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StemCellsandCancer

PieroDalerba,MDHICCC-HerbertIrvingComprehensiveCancerCenter

DepartmentofPathologyandCellBiologyDepartmentofMedicine(DivisionofDiges+veandLiverDiseases)

CELLULAR&MOLECULARBIOLOGYOFCANCER-PATHG4500-001October17,2016

StructureoftheClass

1.   theconceptof“stemcells”

2.   theconceptof“cancerstemcells”

3.   biologicalimplicaYonsofthe“cancerstemcell”model

4.   clinicalimplicaYonsofthe“cancerstemcell”model

Historicaloriginsoftheword“stemcell”[#1]

ErnstHaeckelisfamousforhavingintroducedtheconcept:“Ontogenesisrecapitulatesphylogenesis”

ErnstHaeckel,Anthropoghenie,1874ErnstHaeckel(1834-1919)

Imagesfrom:theWellcomeLibrary,London

Theearlystagesofembryonic

developmentareverysimilaracrossspecies

Historicaloriginsoftheword“stemcell”[#2]Historically, ErnstHaeckel first used theword “stem cell” to idenYfy anancestor/progenitor cell that stands at the “stem” of a genealogic tree,used to depict either evoluYonary (i.e. phylogeneYc) or developmental(i.e.ontogeneYc)processes.

PhylogeneYctreeoflife(i.e.evoluYonoflifeonEarth)

ErnstHaeckel,1866

ectodermmesoderm

endoderm

ICM(innercellmass)

trophoblast

zygote

OntogeneYctreeoflife(i.e.embryonicdevelopment)

Currentuseoftheword“stemcell”

The meaning remains essenYally intact today, where it ismainly used in developmental biology, to idenYfy a cellcapable to generate and sustain over Yme a specific set ofdiversifiedcellpopulaYonswhoseaggregateinteracYonleadstotheformaYonofeither:

a)anenYrelivingorganism:-thezygote(toYpotentcapacity)-embryonicstemcells(pluripotentcapacity)

b)  aspecificsubsetofitsorgansandYssues:-adultstemcells(oligopotentormulYpotentcapacity)

ManyYssueshavea“hierarchical”organizaYonandundergoaconYnuousprocessofcellturnover.

oligolineage precursors

mature cells

long-term self-renewing

stem cells

short-term multipotent progenitors

specializedcelltypesoeenshort-lived

undergoturnoverduringlifeYme(withvariablekineYcs)

long-lived

StemCellProperYes


mature cells


stem cells


StemcellsaredefinedbythreekeyproperYes:

2) The ability to form new stem cells with idenYcal, intact differenYaYonpotenYal,thusmaintainingthestemcellpool(self-renewal);

self-renewal

differen+a+on

1) The ability to give rise to a funcYonally heterogeneous progenyof cells,whichprogressivelyspecializeaccordingtoahierarchicalprocess(differen+a+on);

3) The ability to modulate the two previous properYes according toenvironmentalsYmuliandgeneYcconstraints(homeosta+ccontrol).

ExamplesofnormalYssuesthataresustainedbyadultstemcellpopulaYons[#1].

Hematopoietic system

Dalerbaetal.,Stemcells,celldifferenYaYonandcancer.In:Abeloff’sClinicalOncology-5thediYon-Chapter7(2013)

Intestinal epithelium

Radtke&Clevers,Science307-1904-1909(2005)

ExamplesofnormalYssuesthataresustainedbyadultstemcellpopulaYons[#2].

CK20

MUC2

Tumorsarehistological“caricatures”ofcorrespondingnormalYssues

colon cancer SU#56 colon cancer UM#8 colon cancer UM#17

colon cancer UM#4 colon cancer SU#60 colon cancer MIC#69 normal colon

normal colon

Dalerba et al., Nature Biotechnology, 29:1120-1127, 2011

DavidvonHansemann(1858-1920)

Tumorsarehistological“caricatures”oftheircorrespondingnormalYssues.

Imagesfrom:HistoricBookCollec+on,HumboldtUniversität,Berlin

von Hansemann, Die mikroskopische Diagnose der bösar+gen Geschwülste[Themicroscopicdiagnosisofmalignanttumors](1897)

Figure7.RectalCancer[…]Greatsimilaritywiththenormalmucosa.Gobletcells.

normalcolon coloncancer

TumorYssuesactas“histologicalcaricatures”oftheirnormalcounterparts.

