Cancer Stem Cells Michael Rosemann, Helmholtz-Zentrum Muenchen, Radiation Biology rosemann@gsf.de

1. Cancer Stem Cells Michael Rosemann, Helmholtz-Zentrum Muenchen, Radiation Biology rosemann@gsf.de

2. Long-Term repopulating potential (LTRP)

3. Example: Normal adult tissue stem cell Origin and differentiation of the mesenchymal cells Fibroblast Progenitor / Histiocyte Mesenchymal Stem cell Fibroblast (Connective tissue) Osteopregenitor Osteoblast (forming Bone) Pre-Chondroblast Chondroblast (forming Cartilage) Pre-Adipocyte Adipocyte (Fatty tissue)

4. Asymmetric vs.Symmetric Division of Stem-Cells Tissue Homeostasis (asymetric) Organ growth or Wound Healing (symetric + asymetric) Long-Term Repopulating cell (LTR) Differentiated cell (LTR) Differentiated cell number of LTR cells constant number of LTR cells increases

5. Concepts of Cancer Stem Cell • First formulated in 1875, Julius Cohnheim • proposed that stem cell misplaced during embryonal development were the source of tumors later in the life <embryonal-rest theory> • Decades ago (Xenotransplantation) • only a small minority of cancer cells were able to proliferate extensively • An “operational” & functional term • ability to self-renew (long-term repopulating potential) • dividing to another malignant stem cell and a cancer cell Nature Med 2006;12:296-300 Nature 1963;199:79–80 Nature 2001;414:105–111 Virchows Arch Pathol Anat Physiol Klin Med 1875;65:64-9 Nat Rev Cancer 2003;3:895-902

6. Models of Tumor Heterogeneity (any cell contributes to tumor growth) (only a subset of Tumor-initiating cells contributes to tumor growth) Trends in Cell Biology 2005;15:494-501

7. Models of Tumor Heterogeneity Testing tumor cells in-vitro for their long-term repopulating potential (LTRP) Primary Tumor Plating single cells +10 days 10 000 tumor cells  100 colonies (1% CSCs) (Long Term repopulating potential) Trends in Cell Biology 2005;15:494-501

8. Models of Tumor Heterogeneity Tumor is mixture of few cells with LTRP (CSCs) and many cells without LTRP plating, +10 days each colony = 50 – 100 cells colonies no colonies  Only a small subset of tumor cells have Long Term repopulating potential Trends in Cell Biology 2005;15:494-501

9. What determines LTRP ? replating, +10 days plating, +10 days Tumor 1st colony 2ndary colonies 1st colonies contain again only ~1% cells that form 2ndary colonies

10. What determines LTRP ? replating, +10 days plating, +10 days Tumor 1st colony 2ndary colonies 1st colonies contain again only ~1% cells that form 2ndary colonies Cells with Long Term repopulating potential (CSCs) are not genetically different from non-LTRP cells. LTRP is reversible (epigenetically controlled)

11. Normal Stem Cells (embryonal or adult) asymetric cell division (tissue homeostasis) symetric cell division only during development or wound healing (tightly regulated) Cancer stem-cell like cells (or tumor initiating cells) both symetric and unsymetric cell division (Number of CSCs increases with tumor growth, but differentiated tumor cells determine pathological features)

12. Somatic stem cells • - long-term repopulation potential • - can provide commited precurser cells of different lineages after diff. stimuli • (MSC (multipotent):  precurser fot Fibroblasts, Adipocites, Osteoblasts...) • ESC (totipotent):  all tissues and organs, entire organism • - asymmetric division: SC  SC + precursor (No. of stem cells limitted) • - cell-division controlled by exogeneous stimuly • - genetically stable (high DNA repair capacity, „immortal DNA strand theory“) • - Apoptosis resistant • vs. • Cancer stem-cell like cells (or tumor initiating cells) • unlimited life span, unlimited potential to divide • - only limited potential to differentiate into different cell lineages • symmetric cell division: SC  SC + SC (No. of stem cells increases) • cell division without external stimuli • - cell fate not controlled, high degree of plasticity • - genetically instable (error-prone DNA repair, „immortal DNA strand theory“) • - but apoptosis can be induced

