mechanisms of malignant progression - win consortium

Mechanisms of Malignant

Progression

Robert A. Weinberg Whitehead Institute for Biomedical Research

MIT/Ludwig Center for Molecular Oncology

MIT Department of Biology

Cambridge, Massachusetts, USA

Disclosures

I have nothing to disclose.

What goes on here? Primary tumor formation

Figure 11.10 The Biology of Cancer (© Garland Science 2007)

Primary

tumor

Invasion-metastasis

cascade


Primary

tumor

The invasion-metastasis

cascade:

How do cancer

cells acquire all of these

capabilities?

Implanted (human)

cytokeratin-positive cancer cells (therefore epithelial)

human vimentin-positive (therefore mesenchymal cells

of human origin) recruited mouse stroma

K. Hartwell and T. Ince

A key clue: The behavior of a

BPLER br. ca. xenograft in mouse host

Invasive cell

(human) cytokeratin-positive (therefore epithelial)

human vimentin-positive (therefore mesenchymal)

signals from

mouse stroma

K. Hartwell and T. Ince

EMT

How can one explain

the EMT’s exhibited

by cells at the edges

of epithelial cell islands?

e.g.,

EMT = epithelial-mesenchymal transition

cytokeratins (epithelial)

human vimentin (mesenchymal)

mouse stroma

transformed

human MECs (BPLERs)

mouse stroma

mouse stroma

K. Hartwell & T. Ince

BPLER tx human mammary epithelial cells in mouse host

EMT

EMT

cytokeratins (epithelial)

human vimentin (mesenchymal)

mouse stroma

transformed

human MECs (BPLERs)

mouse stroma

mouse stroma

K. Hartwell & T. Ince

BPLER tx human mammary epithelial cells in mouse host

EMT

EMT

Conclusion:

The microenvironment of the primary tumor can contribute

importantly to the phenotypic conversion occurring

during an EMT , which involves an adaptation of cancer

cells to contextual signals originating in the stroma.

A group of pleiotropically acting transcription factors that

induce EMTs at various stages of embryogenesis

The epithelial-mesenchymal transition (EMT) is a complex,

multi-faceted program involving multiple changes in cell

properties.

epithelial mesenchymal

EMT

epithelial mesenchymal

Twist (an EMT-inducing transcription factor) is essential

for 4T1 tumors to metastasize from the mammary gland to the lung.

Jing Yang

Shut down Twist:

Lung mets down 85%

Primary Tumor Invasion Intravasation Transport

Extravasation Micrometastasis Metastasis

How many steps of the invasion-metastasis cascadecan EMT-inducing

transcription factors program? A speculation:

Blood/lymph vessel

Colonization


Perhaps primary carcinoma cells

possess all of the genetic

changes need to disseminate

and lack only the contextual

signals to activate their

otherwise-latent EMT programs.

EMT

Epigenetic signals to

activate an

To turn to another notion:

When implanted in a mouse

One in 106 has

tumor-initiating

ability

One in 102 has

tumor-initiating

ability

HMLER BPLER

Ductal differentiation No Yes

Invasion Minimal Yes

Stromal recruitment Minimal Yes

Metastasis No Yes

Tumorigenicity 106 cells needed to seed tumor

<102 cells needed

to seed tumor

Cell of Origin Determines

Tumor Morphology and Behavior

Tan Ince

(Standard medium) (Special medium)

Hence, there must be at least two distinct

types of cells in these tumors:

those that can initiate tumors and those that

cannot.

Figure 11.14a The Biology of Cancer (© Garland Science 2007)

al-Hajj et al. 2004

FACS separation

(Fluorescence-Activated

Cell Sorting)

Fractionate cells

on the basis of

their cell-surface

antigens

Figure 11.16b The Biology of Cancer (© Garland Science 2007)

Hierarchical

organization of many

normal tissues


This cell is

qualified to

seed a new

tumor

These cells

are not

qualified

to seed a

new tumor

In the context of

a tumor cell population:

Tumor-initiating

cell (sometimes called

a “cancer stem cell”)


This cell is

qualified to

seed a new

metastasis.

These cells

cannot

seed a

new metastasis (because they

lack self-renewal

capability)

Tumor-initiating

cell

Among the cancer

cells disseminating

from a primary tumor

To summarize:

Immortalized human mammary epithelial cells (MECs)

What’s the connection,

if any, between an

EMT and epithelial

stem cells?

