Cancer stem cells have implications in clinical settings. Only a small portion (<1%) of tumor cells can form new tumors, known as tumor initiating cells or cancer stem cells. These cells express specific cell surface markers that can be identified using flow cytometry and fluorescence-activated cell sorting. Targeting cancer stem cells is important for therapy because they are resistant to chemotherapy and radiation. Combination therapies that target cancer stem cell pathways such as Hedgehog and Wnt signaling or aldehyde dehydrogenase activity may be more effective treatments.

1. Cancer stem cells: implications in the clinical setting Malcolm Alison; m.alison@qmul.ac.uk 4 th April 2011

5. Time Initiator Promoter Initiators & promoters * Tumours Tumours No tumours DMBA TPA

7. SVZ astrocytic ribbon

9. Cancer Stem Cells The idea is not new

10. Hamburger and Salmon 1977, Science The plating efficiency of most 1 0 tumours is <1% PE = macroscopic colonies formed number of tumour cells plated

11. The tumour battleground

12. Markers Cancer stem cells

15. Flow Cytometry

16. Fluorescence Activation Process (or Immunofluorescence) FITC FITC Antibodies recognize specific molecules in the surface of some cells But not others When the cells are analyzed by flow cytometry the cells expressing the marker for which the antibody is specific will manifest fluorescence. Cells who lack the marker will not manifest fluorescence Antibodies are artificially conjugated to fluorochromes Antibodies FITC FITC FITC FITC

17. Fluorescence intensity 10 1 10 4 10 3 10 2 Relative fluorescence intensity Number of Events FITC FITC FITC FITC FITC FITC FITC FITC FITC FITC

18. Y X Z Y Z X Cells are presented to the laser using principles of hydrodynamic focusing Laser optics Laser Beam Flow chamber Sheath Sample

19. 2 Parameter Histogram FITC FL PE FL Negative Population Single Positive FITC Population Single Positive PI Population Double Positive Population

21. Xenografting

26. The pre-eminence of ALDH as a cancer stem cell marker Responsible for the oxidation (detoxification) of cytotoxic aldehydes to carboxylic acids RCHO + NAD + + H 2 0 RCOOH + NADH + H + : a major cellular defence mechanism

28. Vitamin A Differentiation

31. Charafe-Jauffret et al.

32. Only 21 of 203 cases were ALDH1 +

35. Victoria L Bautch

36. How do you increase CSC numbers

38. p53 MDM2 =

39. The cancer stem cells bandwagon

40. Malignant melanoma

44. In Ptc +/- mice, Math1 + /CD15 + (SSEA1) selects for TPCs

45. E-Cadherin

49. Options for therapy • Irradiation • Self-renewal pathways (Wnts, Notch, Hedgehog, PTEN-Akt) • Aldehyde dehydrogenase activity • EMT • ABC transporters • miRNAS and HDACi • Targeting the niche

50. Cancer stem cells are very chemoresistant and radioresistant

51. Front line treatment of pancreatic cancer enriches for CSCs In vitro In vivo

52. Colorectal xenograft treated with cyclophosphamide but Dylla et al.

55. Time Selection pressures: nutrition/oxygen/immunity/therapy Clonal co-existence Clone 1 Clone 3 Clone 2

57. A more complex subclonal architecture in ALL Does genetic diversity affect the nature of tumour propagating cells? Yes, the potency, but not the phenotype (CD34 + CD38 -/low CD19 + )

59. Enrich Less apoptosis Enhanced DDR

60. Targeting L1CAM with shRNA sensitises GSCs to irradiation

62. Induce differentiation = reduced sphere formation Inhibit ALDH = increased sphere formation

63. Dissociated CK20 colonosphere cells treated in vitro for 6 days

69. Hedgehog signalling

70. Keeping things stem-like Mdm2

72. Hedgehog inhibitor: Cyclopamine Corn Lilly Cyclopia Poor oral bioavailability; short half-life

76. Reduces Gli expression Reduces ALDH bri % * Blocked metastasis

80. GFs PI3K PIP3 Akt PTEN ABCG2

81. Knock-down of PTEN increases efficiency of mammosphere formation

82. Combination therapy works

87. Sulforaphane

89. Related to a large reduction in ALDEFLUOR+ cells

90. also inhibits xenograft growth

91. • Cancer is a stem cell disease • Tumours have stem cells: ‘cancer stem cells’ • Therapy should target cancer stem cells