13. In-vivo Model of marrow-derive AML CSCs Transplantation in immunodeficient Nod-Scid mice Blood from AML patient Leukaemic Blast cells AML

14. In-vivo Model of marrow-derive AML CSCs Transplantation in immunodeficient Nod-Scid mice % of recipient mice Developing AML after transplantation 100% 0% 5 20 50 100 200 500 1000 2000 * 103 cells injected

15. Normal Haematopoiesis Myeloid Leukaemia

16. Cancer Stem Cell in human AML (carry MLL-ENL-Translocation in leukaemic myeloid progenitor cells) • MLL-related AML cancer stem cells can be from different stages of cells. • Similar immunophenotype after transformation =>arrest at identical stage of differentiation Transplantation in bone-marrow ablated mice Genes Dev. 17, 3029–3035 (2003)

17. mutation Origin of Cancer Stem Cell environmental effect / mutagenic mechanismchemical/physical carcinogenscause reversion of normal differentiation processDe-differentiation, CSC aquire multipotency(maybe true for multi-lineage Teratoma) Nature Review Cancer 2005;5:899-904

18. Origin of Cancer Stem Cell cell-cell fusion • Mammalian fusogenic factors • CD44, CD47 (macrophage) • CXCR4/SDF1 (osteoblast) mutation Stem Cells 23, 879–894 (2005) Science 308, 369–373 (2005) Nature Review Cancer 2005;5:899-904

19. Tumor stem cells and metastasis (single cell  1cm Metastasis (~ 100 000 000 cells) i.e requires ~ 27 cell divisions  virtually unlimited potential to divide Metastasis same histo-morphology as primary tumor:  Tumor stem cells have the potential to form the same tumor type from a single cell

20. Metastasis and Transdifferentiation CK- (Mesench. Diff.) CK- CK+ (Epithelial Diff.) CK+ Cytokeratin+ cells exhibit epithelial differentiation (typical carcinoma) Cytokeratin - cells loose epithelial differentiation ( mesenchymal), hypoxic areas Nat. Rev. Cancer 2005; 5: 744–749

21. Tumor cell plasticity and the process of Invasion and Metastasis Carcinoma: tumor cells of epithelial origin, cell polarity, homotypic (cell-to-cell) and heterotypic (cell-to-matrix) adherance by adhesion molecules (Cadherins, E-Cam), Form highly structured tumors (squamous epithelium, glandular epithelium, ductal epithelium etc). Invasion of other tissues or metastasis into other organs (local lymph nodes or distant organs) requires change of cell morphology EMT: epithelialmesenchymal transformation Mesenchymal tumor cells: no polarity, higher migration capacity no heterotypic adhesion expression of MMPs to infiltrate normal tissue, blood vessels and lymphatic ducts. After „homing“ of EMT transformed tumor stem cells, they re-transform back into typical epithelial tumor cells (i.e. high plasticity, transdifferentiation)

22. Epithelial-Mesenchymal-Transition (EMT) CK- (Mesench. Diff.) CK- CK+ (Epithelial Diff.) CK+ Primary Tumor metastatic cell distant Metastasis (EMT) (MET) Ep Mes Mes Nat. Rev. Cancer 2005; 5: 744–749

23. Integration of EMT & CSC Process of Metastasis requires CSCs single metastatic cell macroscopic Metastasis  cells forming a metastasis must by CSCs !!! Nat. Rev. Cancer 2005;5: 744–749

24. Stem Cell Pathways • WNT: APC/axin/GSK3-β/Dsh; β-catenin; LEF/LCF gastrointestinal tumors, lymphoid leukaemia, Brain tumors • Hedgehog: sonic(Shh), Desert(Dhh), Indian(Ihh); patched, smoothened, Fused (Fu), SuFu, Gli Skin tumors, BCC, Medulloblastoma, Glioma • Bmi-1: INK4a, ARF, MDM2, Cyclin D, CDK4 Myeloid Leukaemia, B-Cell Lymphoma, Mammary-Tumors • Notch: Dll, SHARP, HDAC, SKIP, CBF-1 Mammary-Tumors, Lymphoid Leukaemia • PTEN: PI3K, AKT, mTOR Lymphoma, Glioma

