microbiota composition of women with breast cancer differs from that from
several gut bacteria, which could be over-expressed during dysbiosis, may be linked with breast cancer
healthy controls, and suggesting that several gut bacteria, which could be over-expressed during
development [83].
dysbiosis, may be linked with breast cancer development [83].
Although there are notable examples of pathogenic microbiota capable of promoting oncogenesis
Although there are notable examples of pathogenic microbiota capable of promoting
through the modulation oncogenic host’s cell pathways or by interfering either with the host’s
oncogenesis through the modulation oncogenic host’s cell pathways or by interfering either with the
hormonal or the host’s immune system, no strong bacterial oncogenic driver has been identified
host’s hormonal or the host’s immune system, no strong bacterial oncogenic driver has been
yet. In particular,
identified yet. Initparticular,
is difficultittoisclearly determine
difficult to clearlywhether microbiota
determine whetherchanges might
microbiota affect might
changes cancer
genesis or the contrary [84]. Additionally, changes in the host’s lifestyle, diet and immune
affect cancer genesis or the contrary [84]. Additionally, changes in the host’s lifestyle, diet and system are
among
immune thesystem
factorsarewhich
amongdeeply
the influence the microbiota
factors which composition
deeply influence and activity
the microbiota [85]. Moreover,
composition and
the very same anti-cancer treatment might shape the patient’s microbiome and, at
activity [85]. Moreover, the very same anti-cancer treatment might shape the patient’s microbiomethe same time, host’s
specific
and, atmicrobiome
the same time, canhost’s
deeply affect patient’s
specific microbiomeresponse to therapy
can deeply affect[19]. A summary
patient’s response ofto
the gut bacteria
therapy [19].
pro-tumoral functions is schematized in Figure 2.
A summary of the gut bacteria pro-tumoral functions is schematized in Figure 2.

Figure2.2.Pro-tumoral
Figure Pro-tumoral effects
effects of
of the
the gut
gut microbiota.
microbiota. Bacteria
Bacteria prominent
prominent during
during gut
gut dysbiosis
dysbiosis can
can
secretetoxins
secrete toxins able
able to
tointerfere
interferewith
with host cellcell
host growth,
growth,finally predisposing
finally the host
predisposing theorganism to cancer
host organism to
development. Schematic of the intestinal layers, from top to bottom: mucus and
cancer development. Schematic of the intestinal layers, from top to bottom: mucus and microbiota, microbiota, gut
epithelium.
gut epithelium.IntoInto
thethe
grey boxes
grey boxes areare
illustrated,
illustrated,from
fromtop toptotobottom,
bottom,thethe microorganism
microorganism species
species
implicatedininthe
implicated thepro-cancer
pro-cancerprocess,
process, thethe molecules
molecules produced
produced and and the
the corresponding
corresponding effects
effectsinduced
induced
withinthe
within thehost.
host. Abbreviations:
Abbreviations: ROS,ROS, Reactive
Reactive Oxygen
Oxygen Species;
Species; CTD, CTD, cytolethal
cytolethal distending
distending toxin;toxin;
IpgD,
IpgD, inositol phosphate phosphatase D; VirA, virulence gene A; CagA, cytotoxin
inositol phosphate phosphatase D; VirA, virulence gene A; CagA, cytotoxin associated gene A; FadA, associated gene A;
FadA, Fusobacterium effector adhesin A; MP Toxin, metalloproteinase toxin;
Fusobacterium effector adhesin A; MP Toxin, metalloproteinase toxin; AvrA, avirulence protein A; AvrA, avirulence
proteinβ-glucuronidase.
β-gluc, A; β-gluc, β-glucuronidase.

3.3.Lactobacillus
Lactobacillusrhamnosus
rhamnosusGG:
GG:AAProbiotic
Probiotic Model
Model in
in Cancer
Due
Duetotoits itsanti-inflammatory
anti-inflammatory properties,
properties, the
the probiotic
probiotic archetype Lactobacillus rhamnosus
archetype Lactobacillus rhamnosus GG GG
(LGG) is one of the most studied and well characterized
(LGG) is one of the most studied and well characterized among probiotics. The probiotics, including
The probiotics, including
Lactobacilli,
Lactobacilli,areare studied
studied as supportive
as supportive treatment
treatment for chemotherapy-associated
for chemotherapy-associated gastrointestinal
gastrointestinal toxicity,
toxicity,
thanks to thanks to their
their ability to ability
restoretogut
restore gut microbial
microbial balance, balance, asdescribed
as further further described in Section
in Section 5 [86].
5 [86]. Among
Among these probiotic species, LGG is one of the first studied specifically
these probiotic species, LGG is one of the first studied specifically in oncology in oncology [87]. LGG
LGG is ais
gut resident bacterium known to have several anti-inflammatory effects within the intestinal
microenvironment [88–90]. In animal models, LGG administered with food attenuates 5-FU-
mediated as well as radiation-mediated gut epithelial injury, therefore helping the preservation of
the gut microbiota balance and the intestinal epithelial barrier functionality [91–93].
Several potential benefits of LGG administration to cancer patients have been foregrounded
Cancers 2019, 11, 38 7 of 26