The three-dimensional architecture and cell composition of neoplastic tissues usually

mirrors that of the corresponding normal tissues of origin, and includes a variety of cell types.

RajendranandDalerba,TheoreYcalandexperimentalfoundaYonsofthe“cancerstemcell”model.In:CancerStemCells(ed.V.K.Rajasekhar)-Chapter1(2014)

Keypoints[#1]•  cancer tissues are frequently heterogeneous in cell

composition; this diversity often mirrors the repertoire of specialized cell types found in normal counterparts;

•  thus, in many instances, cancer tissues are not simply amorphous collections of transformed cells, but complex three-dimensional structures that retain architectural features of their parent tissues;

amorphous tumor mass

architecturally complex tumor mass

vs.

IftumorYssuesare“caricatures”ofnormalones(andretainmanyoftheirarchitecturalfeatures),thendotheyalsocontainapathological(e.g.mutated)stemcellpopulaYonthatsustainstheirlong-termgrowth?

The“CancerStemCell”hypothesis

Inotherwords,aremalignantYssuestheproductofpathologicalstemcells?

Ifthishypothesisiscorrect,whatareitsbiologicalimplicaYonsand

howcanwetestit?

BiologicalimplicaYonsofthe“CancerStemCell”hypothesis

1)OriginofcellheterogeneityincancerYssuesIsthecellulardiversityobservedwithintumorYssuesgeneratedonlybygeneYcmechanisms(e.g.randommutaYons)oralsobyepigeneYcmechanisms(e.g.mulY-lineagedifferenYaYon)?

geneYcvs.epigeneYcdiversity

2)FuncYonalhierarchyincancercellpopulaYons

ArethedifferenttypesofcancercellsfoundwithinmalignantYssuesalsodifferentintheircapacitytosustain

long-termYssueexpansionandproliferaYon?

tumorigenicvs.non-tumorigenicpopulaYons

SourcesofcellheterogeneityincancerYssues[#1]

differences between various types of cancercellsarecausedbydifferencesintherepertoireof geneYc mutaYons (i.e. co-existence ofdifferentgeneYcsub-clones);

“stochasYc”model(randommutaYon)

differences between various types of cancercells are caused by differenYal acYvaYon ofspecialized gene-expression programs that“enforce” specific cell idenYYes (i.e.differenYaYon);

“cancerstemcell”model(mulY-lineagedifferenYaYon)

clone#3

clone#2clone#1

lineagecelltype#2

lineagecelltype#1

lineagecelltype#3

theheterogeneityisgeneYc theheterogeneityisepigeneYc

Monoclonaloriginofhumancancer

StanleyM.Gartler(1923-present)

From:Rubin’sPathology:clinicopathologicfoundaYonsofmedicine-7thediYon(2015)StrayerandRubin–Chapter5“Neoplasia”.

NatureRev.Cancer,10:441-448(2010)

ProfessorEmeritus:UniversityofWashingtonPost-doctoralfellow:ColumbiaUniversity

Inwomen,eachcellrandomly“inacYvates”oneofthetwoXchromosomes.

Inwomen,adultYssuesaremosaicswithregardtoXchromosomeexpression.

WhatabouttumorYssuesin

women?

IndirectevidenceofmulY-lineagedifferenYaYoninhumanhematologicalmalignancies

PhilipFialkow(1934-1996)



stem cells


Image:UniversityofWashington

mature cells

CML(chronicmyelogenousleukemia)

monoclonal expansions of either A or B G6PD alleles

(in female patients)

Dalerbaetal.,AnnRevMed.,58:267-284(2007)

? malignant stem cell

SourcesofcellheterogeneityincancerYssues[#2]

IMPORTANT: the two models are not mutually exclusive, the twomechanismsco-existandacttogethertogeneratecelldiversity…butwithdifferentfuncYonalimplicaYons!



vs.

sub-clone#1

lineagecelltype#2

lineagecelltype#1

lineagecelltype#3

sub-clone#3

sub-clone#2

monoclonalinorigin(inmostcases)

monoclonalinorigin(inmostcases)

ImplicaYonsofdifferentsourcesofcelldiversity

Thedifferenttypesofcancercells(i.e.thedifferentgeneYc clones within the tumor mass) are allendowedwithlong-termgrowthandproliferaYoncapacity. Each of them can expand as a disYnctmonoclonalpopulaYon.


