Paper - Review

DOI: 10.1128/mBio.00692-13

Abstract

❗: Individual (← with colorectal cancer)
→ have → an altered gut microbiome

❓: whether these differences are
→ 1⃣ a response → to tumorigenesis 2⃣ actively drive tumorigenesis

Characterized → the gut microbiome
← in a murine model (← of inflammation-associated CRC (← colorectal cancer))
→ to determine → the role of (the gut microbiome) ← in the development of CRC

Followed
→ the development of (an abnormal microbial community structure)
← associated with (1⃣ inflammation 2⃣ tumorigenesis)
← in the colon

Enrichment in OTUs
← affiliated with 1⃣ Bacteroides 2⃣ Odoribacter 3⃣ Akkermansia genera
Decreases in OTUs
← affiliated with 1⃣ Prevotellaceae 2⃣ Porphyromonadaceae families

Conventionalization ← of (germfree mice)
→ significantly increased tumorigenesis ← in the colon

Germfree mice
← that colonized with microbiota ← from tumor-bearing mice
→ harbored → a higher relative abundance of populations
← which associated with (tumor formation) ← in conventional animals

Manipulation of (the gut microbiome) ← with antibiotics
→ resulted → in a dramatic decrease ← in 1⃣ the number of tumors 2⃣ the size of tumors

❗: Changes in (the gut microbiome)
← associated with 1⃣ inflammation 2⃣ tumorigenesis
→ contribute to tumorigenesis

❗: interventions ← which affecting (the composition of the microbiome)
→ may be → a strategy
→ to prevent → the development of CRC

Importance

Gut microbiome
→ are important → for normal functioning of the intestine

Disruptive changes ← in the gut microbiome
→ are associated ← with the development (CRC)

❓: how (the gut microbiome) changes
← with time during tumorigenesis
❓: whether these changes
→ directly contribute → to disease

(Dramatic & Continual alterations) ← in the microbiome
← during (the development of tumors)
→ which are directly responsible → for tumor development
∵ Using (a mouse model) of (inflammation-driven CRC)

❗: Target (these changes ← in the microbiome)
→ may be → an affective strategy → for preventing (the development of CRC)

Introduction

Significant risk factors (← for CRC)
→ include 1⃣ diet ← rich in (red & processed meat) 2⃣ alcohol consumption 3⃣ chromic inflammation ← of the gastro-intestinal tract

These factors
→ are closely associated with changes
← in 1⃣ composition 2⃣ function of (the complex community ← of microorganisms)
← that inhabits our gastrointestinal tract

The gut microbiome
→ promotes various physiological functions
← that are associated with cancer
← including 1⃣ cell proliferation 2⃣ angiogenesis 3⃣ apoptosis

❓: The 1⃣ composition 2⃣ structure 3⃣ functional capacity
← of the gut microbiome
→ directly affect (tumor development) ← in the colon

Characterizing (the composition ← of the gut microbiome)
→ associated ← with patient with CRC

❗: Observed (a significant shift)
← in the composition of (the gut microbiome)
← in patient with CRC
∵ using culture-independent approaches

Observed
← in 1⃣ the luminal microbiome ← from feces 2⃣ the mucosa-associated microbiome
→ from tumor biopsy specimens

No bacterial populations
← that have consistently been identified
→ can be attributed → to 1⃣ the development 2⃣ the presence ← of CRC

∴ An association
← between 1⃣ abnormalities ← in the gut microbiome 2⃣ CRC

❓: The effect of (the development ← of this abnormal community)
← on colon tumorigenesis
→ remain unclear

1⃣ B. fragilis 2⃣ E. coli
→ can directly affect → tumor development ← in the colon
← through (the production of virulence factors)
← e.g. 1⃣ toxins 2⃣ gene products

Bacterial populations
← that produce (the short-chain fatty acid butyrate)
→ have anti-tumor effects
← by promoting apoptosis of (CRC cells)