a gut resident bacterium known to have several anti-inflammatory effects within the intestinal
microenvironment [88–90]. In animal models, LGG administered with food attenuates 5-FU-mediated
as well as radiation-mediated gut epithelial injury, therefore helping the preservation of the gut
microbiota balance and the intestinal epithelial barrier functionality [91–93].
Several potential benefits of LGG administration to cancer patients have been foregrounded since
long time, by in vitro, in vivo and clinical studies, as recently reviewed by Banna et al. [94]. In line
with these studies, a number of ongoing clinical trials are currently focused on establishing the role of
LGG administration in preventing or ameliorating the toxic effects of anti-cancer therapies (Table 1).
Additionally, two clinical trials have been designed by our research team and approved by the local
ethical committees; while the ClinicalTrials.gov identifiers (NCT numbers) are on their way to be
assigned. The two studies are entitled respectively: “Maintenance of normal gastro-intestinal function
with dietary supplement containing Lactobacillus rhamnosus GG in cancer patients treated with cytotoxic
chemotherapy and/or targeted therapy” and “Maintenance of normal gastro-intestinal function with
dietary supplement containing Lactobacillus rhamnosus GG in patients treated with abdominal or pelvic
radiotherapy”. The two trials are aimed to assess the efficacy of LGG daily oral administration in the
maintenance of normal gastro-intestinal functions in cancer patients treated either with chemotherapy
and/or targeted therapy or abdominal/pelvic radiotherapy. In addition, the effects of such dietary
supplementation on both the patients’ intestinal microbiome composition and also in their circulating
microRNAs pattern will be further evaluated.

Table 1. Clinical trials * describing the efficacy of probiotics dietary supplementation and/or FMT in
cancer patients.

ClinicalTrials.
Status Title Intervention Disease Ref.
Gov Identifier
NCT00936572 C Probiotics in CRC patients DS: probiotic La1 CRC [95]
Impact of probiotics on diarrhea in patients DS: probiotic Various
NCT01839721 C [96]
treated with pelvic radiation Bifilact Cancers
Prevention of irinotecan-induced diarrhea DS: probiotic Colon
NCT01410955 C CRC [97]
by probiotics Dophilus
DS: prebiotics and
Synbiotics and GI function-related quality
NCT01479907 C probiotics Synbiotic CRC [98]
of life after colectomy for cancer
Forte
Impact of probiotics on the intestinal
NCT01609660 C DS: S boulardii CRC [99]
microbiota
Using probiotics to reactivate DS: probiotic
NCT03072641 C CC [100]
tumor-suppressor genes in CRC ProBion Clinica
Effect of probiotics in patients undergoing
NCT01468779 C DS: probiotics PC [101]
surgery for periampullary neoplasms
Impact of probiotics in modulation of
NCT01895530 C DS: S boulardii CRC -
intestinal microbiota
Action of synbiotics on irradiated GI
NCT03420443 C DS: probiotics RC -
mucosa in RC treatment (FIPIREX)
Intestinal microbiota in lung cancer after
NCT02771470 C DS: probiotics LC -
chemotherapy
Influence of probiotics administration
NCT02021253 C before liver resection in liver disease DS: probiotics HCC -
(LIPROCES)
The effect of probiotics on bowel function
DS: probiotic
NCT02751736 O restoration after ileostomy closure in RC -
CJLP243
patients with RC
DS: probiotic
NCT03290651 O Probiotics and breast health BC -
RepHresh Pro-B
Vivomixx for prevention of bone loss in
DS: probiotic
NCT03518268 O women with BC treated with an aromatase BC -
Vivomixx
inhibitor
The effect of yogurt in cancer patient with DS: probiotics in Various
NCT03177681 O -
moderate GI symptoms yogurt Cancers
Cancers 2019, 11, 38 8 of 26

Table 1. Cont.

ClinicalTrials.
Status Title Intervention Disease Ref.
Gov Identifier
Probiotics combined with chemotherapy Drug with DS of
NCT03642548 O NSCLC -
for patients with advanced NSCLC probiotic Bifico
DS: Probiotic
NCT03358511 O Engineering gut microbiome to target BC BC -
Primal Defense Ultra
Probiotic yogurt supplement in reducing
diarrhea in patients with metastatic kidney DS: probiotics in Renal Cell
NCT02944617 O -
cancer being treated with VEGF-TK yogurt Cancer
inhibitor
Probiotics in radiation-treated gynecologic Gynecologic
NCT02351089 O DS: probiotics -
cancer (ProRad) Cancer
Iodine-131 Therapy
Microbiota are associated with Iodine-131 Thyroid
NCT03574051 O with DS of -
therapy and hypothyroidism Cancer
probiotics
Effects of Probiotics in preventing oral
DS: probiotic L Head-and-neck
NCT03552458 O mucositis in patients undergoing head and -
Reuteri Cancer
neck radiotherapy
DS: probiotic
prevention of irinotecan-induced diarrhea
NCT02819960 O Probio-Fixinum CRC -
by probiotics
(including LGG)
Probiotic LGG for prevention of side-effects
NCT01790035 O in patients undergoing chemoradiation for DS: probiotic LGG GI Cancer -
GI cancer
Lactobacillus Rhamnosus in prevention of
NCT00197873 O DS: probiotic LGG CRC -
chemotherapy-related diarrhea
Safety of stool transplant for patients with Various
NCT02770326 O FMT -
difficult to treat C. difficile infection Cancers
Prevention of dysbiosis complications with
autologous FMT in acute myeloid leukemia
NCT02928523 C Autologous FMT AML -
patients undergoing intensive treatment
(ODYSSEE)
FMT in metastatic melanoma patients who
NCT03353402 O FMT Melanoma -
failed immunotherapy
FMT with
NCT03341143 O FMT in melanoma patients Melanoma -
Pembrolizumab
* Registered at ClinicalTrials.gov; Abbreviations: C, Closed; O, Ongoing; GI, Gastrointestinal; DS, Dietary
Supplement; CRC, Colorectal Cancer; CC, Colon Cancer; RC, Rectal Cancer; BC, Breast Cancer; NSCLC, Non-Small
Cell Lung Cancer; PC, Periampullary Carcinoma; LC, Lung Cancer; HCC, Hepatocellular Carcinoma; AML, Acute
Myeloid Leukemia.