Onlyasubsetofcancercells,the“cancerstemcells”(CSC),isendowedwithlong-termgrowthcapacity, while other tumor cell types havel imited proliferaYve capacity. TissuesoriginatedfromCSCcontainmulYplecelltypesasaresultofmulY-lineagedifferenYaYon.


clone#3

clone#2clone#1

monoclonalexpansion#1



long-termgrowthandfullreconsYtuYonofparentYssuediversity

limitedproliferaYon

cell sorting by flow cytometry

Experimentalapproachtotestthe“cancerstemcell”model

primarytumorspecimen

dis-aggregaYonintosingle-cellsuspension

evaluation of differential tumorigenic properties by

injection in NOD-SCID mice +

evaluation of the ability to re-create the phenotypic

heterogeneity of parent tumors +-

laser detector

stainingwithmonoclonalanYbodies

visualizaYonofdifferentcelltypesbyflowcytometry

ExperimentalvalidaYonofthe“cancerstemcell”model

ColonCancer

EpCAM

SelecYonofsurfacemarkersfordifferenYalisolaYonbyflowcytometryofmature/immaturecelltypes

inthehumannormalcolonepithelium

CD44 merge

PieroDalerbaandShigeoHisamori

Sorting and injection strategy for differential tumorigenicity experiments based on EpCAM/CD44 in human Colon Cancer.

2nd sort

P9

P5

2nd sort

subcutaneous injection in immunodeficient mice

(NOD/SCID, NOD/SCID/IL2Rγ-/-)

D

22.6 %

63.8 % A

92.7 %

B

96.7 %

1st sort

1st sort

C

UM-COLON#4 Linneg population

Cell dose and tumor formation

in NOD/SCID mice

dose take

EpCAMhigh/CD44+ 1000 2/3

EpCAMlow/CD44neg 10.000 0/3

CD44

Dalerba et al., P.N.A.S., 104:10158-10163 (2007)

Reconstitution of parental EpCAM/CD44 expression profile in tumors grown from sorted EpCAMhigh/CD44+ cells.

parent xenografts

UM#4

MIC69

tumors grown from [EpCAMhigh/CD44+]

26.42 %

0.74 % *

19.84 %

3.4 %

ES

Ahi

gh/C

D44

+ ce

lls

14 % 19 %

C

F

BA

ED

Dalerbaetal.,PNAS,104:10158-10163(2007)

Morphology and differentiation of human CRC tumors grown from EpCAMhigh/CD44+ cells.

CK20

Muc

Normal epithelium

UM#4 [xeno]

UM#4 [ESA/CD44]

MIC69 [ESA/CD44]

MIC69 [xeno]

A

F

B

G I

DC

H L

E

Dalerbaetal.,PNAS,104:10158-10163(2007)

LimitaYonsofclassictransplantaYonexperimentsbasedonflowcytometry[#1]

Due to obvious ethical reasons, transplantaYonexperimentsusing human cells areperformed inxenogeneichosts(i.e.immuno-deficientmice).

Are differences in the tumorigenic capacity ofdifferenttumorcelltypesduetoanincompaYbilityofspecificcelltypeswiththemousemicroenvironment?

BreastCancer(MMTV-Wnt-1)Choetal.,StemCells,26:364-371(2008)

SkinCancer(squamouscellcarcinoma)Malanchietal.,Nature,452:650-653,(2008)

Mouse epithelial “Cancer Stem Cell” models show that

differences in tumorigenic capacity can exist independently of species-specific differences in

host microenvironment.

CD34+, KRT10neg: tumorigenic CD34neg, KRT10+ : non-tumorigenic

Malanchietal.,Nature,452:650-653,(2008)

DMBA/TPA-induced Skin Carcinoma

Mousemodelsofepithelial“CancerStemCells”

LimitaYonsofclassictransplantaYonexperimentsbasedonflowcytometry[#2]

Mostexperimentsareperformedusinghighnumbersofpurifiedcells(102-103cells/dose).

How can we be absolutely certain that, indeed, thevariouscelltypesfoundinsecondarytumorsarisefromthemulY-lineagedifferenYaYonofasinglecell,andnotfromtheparallelexpansionofmulYplegeneYcclones?

