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Cancer stem cells in head and

neck malignancies: Their role

and possible targeted therapy

Mark PrinceChief, Division of Head and Neck SurgeryDepartment of Otolaryngology-HNSUniversity of Michigan

I have no disclosures

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Andrew Kaczorowski*

Matt Clay*

John Owen*

Owen A. Darr

Steve Chinn*

Thomas Carey

Jacques Nor

Jeffrey Moyer

Qaio Li

Alfred Chang

UM Head and Neck SPORE

University of Michigan Cancer Center

T32 NIH Training Grant

Samantha Davis*

Alice Tang*

Shannon Flynn

Mark Tabor*

Gregory Wolf

Carol Bradford

Doug Chepeha

Vasu Divi

Scott Mclean

Kelly Malloy

Emily L. Bellile, MS

Max Wicha

Mike Clarke

Laurie Ailles

Mike Kaplan

Logenbaugh Foundation

American Academy of Otolaryngology

• Why might this cancer be cured in one patient but persist or possibly recur years later in another?

• Why might this cancer spread in one patient to metastatic sites but remain localized in another?

Cancer stem cells - changing our view of head and neck cancer treatment

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Learning objectives:

1. Understand the cancer stem cell model of

carcinogenesis

2. Be able to discuss the differences between cancer

stem cells and the “other” cancer cells

3. Recognize some potential opportunities to target the

cancer stem cell population.

What are cancer stem cells

o Isolation

o Markers

Models and Culture Techniques

Behaviour

o Metastasis

Clinical Implications

o A Therapeutic Application

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Old concept (1950’s) with new relevance

Cancer Stem Cells: of all the cancer cells present they are the only cells with the capacity to grow cancer. They are the Critical Cells in cancers.

Cancer Stem Cell Defining Featureso Represent a small portion of the cancer cells within a tumor with tumorigenic potential.

o Can be isolated from the other cancer cells.

o Tumors resulting from CSC’s contain the mixed tumorigenic and non-tumorigenic cells of the original tumor.

o Can be serially transplanted through multiple generations, indicating that it is a self-renewing population

First identified in a solid tumor in Mike Clarke’s laboratory (at the University of Michigan) in breast cancer

First identified/isolated in HNSCC at the University of Michigan (M. Prince et. al. 2007)

If we can eliminate the cancer stem cells we can cure cancer!?

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Flow Cytometry

Isolation of human solid tumor cancer stem cells

Patient

Sort for marker(s)

of interest

Mike Clarke - modified

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CD44

ALDH

Side population

Combinations (CD44/ALDH)

CD133

FMRD4A (S. Goldie)

CD271 (J Sunwoo)

CD146, CD 271, Podoplanin (L. Ailles)

A variety of genes: cMET, BMI-1, ABCG2 etc.

a

b c

CD44+ cells are tumorigenic in HNSCC

Prince et. al

2007 PNAS

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Growth of HNSCC tumors in mice injected with CD44+Lineage- or CD44-

Lineage- cells

Sample Population Cell Count

500-650K 200-300K 100-150K 40-50K 20-25K 10-15K 5K 2K

UMHN1*CD44+ 1/1

CD44- 0/1

UMHN2*CD44+ 2/2 1/1

CD44- 0/2 0/1

UMHN3**CD44+ 3/3

CD44- 0/3 0/3 0/3

UMHN4*CD44+ 1/1

CD44- 0/1

UMHN5**CD44+ 1/1 1/1 1/2

CD44- 0/1 0/2

UMHN6**CD44+ 1/1 0/1

CD44- 0/2

UMHN7**CD44+ 1/1 0/1

CD44- 0/1

* Samples that were passaged once through mice prior to sorting for CD44+ and CD44- populations

**Samples that were directly sorted from patient samples for CD44+ and CD44- population.Prince et. al 2007 PNAS

Had been used to isolate Hematopoietic stem cells

Aldehyde dehydrogenase is a polymorphic enzyme

responsible for the oxidation of aldehydes to carboxylic

acids

o Three different classes 1, 2 and 3

The Aldefluor® kit substrate is not class specific (detects all

3 classes of aldehyde dehydrogenase)

ALDH activity may be a common Cancer Stem Cell Marker??

