Cancer Stem Cells

Michael Rosemann, Helmholtz-Zentrum Muenchen, Radiation Biology

rosemann@gsf.de

• Long-Term repopulating potential (LTRP)

Example: Normal adult tissue stem cellOrigin and differentiation of the mesenchymal cells

MesenchymalStem cell Fibroblast (Connective tissue)

Osteoblast (forming Bone)

Chondroblast (forming Cartilage)

Osteopregenitor

Adipocyte (Fatty tissue)

Fibroblast Progenitor / Histiocyte

Pre-Chondroblast

Pre-Adipocyte

Asymmetric vs.Symmetric Division of Stem-Cells

Long-Term Repopulatingcell (LTR)

Differentiated cell

Differentiated cell(LTR)

Tissue Homeostasis(asymetric)

Organ growth or Wound Healing (symetric + asymetric)

number of LTR cells constant number of LTR cells increases

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

Nat Rev Cancer 2003;3:895-902Nature 2001;414:105–111

Nature 1963;199:79–80Virchows Arch Pathol Anat Physiol Klin Med 1875;65:64-9

Nature Med 2006;12:296-300

Models of Tumor Heterogeneity

Trends in Cell Biology 2005;15:494-501

(any cell contributes to tumor growth) (only a subset of Tumor-initiating cellscontributes to tumor growth)

Models of Tumor Heterogeneity

Trends in Cell Biology 2005;15:494-501

Testing tumor cells in-vitro for their long-term repopulating potential (LTRP)

(Long Term repopulating potential)

Plating single cells+10 days

10 000 tumor cells 100 colonies (1% CSCs)

Primary Tumor

Models of Tumor Heterogeneity

Trends in Cell Biology 2005;15:494-501

Tumor is mixture of few cells with LTRP (CSCs) and many cells without LTRP

Only a small subset of tumor cells have Long Term repopulating potential

plating, +10 days

each colony= 50 – 100 cells

colonies no colonies

What determines LTRP ?

replating, +10 days

1st colonies contain again only ~1% cells that form 2ndary colonies

plating, +10 days

Tumor 1st colony 2ndary colonies

What determines LTRP ?

Cells with Long Term repopulating potential (CSCs) are notgenetically different from non-LTRP cells.

LTRP is reversible (epigenetically controlled)

replating, +10 days

1st colonies contain again only ~1% cells that form 2ndary colonies

plating, +10 days

Tumor 1st colony 2ndary colonies

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)

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

In-vivo Model of marrow-derive AML CSCsTransplantation in immunodeficient Nod-Scid mice

Leukaemic Blast cells AML

Blood from AML patient

In-vivo Model of marrow-derive AML CSCs

100%

0%

% of recipient mice Developing AML after transplantation

Transplantation in immunodeficient Nod-Scid mice

5 20 50 100 200 500 1000 2000 * 103 cells injected

Myeloid Leukaemia

Normal Haematopoiesis

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

Genes Dev. 17, 3029–3035 (2003)

Transplantation in bone-marrow ablated mice

Nature Review Cancer 2005;5:899-904

Origin of Cancer Stem Cell

mutation

environmental effect / mutagenic mechanism

chemical/physical carcinogenscause reversion of normal differentiation process

De-differentiation, CSC aquire multipotency(maybe true for multi-lineage Teratoma)

Nature Review Cancer 2005;5:899-904

Origin of Cancer Stem Cell cell-cell fusion

Mammalian fusogenic factors• CD44, CD47 (macrophage)

• CXCR4/SDF1 (osteoblast)

Science 308, 369–373 (2005)Stem Cells 23, 879–894 (2005)

mutation

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

Metastasis and Transdifferentiation

Nat. Rev. Cancer 2005; 5: 744–749

CK+(Epithelial Diff.)

CK-(Mesench. Diff.)

CK-

CK+

Cytokeratin+ cells exhibit epithelial differentiation (typical carcinoma)Cytokeratin - cells loose epithelial differentiation ( mesenchymal), hypoxic areas

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: epithelialmesenchymal 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)

Tumor cell plasticity and the process of Invasion and Metastasis

Epithelial-Mesenchymal-Transition (EMT)

Nat. Rev. Cancer 2005; 5: 744–749

CK+(Epithelial Diff.)

CK-(Mesench. Diff.)

CK-

CK+

Primary Tumor metastatic cell distant Metastasis(EMT) (MET)

Ep

Mes Mes

Integration of EMT & CSC

Nat. Rev. Cancer 2005;5: 744–749

Process of Metastasis requires CSCs

single metastatic cell

macroscopic Metastasis

cells forming a metastasis must by CSCs !!!

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

Stem Cell Pathways in Cancer and embryonal development

WNT Hedgehog Notch

cell fate determination in organ morphogenesis,- thymus medulla vs. cortex - rostral-caudal determination, - somite patterning (vertebrae)-Active Notch pathway promote proliferation

Limb development determination of body axis, left-right symmetry-Active Notch pathway promote proliferation

Neural tube formationDorsal-ventral axisCell-polarityAxon guidance in neurogenesis

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)

Extensively studied Tumor types for CSCs:

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)

CSC markers

Tumor stem cells and Chemotherapy resistance

Problem of Tumor therapy: A single surviving Tumor stem cell can cause relapse by growing a recidive.

3.95% 0.60%

Hoechst 33342 (blue)

Hoe

chst

333

42 (

red

)

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 osteosarcomacell line)

- VP + VP

VP: Verapamil, ABC-Inhibitor

3.95%

Hoechst 33342 (blue)

Hoe

chst

333

42 (

red

)

Side-Popiulation cells - form in-vitro Sarcospheres - highly tumorigenic after injection in recipient mice

- VP

(Red Fluorescence: Osteosarcoma cells were stabily labelled with Cherry-Fluorescence-Protein)

Side population cells have long-term repopulation capacity

(Cherry Fluorescence labeled)(unlabeled)

mixing,plating out

+ 3 days

+ 14 days

MP

SP

Side Polulation Cells are resistant to cytostatica

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)

Neuroblastoma cell linesJF and IMR32

SP

Non-SP

mRNAExpression

Survival

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)

CSCs determine Radiotherapy response

(Short Term in-vitrogrowth inhibition)

(Long Term repopulating potential)

Control + RTx

Short-term inhibition of cell growth not correlated with tumor responseInbibition of LTRP correlates with tumor control by RTx

-5

-4.5

-4

-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

0 1 2 3 4 5

Dose / Gy

Log S/So

RadioresistantTumor

RadioresponsiveTumor

CSCs determine therapy response

Clonogenic survival assay is a stem-cell assay

Inhibition of clonogenic survivalcorrelates 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.

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