Lineagetracingexperimentson“CancerStemCells”

Analysis of a well-differentiated human colon cancer tissue

reveals the presence of multiple epithelial cell types

Analysis of monoclonal EGFP+ colon cancer xenograft tissues reveals the presence of multiple

epithelial cell types, closely recapitulating the full cellular diversity of the parent tissue

Infection of cancer cells with a lentivirus

encoding EGFP

Subcutaneous injection into a NOD/SCID/IL2Rϒ-/- mouse

and generation of a monoclonal EGFP+ colon

cancer tissue

Monoclonality of cancer tissues is verified by

confirmation of a unique lentivirus integration site

Isolation of a single (n= 1) EGFP+ human colon CSC

(EpCAMhigh/CD44+)

Dissociation into a single-cell

suspension


UM-COLON#4 clone 8 Lin- phenotypic populations

Tested for tumorigenicity

Cell dose and tumor formation in NOD/SCID/

IL2Rγ-/- mice

500 cells 100 cells

EGFP+/EpCAM+/CD44+ 5/5 (100%) 5/5 (100%)

EGFP+/EpCAM+/CD44- 0/5 (0%) 0/5 (0%)

Cell dose and tumor formation in NOD/SCID/IL2Rγ-/- mice (EpCAM+/CD44+)

Xenograft line 10,000 5,000 1,000 500 100 50 30 10 5 1 frequency (95%CE)

UM-COLON#4 - - 3/3 12/12 12/14 3/4 13/30 2/34 0/4 1/132* 1/62 (1/44 – 1/89)

UM-COLON#8 4/4 4/4 4/4 3/4 4/10 2/10 - 1/6 - - 1/223 (1/117 – 1/427)

Clo

ne 8

CD

44-

Clo

ne 8

CD

44+

pare

nt C

D44

- pa

rent

CD

44+

A

B C

F G H

KRT20 Ki67

D E

Generation and characterization of a monoclonal colon cancer xenograft, from a single (n =1) “cancer stem cell”


Keypoints[#2]

•  the cellular diversity of cancer tissues frequently recapitulates the physiological lineage composition of their normal counterparts;

•  the cellular diversity of cancer tissues is frequently epigenetic in origin, and arises as the result of a multi-lineage differentiation process, reminiscent of a stem cell hierarchical system;

•  often, within cancer tissues, only a specific subset of cancer cells is endowed with the capacity to initiate the growth of a secondary tumor when transplanted; tumors originated from these cells, however, recapitulate the repertoire of heterogeneous cells types found in the parent tissues; these cells are defined “cancer stem cells”;

IntegratedviewofcellheterogeneityincancerYssues



geneYcsub-clone#3

geneYcsub-clone#2

geneYcsub-clone#1

lineagecelltype#2

lineagecelltype#1

lineagecelltype#3

Integratedmodelsub-clone#1

(undergoingmulY-lineageepigeneYcdifferenYaYon) sub-clone#2

(undergoingmulY-lineageepigeneYcdifferenYaYon)

sub-clone#3(undergoingmulY-lineageepigeneYcdifferenYaYon)

CharlesDarwin(1858-1920)

Imagesfrom:theWellcomeLibrary,London

ConceptualimplicaYonsofthe“cancerstemcell”theoryfor“evoluYonary”or“darwinian”modelsoftumorprogression

the“unitsofselecYon”incancerYssuesareindividual“cancerstemcells”

(i.e.thecellswithself-renewalcapacity)asopposedtoanyindividualcell

clone#1(sustainedby“cancerstemcells”withgeneYc

background#1)


background#2)


background#3)

OriginandbiologicalidenYtyof“cancerstemcells”

1)“cancerstemcells”vs.normalstemcellsAre“cancerstemcells”mutatedvariantsofnormalstemcells

(thathavelostnormalhomeostaYccontrols)ordotheycorrespondtomoremature,differenYatedcelltypes(thathave

aberrantlyacquiredthecapacitytoself-renew)?

2)onevs.mulYple“cancerstemcell”idenYYesAre“cancerstemcell”populaYonshomogeneousor

heterogeneousintheirepigeneYcidenYYes(i.e.dotheyencompassoneormulYplecelltypes)?

TheoreYcalconsideraYonsinsupportofastemcelloriginofcancerYssues.