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Growth of HNSCC tumors in mice injected with ALDHhighLineage- cells or

ALDHlowLineage- cells

Sample Population

# Cells Injected

5000+ 1001-5000 501-1000 101-500 50-100

HN72

ALDHhigh 1/2 0/1 1/1 2/3

ALDHlow 0/1 0/2 0/4

HN76

ALDHhigh 1/3 2/2 0/2

ALDHlow 1/2

HN78

ALDHhigh 3/4 2/4 2/4

ALDHlow 0/4 1/4 0/4

HN79

ALDHhigh 1/1 1/1 1/1 2/4

ALDHlow 0/1 0/2 0/4

HN80

ALDHhigh 0/2 1/2 2/4

ALDHlow 0/3 0/2

HN84

ALDHhigh 1/2 1/2

ALDHlow 1/2 0/2

24/45 ALDHhigh 3/37 ALDHlow Prince et al 2010, Head and Neck

Meyers et al. (Nör), unpublished data

ALDH & CD44 select for a sup-population of

cells that is highly tumorigenic in vivo

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We do not know the relevance (if any) of the different

subpopulations of cancer stem cells

CD44

SP

ALDH

CD133

v1

v11OTHERS

Models

o Mainstay

• Immuno-deficient mouse – still the best ( in my opinion)

o Spheroids

• Can it replace the mouse models?

o 3D culture platforms - add cells of interest and place in growth environment

Culture Techniques

o Passage in an animal (immuno-deficient mouse)

• Perhaps the best, also the most cumbersome and expensive

o Spheroids

o Traditional culture (cell lines)

o Primary tumor should still be used when possible to confirm results obtained using other methods

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Derived from the ability to culture some normal stem cells in low attachment conditions with growth factors added

o mammospheres, neurospheres,

Similar growth conditions have been applied to cancer stem cells

o Breast cancer best studied

o In HNSCC squamospheres”/”orospheres” can be grown

Some investigators report spheroids can be used to test the tumorigenicity of subpopulation of cells as a surrogate for the immune compromised mouse models

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Cells with stem cell like features are present in cell line

o Are tumorigenic

o Express stem cell genes

o Recreate cell line (tumor) heterogeneity

Primary tumor

Microenvironment

o Endothelial cells

o Tumor associated fibroblasts

o Cytokines

Metastasis

HPV +/-

Gene expression

o Notch

o Stat3

o cMet

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Metastasis are deadly in HNSCC

The development of metastasis is a complex multistep

process.

Cancer stem cells might be critical to this multistep process.

We hypothesized that Cancer stem cells have greater

metastatic potential than non-Cancer stem cells

Metastatic potential of Head and Neck Cancer Stem Cells

CD

44-

CD

44+

In vivo model of metastases. Bioluminescence imaging of NOD-SCID mice 8 weeks post-tail vein injection with

squamous cell carcinoma cell lines transduced with luciferase. Left: injected with 5x104 cells with low CD44

expression from UMSCC-47. Middle: injected with 5x104 cells with high CD44 expression from UMSCC-47. Right:

no injection. Davis (Prince) et al. Arch Otolaryngol HNS 2010

Control

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Orthotopic Mouse Model

Luciferase transfected CSC and Non-CSC tip of tongue injections

Bioluminsecent Imaging (BLI) of Luciferase activity (Photons/sec) over time

Hematoxylin & Eosin Confirmation of tumor growth

Chinn, Prince et al Head and Neck epub

0

500

1000

1500

2000

2500

3000

3500

4000

7 14 21 28 36 43

Ph

oto

ns/se

co

nd

Time (days)

CSC

Non-CSC

Unsorted

CSC

Non-CSC

CSC have a significantly greater rate of primary

tumorigenesis/bioluminescence compared to non-CSC (p=0.0011)