Given the beneficial role of LGG in ameliorating the anti-cancer therapy-related side effects,
many groups are investigating also the potential role that LGG might have in the direct modulation of
cancer development. In particular, it has been observed that LGG exerts its effect either directly on
cancer cells or indirectly through the modulation of the immune system, both in vitro and in vivo.
Firstly, LGG is capable of counteracting cancer growth. It has been demonstrated within several
in vitro tumor models (including colorectal, ovary, breast, cervical, hepatic and oral squamous) that
LGG is able to exert either anti-proliferative effects or anti-metastatic effects [102–106]. This could
be mediated through the direct modulation of several host’s proliferation pathways, such as mTOR
or WNT [107]. In addition, LGG prevents polyps’ formation in a colorectal APC/min mouse cancer
model, and reduces colitis-associated cancer in mice [108,109].
Secondly, LGG can influence host’s immune system, therefore helping the host to eliminate newly
developing cancer cells. In fact, treatment with LGG in a rat dimethyl hydrazine-induced colon cancer
model, is able to reduce the tumor mass through the modulation of the commensal gut microbiome
and the downregulation of pro-inflammatory molecules produced by both gastrointestinal cells and
gut-resident immune cells [110]. Additionally, host’s DC exposed to LGG induce TH1 immune cells
polarization and, in turn, antitumor immune-response potentiation [111].
Cancers 2019, 11, 38 9 of 26

LGG triggers the immune response also within the normal not transformed gut epithelium,
thus protecting towards inflammation, which can support the formation of a cancer-favorable
milieu [112]. On this line, it has been recently observed how LGG attenuates NLRP6-mediated
inflammasomes activation in the intestine [113]. LGG administration can also change gene expression
in intestinal porcine epithelial cells and intestine myo-fibroblasts towards an anti-inflammatory
profile [114,115]. A complete list of the latest in vitro and in vivo studies untangling the complex
role of LGG in cancer development is reported in Table 2.

Table 2. The last three-years in vitro and in vivo studies describing the role of LGG in cancer.

LGG-Mediated Effect Experimental Model Target Cells Reference

Anti-inflammatory and anti-cancer effect in colon DMH
rats cancer cells [110]
cancer model
Dendritic cells exposed to LGG induce TH1 polarization and cancer and
ex-vivo immune cells [111]
antitumor response potentiation immune cells
Anti-proliferative effects on colon adenocarcinoma cells cell culture cancer cells [102]
Anti-metastatic effects on malignant cells cell culture cancer cells [103]
Promotion of IgA production through upregulation of APRIL cancer and
cell culture and mice [89]
expression in intestinal epithelial cells normal cells
Change in transcriptome of small intestine cells mice normal cells [112]
Attenuation of the NLRP6-mediated inflammasomes in the
pigs normal cells [113]
intestine
Prevention of polyp formation in colorectal APC/min cancer
mice cancer cells [108]
model
Modulation of mTOR and Wnt/β-catenin pathways genes in
cell culture cancer cells [107]
cancer cell lines (colon, cervical, breast)
Anti-oxidative effects on CC cells cell culture cancer cells [104]
Inhibition of growth of hepatic cancer cells cell culture cancer cells [105]
Anti-cancer effect on oral squamous cell carcinoma cell culture cancer cells [106]
Reduction of colitis associated cancer mice cancer cells [109]
Change of gene expression towards anti-inflammatory profile
cell culture normal cells [114]
in intestinal porcine epithelial cells
Anti-inflammatory effects in myo-fibroblast colonic cells but
cell culture and mice normal cells [115]
not in cancer cells
Attenuation of 5-FU-mediated intestinal injury mice normal cells [91]
Protection of gut epithelial cells from radiation injury mice normal cells [92]
Preservation of the gut microbiota balance and intestinal
pigs normal cells [93]
epithelial barrier

Altogether, the currently ongoing clinical studies (dissecting the beneficial effects of LGG
administration during anti-cancer therapy), coupled with the in vitro and in vivo studies (supporting
LGG as a direct cancer modulator), make LGG a suitable candidate to be further characterized as
possible adjuvant in integrated anti-cancer therapies.