❗: dysbiosis (← of the gut microbiome)
→ leads
→ 1⃣ enrichment ← of cancer-promoting bacterial populations 2⃣ loss ← of protective populations

The gut microbiome
→ contributes → to CRC
← through (the initiation ← of inflammation)

The link
← between 1⃣ inflammation 2⃣ cancer
→ is well established

The risk for cancer
→ is related
→ to 1⃣ the duration 2⃣ the severity of inflammation
← in the case of (ulcerative colitis)

Chronic inflammation (← of the colon)
→ leads → to the production of (1⃣ various inflammatory cytokines 2⃣ reactive oxygen species)
→ to generate (a tumor micro-envrionment) ← that promotes carcinogenesis

❓: how (the normally beneficial) (gut microbiome)
→ becomes inflammatory

Used → well-established model (← of colitis-associated CRC)
← that recapitulates (the progression ← from chronic inflammation)
→ to 1⃣ dysplasia 2⃣ adenocarcinoma
→ to determine → the role of the gut microbiome

Characterized → the dynamics of the gut microbiome
Demonstrated → community-wide changes promote tumorigenesis

Results

Inflammation-associated colon tumorigenesis

Replicate
→ an inflammation-based murine models of tumorigenesis
← in SPF (← specific-pathogen-free) mice
← using (an intraperitoneal injection) ← of (the chemical carcinogen azoxymethane)

The mice showed
→ a consistent pattern ← of (weight loss)
→ following each round of (DSS treatment)

Did NOT observe
→ macroscopic tumors
→ following (the first round ← of DSS administration)

Observed
→ increased infiltrations
← by 1⃣ immune cells 2⃣ lytter effsignificant epithelial damage 3⃣ submucosal edema

A significant increase
← in 1⃣ MIP-2 (← macropahge inflammatory protein 2) 2⃣ IFN-γ (← gamma interferone) 3⃣ TNF-α (← tumor necrosis factor alpha) 4⃣ IL-6 5⃣ IL-1β

1⃣ Macroscopic tumors 2⃣ epithelial hyperplasia
→ were apparent
→ following (the second round of DSS)

❗: the cohort had → a median of 14.5 tumors/mouse

Our cohort of (AOM/DSS-treated mice)
← developed → a significant number of (colonic tumor)
→ could be detected ← as early as 7 weeks

Treated mice
← with (an antibiotic cocktail) ← of 1⃣ metronidazole 2⃣ vancomycin 3⃣ streptomycin
→ ❓: to determine → whether (tumor incidence & penetrance) → were dependent on (the gut microbiome)

∴ Antibiotic-treated mice
→ had significantly fewer tumors

Tumors
← that were present in antibiotic-treated mice
→ were also significantly smaller

❗: specific populations (← within the microbiome)
→ were essential → for tumorigenesis

Performed → quantitative PCR ← with the 16S rRNA gene
→ to determine → ❓: whether the relative change in (bacterial density)
∵ a change in the bacterial load

The number (← of 16S rRNA gene copies / feces)
→ was NOT significantly different

Changes to (the structure of the community)
→ affected tumorigenesis

Significant shifts in the microbiome are associated with colon tumorigenesis

Examine → the dynamics of (the gut microbiome)
← using stool samples
→ to test → specific changes
← in (the microbial community structure)
→ were associated ← with 1⃣ inflammation 2⃣ tumorigenesis

Used → the fecal sample
→ as a baseline control → for each mouse
Took samples → following each subsequent round of (DSS administration)

Mice
→ showed (a significant decrease)
← in microbial diversity ← in the gut microbiome
→ following (the first round of DSS administration)

Ordination
← of the distance between (fecal samples)
→ showed that → at the time of euthanization
Tumor-bearing mice
→ developed (a significantly altered microbiome)
→ that clustered separated ← that in baseline samples