Sendurai Mani

Induction of EMT by TGF-b 1 generates

CD44hi and CD24lo HMLE human immortalized human MECs

CD24

CD

44

Day 0

5 ng/ml TGF-b1

induce EMT

Day 12

S.A.Mani & W. Guo

CD44hi/CD24lo

(position of stem

cells)

CD44lo/CD24hi

(non-stem cells)

+TGF- b1

Is there any connection between the EMT and stem cells?

Induction of EMT by Snail and Twist EMT-TFs

also generates CD44hi CD24lo cells

HMLE-Snail HMLE-Twist HMLE

Vector Twist Snail

CD24

CD

44

S.A.Mani &

W. Guo

CD44lo/CD24hi

(position of non-

stem cells)

CD44hi/CD24lo

(position of stem

cells)

÷

Naturally present CD44hi/CD24lo (stem-like) cells isolated from

immortalized human mammary epithelial cells: What mRNAs do they express?

CD44hiCD24lo/CD44loCD24hi

E-c

ad

N-c

ad

Vim

FN

1

FO

XC

2

Slu

g

SIP

1

Sn

ail

0.01

0.1

1.0

10.0

100.0

EMT markers (measured by RT-PCR)

non-stem

cell

fraction

stem

cell

fraction

epithelial marker

mesenchymal markers

Tw

ist

CD44hiCD24lo

CD44loCD24hi

EMT-TFs

CD44hi/CD24lo

(position of stem

cells)

CD44lo/CD24hi

(position of non-

stem cells)

Prepare mRNAs from the

non-stem-cell fraction and

compare with those in the

stem-cell fraction

÷

Naturally present CD44hi/CD24lo cells isolated from

immortalized human mammary epithelial cells express mesenchymal markers

CD44hiCD24lo/CD44loCD24hi

E-c

ad

N-c

ad

Vim

FN

1

FO

XC

2

Slu

g

SIP

1

Sn

ail

0.01

0.1

1.0

10.0

100.0

EMT markers (measured by RT-PCR)

non-stem

cell

fraction

stem

cell

fraction

“stem cell” ÷ “non-stem cell”

epithelial marker

mesenchymal markers

Tw

ist

CD44hiCD24lo

CD44loCD24hi

EMT-TFs

EMT and cancer progression

1. Normal cells induced to undergo an EMT acquire stem-cell properties.

a. CD44hiCD24lo antigenic phenotype

b. Ability to form mammospheres indefinitely

2. Same outcome with cancer cells

3. Epithelial cells in culture that are naturally CD44hiCD24lo show mesenchymal

morphology; same is true of cells from reduction mammoplasty

4. Transient exposure to Snail or Twist causes descendants of exposed cells to form

mammospheres indefinitely (as gauged by serial passage).

Hence, induction of an EMT may allow cancer cells

a. to disseminate

b. to become self-renewing

c. to acquire resistance to therapy

But can one produce more compelling biological proofs

that EMTs generate epithelial stem cells?

One experimental solution: Switch from human breast

cancer cells to normal murine mammary epithelial cells

Can one PROVE that EMT SCs??

A robust in vivo mammary stem cell assay

remove mammary

epithelial rudiment

transplant mammary

epithelial cells

into cleared fat pad

regeneration of

mammary ductal tree

reconstituted

mammary ductal tree

endogenous

mammary ductal tree

Cleared mammary fat pad transplantation

Wenjun Guo

Enriched mouse mammary stem cells show mesenchymal attributes

E - cad N - cad Vim Slug Twist

CD49fhiCD24med 0.10 10.42 6.03 36.23 1.28

0.01

0.10

1.00

10.00

Re

lati

ve m

RN

A le

vels

CD49fhiCD24med / the other cells

1K cells

1K cells

CD49f

CD

24

Lin– MECs

Slug DAPI

Wenjun Guo

stem cells non-stem cells

Implant these separately into cleared mammary stromal fat pads

Visvade

Wenjun Guo

Primary

MECs

Tet-Slug or

control viral vector

Cells exposed to Slug

Early time points late time points

Equal no. of control

cells

competitive reconstitution assay

Dox induction

in culture for 1 week

then remove Dox

Wenjun Guo

Inject mixed

populations in cleared

mammary stromal fat

pad

mix

?