4. Gut Microbiota and the Inflammasomes

In addition to the role of gatekeeper with the GBA, the gut represents also the interface between
the microbiome and the host’s immune system. The gut microbial population plays a key role in
training, functioning and stimulating the host immune system, with the final effect of developing
tolerance towards beneficial microbiota and eliciting immune response against gut pathogens [76].
Within the Sections 2 and 3 of this review, how the gut microbiome is able to deeply influence
the host’s immune and inflammatory responses, with either protective or detrimental effects on the
oncogenesis, depending on the nature of the bacteria and the host’s immune cells involved, has been
described. Additionally, gut microbiome interacts both with immune cells and gut cells through the
activation of the so-called inflammasomes. Inflammasomes are multiprotein intracellular complexes
Cancers 2019, 11, 38 10 of 26

expressed by both immune cells and epithelial cells. They can detect pathogenic and non-pathogenic
microorganisms-derived molecules, as well as sterile stressors molecules, via a subset of cytoplasmic
pattern recognition receptors (PRRs), called NOD-like Receptors (NLRs) [116].
Given the ability of such multiprotein complexes to sense both microbial and endogenous stimuli,
inflammasomes are regarded as the guardians of cellular and tissue integrity. Upon disruption of host’s
homeostasis and activation of inflammasomes sensors, a strong inflammatory response is activated.
In detail, the inflammasomes complex mediates the activation of caspase-1 which, in turn, triggers the
secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) as well
as pyroptosis, a form of programmed cell death [117]. Dysregulation of inflammasomes results in a
variety of diseases, ranging from autoimmunity to cancer [118]. Importantly, among all the stimuli,
inflammasomes are also capable of sensing host-microbiota interactions, thus taking an active role in
response to commensal and pathogenic bacteria [116].
The role of the inflammasomes in tumorigenesis is controversial. In fact, the inflammasomes
have been shown to be either tumor-promoting or tumor-suppressive, depending on the nature
of both the tumor and its microenvironment. The exact outcome of the inflammasomes activation
depends on multiple factors, including its expression pattern and effector molecules, together with
the tumor nature and stage. Importantly, the gut microbiome may also influence the outcome
of the specific inflammasomes activation [119]. Currently, we have very limited knowledge on
the mechanisms responsible for inflammasomes activation during tumor development. Multiple
studies using different mice deficient for inflammasomes components (including NLRP3, NLRP1,
NLRP6, NLRC4 and Caspase-1) found that the inflammasomes protect mice from colitis-associated
CRC [120–124]. Importantly, the inflammasomes effector IL-18, but not IL-1β, plays a pivotal role in
suppressing colitis. In fact, IL-18 KO or IL-18R KO mice are also highly sensitive to colitis-associated
CRC [124,125]. All these studies highlight the importance of inflammasomes-dependent IL-18
production in suppressing CRC. On the contrary, inflammasomes activation results detrimental to
the development of lung, skin, breast and pancreatic cancer. In those cases, IL-1β is the key player,
acting as pro-inflammatory, tumor-promoting trigger [126–130].
The commensal microbiota and their bioproducts are sensed by epithelial cells and innate immune
cells via innate receptors, including the inflammasomal NLRs. In particular, inflammasomes-mediated
IL-18 is critical for intestinal tissue remodeling and gastro-intestinal barrier maintenance. Commensal
bacteria and their bioproducts induce inflammasomes activation and IL-18 production in the gut,
which, in turn, prevents intestinal barrier disruption and dysbiosis [121,124,131–133]. Deficiency
in inflammasomes components leads to reduced production of IL-18, resulting in an intestinal
barrier impairment. Such damage causes larger commensal bacteria penetration, and increased
inflammation, which may finally trigger tumorigenesis. Dysbiosis has been observed in mice deficient
for inflammasomes components, including NLRP6, ASC, caspase-1, and IL-18 [131–134]. Importantly,
it has been proposed that inflammasomal NLRP6 is required for the commensal bacteria homeostasis.
NLRP6 KO mice show dysbiosis and increased incidence of inflammation-associated CRC [135–137].
Inflammasomes and IL-18 are protective in inflammation-induced CRC. Further studies are needed
to assess whether inflammasomes and/or IL-18 inhibit CRC development in genetic CRC models
(such as the APC/min mice), as well as in human CRC. Relevantly, Lactobacilli are able to activate the
inflammasomes in human primary macrophages, as well as in primary mammalian gut epithelial cells,
as defense mechanism against viral infection or epithelial injury [138].
Therefore, inflammasomes represent a double-edged sword in tumorigenesis and the gut
microbiome may influence the outcome of the specific inflammasome activation during tumorigenesis.
In CRC inflammasomes activation has a protective role, on the contrary in breast and skin cancers their
triggering leads to detrimental outcomes [119]. Regarding the correlation between inflammasomes,
microbiome and cancer, NRPL6 plays a key role in colorectal carcinogenesis and, in particular, NLRP6
regulates susceptibility to intestinal inflammation through its microbiome-modulatory activity [139].
Cancers 2019, 11, 38 11 of 26

