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

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. Cancer stem cells models Acute myelogenous leukemia: [CD34+,CD38-] Breast Cancer: [CD44+, CD24-/low] Brain tumor: [CD133+] Prostate cancer: [CD44+] Colon cancer: [CD133+]

9. 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

10. 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

11. Resistance to radiation: → given by CD133+

12. Glioma xenograft D456MG: in vivo CD133+ enrichment after radiation →no significant difference between sc and ic →enriched CD133+ population 48h after radiation (3-5x)

13. in vitro CD133+ enrichment after radiation Cultures from human glioma xenograft (D54MG): →48h after radiation: 3x enrichment Patient glioblastoma samples:

14. 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+)

15. CD133+ enrichment due to clone selection CD133+ expression is not induced by irradiation

16. Irradiation effects at molecular level DNA damage (alkaline comet assay): CD133+ cells repaired the DNA damage more efficiently than CD133-

17. 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:

18. Radioresistance at molecular level Activation of cleaved caspase-3 (apoptosis) assessed after 24h in vitro irradiation in vivo irradiation

19. Radioresistance at molecular level Activation of apoptosis assessed after 20h in vitro irradiation

20. Radioresistance: proof of principle at cellular level Cell survival as assessed by colony formation assay

21. Radioresistance: proof of principle in vivo DNA repair machinery induced by DNA damage is as promizing drug target to overcome radioresistance.

22. CD133+ subpopulation have cancer stem cell properties

23. in vivo tumorigenic potential

24. tumorigenic potential proportional to CD133+ Increased CD133+ cell fractions dose-dependently decreased tumor latency increased tumor growth and vascularisation

25. 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

26. serial propagation of tumor with selected CD133+ CD133+ cells derived from xenografts are patient sample show tumorigenic potential independently of prior irradiation.

27. in vivo tumorigenic potential of selected CD133+ tumor cells D456MG CD133- (2 x 10 6 ) formed small tumors in 2 out of 5 xenotransplanted in immunocompromised mice. CD133+ cells (10 4 ) from patient sample or xenograft transplanted into brains of immunocompromised mice. Brain observed at appearence of neurological signs or after 8 weeks. in vitro irradiation

28. Self-renewal potential

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

30. Expression of specific surface markers Multi-lineage differentiation ability

31. Stem cell-specific markers Identified on neurospheres formed from CD133+ tumor cells from glioma xenografts (D456MG) and patient samples (T3379) by immunofluorescence.

32. Markers of differentiated cells: in vitro in vitro irradiation

33. Markers of differentiated cells: in vivo Immunofluorescent staining of frozen sections of tumors generated by CD133+ (source not specified)

34. 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

36. Haematoxylin:blue staining of the nuclues Eosin:pink staininig of cytoplasm

37. CD133+ enrichment due to clone selection

38. 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