Significant alteration ← in the microbiome
→ could be observed ← as early as the first round of (DSS administration)
∵ Further examination of (fecal samples)
← collected at various (time points)
← during the AOM/DSS tumor induction protocol

Fecal samples
→ taken from tumor-bearing mice
← until the time of euthanization
→ clustered separate ← from earlier samples

These clusters
→ were observed ← using 1⃣ operation taxonomic unit 2⃣ phylogenetic-based metrics of β-diversity
→ could be distinguish from one another
← using the Random Forest machine learning algorithm

❗: the association
← between 1⃣ a dramatically altered microbiome structure 2⃣ the presence of tumors

Treated mice
← with three rounds of DSS
← without the AOM injection
→ ❓: determine (the effect of inflammation) ← on the microbial community → independent of tumorigenesis

There was (an initial community shift)
→ following the first round of DSS
The subsequent stepwise shifts
→ were NOT observed ← in mice treated with DSS only

Inflammation
→ is not sufficient
→ to cause microbial community changes

AOM/DSS model
→ are necessary
→ for 1⃣ development of the altered microbiome structure 2⃣ tumorigenesis

Identified
→ which OTUs were responsible
→ for the dramatic shifts ← in the microbial community structure
← during 1⃣ inflammation 2⃣ tumorigenesis

Observed changes
← in 37 bacterial population
← during (the time course) of the model
→ relative to those in baseline samples

Fecal samples
← which taken after the first round of DSS
→ were enriched ← with members of (the genus Bacteroids)

Observed
→ a significant decrease ← in the relative abundance of OTUs
← with 1⃣ Prevotella genus 2⃣ Porphyromadaceae family

Mice
← which following the 3rd round of DSS
→ showed significant differences
← compared to following 1⃣ the 1st round of DSS 2⃣ healthy baseline mice

Tumor-bearing mice
→ showed enrichment ← in 1⃣ Bacteroides 2⃣ Odoribacter 3⃣ Turicibacter

Detected → a marked bloom of (a member of the Ery. family)
→ which was undetectable ← when tumors are not evident

A significant decrease
← in the relative abundance of OTUs
← with members of 1⃣ Prevotella genus 2⃣ Porphyromanadaceae family

❗: 1⃣ inflammation 2⃣ tumorigenesis
→ promote → gut microbiome dysbiosis

❓: the variability in (tumor burden)
← among AOM/DSS-treated mice
→ was associated ← with variability in (the gut microbiome)

The relative abundance of (this bacterial population)
→ decreased ← with each round of DSS
This drop
→ was more pronounced
← in mice ← with higher tumor burdens

Alterations
← in the relative abundances of (specific bacterial populations
→ were associated → with 1⃣ the incidence of tumors 2⃣ prevalence of tumors

Tumor-associated alterations in the microbiome increase tumorigenesis in germfree mice

Conventionalized → germfree mice
←with 1⃣ the healthy microbiome 2⃣ the microbiome of (tumor-bearing mice)
→ to determine → ❓: whether the community changes → contribute to (tumor incidence) ← in the colon

Transferred → 1⃣ fresh feces 2⃣ bedding
→ to two groups of (germfree mice)
→ ensure → mice were (1⃣ repeatedly inoculated 2⃣ stably colonized)

Each group
→ was comprise of (two cages) of 5 mice
← collected from separate litters
← that were randomly assigned to each of the cages

(All bacterial phyla) & (90% of genus level taxa)
→ were detected ← within the recipient germfree mice
← which is higher than has been previously reported

81% of the sequences
← that obtained from the donor mice
→ belonged to OTUs ← that were found in (the recipient germfree mice)

Mice
← conventionalized with the microbiome of (tumor-bearing mice)
→ had a 2-fold increase in tumor burden

Tumors ← from these mice
→ were significantly larger
← than those observed in recipients of (a healthy microbiome)

Germfree mice
← conventionalized with the community of (tumor-bearing mice)
→ had a significantly less diverse (gut microbiome)

Conventionalization
← with these two treatments of bedding
→ resulted → in two distinct (microbial community structures)
∵ community-wide β-diversity analyses

Germfree mice
← which conventionalized with (the microbiome of tumor-bearing mice)
→ showed significant enrichment
← in the relative abundance of OTUs ← with 1⃣ the genera Bacteroides 2⃣ the family Ery.