5. Gut Microbiota and Anti-Cancer Therapy

Anti-cancer therapies are designed with the final goal of being effective in the eradication of
the targeted malignancy. Because almost every available anti-cancer treatment is toxic also towards
normal cells, their use may be coupled with side effects, some of which can compromise the overall
survival of the patients [140].
Additionally, tumors are intrinsically complex: as they attempt to accumulate mutations, cancers
evolve and adapt to the hosting organism [141]. In fact, cancers derive from the stochastic acquisition
of driver mutations within genes involved in key processes, including DNA duplication, DNA repair,
oxidative stress response. Such accumulation finally allows the transformation of a normal cell into a
malignant one [141]. Both the initiation and the progression of a tumor may be viewed as a blended
impairment of such fundamental cellular processes, meaning that from one original cancer cell might
derive a molecularly varied bulk tumor made of multiple clones of cancer cells, each one displaying
a differential intrinsic sensitivity to the anti-cancer therapies [142]. This heterogeneity derives from
intrinsic tumor cellular genomic instability, ranging from microsatellite instability (due to impairments
of the DNA mismatch repair system) to chromosomal instability (arising from segregation errors during
cell mitosis) [143,144]. On top of that, such genetic mechanisms might be coupled with epigenetics,
transcriptional and post-transcriptional intracellular changes, finally leading to a growing tumor
complexity, through time and space [145].
Importantly, this intra-tumoral variety is tightly linked with the development of the resistance
to therapy, considered the first cause of failure of the available anti-cancer treatments, as well as
subsequent tumor relapses [146]. To fight such resistance, integrated therapies and personalized
approaches, based on the specific genetic features of the malignancy, are in continuous progress [146].
Developing malignant cells not only are subjected to their intrinsic heterogeneity, additionally they
are recognized and eliminated by the host’s immune system [147]. On their side, tumor cells, thanks to
their genetic instability, constantly evolve novel strategies to escape from such immunosurveillance
and expand within the host [147]. Along with chemotherapy and radiotherapy, a novel anti-cancer
approach is considered the so-called targeted immunotherapy, bearing the dual role of both boosting
the host anti-tumor immune response, and, at the same time, helping to hit cancer resistance and
recurrence mechanisms [148,149].
Modulating gut microbiome may deeply influence the outcome of anti-cancer therapies. In fact,
radiotherapy, chemotherapy and immunotherapy treatments can all modify patients’ microbiome and,
at the same time, microbiome composition can deeply affect patients’ response to such therapies [150].
It is therefore fundamental to identify which are the factors able to influence the gut microbiome
and, in turn, to find novel strategies to manipulate the gut microbiome, with the main goal of
finally improving patients’ therapeutic outcome. Specifically, interventions on microbiome may
be pivotal to ameliorate anti-cancer therapy-related toxicity, as well as to improve anti-cancer therapy
efficacy [151,152]. The emerging role of the gut microbiome in cancer therapy is summarized
in Figure 3.
Back in 1890 two heat-inactivated microorganisms (Streptococci) were injected intratumorally for
the very first time in humans as an attempt to cure cancer [153,154]. Furthermore, several decades
later, Mycobacterium bovis was successfully injected into bladder in patients, following the resection of
a bladder tumor. It has been observed that the bacteria, by inducing a local immune response, helped
to reduce the relapse of the tumor [155]. Moreover, it has been discovered how oral administration
of Lactobacillus casei decreased superficial bladder cancer recurrence [156]. The mechanism behind
involves the direct bacterial stimulation of host’s NK cells and macrophages, in turn responsible of a
strong antitumoral immune response [157].
These observations paved the way for many published, as well as ongoing, clinical trials, based on
the usage of gut bacterial attenuated strains in anti-cancer therapy. These trials are shedding light on
the key role of such bacteria on triggering anti-tumor immune response [158]. For example, it has
been observed that the intradermal injection of Mycobacterium obuense in melanoma and in pancreatic
they are recognized and eliminated by the host’s immune system [147]. On their side, tumor cells,
thanks to their genetic instability, constantly evolve novel strategies to escape from such
immunosurveillance and expand within the host [147]. Along with chemotherapy and radiotherapy,
a novel anti-cancer approach is considered the so-called targeted immunotherapy, bearing the dual
role of2019,
Cancers both11, boosting
38 the host anti-tumor immune response, and, at the same time, helping12toofhit 26
cancer resistance and recurrence mechanisms [148,149].
Modulating gut microbiome may deeply influence the outcome of anti-cancer therapies. In fact,
ductal carcinoma
radiotherapy, activates antitumoral
chemotherapy immune response,
and immunotherapy acting
treatments canonallhost’s
modifyantigen presenting
patients’ cells
microbiome
(APCs) and cytotoxic T cells [159,160]. Additional clinical trials further revealed
and, at the same time, microbiome composition can deeply affect patients’ response to such therapies how attenuated
bacteria
[150]. Itinjected directlyfundamental
is therefore into the tumor to mass are able
identify to both
which are stimulate anti-tumoral
the factors immune response
able to influence the gut
and also have a direct cytotoxic effect on the tumor cells, because their
microbiome and, in turn, to find novel strategies to manipulate the gut microbiome, with capability of colonizing
the main
tumors, as observed
goal of finally in several
improving different
patients’ refractory
therapeutic solid tumor
outcome. studies,interventions
Specifically, towards the onadministration
microbiome
of attenuated and/or genetically modified Salmonella typhimurium [161–163]. Although
may be pivotal to ameliorate anti-cancer therapy-related toxicity, as well as to improve anti-cancer these results
are encouraging, many clinical trials are currently ongoing in order to ameliorate the
therapy efficacy [151,152]. The emerging role of the gut microbiome in cancer therapy is summarized patients’ clinical
outcomes,
in Figure 3.given bacteria-associated toxicity, mainly correlated to their long half-life [163].