0

5

10

15

20

25

30

35

Tet-on Slug

RFP control

Vector control

RFP control

1 d

ay p

ost in

jectio

n

1 w

ee

k

7 w

ee

ks

Rela

tive

reco

nstitu

tio

n e

ffic

iency (

GF

P c

ells

/R

FP

ce

lls)

1 week 7 weeks 1 day

Vector / RFP

Slug / RFP

Wenjun Guo

+

Mammary epithelial cells that experience transient

Slug expression exhibit a >25X increase in mammary

gland-reconstituting activity

Screening for Slug co-factor(s)

Slug/Snail

Sox9

neural crest

neural

tube

Neural crest formation

Adapted from Knecht & Bronner-Fraser. 2002.

Slug + vector Slug + Sox9

0

5

10

15

20

25

ctrl sox2 Sox4 Sox9 Myc Klf4 FoxD3 Hes1 b-cat Bmi1b-catenin

N90

Org

an

oid

s /

1K

ce

lls

+ Slug

0 10 2 10 3 10 4 10 5

0

10 2

10 3

10 4

10 5

CD49f

CD

61

stem cells &

basal cells

luminal progenitors

mature

luminal cells

Dox

5 days

TetO- Slug + other TFs organoid culture

without Dox

Wenjun Guo

Transient expression of Slug & Sox 9 (a 2nd

EMT-inducing transcription factor results

In a >100x increase in mammary gland-repopulating

activity: effects of treating unfractionated population

of MECs with Slug+Sox9 expression vector.

stem cell

myoepithelial

progenitor

mature

myoepithelial cells

luminal

progenitor

Differentiated

luminal cells

+Slug

&Sox9

+ Sox9

Slug & Sox9

normal mammary gland

breast cancer

Slug

Sox9 Slug

Sox9

tumor

Sox9 Slug

+ Slug

A genetic pathway to the SC state


Ostensibly primary carcinoma

cells possess all the genetic





EMT


activate an

Revisiting this hypothesis

EMT Control

Z. Keckesova

& J. DeCock

Tumors become invasive

Z. Keckesova

& J. DeCock


Ostensibly primary carcinoma

cells possess all of the genetic





EMT


activate an

IRES-based bicistronic expression

of yellow fluorescent protein

reporter from the endogenous

genetic loci of EMT-TFs

(Slug, Snail, and Twist)

EMT-

TF YFP

IRES

Genetically Engineered Knock-in Reporters for

EMT-inducing Transcription Factors

Tsukasa

Shibue

Sensitive detection of transcription by

immunofluorescence

Isolation of positive cells by FACS

What is the role, if any, of EMT-inducing TFs in normal mammary

epithelium and how do they function during tumor progression?

EMT-TF = EMT-inducing transcription factor

Xin Ye

DAPI YFP(Snail) CK14

CK8

DAPI YFP(Twist1) CK14

CK8

DAPI YFP(Slug) Zeb1

Pan-cytokeratin

Ducts containing marker

positive MECs

Total number of ducts

examined

Slug-YFP 100 100

Snail-YFP 0 100

Twist-YFP 0 100

Zeb1 0 100

EMT-TF

Differential Expression of Snail and Slug in Normal Mouse

Mammary Gland

Ducts containing marker

positive MECs

Slug-

YFP

100/100

Snail-

YFP

0/100

Zeb1 0/100

Xin

Ye

Snail in stroma (green)

ZEB1 in stroma (red/pink)

DAPI Pan-Cytokeratin E-Cad YFP(Snail)

E-Cadherin (epithelial

marker)

Xin Ye

Slug-YFP+ Cells are Located at Comparable Locations in Early

Stage Mammary Tumors and Normal Mammary Epithelium

normal early stage ca.

Xin Ye

Snailhi but not Slughi Cells Display Strong Mesenchymal

Phenotypes and are Enriched with Metastatic Potential

Xin

Ye

(Slughi

Enriched)

(Snailhi

Enriched)

(Slughi Enriched) (Snailhi Enriched)

(Snailhi

Enriched)

(Snailhi

Enriched)

(Slughi

Enriched)

(Slughi

Enriched)

Hence, breast carcinoma arises in a cellular compartment

distinct from the normal stem cell compartment.

2D cultured

HMEs

2D cultured

HME-flopcs

Collect floating

population of cells

(flopcs)

Isolation of HMEC subpopulations

HME-flopc

CD

44

CD24 Christine Chaffer

stem-like

non-stem-like

C. Chaffer

CD44low

(non-stem cells)

CD44high

(stem cells)

The CD44lo cells generate CD44hi

populations (a surprise)

C. Chaffer

Non-stem cells can

generate new stem cells!