25. Stem Cell Pathways in Cancer and embryonal development Neural tube formation Dorsal-ventral axis Cell-polarity Axon guidance in neurogenesis • Limb development determination of body axis, • left-right symmetry • Active Notch pathway promote proliferation • cell fate determination in • organ morphogenesis, • - thymus medulla vs. cortex • - rostral-caudal determination, • somite patterning (vertebrae) • Active Notch pathway promote proliferation WNTHedgehogNotch

26. Extensively studied Tumor types for CSCs: AML Glioblastoma Pancreas Carcinoma Mammary Carcinoma Background: These Tumors have a high rate of therapy relapse. Idea was: „Therapy resistance is associated with high content of stem cells“ (hypothesis later rejected)

27. CSC markers cell surface markers CD34+ / CD38 - AML CD44+ / CD24– Mammary Tumor CD133+ Glioblastoma, Osteosarcoma transcription factors Sox2 + Osteosarcoma Functional markers +++ ABC-transporter genes / Eflux-pump +++ Aldehyd-Dehydroxygenase --- Low Proteasome activity / reduced Protein turnover +++ Growth in spheroids (neurospheres, mammospheres, sarcospheres)

28. Tumor stem cells and Chemotherapy resistance Problem of Tumor therapy: A single surviving Tumor stem cell can cause relapse by growing a recidive.

29. High Expression of A(TP)-B(inding)-C(asette)-Eflux Pump (MDR) in TSCs  highly efficient excretion of DNA-binding dyes and toxins  TSC form a side population with reduced Hoechst-staining (murine osteosarcoma cell line) 0.60% 3.95% Hoechst 33342 (red) - VP + VP Hoechst 33342 (blue) VP: Verapamil, ABC-Inhibitor

30. Side-Popiulation cells - form in-vitro Sarcospheres - highly tumorigenic after injection in recipient mice (Red Fluorescence: Osteosarcoma cells were stabily labelled with Cherry-Fluorescence-Protein) 3.95% Hoechst 33342 (red) - VP Hoechst 33342 (blue)

31. Side population cells have long-term repopulation capacity (Cherry Fluorescence labeled) (unlabeled) MP SP mixing, plating out + 3 days + 14 days

32. SP Non-SP Side Polulation Cells are resistant to cytostatica Neuroblastoma cell lines JF and IMR32 • Cancer stem cell (SP) have high expression of ABC transporters with drug-eflux capacity and are relatively resistant to Mitoxantrone and other cytostatica (MDR) Survival mRNA Expression Proc. Natl. Acad. Sci. USA 101, 14228–14233 (2004)

33. Targeting CSC resistance by novel drugs: - ABC eflux pump inhibitors (Verapamil) - Transiently induce stem-cell proliferation  more S-phase cells, sensitivity to toxic DNA analoga (5-FU) or replication-specific drugs (Etoposide) - Target the high DNA repair capacity of CSCs with inhibitors (siRNA) (problem of specifity unsolved)

34. CSCs determine Radiotherapy response (Short Term in-vitro growth inhibition) + RTx Control (Long Term repopulating potential) Short-term inhibition of cell growth not correlated with tumor response Inbibition of LTRP correlates with tumor control by RTx

35. CSCs determine therapy response Dose / Gy Clonogenic survival assay is a stem-cell assay Inhibition of clonogenic survival correlates with curability of tumor by RTx  CSC sensitivity is important for therapy response of tumors But: Little correlation of tumor response with initial number of CSCs  Inherent CSC sensitivity is important, not their initial number. Radioresistant Tumor Log S/So Radioresponsive Tumor

36. Summary: Understanding CSCs is important - to successfully fight metastasis - to overcome therapy resistance / tumor relapse - to design novel therapeutic strategies (Immunotherapy, Gene-Therapy, Antibody-based Therapy etc) Future Potential to improve Tumor Diagnostics / Prognosis / Individualized Therapy Still unclear how CSCs are involved in the process of Tumor Induction