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Cancer stem cells. IOSI Journal Club Giulia Poretti January 19, 2007. stem cells (sc). Stem cells → progenitor cells → mature cells. SELF-RENEWAL i.e. replenish the repertoire of identical stem cell DIFFERENTIATION

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Cancer stem cells l.jpg
Cancer stem cells

IOSI Journal Club

Giulia Poretti

January 19, 2007


Stem cells sc l.jpg
stem cells (sc)

Stem cells → progenitor cells → mature cells

  • 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 CONTROLaccording to the influence of microenvironment.

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


Cancer stem cells csc l.jpg
cancer stem cells (csc)

Minority of cancer cells with tumorigenic potential

NORMAL

  • SELF-RENEWAL

  • DIFFERENTIATION

  • PROLIFERATIVE ABILITY

    ABERRANT REGULATION

TUMORAL

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


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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


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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


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Assays in stem cell research

Surrogate 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


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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


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Cancer stem cells models

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

  • Breast Cancer: [CD44+, CD24-/low]

  • Brain tumor: [CD133+]

  • Prostate cancer: [CD44+]

  • Colon cancer: [CD133+]


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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


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Experimental models

in vitro models (ex vivo )

  • Cultured cell from human glioma xenograft:

    D456MG

    D54MG

  • Patient glioblastoma samples

    in vivo models

  • Human xenograft models in immunocompromised mice


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Resistance to radiation:→ given by CD133+


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in vivo CD133+ enrichment after radiation

  • Glioma xenograft D456MG:

→no significant difference between sc and ic

→enriched CD133+ population 48h after radiation (3-5x)


In vitro cd133 enrichment after radiation l.jpg
in vitro CD133+ enrichment after radiation

  • Cultures from human glioma xenograft (D54MG):

→48h after radiation: 3x enrichment

  • Patient glioblastoma samples:


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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+)


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CD133+ enrichment due to clone selection

CD133+ expression is not induced by irradiation


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Irradiation effects at molecular level

DNA damage (alkaline comet assay):

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


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Irradiation effects at molecular level

Early DNA damage checkpoint responses:

Early DNA damage checkpoint responses (phosphorylation) checked before treatment and after 1h.

Higher amount of phosphorylated proteins in CD133+.


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Radioresistance at molecular level

in vitro

irradiation

in vivo

irradiation

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


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Radioresistance at molecular level

in vitro

irradiation

Activation of apoptosis assessed after 20h


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Radioresistance:proof of principle at cellular level

Cell survival as assessed by colony formation assay


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Radioresistance:proof of principle in vivo

DNA repair machinery induced by DNA damage is as promizing

drug target to overcome radioresistance.


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CD133+ subpopulation have cancer stem cell properties


In vivo tumorigenic potential l.jpg
in vivo tumorigenic potential


Tumorigenic potential proportional to cd133 l.jpg
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 l.jpg
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 l.jpg
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 potential of selected cd133 tumor cells l.jpg
in vivo tumorigenic potentialof selected CD133+ tumor cells

in vitro

irradiation

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.

D456MG CD133- (2 x 106) formed small tumors in 2 out of 5

xenotransplanted in immunocompromised mice.



Cancer neurospheres assay l.jpg
„Cancer neurospheres“ assay

Purified CD133+ tumor cells from glioma xenografts (D456MG)

and patient samples (T3379, T3317) form neurospheres.


Slide30 l.jpg


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Stem cell-specific markers

Identified on neurospheres formed from CD133+ tumor cells

from glioma xenografts (D456MG) and patient samples (T3379)

by immunofluorescence.


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Markers of differentiated cells: in vitro

in vitro

irradiation


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Markers of differentiated cells: in vivo

Immunofluorescent staining of frozen sections of tumors

generated by CD133+ (source not specified)


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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


Slide36 l.jpg

Haematoxylin: blue staining of the nuclues

Eosin: pink staininig of cytoplasm



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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


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