Revisiting Kochs postulates from a

microbial community perspective

Fusobacteria, colon cancer, & a protozoal amuse-bouche

Wendy S. Garrett
Harvard T H Chan School of Public Health
Animal Models for Microbiome Research: Advancing Basic and Translational Science
December 19th 2016

GarrettLab

AurumexStercore

Causality theory from miasma to the microbiome

Fracastoro

Klebs

Koch

Bradford

Rivers

Evans

Fredricks

Relman

How do microbes contribute to colon cancer?

SearsandGarrett.2014.Microbes,Microbiota,andColonCancer.CellHostandMicrobe.
Garrett.2015.CancerandtheMicrobiota.Science.
BrennanandGarrett.2016.GutMicrobiota,Inflammation,&ColonCancer.AnnualRevofMicro

Fusobacteria are enriched in CRC

Enriched in tumors
Decreased in tumors

n=9,deepDNASeq
Kostic AD et al. 2012. Genome Research.
PathSeq, Meyerson Lab
LEfSe, Huttenhower Lab

LDA score (log10)

TUMOR

NORMAL

n=95,16SrRNA geneampliconsurvey

Approaching causality using mouse models

Alex Kostic Eunyoung Chun

Kostic andChunetal.2013.CellHost&Microbe.

Fusobacterium nucleatum potentiates intestinal

tumorigenesis

F. nucleatum expands intra-tumoral myeloid

immune cells at the earliest stages of colorectal
tumor development

Combining human data and mouse models

to determine Fusobacterium-associated colon cancer
signatures

Fusobacterium
abundance

APCMin/+

Host gene
expression

Gene Rank
PTGS2 (COX-2)
IL8
IL6

Decreased in tumors

MMP3

Enriched in tumors

TNF (TNF-)

Fusobacteria and Human Colorectal Cancer Cohorts

Mima etal.2015.Fusobacteriumnucleatum andTcellsincolorectalcarcinoma.

Mima etal.2015.F.nucleatum inColorectalCarcinomaTissue&Prognosis.
Mima etal.2016.F.nucleatum inCRCTissueaccordingtoTumorLocation.

Kosuke Mima,M.D.Ph.D.
Ogino Lab

F. nucleatum is culturable from

human colorectal tumors

F. nucleatum clinical tumor isolates differ in their

ability to suppress NK-cytotoxicity
30

Killing (%)

25
20
15

none
CTI-2

10

CTI-7

E:T ratio

1.25:1

2.5:1

5:1

0
Chamutal Gur
CollaborationwithMandelboim &Bachrach Labs
HebrewUniversityofJerusalem
Gur etal.Immunity2015

10:1

Screening of a library of F. nucleatum 23726

identified two clones that did not impair NK
cytotoxicity

70
50
40

721.221

30
20

70

721.221
+ FN726

10
2.5:1

1.25:1

0
5:1

60

E:T ratio

50
40
30
20
10

#24

D22

#19

#18

#16

#15

#13

#12

#11

#10

#6

#4

#2

K50

FN726

0
721.221

Killing%

Killing (%)

60

Sequencing of the F. nucleatum clones identified fap2, an adhesin,

as the disrupted gene and numerous NK cell clone studies identified
TIGIT, an immune checkpoint inhibitor, as fap2s binding partner
40
35
721221

25
20

721221+FN726
+TIGIT-Ig
721221+FN726
+C-Ig

15
10
5

E:T ratio

2.5:1

5:1

0
10:1

Killing (%)

30

Fap2 Mediates F. nucleatum Colorectal Adenocarcinoma

Enrichment by Binding to Tumor-Expressed Gal-GalNAc

JawadAbed,D.M.D.,Ph.D.
Abedetal.2016CellHost&Microbe

Fusobacteria and other bacteria may exploit shared immune

effectors to ensure their own survival and promote cancer
growth and spread

Eunyoung Chun,Ph.D.
Chunetal.2015.CCL2PromotesColorectalCarcinogenesisbyEnhancingPolymorphonuclear
MyeloidDerivedSuppressorCellPopulationandFunction.CellReports

Fusobacteria and Cancer

Live and Let Live Hypothesis

Createaimmunemicroenvironmentthatispermissiveformicrobeandtumor
1.Recruitandreconditionimmunecelltypes.Ccl2
Chunetal.2015.CCL2PromotesColorectalCarcinogenesisbyEnhancingPolymorphonuclear MyeloidDerived
SuppressorCellPopulationandFunction.CellReports.

2.InhibitImmuneCellMediatedKillingofBacteria.Fap2andTIGIT
Gur etal.2015.BindingoftheFap2proteinofFusobacterium nucleatum tohumaninhibitoryreceptorTIGIT
protectstumorsfromimmunecellattack.Immunity.

