OncoRay – National Center for

Radiation Research in Oncology, Dresden

Radiotherapy and cancer stem cells

Anna Dubrovska

Workshop: Biological basis of radiotherapy: where do we stand?

4-5 September 2014, Stockholm, Sweden

The battle with cancer

Rudolf Virchow (1821-1902)

Tumour are heterogeneous

Tumour heterogeneity in diagnostics

Marusyk et al., Nature Reviews Cancer 12, 323-334, 2012

Intratumour heterogeneity depends on the number of the cells with

tumorigenic potential which contribute to tumor growth

Meacham C & Morrison S, 2013, Nature 501, 328–337

Models of tumor heterogeneity

Dick JE, 2009, Nature Biotechnology 27, 44 - 46

Properties Sometimes Ascribed to Cancer

Stem Cells but Not Required by the Model

Properties of cancers that follow CSC models

Cancer Stem Cell Model

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-

-

-

-

-

- ?

Magee et al. Cancer Cell, 21, 2012

CSCs: tools and models

Adapted from Charafe-Jauffret et al., BMC Cancer 2009, 9:202

Surface markers (CD44, CD133, CD24 etc.)

Enzymatic activity (ALDH)

Efflux activity (side population)

Slow proliferation (PKH26 retaining capacity)

Low 26S proteasome activity (GFP reporter)

Xenograft assays to measure human CSCs

Beck B & Blanpain C, Nature Reviews Cancer 13, 727–738 (2013)