Germfree mice
← conventionalized with a healthy microbiome
→ recapitulated → the community dynamics

❗: alterations to (the gut microbiome)
→ were associated ← with 1⃣ chronic inflammation 2⃣ tumorigenesis
→ can exacerbate (colon tumorigenesis)

Discussion

Established → a causal role
→ fro the gut microbiome ← in exacerbating tumor formation
← in an inflammation based model of tumorigenesis

Manipulation of the microbiome
← using anti-biotics
→ reduce (tumor formation)
∴ importance of (bacterium-driven factor) ← in tumorigenesis

Dynamic changes
← in the microbial community structure
→ associated ← with dysbiosis
❗: Prior → to the first sign of (macroscopic tumor formation)

Transfer of microbiota
← from tumor bearing mice
→ significantly increased → 1⃣ the number of tumors 2⃣ the size of tumors

Dramatic shifts
← in the relative abundances of (bacterial populations)
→ to the Bacteroids genus
→ were associated ← with increased tumorigenesis

❗: significant difference
← in the microbial community structure
❓: 1⃣ the specific gut microbiome composition 2⃣ profile → associated with CRC

Inability
→ to identify 1⃣ a consensus community profile 2⃣ etiological agent
∵ 1⃣ the large variation in (the structure of the microbiome) ← across individuals 2⃣ the improbability of there being ← that is associated with all CRCs

Reduce
→ 1⃣ the inter-individual variation 2⃣ the diversity of cancer types
← using (a murine model) ← of (inflammation-induced CRC)

❗: Dynamics changes ← in the microbiome
← during (1⃣ the development of inflammation 2⃣ tumorigenesis)
∴ These changes directly → cause disease

The gut microbiome
→ complements → the activity of 1⃣ AOM 2⃣ DSS
→ to cause tumorigenesis

❓: mechanisms
← driving microbially mediated tumorigenesis
→ remain to be elucidated

❓: whether there is (an increase ← in bacterial populations)
→ that induce inflammation

An increasingly (inflammatory environment)
→ generate → a self-reinforcing pathogenic cascade
← between 1⃣ the gut microbiome 2⃣ the host

∴ Fostering → the development of cancer

Changes
← medicated (1⃣ chronic inflammation 2⃣ tumorigenesis)
→ lead → to the enrichment of (bacterial populations)
← through 1⃣ the production of metabolites 2⃣ antigens 3⃣ virulence factors 4⃣ other potential tumor-promoting gene products

Colonics inflammation
← in the IL-10 deficient mouse
→ impacts → the composition of (the gut microbiome)
∴ An enrichment of (tumor-promoting E. coli strains)

Our tumor model
→ are enriched
← with populations → that fill a similar role

ETBF
→ has been shown → to strongly induce colonic tumors
← in multiple-intestinal-neoplasia mice
→ through secretion of (a metalloprotease toxin)

Similar processes
→ are occurring ← during tumorigenesis in mice

1⃣ Chronic inflammation 2⃣ tumorigenesis
→ lead to (the loss of members) of (the gut microbiome)
← which are important for maintaining 1⃣ epithelial health 2⃣ immune homeostasis

These bacterial populations
→ serve a protective role
→ are important mediators of (gut health)
← in the murine gut microbiome

Loss of (butyrate-producing populations)
→ increase → 1⃣ inflammation 2⃣ tumorigenesis
∵ Extensive epidemiological data
← demonstrate a link ← between 1⃣ diets in fiber 2⃣ CRC risk

The Por. family
→ are importance mediators
← of anti-inflammatory signals ← in the gut

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