3.TumorAdherenceandEnrichment.Fap2andGalGalNAc
Abedetal.2016.Fap2MediatesFusobacteriumnucleatum ColorectalAdenomaandadenocarcinomaEnrichment
byBindingtoTumorExpressedGalGalNAc.CellHost&Microbe

For every project, there is a time to seq

and a time not to seq

Tuft cells, taste-chemosensory cells, orchestrate

parasite type 2 immunity in the gut
Tuftcells

Tritrichomonas muris

Symbioticprotozoaorhelminths increaseguttuftcellabundance
Tuftcellsinfluencetype2immunityviaTrpm5
TuftcellsexpressIl25andelicitILCsviaTrpm5
ILCsandIl13increasetuftcellabundance
TasteReceptorLigandsand..
AllergyandType2Immunity
MichaelHowitt,Ph.D.
Howittetal.2016.Science.

Tuft Cell

Enteroendocrine
Cell

Absorptive
Enterocyte

Goblet Cell

Paneth Cell

Intestinal Stem Cell

Trier et al. Anat. Rec. (1987)

Crosnier et al. Nature Reviews Genetics (2006)

Tuft cells
WT
(Bred In House)

E-cadherin

% Tuft cells in total epithelium

DAPI

WT
(JAX)

WT
(BIH)

WT
(JAX)

Tuft cells
WT
(Bred In House)

E-cadherin
DAPI

Feed the cecal

contents

WT
(JAX)

Sacrifice mice
3 weeks later
WT (JAX)
+
cecal contents
(BIH)

WT (Bred In House)

Relative T. muris 28S abundance

Tritrichomonas muris

WT (JAX)

ND
WT
(BIH)

WT
(JAX)

Colonize mice with Tritrichomonas muris

WT +Tritrichomonas muris (Tm)

% Tuft cells in total epithelium

WT

WT

Tuft cells

E-cadherin

DAPI

WT
+ Tm

Heligmosomoides polygyrus (Hp)

Trichinella spiralis (Ts)

% Tuft cells in total epithelium

Nippostrongylus brasiliensis (Nb)

WT

WT
+Hp

WT
+Ts

WT
+Nb

Tuft cells, taste-chemosensory cells,

orchestrate parasite type 2 immunity in the gut
Tuftcells

Tritrichomonas muris

Symbioticprotozoaorhelminths increaseguttuftcellabundance
Tuftcellsinfluencetype2immunityviaTrpm5
TuftcellsexpressIl25andelicitILCsviaTrpm5
ILCsandIl13increasetuftcellabundance

Unravelinghostmicrobiotainteractionoffersendlessopportunity
andsheerwonderanddelightinscience
MichaelHowitt,Ph.D.
Howittetal.2016.Science.

Animal Welfare

because mice are a sine qua non

Movingbeyondmonitoringforsignsofdistress2ndto
procedurestoincluderoutineworkflowsthatmonitorfor
signsofdistress2nd todisease
Recognizingsignsofsufferingandtakingaction
Incolonictumormodels,sometimesassimpleas
changingthetypeofbedding
Refininghumanemonitoringandendpoints
Invasivemonitoring(colonoscopy)andnoninvasive
monitoring(luminescencebasedinvivoimaging)

Innovation,futuredirections,challenges
Buildingabetteravatar
Definingthemicrobiota
Definingtumorgeneticsand
hasteningtimelines
Organoids(geneticperturbation)
coupledwithorthotopic colonoscopy
guidedtransplantation

Tumorandtumorimmunemonitoring
Assessingperturbations
Diet,drugs,immunotherapy

Opennessandsharingofmodels
andtheirmethods
Improvingreproducibility
Advancingthepaceofscientificdiscovery
Helpingpatients

Acknowledgements
AurumexStercore

Past

Present
Caitlin Brennan
Grace Cao
Eunyoung Chun
Diogo Da Fonseca Pereira
Kathrin Fenn
Carey Gallini
Georgina Hold
Michael Howitt
Jessica Lang
Sydney Lavoie
Lior Lobel
Monia Michaud
Nora Ou
Sahitya Raja
Michelle Rooks
Patrick Veiga

Collaborators

Emma Allen-Vercoe, U Guelph

Gilad Bachrach, Hebrew U
Dean Brenner, U Mich
Andrew Chan, MGH
Thomas Clancy, BWH & DFCI
Cammie Lesser, MGH
Marie-Pierre Chapot, INRA
Dan Chung, MGH
Ashlee Earl, Broad Institute
Charles Fuchs, DFCI
Dirk Gevers, Broad Institute
Jonathan Glickman, BWH
Curtis Huttenhower, Harvard Chan
Paul Lochead, MGH
Saulius Kulakauskas, INRA
Ofir Mandellboim,Hebrew U
Robert Margolskee, Monell Institute
Funding- Current and Past
Matthew Meyerson, DFCI & Broad
DFCI GI SPORE, Burroughs Wellcome Fund, Cancer Research Institute,
Shuji Ogino, DFCI
Damon Runyon Cancer Research Foundation, Euroscreen, Hoffman LaRoche,
NIH (NIAAA, NIAID, NCI, NIDDK, NIGMS), Nutraceutix, Searle Scholars Fund, V Foundation,
Merkin Family Foundation.
Sonia Ballal
Robbie Eaton
Han Gil Jeong
Jason Kim
Alex Kostic
Laura Kvenvold
Maeva Metz
Grace ONeale
Emily Patterson
Kayla Putnam
Patrick Smith
Leslie Wardwell-Scott