•  •  •  • 

•  •  •  •  •  •  •  • 

•  •  •  • 

CanceroriginatesastheresultoftheprogressiveaccumulaYonofmulYplegeneYcmutaYonsover several years (FearonandVogelstein,1990).Thus,stemcellsrepresenttheidealtargetsformalignanttransformaYon,astheyare long-lived and have the opportunity to accumulate sequenYalmutaYonsoverYme.

•  •  •  • 

•  •  •  •  •  •  •  • 

•  •  •  •  •  •  •  • 

•  •  •  •  •  •  •  • 

•  •  •  • 

stemcells(long-lived)

maturecells(oeenshort-lived)

1st mutation 2nd mutation 1+2 1

1+2

1+2

1 1

1 1

1

1 1

1

1+2

1+2

1+2 1+2

1+2

2

“Cancerstemcells”isanoperaYonaldefiniYon.

1. Only a subset of cancer cells within each tumor is endowed with tumorigeniccapacityandcanbeseriallytransplantedintoimmunodeficientmice.

ThreeobservaYonsdefinetheexistenceofa“CancerStemCell”populaYon:

3. Tumors grown from tumorigenic cells contain mixed populaYons of bothtumorigenic and non-tumorigenic cancer cells, thus recreaYng the full phenotypicheterogeneityoftheparenttumor.

2.TumorigeniccancercellsarecharacterizedbyadisYncYveprofileofsurfacemarkersandcanbedifferenYallyandreproduciblyisolatedfromnon-tumorigeniconesbyflowcytometry.

1)itisa“workingtool”todissectthefuncYonalhierarchyofcancerpopulaYons;2) it implies the fulfillment of two stem-cell properYes (i.e. self-renewal anddifferenYaYon), but it does not imply the origin of “cancer stem cells” from normalstemcells.

Thus,theconceptof“CancerStemCells”…

a)aretheyhomogeneousorheterogeneouspopulaYons?

b)whichcelltypesdotheycontain?

c)aretheycomposedbyimmatureormaturecelltypes?

LimitaYonsoftransplantaYonexperimentsbasedonflowcytometry[#3]

Flow cytometry provides only limited knowledge aboutmolecular idenYty, funcYonal status and heterogeneouscomposiYonof“cancerstemcells”.

+-

3) Single-cells from selected phenotypic populations are sorted into individual 96-well PCR plates

1 cell/well (96-well PCR plate)

6) Individual cell real-time PCR curves are analyzed and converted into gene-expression levels. Individual cells are associated into distinct subsets using

statistical clustering algorithms.

5) 9,216 (96x96) single-cell PCR reactions are run in parallel using a microfluidic chip

1) Primary tissues are collected from surgical specimens and dis-aggregated into single-cell suspensions.

2) Single-cell suspensions are stained with fluorochrome-conjugated monoclonal antibodies and analyzed by flow cytometry

4) RNA is reverse transcribed and loaded into a microfluidic chip

Dalerbaetal.,NatureBiotechnology,29:1120-1127(2011)

AnalysisofYssuecellcomposiYonusing“single-cellgenomics”

Each row identifies an

individual single-cell

Each column identifies an

individual gene

genes

cells

Fluo

resc

ence

sign

al

Ct value

1) For each of the 9,216 (96x96) SINCE-PCR reac;ons, an amplifica;on curve is generatedandanindividualthresholdcycle(Ct)valueiscalculated.

2) Ct values are normalized and color-coded. Normalized Ct values (Ctnorm) are obtained bysubtrac;ngfromtherawCtvalue(Ct)themeanCtvalueforthesamegeneonthewholesample(Ctmean)andthenbydividingbythree;mesthestandarddevia;onofthesamegene’sCtvaluesdistribu;on (3SDCt). Results are color-coded using increasingly darker shades of red for highexpressionvalues(Ct<Ctmean),increasinglydarkershadesofgreenforlowexpressionvalues(Ct>Ctmean)andgreyforlackofexpression(Ct>40).

3) Gene-expression results are ploUed using hierarchicalclustering algorithms available in the MATLAB so[ware(MathWorks Inc.). Hierarchical clustering is performed on bothcells and genes, to visualize simultaneously cells with similarexpressionpaUernsandgeneswithsimilarexpressionprofiles.