CD44low

CD44high

CD44hi

CD44lo

HME-flopc

FACS sort

Day 14

Non-stem cells can switch to a SC-like state

Day 0

00

CD24

CD

44

CD44hi

CD44lo

If immortalized mammary epithelial cells (MECs) can generate CD44 hi cells

And if immortalized contextual signals can induce tumorigenic MECs to undergo an EMT

And if EMT-inducing regulators create CD44 hi immortalized MECs

It follows that in a variety of non-stem-cell MECs can be converted into stem cells

C. Chaffer

Compounds from chemical screen

2. The CD44low fraction gives rise to CD44high cells

CD44 high

CD44 low

need to add new arrow!!

This suggests that populations of more differentiated epithelial cells can spontaneously de-differentiate into stem-cell-like cells.

Sorting breast cancer cell lines based on CD44 expression

Luminal-CD44lo Basal-CD44lo Basal-CD44hi

CD

44

Luminal

CD

44

Basal

seed

tumors

Christine Chaffer

Analysis of

resulting

tumors

Use FACS to isolate

CD44lo non-CSCs s

CD44hi

CSCs

Basal CD44lo cells give rise to CD44hi cells in vivo

Digest and analyze CD44lo-

derived tumors for CD44

expression

Luminal-CD44lo Basal-CD44lo

CD

44

MCF7-

lo

MCF7R

-lo

T47D-lo

0.0

0.5

1.0

2

3

Tum

or

(g)

Luminal CD44lo

MCF7-

lo

MCF7R

as-lo

T47D-lo

SUM

149-

lo

SUM

159-

lo

BPLE

R-lo

HCC38

-lo

0

2

4

6

8

10

20

30

% C

D4

4h

i ce

lls

MCF7-

lo

MCF7R

-lo

T47D-lo

0.0

0.5

1.0

2

3

Tum

or

(g)

Luminal CD44lo

HM

LE

R- l

o

BP

LE

R- l

o

HC

C3

8- l

o

SU

M1

49

- lo

SU

M1

59

- lo

HM

LE

R- h

i

BP

LE

R- h

i

HC

C3

8- h

i

SU

M1

49

- hi

SU

M1

59

- hi

0 . 0

0 . 5

1 . 0

2

3

p = 0 . 0 2 9

p = 0 . 0 4 2 3

Tu

mo

r (

g)

B a s a l C D 4 4l o

B a s a l C D 4 4h i

p = 0 . 0 0 0 1

p = 0 . 0 1 3

p = 0 . 0 0 8 6

Inject CD44lo luminal or CD44lo basal cells

into mice

% of CD44hi CSCs in

resulting tumors

0 4 8 12 160

4

8

12Control (-/-)

Control (+/-)

Control (+/+)

Days

%C

D44hi cells

0 4 8 12 160

4

8

12zeb1-25 (-/-)

zeb1-25 (+/-)

zeb1-25 (+/+)

Days

%C

D44hi cells

0 4 8 12 160

4

8

12zeb1-63 (-/-)

zeb1-63 (+/-)

zeb1-63 (+/+)

Days

%C

D44hi cells

zeb1

-25

(-/-)

zeb1

-25

(+/-)

zeb1

-25

(+/+

)

zeb1

-59

(-/-)

zeb1

-59

(+/-)

zeb1

-59

(+/+

)

zeb1

-63

(-/-)

zeb1

-63

(+/-)

zeb1

-63

(+/+

)

0

50

100

*

**

*

** ***%

Inhib

ition

No spontaneous

dedifferentiation

into SC state Christine Chaffer w. Nemanja Marjanovic

Doxycycline-inducible shRNAs

-/- = 8 days OFF + 8 days OFF

+/- = 8 days ON + 8 days OFF

+/+ = 8 days ON + 8 days ON

Use ZEB1

shRNA tp

block ZEB1

expression

Transformed derivatives of

immortalized mammary

epithelial cells

EMT-TF = EMT-inducing transcription factor

% of spontaneously

arising CD44hi CSCs

% C

D44

hi c

ells

zeb1-25 zeb1-25 zeb1-59 zeb1-590

10

20

30Control

0.2 ng/ml

2 ng/ml

20 ng/ml

(- dox) (+ dox) (- dox) (+ dox)

HME-flopc (basal)-CD44lo

If you block Zeb1 induction , you prevent TGF-b from

promoting basal CD44lo(non-CSCs) into CD44hi (CSCs)

(TGF-b is major EMT inducer)

Christine Chaffer

TGF-b

Dox-inducible

anti-ZEB1

shRNA

% C

D44

hi c

ells

Con

trol

0.02 0.

2 2 200.

02 0.2 2

Con

trol

0.02 0.