Figure 3.
Figure 3. Role
Role of probiotics
probiotics in
in anti-cancer
anti-cancer therapy.
therapy. Probiotics
Probiotics and
and Fecal
Fecal Microbiome
Microbiome Transplantation
Transplantation
(FMT) are currently studied
(FMT) are currently studied as anti-cancer adjuvants to fight dysbiosis following anti-cancer
anti-cancer therapy,
therapy,
to increase
to increase chemotherapy
chemotherapy and immunotherapy
immunotherapy efficacy and to bothboth reduce
reduce tumor
tumor mass
mass and
and prevent
prevent
tumor recurrence.
tumor recurrence.

5.1. Modulation of Gut Microbiota to Enhance Chemotherapy and Immunotherapy Efficacy

The microbiota, when affected by dysbiosis, can deeply influence both cancer pathogenesis
(as reported in Section 2) and its therapeutic outcome. In particular, the regulation of such therapeutic
outcome is tightly connected with the ability of the gut microbiota to metabolize anti-tumoral
compounds, as well as to modulate host’s immune response and inflammation pathways [164].
These two effects combined together may explain the strong involvement of the patient’s microbiome
composition in affecting the efficacy of both chemotherapy and immunotherapy [19].
With regard to chemotherapy, it has been reported how tumor-bearing mice, either germ free
or having their gut microbiota depleted after antibiotics therapy, do not respond to oxaliplatin drug
treatment. The explanation is that commensal microbiome members within the gut of the mice might
produce TLR agonists, thus promoting the rise of an oxidative stress milieu and tumor cell death.
As a direct consequence, without a healthy gut microbiota there is a decreased microbiota-dependent
ROS production, thus a less effective chemotherapeutic response [165]. Consistently, mice bearing
lung tumors treated with cisplatin coupled with antibiotics, survive less and develop bigger tumors.
If cisplatin is combined with probiotics, such as Lactobacilli, mice show an improved response to
therapy. The mechanism involves the induction of pro-apoptotic genes within the tumor mass and the
enhancement of host’s immune response [166].
Another widely used anti-cancer molecule, cyclophosphamide, coupled with oral bacterial
administration (Lactobacillus johonsoni and Enterococcus hirae), leads to the conversion of T cells from
naïve to pro-inflammatory T helper 17 (TH17), with the final effect of improving cyclophosphamide
efficacy in tumor-bearing mice [167,168].
Cancers 2019, 11, 38 13 of 26

With reference to immunotherapy, the administration of CpG oligodeoxynucleotides, synthetic

molecules mimicking bacterial DNA, strongly stimulate the immune system, therefore showing
anti-tumor activity in several cancers [169]. Along this line, the intra-tumoral injection of CpG
oligodeoxynucleotides administered together with an anti-interleukin-10 receptor (IL-10R) antibody,
induce TNF production from tumor infiltrating myeloid cells and, in turn, reduce the growth several
types of tumors in mice [165]. Moreover, the administration of a specific bacteria, Alistipes shahii,
to antibiotic-treated tumor bearing mice, restores TNF production with a notable improvement in the
therapeutic outcome [165].
Given the multiplicity of effects that gut microbiome may play on the host’s immune system, it is
not surprising that emerging studies strongly linked the patients’ microbiome composition with the
intrinsic efficacy of immune checkpoint inhibitors-based immunotherapy, in the treatments of different
solid tumors [170–172]. Immune checkpoint inhibition consists in the administration of therapeutic
agents able to block the immune-inhibitory pathway, thus modulating T cell activation against tumor
target cells. The currently marketed checkpoint inhibitors are in monoclonal antibodies targeting
cytotoxic T lymphocyte-associated protein 4 (CTLA4) or the programmed death 1 (PD1) located
on T cell surfaces, or its ligand, programmed death ligand 1 (PD-L1), expressed by the APCs [173].
While CTLA-4 regulates T cells proliferation early in the immune response within the lymph nodes,
PD-1 suppresses T-cell activation later, within the body periphery [174].
A few years ago, two studies suggested the potential involvement of the gut microbiome in
modulating the efficacy of such anti CTLA4 and anti-PD1 based therapies [175,176]. Vetizou et al.
demonstrated that the efficacy of anti-CTLA4 antibodies in reducing sarcoma tumor growth in mice
is significantly increased when the gut microbiome is enriched in Bacteroides fragilis and Burkholderia
cepacia [175]. On the same line, Sivan et al., found that the efficacy of PD-L1 targeting-antibody in the
cure of melanoma in mice is improved in the presence of gut microbiome enriched in Bifidobacterium
species [176]. In fact, they demonstrated that oral administration of a cocktail of Bifidobacterium species
combined with the anti-PD-L1 antibody, specifically boosts T cell response and blocks the melanoma
growth [176].
Multiple translational studies, published in 2018, further support the pivotal role of
the gut microbiome in modulating the response to immune checkpoint blockade [170–172].
In particular, Routy et al. [170] found that melanoma patients treated with antibiotics along with the
anti-PD1/anti-PD-L1 immunotherapy had a lower survival rate. Importantly, metagenomics analysis
of patients’ fecal gut microbiome showed a difference in the composition of their gut microbiome.
Anti-PD1 responders were enriched in two phyla (Akkermansia and Alistipes). Performing FMT from
patients to germ free mice, the authors found that Akkermansia muciniphila (alone or in combination
with Enterococcus hirae) was able to increase intra-tumoral cytotoxic T cell infiltrates, thus increasing
the PD-1 blockade response in mice [170]. In parallel, Gopalakrishnan et al. [171] demonstrated
through metagenomics analysis of melanoma patients’ fecal samples that the anti-PD1 responders’
microbiome was different in composition compared with that of non-responders. The authors
observed in patient’s gut microbiome an increase in the abundance of Clostridiales, Ruminococcaceae and
Faecalibacteriae. Functional studies performed with FMT in germ free mice further demonstrated how
treating mice with the identified bacteria, along with the anti-PD1 therapy, enhanced the anti-cancer
effects and reduces the melanoma growth [171]. Along the same line, Matson et al. [172] performed
a metagenomics characterization of stools samples from melanoma patients treated with immune
checkpoint inhibitors, further corroborating the finding that responders showed a different microbiome
compared to those not responding to therapy. They identified and functionally proven in vivo the
importance of Bifidobacterium longum, Enterococcus faecium and Collinsella aerofaciens in ameliorating
anti-PD-L1 efficacy [172].
Although the immune checkpoint inhibitors have success in treating various malignancies,
there is still a significant number of patients that can use such therapy only for a limited amount
of time, given the occurrence of strong toxic side effects, including gut inflammation, due to the
Cancers 2019, 11, 38 14 of 26