Cancer Animal Injection Percentage

of CSCs Markers Reference

ALL (B-ALL) NOD/SCID/IL2rγc−/−

newborns Intravenous 82.50% CD34+/CD19+ Kong et al., 2008

AML NOD/SCID Intravenous 0.75% CD34+/CD38− Bonnet and Dick, 1997

Bladder Rag2γcDKO Intradermal 3–36.3% CD44 Chan et al., 2009

Breast NOD/SCID Mammary fat pad 11–35% ESA+/CD44high/

CD24low-neg Al Hajj et al., 2003

Breast NOD/SCID Humanized mammary fat

pad 3–10% ALDH-1+ Ginestier et al., 2007

Brain NOD/SCID Intracranial 6–29% CD133+ Singh et al., 2004

Colorectal NOD/SCID Renal capsule 1.8–24.5% CD133+ O’Brien et al., 2007

Colorectal NOD/SCID Subcutaneous 2.60% ESAhigh/CD44+ Dalerba et al., 2007

Colorectal NOD/SCID Subcutaneous 3.50% ALDH-1+ Huang et al., 2009

Head and neck

squamous cell

carcinoma

NOD/SCID and

Rag2γcDKO Subcutaneous 10–12% CD44+ Prince et al., 2007

Liver SCID Intrahepatic 2.50% CD45−/CD90+ Yang et al., 2008

Lung SCID and NUDE Subcutaneous, after in

vitro expansion 0.4–1.5% CD133+ Eramo et al., 2008

Lung NOD/SCID/IL2rγc−/− Subcutaneous Median 15% lin-/CD166+ Zhang et al., 2012

Melanoma NOD/SCID Subcutaneous 1.6-20.4% ABCB5+ Schatton et al., 2008

Melanoma Rag2γcDKO Intradermal 2.5–41% CD271+ Boiko et al., 2010

Melanoma NOD/SCID/IL2rγc−/− Subcutaneous NA NA Quintana et al., 2010

Pancreatic NOD/SCID Subcutaneous and

intrapancreatic 0.2-0.8%

ESA+/CD44+/

CD24+ Li et al., 2007

Baccelli and Trumpp, JCB, 198 (3): 281

Identification of CSC biomarkers using in vivo assays

Cancer Animal Injection Percentage

of CSCs Markers Reference

ALL (B-ALL) NOD/SCID/IL2rγc−/−

newborns Intravenous 82.50% CD34+/CD19+ Kong et al., 2008

AML NOD/SCID Intravenous 0.75% CD34+/CD38− Bonnet and Dick, 1997

Bladder Rag2γcDKO Intradermal 3–36.3% CD44 Chan et al., 2009

Breast NOD/SCID Mammary fat pad 11–35% ESA+/CD44high/

CD24low-neg Al Hajj et al., 2003

Breast NOD/SCID Humanized mammary fat

pad 3–10% ALDH-1+ Ginestier et al., 2007

Brain NOD/SCID Intracranial 6–29% CD133+ Singh et al., 2004

Colorectal NOD/SCID Renal capsule 1.8–24.5% CD133+ O’Brien et al., 2007

Colorectal NOD/SCID Subcutaneous 2.60% ESAhigh/CD44+ Dalerba et al., 2007

Colorectal NOD/SCID Subcutaneous 3.50% ALDH-1+ Huang et al., 2009

Head and neck

squamous cell

carcinoma

NOD/SCID and

Rag2γcDKO Subcutaneous 10–12% CD44+ Prince et al., 2007

Liver SCID Intrahepatic 2.50% CD45−/CD90+ Yang et al., 2008

Lung SCID and NUDE Subcutaneous, after in

vitro expansion 0.4–1.5% CD133+ Eramo et al., 2008

Lung NOD/SCID/IL2rγc−/− Subcutaneous Median 15% lin-/CD166+ Zhang et al., 2012

Melanoma NOD/SCID Subcutaneous 1.6-20.4% ABCB5+ Schatton et al., 2008

Melanoma Rag2γcDKO Intradermal 2.5–41% CD271+ Boiko et al., 2010

Melanoma NOD/SCID/IL2rγc−/− Subcutaneous NA NA Quintana et al., 2010

Pancreatic NOD/SCID Subcutaneous and

intrapancreatic 0.2-0.8%

ESA+/CD44+/

CD24+ Li et al., 2007

Baccelli and Trumpp, JCB, 198 (3): 281

Identification of CSC biomarkers using in vivo assays

Local tumour control depends on the eradication of all CSCs

Baumann M, Krause M, Hill R, Nature Reviews Cancer 2008

Importance of cancer stem cell survival for local tumor control after irradiation

Correlation of tumor transplantability and radiocurability

Hill, R. P. & Milas, L. Int. J. Radiat. Oncol. Biol. Phys. 16, 513–518 (1989)

TCD50 - a tumor control rate of 50%

TD50 - number of cells providing tumor development in 50% of the animals

13 different experimental

murine tumor models

Tumor

Tumor

Tumor

Tumor

TCD50

Dose (Gy)

Tu

mo

r c

on

tro

l

pro

bab

ilit

ies

(%

)

Density of CSC

before radiotherapy

Radiosensitive tumors

Radioresistant tumors

Tumor

Density of CSCs in tumors might be related to tumor radiocurability

Mäbert K et al. Dubrovska A, Int J Radiat Biol. 2014

- Tumor initiating cells

Sam

e tum

or

volu

me

Cancer, (n) Treatment Markers Method Clinical parameters Reference

Glioblastoma

(172)

Complete or partial tumorectomy followed

by radiotherapy combined with

temozolomide treatment

CD133/Ki 67 IHC, IFC

Affymetrix

GeneChip

Microarray,R

T-PCR, IHC

Shorter overall and

progression free survival

Metellus et al. 2011

Murat at al. 2008

Pallini et al. 2008

Larynx cancer

(52)

Radiotherapy CD44 RT-PCR, IHC Local relapse de Jong et al. 2010

HNSCC (74) Accelerated platinum-based radiotherapy CD44, Oct4,

integrin ß1

IHC Local relapse,

Distant metastasis,

Shorter overall survival

Koukourakis et al.

2012

Uterine cervical

squamous cell

carcinoma (73)

Radiotherapy alone or with concurrent

cisplatin-based chemoradiotherapy

CD24 IHC Distant metastasis Kwon et al. 2007

Rectal

carcinoma (551)

Preoperative radiotherapy

Preoperative 5-fluorouracil-based

chemoradiotherapy

5-fluorouracil and tegafur-uracil (UFT) with

concurrent radiotherapy followed by

surgery

CD133

OCT4

SOX2

CD44v6

CXCR4

CXCL12

IHC

RT-PCR

Shorter overall and

progression free survival

Distant metastasis

Shorter progression free

survival

Wang et al. 2009

Saigusa et al. 2011

Sprenger et al. 2013

Saigusa et al. 2009

Saigusa et al. 2010

Avoranta et al. 2012

NSCLC (50)

Preoperative docetaxel/cisplatin - based

chemoradiotherapy

CD133

ALDH1

IHC

Shorter overall survival,

shorter progression free

survival

Shien et al. 2012

EHBD cancer

(84)

Surgery followed by adjuvant 5-

fluorouracil-based chemoradiotherapy

CD24 IHC Distant metastasis Kim et al. 2013

SCC (140) Radical hysterectomy followed by

adjuvant radiotherapy or

chemoradiotherapy with cisplatin

CD24 Distant metastasis (CRT

group) or shorter locoregional

failure-free survival (RT group)

