cancer stem cells michael rosemann, helmholtz-zentrum muenchen, radiation biology [email protected]
TRANSCRIPT
• 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