Cancer stem cells IOSI Journal Club Giulia Poretti January 19, 2007.

Cancer stem cells

IOSI Journal Club

Giulia Poretti

January 19, 2007

stem cells (sc)

• SELF-RENEWAL i.e. replenish the repertoire of identical stem cell

• DIFFERENTIATION i.e. create a heterogeneous progeny differentiating to mature cells

• EXTRAORDINARY PROLIFERATION POTENTIAL

HOMEOSTATIC CONTROL according to the influence of microenvironment.

Modified from Clarke MF et al. Cell. 2006;124:1111-1115

Stem cells → progenitor cells → mature cells

cancer stem cells (csc)

• SELF-RENEWAL• DIFFERENTIATION• PROLIFERATIVE ABILITY

ABERRANT REGULATION

Modified from Bjerkvig R et al. Nat Rev Cancer. 2005;5:899-904

Minority of cancer cells with tumorigenic potential

NORMAL

TUMORAL

stem cells: identifying properties

• SELF-RENEWAL• DIFFERENTIATION• EXTENSIVE PROLIFERATION POTENTIAL

• Are the minority subpopulation in a given tissue • Mainly appear to be in a quiescent cell-cycle state• long-lived cells giving rise to short-lived, differentiated cells• Highly influenced by signals form their microenvironment• Characterized by specific surface markers

Therapeutic implications• Resistance to treatment → absence of the targeted biological property (imatinib mesylate in CML)→ quiescent state→ expression of efflux proteins protecting vs xenobiotic toxins

• Relapse

• Metastasis

Strategies to target cancer stem cells:

• Immunotherapy against stem-cell-specific markers• Combination of treatment vs tumor burden and

treatment vs cancer stem cells• Therapies promoting differentiation of cancer stem cells

Assays in stem cell researchSurrogate in vitro and in vivo studies

• Clonogenic assays• Repopulation experiments in immunodeficient mice strains

STEM CELLS• 1960s: transplantation experiments in immunodeficient mice

→very small population of cells responsible for reconstitution

→surface marker phenotype negative for lineage-specific antigen

CANCER STEM CELLS• 1990s: AML cells transplanted in immunodeficient mice

→cells able to sustain tumor growth are a minority subpopulation

→reconstitution of the phenotypic heterogeneity of donor tumor

Brain tumor:„Neurosphere“ assay

• Cell culture system for normal neural stem cells → long-term self-renewing→ multi-lineage-differentiating

• Galli R et al. Cancer Res. 2004;64:7011-7021:

isolation and serial propagation of „cancer neurospheres“ → long-term self-renewing→ multi-lineage-differentiating→ in vivo tumorigenicity

• Singh SK et al. Nature. 2004;432:396-401:

Cell surface marker CD133 identifies glioma stem cells

Cancer stem cells models

• Acute myelogenous leukemia: [CD34+,CD38-]

• Breast Cancer: [CD44+, CD24-/low]

• Brain tumor: [CD133+]

• Prostate cancer: [CD44+]

• Colon cancer: [CD133+]

Cancer stem cells models

• Glioma stem cells are identified by CD133+ cell-surface marker• Glioma CD133+ cells are resistant to radiation• Radioresistance due to more efficient activation of DNA damage checkpoint

• Proof of principle: radioresistance of CD133+ glioma stem cells can be reversed with inhibitor of DNA damage checkpoint

• Biological explanation of the long-term failure of radiation therapy:tumorigenic subpopulation of CD133+ glioma cells is not eliminated

Experimental models

in vitro models (ex vivo )

• Cultured cell from human glioma xenograft:D456MGD54MG

• Patient glioblastoma samples

in vivo models

• Human xenograft models in immunocompromised mice

Resistance to radiation:

→ given by CD133+

• Glioma xenograft D456MG:

in vivo CD133+ enrichment after radiation

→no significant difference between sc and ic→enriched CD133+ population 48h after radiation (3-5x)

in vitro CD133+ enrichment after radiation

• Cultures from human glioma xenograft (D54MG):

→48h after radiation: 3x enrichment

• Patient glioblastoma samples:

in vitro CD133+ enrichment after radiation

• CD133+ and CD133- cells derived from patient glioblastoma sample:

→ separately dye-labeled CD133+ (green) CD133- (red)

→ mixed (5%CD133+)

CD133+ enrichment due to clone selection

CD133+ expression is not induced by irradiation

Irradiation effects at molecular level

DNA damage (alkaline comet assay):

CD133+ cells repaired the DNA damage more efficiently than CD133-

Irradiation effects at molecular level

Early DNA damage checkpoint responses (phosphorylation) checked before treatment and after 1h.Higher amount of phosphorylated proteins in CD133+.

Early DNA damage checkpoint responses:

Radioresistance at molecular level

Activation of cleaved caspase-3 (apoptosis) assessed after 24h

in vitroirradiation

in vivoirradiation

Radioresistance at molecular level

Activation of apoptosis assessed after 20h

in vitroirradiation

Radioresistance:proof of principle at cellular level

Cell survival as assessed by colony formation assay

Radioresistance:proof of principle in vivo

DNA repair machinery induced by DNA damage is as promizing drug target to overcome radioresistance.

CD133+ subpopulation have

cancer stem cell properties

• in vivo tumorigenic potential

tumorigenic potential proportional to CD133+

Increased CD133+ cell fractions dose-dependently • decreased tumor latency• increased tumor growth and vascularisation

serial propagation of tumor (secondary tumor formation)

Tumor cells derived from irradiated xenografts are enriched in CD133+ tumor cells and show increased tumorigenic potential when xenotransplanted in immunocompromised mice

serial propagation of tumor with selected CD133+

CD133+ cells derived from xenografts are patient sample show tumorigenic potential independently of prior irradiation.

in vivo tumorigenic potentialof selected CD133+ tumor cells

D456MG CD133- (2 x 106) formed small tumors in 2 out of 5 xenotransplanted in immunocompromised mice.

CD133+ cells (104) from patient sample or xenograft transplanted into brains of immunocompromised mice. Brain observed at appearence of neurological signs or after 8 weeks.

in vitroirradiation

• Self-renewal potential

„Cancer neurospheres“ assay

Purified CD133+ tumor cells from glioma xenografts (D456MG) and patient samples (T3379, T3317) form neurospheres.

• Expression of specific surface markers

• Multi-lineage differentiation ability

Stem cell-specific markers

Identified on neurospheres formed from CD133+ tumor cells from glioma xenografts (D456MG) and patient samples (T3379)by immunofluorescence.

Markers of differentiated cells: in vitro

in vitroirradiation

Markers of differentiated cells: in vivo

Immunofluorescent staining of frozen sections of tumors generated by CD133+ (source not specified)

Concluding remarks

• Glioma cell lines D456MG and D54MG are p53 wild-type

• Radiation on individual cells ex vivo:

→ absence of specific microenvironment

• Lack of conservation in the experimental models adopted for the different assays

Haematoxylin: blue staining of the nucluesEosin: pink staininig of cytoplasm

CD133+ enrichment due to clone selection

Remarks

• Glioma cell lines D456MG and D54MG are p53 wild-type

• Radiation on individual cells ex vivo:

→ absence of specific microenvironment

• CD133+ glioma stem cells treated with ChK inhibitor DBH were not xenotransplanted to evaluate tumorigenicity