subsequent immune-dysregulation (i.e., autoimmunity) [177]. In animals, oral gavage of Bacterioides

fragilis and Burkholderia cepacia demonstrated an amelioration of such immunotherapy-associated
toxic side effects [175]. In line with this observation, it has been seen that patients treated with
anti-CTLA4 antibody, toxic side effects are mediated by an increased abundance of Firmicutes,
such as Faecalisbacterium, and a decreased abundance of Bacterioides [178,179]. Altogether, these
data provide a strong evidence of the role of gut microbiota composition in modulating the effect of
both immunotherapy response and toxicity.
Even if the last decade witnessed massive advances in unveiling the role of gut microbiome in
cancer and other diseases, there are still many obstacles for translating basic microbiome research into
therapeutic applications. Among the gut bacteria can develop potential pathogens and that could
limit, or at least slow down, the translation of the in vitro and in vivo results to the clinic. In light of
the novel studies reported above, any antibacterial therapy altering the intestinal equilibrium, during
anti-cancer therapy, needs to be carefully evaluated. In fact, the heterogenous patients’ microbiome
can either be detrimental or beneficial to tumor progression and therapy, depending on its composition
and prevailing species. As further discussed below, looking at the effects of probiotics treatments in
anti-cancer therapy, it might be necessary in the future to pursue a personalized approach, based on
the specific patient’s microbiome composition.

5.2. Use of Probiotics in Oncology

As discussed above, chemotherapy, targeted therapy, immunotherapy and radiotherapy represent
the pillars of the currently available anti-cancer treatments. Such treatments may cause diverse and
even drastic side effects in patients [180–186]. Several preclinical studies and clinical trials share the
common goal of evaluating the overall efficacy of probiotics in decreasing the risk and the severity of
such anti-cancer treatments related-toxicity, mainly diarrhea and mucositis [187,188]. In fact, the aim of
administering probiotics to cancer patients, principally Lactobacilli, is to re-populate the compromised
patients’ gut microbiota, thus re-establishing the levels and functionality of the commensal bacteria,
depleted after the treatments [189]. Although probiotics are generally regarded as safe, the main
concerns of administering them to immunocompromised cancer patients are both the potential risk
of opportunistic infection development and the transfer of antibiotics resistance [190,191]. Despite of
that, probiotics administration in multiple trials has shown beneficial effects on ameliorating diarrhea
and other gut-related damages following anti-cancer therapy, thus re-establishing a healthy intestinal
microbiota composition [192]. Moreover, within the Multinational Association of Supportive Care in
Cancer and International Society of Oral Oncology (MASCC/ISOO) and European Society of Medical
Oncology (ESMO) Clinical Practice Guidelines for Gastrointestinal Mucositis, probiotics containing
Lactobacillus species are suggested be used to prevent diarrhea in patients receiving chemotherapy
and/or radiation therapy for a pelvic malignancy (Level of evidence III) [193,194].
Given that a growing body of studies corroborated the fundamental role of microbiome in cancer,
many clinical studies are currently ongoing with the common aim of investigating the therapeutic
potential of manipulating gut microbiota in cancer patients. Results from early clinical trials are
promising. In 2010, it was assessed for the first time the interaction between probiotic administration,
variation of gut microbiota composition, and regulation of intestinal immune-functions in cancer
patients undergoing colorectal resection [95]. A mixture of two probiotic species Bifidobacterium
longum (BB536) and Lactobacillus johnsonii (La1) was administered to the patients in the double-blind
study, finding that one of that, La1, was able to adhere to the colonic mucosa, thereby reducing
the concentration of gut pathogens and modulating the local immunity [95]. Subsequently, in 2014,
a randomized double-blind controlled trial assessed the beneficial administration of the probiotics
Bifilact (Lactobacillus acidophilus LAC361 and Bifidobacterium longum BB536) on significantly reducing
moderate and severe treatment-induced diarrhea during pelvic radiation [96]. On the same line,
in 2015, for the first time, a clinical trial evaluated the probiotic formula Colon Dophilus (mixture of
10 different probiotic strains) in the prevention of diarrhea in patients with metastatic CRC, treated with
Cancers 2019, 11, 38 15 of 26