Sung et al. 2010

Cancer stem cells (CSCs) and tumor radioresistance

CSC microenvironment

Normoxia Hypoxia

Brunner TB et al., 2012, Semin Radiat Oncol 22:151-174

Marie-Egyptienne DT, Lohse I, Hill RP. Cancer Letters 341 (2013) 63–72

Deadly teamwork: hypoxia and CSCs

ROS links glycolysis to breast cancer stem cell and EMT phenotype

Schieber M, Chandel NS, Cancer Cell 23, 2013

Glucose + Oxygen = Carbon dioxide + Water + Energy

C6H12O6 + 6O2 = 6CO2 + 6H2O + 2900 kJ/mol (38 ATP molecules)

Aerobic Respiration

Anaerobic Respiration Glucose = Lactic acid + Energy

C6H12O6 = 2C3H6O3 + 120 kJ/mol (2 ATP molecules)

Cells need to consume more glucose

Ito K and Suda T, Nature Reviews Cancer, 2014

Glycolysis and hypoxic conditions

The potential importance of stem cell niches for radiotherapy treatment planning

Baumann M, Krause M, Hill R, Nature Reviews Cancer 2008

2-[18F]fluoro-2-deoxyglucose(FDG)-PET

Hypoxic volume (FMISO and FAZA PET)

Tang DG, Cell Research (2012) 22:457–472.

Stem cell proliferation, self-renewal, differentiation, and transformation

Radiation-induced CSC redistribution and accelerated tumor repopulation

Kyle AH et al., Cancer Res, 2012;72:801-809

Radiation-induced CSC redistribution and accelerated tumor repopulation

Tumor repopulation

G0

Notch,

WNT,

PI3K/AKT

Reoxygenation Hypoxia

Tang DG, Cell Research (2012) 22:457–472

Intrinsic and induced plasticity in CSCs and their progeny

Evolution of cancer stem cells during genotoxic therapy

Stem cell model

Evolution of Cancer Stem Cells

Unified model of cancer stem cells and clonal evolution

Stemness features influence

clinical outcome Tumor heterogeneity is based on

clonal evolution and CSCs

- CSCs are not static entities but can evolve over the lifetime;

- Aquisition of favourable mutations in CSCs drives clonal expansion;

- Genetic changes in CSCs are the source of tumor heterogeneity

Three factors can influence stemness simultaneously:

• Genetics, • Epigenetics,

• Microenvironment

Kreso & Dick 2014, Cell Stem Cell

CSCs

metastatic

cells

IR resistant

cells

Cells causing

relapse

Are CSCs radioresistant?

Properties Sometimes Ascribed to Cancer

Stem Cells but Not Required by the Model

Properties of cancers that follow CSC models

Testing the Cancer Stem Cell Model

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Hewitt and Wilson, Br J Cancer. Mar 1959; 13(1): 69–75

murine tumors of spontaneous origin

TD50 => 5 cells sarcoma leukemia

Xenograft assays to measure human CSCs

Expectation Reality

Magee et al. Cancer Cell, 21, 2012

after a two-year study by the National Toxicology Program of 612 mice, there were

no spontaneous cases of carcinoma in the prostate (S. B. Shappell, G. V. Thomas, R. L. Roberts et

al., “Prostate pathology of genetically engineered mice: definitions and classification. The consensus report from the Bar

Harbor meeting of the mouse models of human cancer consortium prostate pathology committee,” Cancer Research, vol. 64,

no. 6, pp. 2270–2305, 2004)

Alternative animal models to measure human CSCs?

Models based on murine tumors of spontaneous origin don’t work for all types of cancer

Lineage tracing of CSCs in mouse models: the relevance of the results to

humans still needs to be validated by transplantation and in vitro assays

J Chen et al. Nature 1-5 (2012)

CSCs: tools and models

Adapted from Charafe-Jauffret et al., BMC Cancer 2009, 9:202

Surface markers (CD44, CD133, CD24 etc.)