2 2 200.

02 0.2 2

Con

trol

0.02 0.

2 2 200.

02 0.2 2

Con

trol

0.02 0.

2 2 200.

02 0.2 2

0.0

0.2

0.4

0.6

5

10

15

20

25

MCF7Ras

ZR-75-1

HMLER

HCC38

TGFbeta SB431542 TGFbeta SB431542 TGFbeta SB431542 TGFbeta SB431542

Basal CD44lo cells but not luminal CD44lo cells respond

to

TGF-beta by generating CD44hi cells

Christine Chaffer

TGF-b

luminal

basal

luminal

basal

Chromatin configuration at the ZEB1 promoter

Zeb1

Repressed

Zeb1

Poised

Zeb1

Active

H3K27me3 + H3K4me3 H3K4me3 H3K27me3


% P

os

itiv

e C

on

tro

l

K 2 7 m e 3 K 4 m e 3

0

2 0

4 0

6 0

8 0

1 0 0

5 0 0

1 0 0 0

K 2 7 m e 3 K 4 m e 3

0

2 0

4 0

6 0

8 0

1 0 0

5 0 0

1 0 0 0

K 2 7 m e 3 K 4 m e 3

0

2 0

4 0

6 0

8 0

1 0 0

5 0 0

1 0 0 0

K4me3 K27me3 Repressed Poised Active

K4me3 K27me3 K4me3 K27me3


Zeb1

Repressed

Zeb1

Poised

Zeb1

Active


Luminal-CD44lo

(non-CSCs) Basal-CD44lo

(non-CSCs) Basal-CD44hi

% P

os

itiv

e C

on

tro

l

K 2 7 m e 3 K 4 m e 3

0

2 0

4 0

6 0

8 0

1 0 0

5 0 0

1 0 0 0

K 2 7 m e 3 K 4 m e 3

0

2 0

4 0

6 0

8 0

1 0 0

5 0 0

1 0 0 0

K 2 7 m e 3 K 4 m e 3

0

2 0

4 0

6 0

8 0

1 0 0

5 0 0

1 0 0 0

K4me3 K27me3 Repressed Poised Active



Zeb1 (not expressed)

Repressed

Zeb1

Poised

Zeb1(expressed)

Active



% P

os

itiv

e C

on

tro

l

K 2 7 m e 3 K 4 m e 3

0

2 0

4 0

6 0

8 0

1 0 0

5 0 0

1 0 0 0

K 2 7 m e 3 K 4 m e 3

0

2 0

4 0

6 0

8 0

1 0 0

5 0 0

1 0 0 0

K 2 7 m e 3 K 4 m e 3

0

2 0

4 0

6 0

8 0

1 0 0

5 0 0

1 0 0 0

K4me3 K27me3 Repressed Bivalent/Poised Induced


repressive + inductive

(not

expressed)

BASAL CD44lo non-CSCs

Zeb1

AAAA A

Bivalent/Poised

Microenvironmental

cues e.g.TGF-b

Zeb1

AAAA AAAA

AAAA AAAA

AAAA AAAA

Active

LUMINAL CD44lo non-CSCs

Zeb1

AAAA AAA

Monovalent/Repressed

No effect

Model of CD44lo-to-CD44hi plasticity

inductive histone modification

repressive histone modification

Coexisting inductive &

repressive histone marks

(CSCs)

more aggressive

br.ca.

more benign

br.ca.

ZEB1 = EMT-TF

Christine

Chaffer

H3K4me3

H3K27me3

TGF-b

TGF-b

Sensitive Resistant

Claudin 7

Claudin 4

Actin

Vimentin

EPITHELIAL (nonSC) MESENCHYMAL/SC

Epithelial

Vs.

Mesenchymal

“fingerprint”

epitheliaL

markersl

mesenchymal

markerl

Mesenchymal carcinoma cells are generally

more resistant to therapy


Putative effects of eliminating the non-CSCs therapeutically

clinical response:

Most currently used chemotherapeutics kill non-CSCs preferentially.

non-CSCs

CSC = cancer stem cell


Putative effects of eliminating the non-CSCs therapeutically

clinical response:

Most currently used chemotherapeutics kill non-CSCs preferentially.

non-CSCs

CSC = cancer stem cell

Can one therefore target

the CSCs preferentially?

High throughput screen: Screen 16000 compounds

for those that preferentially target SCs/CSCs

Conventional chemotherapeutics

CSC-targeted agents

stable CSCs control

Salinomycin (1uM)

Paclitaxel (2.5nM) (Standard chemotherapeutic)

Vehicle

control

P. Gupta (Lander) & T. Onder

CSCs

Non-CSCs

higher drug conc.



