1. Cancer Stem Cells
Michael Rosemann, Helmholtz-Zentrum Muenchen, Radiation Biology

2. Long-Term repopulating potential (LTRP)

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

4. Long-Term Repopulating
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:
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:

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:

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

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:

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

10. What determines LTRP ? replating, +10 days plating, +10 days
Tumor st colony ndary 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%
* 103 cells injected

15. Normal Haematopoiesis
Myeloid Leukaemia

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

17. Origin of Cancer Stem Cell
mutation
environmental effect / mutagenic mechanism chemical/physical carcinogens cause reversion of normal differentiation process De-differentiation, CSC aquire multipotency (maybe true for multi-lineage Teratoma)
Nature Review Cancer 2005;5:

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:

19. Tumor stem cells and metastasis
(single cell  1cm Metastasis (~ 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
WNT Hedgehog Notch
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

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 transcription factors Functional markers
cell surface markers
CD34+ / CD AML
CD44+ / CD24– Mammary Tumor
CD Glioblastoma, Osteosarcoma
transcription factors
Sox 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 (red)
- VP
+ VP
Hoechst (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 (red)
- VP
Hoechst (blue)

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

32. Side Polulation Cells are resistant to cytostaticaSP
Non-SP
Neuroblastoma cell lines
JF and IMR32
Survival
mRNA
Expression
Cancer stem cell (SP) have high expression of ABC transporters with drug-eflux capacity and are relatively resistant to Mitoxantrone and other cytostatica (MDR)
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