Cancer stem cell assays

Dr. Serdar SivginSeptember 2010

Kayseri

Cancer stem cells

Rare cells within tumors with the ability to self-renew and give rise to the phenotypically diverse

tumor cell population to drive tumorigenesis

Normal stem cells

Rare cells within organs with the ability to self-renew and give rise to all types of cells within the

organ to drive organogenesis

Maria M. (Marj) Peña

In 1963: Robert Bruce and colleagues used the spleen colony-forming assay (CFU-S) to show that only a small subset of primary transplanted mouse lymphoma cells formed colonies in spleens of recipient animals.

Demonstration of cancer stem cells

Normal and Cancer stem cells

– Self renewal• Tissue-specific normal stem cells must self-

renew throughout the lifetime of the animal to maintain specific organs

• Cancer stem cells undergo self-renewal to maintain tumor growth

– Differentiation into phenotypically diverse mature cell types• Give rise to a heterogeneous population of cells

that compose the organ or the tumor but lack the ability for unlimited proliferation (hierarchical arrangement of cells)

Maria M. (Marj) Peña

• Regulated by similar pathways

– Pathways that regulate self-renewal in normal stem cells are dys-regulated in cancer stem cells

Normal and Cancer stem cells

Developmental signaling pathways involved in HSC selfrenewal. Nature 453, 306-313 (15 May 2008)

What controls self-renewal?Intrinsic control Extrinsic control

Self-renewal is driven intrinsically by gene expression in a cell type specific manner and is modulated through interactions with extrinsic cues from the environment, the stem cell niche. The self-renewal property of CSCs may be regulated by the CSC niche.

Functional comparison between NSCs, pCSCs and CSCs

Self- Renewal

Multi-potency ofdifferentiation

Genomicinstability

Differentiation inducedcelldeath (DICD)

Piwil2 Tumourigenesis in recipients

Benign MalignNSCs + + - - - - -pCSCs + + + +(MIN) + +++ SCID but not

BMR mice

CSCs + - + +/++? (MIN/CIN?)

–/+? ++ SCID & IC mice

NSCs: Normal ctem cellspCSCs:Pre-cancerous stem cellsCSCs:Cancer stem cellsMIN: micro-satellite instabilityCIN: chromosomal instability.

Gao JX. Cancer stem cells: the lessons from pre-cancerous stem cells. J Cell Mol Med. 2008;12(1):67-96.

Isolation of CSCs from hematological malignancies

Peripheral blood or bone marrow

LSCs CD34+, 38– isolated by magnetic beads

Culture in methyl cellulose

+ RPMI

+ Penicillin, Streptomycin

+ 10% FCS

+ L-glutamine

+ 1% BSA

+ EPO

+ GM - CSF

+ IL3

+ SCF

Kassem NM. Cancer Stem Cells: From Identification To Eradication . J Egypt Natl Canc Inst. 2008;20(3):209-15.

Isolation of CSCs from solid tumors

Tumor tissue

Washed then subjected to enzymatic dissociation CSCs CD44+, 24– isolated by magnetic beads

Culture in growth factor cocktail

+ EGF

+ bFGF

+ FBS

+ LIF

Kassem NM. Cancer Stem Cells: From Identification To Eradication . J Egypt Natl Canc Inst. 2008;20(3):209-15.

Assays in stem cell research

in vitro models

Clonogenic assays

in vivo models

Experiments in immunodeficient mice (SCID or NOD/SCID mice.)

SCID(Severe combined immunodeficiency) model= deficient in B and T- cells

NOD (Non-obese diabetic) model=deficient in NK cells

Clonogenic assays in stem cell research in vitro model

Neurosphere formation in brain tumor= clonogenic potential

Galli R et al Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res. 2004;64:7011-7021Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB. Identification of human brain tumour initiating cells. Nature. 2004;432:396-401

Normal neural stem cells

Cancer neurospheres

Long-term self-renewing

+ +

Multi-lineage-differentiating

+ +

In vivo tumorigenicity

- +

CD133 - +

Demonstration of cancer stem cells in vivo models

1990s: John Dick and colleagues- Immunocompromised mice (NOD/SCID mice) and xenotransplantation stem-cellassay systems- The AML, ALL and CML cellisolated from patients couldproliferate in the animals.- Leukemic cells recapitulatedhuman leukemias in recipientmice.

Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997, 3 (7): 730-7.

in vivo tumorigenic potentialof selected CD133+ tumor cells

Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 2006 Dec 7;444(7120):756-60.

Verification of macroscopic and microscopic metastases by fluorescence histology

Maria M. (Marj) Peña

Properties of CSCs

• Tumourigenesis in recipients• CSC specific antigens• Genomic instability• Drug resistance• Radiation resistance

Acute myelogenous leukemia

Normal cell Leukemic cell

CD34⁺ CD34⁺

CD38¯ CD38¯

Thy-1⁺ Thy-1¯

c-kit⁺ c-kit¯

IL-3Rα¯ IL-3Rα⁺

Acute myelogenous leukemia

Normal cell Breast cancer cell Breast cancer stem cell

CD44− CD44+ CD44+

CD24+ CD24−/low CD24−/low

CXCR4 BMI-1

CCR7 ESA/ Flotilin2

EpCam Sca-1/ Ly6

ErbB2/ HER2

ErbB2/ HER2

NCAM-L-1/ CD171

Breast Cancer

• CD44+ enhances invasion and metastasis• Piwil 2 enhances CD44+expresion in fibroblasts

1. The magnetic activated cell sorting system (MACS)

2. Immunocytochemistry

3. FACS

4. Immunomagnetic beads and other beads- based detection methods

5. Epitelial immunospot technique

Detection and characterization of CSCs

Minn, A. J. et al. J. Clin. Invest. 2005;115:44-55

SCPs from MDA-MB-231 cells have a poor-prognosis gene expression signature

Maria M. (Marj) Peña

Technical challenges in CSCs

• Tumors from different patients are different (morphology, cell surface markers, genetic lesions, response to therapy)

• Within an individual tumor, all cancer cells are not equal (variation in genetic and epigenetic abnormalities, distinct proliferative and differentiative capacity

*In patients in remission, the AML1-ETO transcripts were found in a fraction of normal HSCs in the marrow, indicating that the translocation occurred originally in normal HSC and that additional mutations subsequently lead to leukaemia.

(CD34 ＋ Cd38 － Thy-1 ＋ → CD34 ＋ Cd38 － Thy-1 － )

• Absence of specific microenvironment• The method for prospective isolation of CSCs from various tumors

(identification of markers). • The assays to demonstrate the populations are truly CSCs

*Miyamoto, T et al, Proc . Natl. Acad. Sci. USA 97: 7521-7526, 2000