CSC-targeted agents

stable CSCs control

Salinomycin (1uM)


Vehicle

control


CSCs

Non-CSCs

higher drug conc.

% liv

ing c

ells


Salinomycin selectively kills cancer stem cells


CSC-targeted agents

stable CSCs control

Salinomycin (1uM)


Vehicle

control


CSCs

Non-CSCs

But is this the answer to cancer??

Compounds from chemical screen

2. The CD44low fraction gives rise to CD44high cells

CD44 high

CD44 low

need to add new arrow!!

This suggests that populations of more differentiated epithelial cells can spontaneously de-differentiate into stem-cell-like cells.

clinical response:

cure

This is the desired response to an anti-CSC treatment:

What if this happens instead? clinical response:


Invasion-metastasis

cascade

What happens to cancer cells after they

extravasate and attempt to proliferate

within the parenchyma of tissue they have

invaded?

Filopodium-like structures contribute to cell-matrix adhesions in 3D

few/no FLP

few/no mature

adhesion

plaque

minimal FAK

activation

slow/no

proliferation

abundant FLPs

abundant mature

adhesion

plaques

potent FAK

activation

rapid

proliferation

nonmetastatic

cells

metastatic

cells

FLPs = filopodium-like

structures Tsukasa Shibue

What happens to carcinoma cells

after they extravasate? Rapid

anchorage to the extracellular

matrix of the host tissue is critical

mature adhesion plaques = stable anchorage of

cell via integrins to adjacent extracellular matrix

FLPs contribute to cell-matrix adhesions in 3D

few/no FLP

few/no mature

adhesion

plaque

minimal FAK

activation

slow/no

proliferation

abundant FLPs

abundant mature

adhesion

plaques

potent FAK

activation

rapid

proliferation

nonmetastatic cells metastatic cells

mature adhesion plaques = stable anchorage of

cell via integrins to adjacent extracellular matrix

(integrins are almost entirely a5b1)

Abundant FLP formation is a common attribute of metastatic cells

Metastatic cells, but not nonmetastatic cells, form

abundant FLPs both in in vitro 3D culture and in

vivo following dissemination into the lungs.

Tsukasa Shibue

FLP formation is governed by two cooperating signal pathways

Integrin-associated, ILK/

b-parvin/cofilin signaling controls the

lifetime of already-formed FLPs.

Initial assembly of FLPs is

governed by Rif/mDia2-

actin nucleating/

polymerizing machinery.

Tsukasa

Shibue

FLP formation is governed by two cooperating signal pathways

Integrin-associated, ILK/

b-parvin/cofilin signaling controls

the lifetime of already-formed FLPs.

Initial assembly of FLPs is

governed by Rif/mDia2-

actin nucleating/

polymerizing machinery.

Tsukasa

Shibue

12-15x up following

EMT 3-12x up following EMT

3-9x up following EMT

1. Control cells have ~1 TIC per 500 cells

2. If you knock down either Rif or b-parvin, a ~103 loss of TICs

(tumor-initiating cells)

3. These knock downs have no effect on proliferation in 2D culture

monolayer

Orthotopically (fat pad) implanted cells also form FLPs, which

contribute to subsequent cell proliferation and ultimate

establishment of primary tumors.

44.5 % 22.6 % 19.7 %

(Ki67 positivity)

~103-fold

difference

Limiting dilution analyses of

tumor-initiating cell frequency

Tsukasa Shibue

EMT induction in HMLER cells stimulate their FLP-forming and tumor-

initiating abilities, which is attributable in part to the elevated b-parvin

expression.

0.5 2.8 (average #

FLPs/cells) 3.1

Tsukasa Shibue

~120-fold

difference

FLP formation by orthotopically-implanted cancer cells: if you induce

an EMT but block FLP formation, you no longer increase CSC

formation! EMT

The formation of FLPs predicts, and contributes

functionally to, the subsequent proliferation of

cancer cells both after metastatic dissemination

and experimental implantation.

Tsukasa Shibue

EMT

Proliferation

following exptl.

implantation

tumor-

Initiating

ability

proliferation

following

dissemination

&

extravasation

metastasis-

Initiating

ability

CSC = cell able to initiate tumor formation following exptl.

implantation

A mechanistic link between EMT & the CSC state:

FLP formation empowers initial proliferation of cancer cells

mechanisms of malignant progression - win consortium

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