High expression

Low expression

Normalization of SINCE-PCR threshold cycles (Ct)

No expression

Ct >40 Ct mean Ct -3SD

Analysisandgraphicdisplayofsingle-cellPCRdata.

Hierarchical clustering associates genes with

similar expression patterns

Hierarchical clustering

associates cells with similar expression

profiles

For each gene, Ct values are normalized

and color-coded

red: Ct < Ct mean green: Ct > Ct mean

grey: Ct > 40

Ct value for gene “X”

n. o

f sin

gle-

cells

Ct norm = Ct - Ct mean

3 SD Ct

Ct +3SD

3SD Ct

TaqMan PCR cycle

M96 chip

Dalerbaetal.,NatureBiotechnology,29:1120-1127(2011)

CD44

EpCAM

CD66a CEACAM1

Analysisbyflowcytometryofthehumancolonepithelium:purificaYonof“top-crypt”vs.“bouom-crypt”cellpopulaYons.


KRT20 CEACAM1 MUC2 Ki67

C DA B E F

Single-cellPCRanalysisofhumannormalcolonepithelium

color key - normalized C

t values

high expression

low expression

CA1+ SLC26A3+

MUC2+ TFF3high

OLFM4+ CA2high

LGR5+ ASCL2+

GUCA2B+

Goblet cells

Enterocytes

CBC (columnar basal cells)

Novel populations


Sorting gate

MUC2 KRT20

UM-COLON#4 Clone 8

OLF

M4+

C

A2h

igh

LG

R5+

A

SCL2

+

MUC2+ TFF3high

Single-cellPCRanalysisofamonoclonalcoloncancerxenograeoriginatedfromasingle(n=1)“cancerstemcell”


Keypoints[#3]

•  “cancer stem cell” populaYons, as currently defined usingflow cytometry and operaYonal criteria, are oeenheterogeneous and contain mulYple cell types, includingimmatureprogenitorpopulaYons;

•  itisunclearwhetheralloronlysomeofthecelltypeswithinthe currently defined “cancer stem cell” populaYons areendowedwith“cancerstemcell”properYes;

•  it also remains unclearwhether the epigeneYc idenYty of“cancerstemcells”correspondstothatofnormalstemcellsorofmoredifferenYated,specializedcelltypes.

“EvoluYon”of“CancerStemCells”overdiseaseprogression?

Dalerbaetal.,Stemcells,celldifferenYaYonandcancer.In:Abeloff’sClinicalOncology-5thediYon-Chapter7(2013)

Mostlikely,themolecularidenYtyof“cancerstemcells”

evolvesduringdiseaseprogression.

While,intheearlystagesofneoplasYctransformaYon,thefirstroundofmutaYonsislikelytooccurinthelong-livedstemcellcompartment,itisalsopossiblethat,duringdiseaseprogression,asecondroundof

mutaYonsmightdisablecontrolsinself-renewal

pathwaysand“unleash”self-renewalinrapidlydividingmulY-potentprogenitors.

Doesthe“CancerStemCell”hypothesishaveimplicaYonsforthemodelingof

metastasis?

TradiYonalmodelofMetastasis.

Dalerbaetal.,Ann.Rev.Med.,58:267-84(2007)

primary tumor

apoptosis

Clone#1

Clone#2

Clone#3

The “classic” model predicts that primary tumors andautologousmetastaseswilldisplaysubstanYaldifferences(e.g.differentrepertoiresofgeneYcmutaYons).

primary tumor

Cancer Stem Cell

Differentiated cancer cells

“CancerStemCell”modelofMetastasis.

metastasis

The “cancer stem cell” model predicts thatprimary tumors and autologous metastases willshowsubstanYaldegreesofsimilarity.

• gene-expressionprofiling(Weigeltetal.,Nat.Rev.Cancer,5:591-602,2005)• histopathology(Brabletzetal.,Nat.Rev.Cancer,5:744-749,2005)

apoptosis (e.g. anoikis)

Dalerbaetal.,AnnualReviewofMedicine,58:267-284(2007)

Indeed, this has been a common (thoughsomewhatunexpected)experimentalfinding:

ClinicalimplicaYonsofthe“cancerstemcell”hypothesis

1)“Cancerstemcells”andtumoraggressivenessIsthecellcomposiYonoftumorYssuesassociatedtoamoreorlessaggressivedisease?Aretumorswithabundant“cancer

stemcells”moreaggressive?