Enzymatic activity (ALDH)

Efflux activity (side population)

Slow proliferation (PKH26 retaining capacity)

Low 26S proteasome activity (GFP reporter)

The mechanisms underlying CSC

radioresistance

AKT

PI3K

FOXO NICD

Integrins

GPCRs

RTK Notch

WNT

β-catenin

Bao et al. Nature 2006;

Ropolo et al. Mol. Cancer Research 2009;

Phillips et al. J Natl Cancer Inst. 2006;

Zhang et al. PNAS 2007;

Yin et al. PloS ONE 2011

CD44+/CD24-/low human breast cancer

CD133+ glioma

CD29high/CD24high mouse mammary tumor

Enhanced DNA repair capability

AKT

PI3K

FOXO NICD

Integrins GPCRs

RTK Notch

WNT

β-catenin

Diehn et al. Nature 2009;

Ishimoto et al. Cancer Cell 2011

CD44+/CD24-/low human breast cancer

Thy+/CD24+ mouse mammary tumor

CD44+ human gastric cancer

Protection from oxidative DNA damage

The mechanisms underlying CSC

radioresistance

AKT

PI3K

FOXO NICD

Integrins GPCRs

RTK Notch

WNT

β-catenin

Wang et al. Stem Cells 2010;

Zhang et al. PNAS 2010;

Woodward et al. PNAS 2007

SP+ human breast cancer

CD133+ glioma

CD29high/CD24high mouse mammary tumor

Activation of the survival pathways

The mechanisms underlying CSC

radioresistance

Colony formation: lack of the

microenvironment-derived signals

that regulate CSC properties

Cell survival assay

cell plating

Tumor cells

Single cell suspension

(sorted cells)

Colony formation assay: it is an appropriate read-out for CSC cell activity?

SF2 as prognostic factor for patient response

to radiotherapy of carcinoma of the cervix

West at al., Br J Cancer. 1997; 76(9): 1184–1190

Cell survival assay

cell plating

Tumor cells

Cell staining

and sorting

0-8 Gy

0-8 Gy

Matrigel (3D) Colonies (2D)

HNSCCcells

ALDH+ cells retain their clonogenicity after irradiation in vitro

Kim et al., Journal of Endocrinology, 2011

Kurth I. et al and Dubrovska A, submitted

CSCs

non-CSCs

Development/validation of predictive biomarkers for radiotherapy

using primary tumor tissues

CSC Biomarkers are analyzed

before and after irradiation in

combination with signatures for

residual DNA damage (γH2A.X)

and apoptosis (Annexin V and

TUNEL assay)

(3D)

Treatment with multiple doses 120 days

(30 fractions/6 weeks)

Follow-up analysis

of tumor uptake

TCD50

Dose (Gy)

Tu

mo

r c

on

tro

l

pro

bab

ilit

ies

(%

)

TCD50 analysis

Validation of predictive biomarkers for radiotherapy using in vivo model

Tumor

Tumor

Tumor

Tumor Curability by irradiation

Biomarker signature 1 (radioresistance)

Biomarker signature 2 (radiosensitivity)

Biomarker signature 3 (no correlation)

TCD50

Dose (Gy)

Tu

mo

r c

on

tro

l

pro

bab

ilit

ies

(%

)

Analysis of tumor curability vs.

biomarker expression

Analysis of biomarkers

before therapy

Radiosensitive tumors

Radioresistant tumors

Tumor

Validation of predictive biomarkers for radiotherapy using in vivo model

Mäbert K et al., Int J Radiat Biol. 2014

Biomarkers validation using clinical trials

Mäbert K et al., Int J Radiat Biol. 2014

Patients‘ analytes

- Must be clinically approved;

- Evidences of the utility of the biomarkers is provided by the prospective clinical trial;

- A standartized protocol of the sample collection and biomarker analysis is available

Molecular imaging in tracking tumor stem-like cells

Xia et al., Journal of Biomedicine and Biotechnology (2012)

Molecular imaging in tracking tumor stem-like cells

Currently, no CSC specific imaging is available in the clinic;

A high accumulation in the healthy organs and nonspecific binding to the blood

cells may impede the use of these imaging tools in the clinic.

Xia et al., Journal of Biomedicine and Biotechnology (2012)

CSC Non-CSC

Su

rviv

ing

f

raction

Survival assays

Dose (Gy)

Functional characterization of the CSC biomarkers

Tumorigenicity Multipotency Self-renewal

Identification of surface biomarkers for CSC tracking and targeting

RT RT CSC-TT + normal tissue

tumor

CSCs

CSC-targeted therapies may lead to more effective cancer treatments

Tumor

bulk

Radioresistant

and tumorigenic

cells

Tumor

B

F G

E

A

D

C

H

Putative

biomarkers

Patient’s

prognosis

Tumor

diagnostics

and staging

Prediction of

response

to therapy

and treatment

selection

Biomarker

analysis

Molecularly

targeted

therapy

Conventional

therapy

Mäbert K et al.& Dubrovska A. Int J Radiat Biol. 2014

Peitzsch C et al. & Dubrovska A. Radiother Oncol. 2013

CSC biomarkers: how it is expected to work