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CSC have a significantly greater rate of regional metastatic

growth/bioluminescence compared to non-CSC (p=0.001)

0

500

1000

1500

2000

2500

7 14 21 28 36 43

Ph

oto

ns/se

co

nd

Time (days)

CSC

Non-CSC

Unsorted

Lung Histology

LN Histology

Right

LN

Left LNVivesection

BLI

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Cancer stem cells seem to be critical to the development of

metastasis

The biological factors controlling this activity need to be

better defined

o The cancer stem cell biology

• What are the critical pathways

o The tumor niche

• Why do metastasis preferentially arise in certain tissues

• Why do some tumors metastasize where others do not

o Other signaling from the tumor, inflammatory cells etc.

J. Moyer A. Chang

M. Prince Q. Li

J. Owen

T. Carey

D. Chepeha

S. McLean

V. Divi

G. Wolf

C. Bradford UM Cancer Center

E. McKean UM Head and Neck Spore

K. Malloy Logenbauch Foundation

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Immune modulating anticancer therapy (anticancer

vaccines) has to date not fulfilled its promise.

The idea of stimulating the patient’s own immune system to

mount on effective response against the tumor is highly

attractive.

Hypothesis: A Dendritic Cell (DC) based vaccine specifically

targeting the cancer stem cell population will be produce a

vigorous anti-tumor effect.

o DC chosen as these are antigen presenting cells capable of

stimulating both a immunoglobulin and cellular response.

(1) Harvest

tumour

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We successfully developed an animal (mouse) model to test

our hypothesis.

o Uses an animal model and a syngeneic scca

o Uses tumor lysate to stimulate DC’s

• Compare DC primed with CSC tumor lysate to whole tumor lysate

We were able to produce a DC vaccine directed against

murine HNSCC cancer stem cells

o The vaccine induced a CSC specific immune response.

o The anti-CSC vaccine was significantly more potent than vaccine

produced against whole cancer lysate, in a protective model of

cancer formation.

Cancer stem cell vaccination confers significant anti-tumor immunity by selectively targeting cancer stem

cells. N Ning, S Teitz-Tennenbaum, J Yet, C Ginestier, MS Wicha, JS Moyer, ME Prince, Y Xu, AE. Chang, Q Li.

Cancer Research 2012.

Cancer stem cell vaccination confers significant anti-tumor immunity by selectively targeting cancer

stem cells. N Ning, S Teitz-Tennenbaum, J Yet, C Ginestier, MS Wicha, JS Moyer, ME Prince, Y Xu, AE.

Chang, Q Li. Cancer Research 2012.

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CSC–pulsed DCs were able to sensitize HNSCC

patient B cells to produce significantly higher

amounts of IgG and IgM in response to CSCs

Cytotoxic lymphocytes are can specifically target

and kill autologous CSC targets

Measurable differences seen between CSC-DC and

non CSC DC

Human data consistent with preclinical mouse data

Clinical Trial is next

Head and neck cancer “cancer stem cells”

o Can be separated from the other cancer cells

o Make up a small fraction of the total number of cancer cells

o Reproduce the original tumor heterogeneity

o Can be passaged in an animal model

o Express some genes associated with normal stem cells

The animal model is still the best

Many factors are critical to cancer stem cell behaviour.

CSC are important to the development of metastasis and

probably are responsible for resistance to therapy and recurrence

We still have a very limited grasp on even the basic biology and

behaviour of the cancer stem cell population

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Are the “CSCs” the only tumorigenic cells in solid tumor

How specific are CSC markers/Is there a universal CSC marker

How can we culture CSC, how close are they to the original

Is the CSC population homogeneous

Is the CSC subpopulation static

What is the cell of origin

What are the biologic differences between CSC, the other cancer cells and normal cells

Are treatment failures caused by CSC

Are CSC critical to the development of metastasis

What is the role of the microenvironment in sustaining and influencing CSC behaviour

Will targeted therapy against cancer stem cells improve patient outcomes?