2)“Cancerstemcells”andresponsetotreatmentsArethedifferenttypesofcancercellsfoundwithinmalignantYssuesalsodifferentintheirsensiYvitytovarioustypesof

anY-tumortherapies?

Exploringa“cancerstemcell”approachtotheprognosYcstraYficaYonofhumanmalignancies.

normal colon

KRT20

Dalerba et al., Nature Biotechnology, 29:1120-1127 (2011)

colon cancer

Is the cell composition of malignant tissues, in terms of mature vs. immature cell types, associated with different prognosis?

Gene-expression arrays:

Breast Cancer Stem Cells

Vs Normal Breast Epithelium

Strategy designed to maximize the content of prognostically relevant information, including:

1) tumor-specific genes

2) cancer stem cell-specific genes

ExploiYng“cancerstemcells”todevelopaprognosYctoolforhumanBreastCancerpaYents

186 genes Invasiveness Gene Signature

(IGS)

Liu et al., NEJM, 357:217-226 (2007)

TheIGSisassociatedwithpoorprognosisinhuman

BreastCancer

Breast Cancer patients whose tumor is characterized by a transcriptional profile similar to the IGS are characterized by reduced overall and disease-free survival.

population:

early breast cancer patients

from the

Netherlands Cancer Institute (NKI database)

Liuetal.,NEJM,357:217-226(2007)


normal colon colon adenoma colon carcinoma

Using differentiation markers to classify human malignancies.

KRT20 Cytokeratin 20

CA1 Carbonic

Anhydrase 1 [The Human Protein Atlas]

Sparsity test FRD < 0.0001

ExpressionofdifferenYaYongenesassociateswithpaYentsurvival.


X

ALCAM (CD166)

neg +

neg

+

Using Boolean logic to identify early markers of epithelial differentiation in the human colon epithelium

Boolean condition: “Xlow implies ALCAMhigh”

Levin et al., Gastroenterology, 139:2072-2082 (2010)

Dalerba et al., NEJM, 374:211-222 (2016)


Relationship between CDX2 and ALCAM mRNA expression values in human colon cancer.

Genes fulfilling the “Xlow implies ALCAMhigh”

Boolean implication (FDR: < 10-4)

PrognosYcroleofCDX2mRNAexpression(discoverydataset:GSE14333,GSE17538,GSE31595,GSE37892)


ImmunohistochemistryforCDX2proteinexpression


C

PrognosYcroleofCDX2inStage-IIpaYents

CDX2mRNA CDX2protein


PredicYveroleofCDX2expressiononpreferenYalbenefitfromadjuvantchemotherapy(Stage-II)

CDX2negtumors CDX2+tumors


TumorprogressivelyexhaustsitsgrowthpotenYal

Treatmentisbroadlycytotoxic,butdoesnotspecificallytargetself-

renewingcancercells

Treatmenttargetsallself-renewingcancercells

Tumorisgrosslyreducedinsize,buteventuallyrelapsesdrivenbyself-

renewingcancercells.

a)

b)

long-termoutcome

typeoftreatment

ImplicaYonsoftheCSCmodelforthedesignandevaluaYonofanY-tumortreatments


short-termoutcome

“StemCell”workingmodelstoexplaintumorresistancetoclassicanY-neoplasYcagents(i.e.chemotherapy,radiotherapy)

a) quiescent - G0 state (leukemia)

b) high-level expression of:

- drug pumps (leukemia) - enzymes for DNA repair (brain cancer) - scavengers of ROS (breast cancer) - anti-apoptotic pathways

a) multi-potent progenitors: - high proliferation rates b) mature - differentiated cancer cells: - exposed to high intra-cellular concentrations of cytotoxic drugs - exposed to high levels of ROS (“reactive oxygen species”) - vulnerable to extensive DNA damage - sensitive to apoptosis

Classical anti-tumor agents (e.g. agents toxic to proliferating cells in the S or M phase of the cell cycle)

Examples:

- Vinca alkaloids - Anti-metabolites - Topo-isomerase inhibitors - Ionizing radiation

Cancer Stem Cell (CSC)

a) multi-potent progenitors (not self-renewing)

b) mature - differentiated cancer cells

Tumor is disrupted in architecture, but eventually

relapses driven by self-renewing cancer cells

Treatment targets a specific lineage of cells, not encompassing

all self-renewing cancer cells

ImplicaYonsoftheCSCmodelforthedesignandevaluaYonofanY-tumortreatments

“Targeted” therapies might be aimed at cell populations devoid of self-renewal capacity, leading to inconsequential clinical results.