irinotecan-based chemotherapy, suggesting that the administration of such probiotics is safe and leads
to a reduction in the incidence and severity of diarrhea and chemotherapy induced gastrointestinal
toxicity [97]. In 2016, another double-blind, randomized trial demonstrated that the administration
of a combination of prebiotics and probiotics to patients subjected to CRC resection may alleviate
irritable bowel syndrome (IBS), often following the operation [98]. In the same year, another trial
further analyzed the effects of randomized oral administration of the probiotic Saccaromices bulardii
in CRC patients. The authors found that this probiotic was able to downregulate pro-inflammatory
cytokines in treated patients, although with lacking effects on the post-operative infection rates [99].
Moreover, according to the result of a trial published in 2017, the randomized administration of
Bifidobacterium lactis and Lactobacillus acidophilus to CRC patients, can change the epigenetic patterns
of tumor tissue from its baseline, with potential therapeutic benefits in CRC by manipulation of the
gut microbiota [100]. The same year a randomized clinical trial with CRC patients demonstrated
that the perioperative administration of a mixture of prebiotics and probiotics, significantly reduced
postoperative infection rates in patients with CRC [101].
Regardless the observed beneficial effects, larger and controlled clinical trials are further needed
to truly endorse both the efficacy and the safety of administering selected species of probiotics during
or following anti-cancer treatments (Table 1).

5.3. Use of Fecal Microbiota Transplantation (FMT) in Oncology

The exchange of gut microbiota between individuals has been used to cure pathogens infections
or in the treatment of gut inflammatory disease and dysbiosis. For example, FMT has been used
to cure recurrent Clostridium difficile duodenal infection [195,196]. Moreover, FMT has been used
in a Graft Versus Host Disease (GVHD) after allogeneic stem cell transplantation [197]. Regarding
anti-tumor therapeutic applications, preclinical studies performed in mice demonstrated the efficacy
of FMT in reducing colon tumorigenesis, although the efficacy in clinical trials still needs to be further
proven [198]. Several clinical trials, designed to evaluate the use of FMT in cancer patients are currently
ongoing, with the common goal of preventing and/or ameliorating intestinal side-effects of anti-cancer
therapies in cancer patients (Table 1).
Despite the success of FMT, there is still a lack of control in this procedure because the whole gut
microbiota is transferred along with the therapeutic bacteria species. Therefore, it is of key importance
the careful control of the donors’ health and their gut microbiome specific composition [199].

6. Conclusions and Future Perspectives

The relationship between gut resident microbiota and their host is complex. Each individual
inherited a specific gut microbiota footprint since their birth, and their intestinal microbiota develops
and changes with aging, diet and lifetime exposure to the heterogeneous environment. Indeed,
this balance is very delicate and subjected to multiple changes during the entire lifespan.
Nowadays, there is a growing attention towards the characterization of the gastro-intestinal
microbiota composition and functionalities. Genetics, together with functional studies, highlighted
a dual role played by the gut microbiome in cancer. Some bacterial subpopulations are able to rise
during gut dysbiosis and, in turn, to trigger the formation of an inflammatory and pro-cancerogenic
environment. On the other hand, many gut derived probiotics are able to protect the host,
re-establishing the conditions of a healthy intestinal microbiota within dysbiotic patients, including
cancer patients.
LGG is a very good example of a probiotic well studied in cancer, often administered as
complementary therapeutic to cure dysbiosis. Given the observed functions as anti-inflammatory and
anti-cancer agent in both cellular and animal models, this probiotic may be suggested to be further
characterized as adjuvant in integrated anti-cancer therapies in the future.
In line with that observation, this year have witnessed novel breakthrough studies on the
association between probiotics and anti-cancer therapy efficacy, as three parallel studies identified
Cancers 2019, 11, 38 16 of 26

specific gut species populating the gastro-intestinal tract of cancer immunotherapy responders, able to
improve the efficacy of immunotherapy treatments.
That questions the usage of both probiotics and FMT in cancer therapy, either as tools to
repopulate cancer patients’ damaged intestine or even as proper adjuvants in immunotherapy
and other kinds of anti-cancer therapies. Correspondingly, care needs to be pursued as patients
are often immunocompromised, therefore it is important to evaluate the specific side effects of
administering selected bacterial species to such sensitive individuals. In the future, the design of
novel experimental trials may undertake a personalized-integrated approach, considering the specific
clinical and pathological background of each single patient to be treated, in order to gain only the
positive outcomes of probiotics administration and/or fecal transplants, possibly without any harmful
side effect.

Author Contributions: Conceptualization: M.L., S.V.; Writing—Original Draft Preparation: S.V.; Figures and
Tables: S.V.; Writing—Review & Editing: S.V., R.S., S.C., L.F., M.S., S.S., F.T., G.L.B, G.T., M.L. All authors have
approved the final version of the manuscript.
Funding: S.V. has been funded by a fellowship from the Italian League Against Cancer (LILT), Catania.
Acknowledgments: S.V. thanks the Italian League Against Cancer (LILT), Catania for its support.
Conflicts of Interest: M.L. is the PI of a research grant founded by Dicofarm Spa to his University Department.
The other authors declare that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest.

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