Keypoints[summary#1]

1) tumor tissues are frequently heterogeneous in cell composition; this diversity: a) is not only genetic, but also epigenetic in origin; b) it often mirrors the physiological diversity of specialized cell types found their normal counterparts; c) can arise as the result of a multi-lineage differentiation process, reminiscent of a stem cell hierarchical system;

2) in tumor tissues, the capacity to form tumors upon transplantation is frequently restricted to a subset of cancer cells, operationally defined as “cancer stem cells”; the precise molecular identity of “cancer stem cells” is still under study, and probably evolves over time, during disease progression;

Keypoints[summary#2]

3) analysis by gene-expression arrays of a bulk tumor’stranscripYonal profile and the idenYficaYon of a highdegree of similarity with a gene-expression signaturecharacterisYc of “cancer stem cells” is usually associatedwithamoreaggressivediseaseand,possibly,adifferenYalresponsetoanY-tumordrugs;

4) anY-tumor treatments unable to target and eradicate“cancerstemcell”populaYonsare likelyunabletoachievelong-termeradicaYonoftumorYssues.

“Not everyone accepts the hypothesis of cancerous stem cells.SkepYcs say proponents are so in love with the idea that theydismissor ignoreevidenceagainst it. […]thehypothesiswasmoreakintoreligionthantoscience.”

GinaKolata“Scien+stsWeighStemCells’RoleasCancerCause”TheNewYorkTimes(December21,2007)

Finalremarks

Suggestedreadings

•  Ramalho-SantosM.andWillenbringM.Ontheoriginoftheterm“stemcell”CellStemCell,1,35–38(2007)

•  WeinbergR.A.MulY-stepTumorigenesisChapter11-In:TheBiologyofCancer(2ndediYon),439-509(2014)

•  RajendranP.S.andDalerbaP.TheoreYcalandexperimentalfoundaYonsofthe“cancerstemcell”model.Chapter1-In:CancerStemCells,3-16(2014)

•  DalerbaP.,ClarkeM.F.,WeissmanI.L.,DiehnM.StemCells,CellDifferenYaYonandCancerChapter7-In:Abeloff’sClinicalOncology(5thediYon),98-107(2013)

Conflictofinterest(COI)disclosures

•  MyspouseisanemployeeofAmgen,apharmaceuYcalcompanywhichmanufacturesandsells an anY-EGFRmonoclonal anYbody (panitumumab) approved for the treatment ofhuman colon cancer. As a result of her employment, my spouse owns stock in thecompany.Asidefromthis,Ihavenootherfinancialinterestsinthecompany(e.g.nopaidconsultant agreements, no industry-sponsored research grants, no direct ownership ofstockopYons).

•  Someof the“single-cellgenomics” techniquesdescribed in thispresentaYonhavebeendescribedinapatentheldbyoneofmyformeracademicinsYtuYons(StanfordUniversity)andlicensedtoastart-uppharmaceuYcalcompany(Quan+celPharmaceu+calsInc.).Asaresult of the licensing agreement negoYated by Stanford University, I was awarded analiquotofrestrictedstockinthecompany,andIreceiveroyalYesinrecogniYonofmyroleasaco-inventor.Asidefromthis,Ihavenootherfinancialinterestsinthecompany(e.g.nopaidconsultantagreements,noindustry-sponsoredresearchgrants).

•  Some of the “cancer stem cell” markers described in this presentaYon have beendescribed in a patent held by one of my former academic insYtuYons (University ofMichigan)andlicensedtoastart-uppharmaceuYcalcompany(OncoMedPharmaceu+calsInc.).As a result of the licensing agreementnegoYatedby theUniversityofMichigan, Ireceive royalYes in recogniYon ofmy role as a co-inventor. Aside from this, I have nootherfinancialinterestsinthecompany(e.g.nopaidconsultantagreements,noindustry-sponsoredresearchgrants,noownershipofstock-opYons).

PieroDalerba

stem cells and cancer - columbia university · pdf filedis-aggregaon into single-cell...

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