Cancer Stem Cells & Glioblastoma JP Hugnot «Brain plasticity, Neural stem cells and Glial tumors» INSERM U1051-UM2 Institut des Neurosciences de Montpellier Montpellier
1-Stem cells and Brain Stem Cells 2-Brain tumors 3-Tumoral heterogeneity and cancer stem cells 4-Regulation of cancer stem cell fate? How tumoral stem cells differentiate or are maintained? 5-Cancer Stem Cells and Treatment Resistance 6-Strategies to eliminate cancer stem cells
1-Stem cells and Brain Stem Cells
Self-renewal of adult organs Skin: 4 weeks Intestine: 3 to 5 days Blood Red blood cells: 200 billions /d Neutrophiles 40 billions /d Platelet 200 billions /d Every year, we produce new cells equalling our body weight
Stem cells
Stem cells are located in niches Bone
Pathways involved in self-renewal of stem cells
And the brain?
Defined media with 2 growth factors (EGF/FGF2) No adherence Identification of Neural stem cells with the Neurosphere assay differentiation Self renewing Neurones Astrocytes Oligodendrocytes Formation of multipotent and self-renewal neurospheres at clonal density
Localisation of stem cells and progenitors in the adult brain Stem Cells Stem Cells
The subventricular zone stem cell niche ansc: activated neural stem cells qnsc: quiescent neural stem cells TAC: transit amplifying cells NB: neuroblast E: ependymocytes
Structural complexity of the neural stem cell niche
Oligodendrocyte progenitor cells 3-5% of brain cells and most proliferative cells in the brain Provide Adult Myelinisation OPC NG2+ cells
2-Brain tumors
3 main types of cerebral tumors
Glial Tumors Most frequent primary brain tumors 3000 new cases each year in France, rare disease equivalent to death rate in car accident in France Adult Children
Glioma Grading Diffuse Low-grade Young patients Slow-growing (2 mm per year) survival 5-15 years Frequent Degeneration to Glioblastomas High-grade Glioblastoma Rapidly growing Very poor prognosis survival < 2 years
Treatments
Glioblastomas are very heterogeous tumors Pseudopallisades Necrosis
3-Tumoral heterogeneity As any tissu, tumoral tissu is often heterogenous A diversity of non tumoral cells: Vessels (endothelial cells, smooth muscle cells, adventitial fibroblast) Normal cells of the parenchyma Cells from immune system
A diversity of tumoral cells: Accumulation of mutations during the tumoral expansion from 1 to hundred of mutations (can take several years, decade) Different tumoral cellular phenotypes & Different tumoral cells level of immaturity Cells with different genetic abnormalities
Where does the difference in phenotype come from? As many cell type, tumoral cells can adopt different phenotypes Through acquisition of senescent features Depending on their enviroment, their metabolic status, their migratory status, their level of activation by several signaling Activation vs Dormancy of cancer cells Through a process of differentiation; there exist a variety of state of differentiation for cancer cells Cancer stem cell concept
Cancer stem cell concept Three Potentials: y proliferation y differentiation y tumorigenic Cancer Stem cells High Y p, y d, y t Cancer Progenitor cells Moderate Yp,yd, yt Cancer Differentiated Cells Low Yp,yd, yt
Cancer stem cells? Similar to the queen and workers in ant colony?
Properties of cancer stem cells Capable of generating several cell types in vitro Capable of generating tumors in animals with several tumoral cell subtypes (phenocopy of real tumor) Capable of self-renewal Capable of generating tumors in animals (xenotransplantation) with a high frequency
Evidence for Cancer Stem cells in Glioblastoma Galli, R. et al. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64, 7011-21 (2004). Yuan, X. et al. Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 23, 9392-400 (2004). Singh, S. K. et al. Identification of human brain tumour initiating cells. Nature 432, 396-401 (2004). CD133+ Neurospheres Differentiation GFAP Oligodendrocyte-like Neuronal-like Astrocyte-like Gal C Tubb3 GFAP
Glioblastoma stem cells induce tumors at a higher rate CD133+ CD15+ High incidence of Tumour in Nude or NOD/SCID mice CD133- CD15- Less tumorigenic Not all Glioblastoma contain CD133+ cells Cancer Res. 2010 Mar 1;70(5):2030-40..
Two models to explain the presence and maintenance of cancer stem cells in tumors Hierarchical Model Equilibrium/Stochastic Model Y p, y d, y t Y p, y d, y t Y p, y d, y t
Growing Evidence for a dynamic model Cell. 2011 Aug 19;146(4):633-44. doi: 10.1016/j.cell.2011.07.026. Stochastic state transitions give rise to phenotypic equilibrium in populations of cancer cells. 1-stem-like (CD44 hi CD24 neg EpCAM lo ) 2-basal (CD44 hi CD24 ne g EpCAM neg ) 3-luminal (CD44 lo CD24 hi EpCAM hi )
Clarke Nature 2004 Origin of Glioblastoma Stem Cells Neural Stem cells Progenitor cells (OPC) Differentiated cells NG2 CSC are derived from stem or progenitor cells Cancer derived from dedifferentiated cell CSC are derived from dedifferentiated cells (hypoxia, mutations, cytokines ) Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice. Friedmann-Morvinski D, Bushong EA, Ke E, Soda Y, Marumoto T, Singer O, Ellisman MH, Verma IM. Science. 2012 Nov 23;338(6110):1080-4.
How to identify and characterize cancer stem cells from tissue? 1. Marker analysis (CD133, CD15, CD44, 2. Label-retaining cells (LRC): Pulse-chase exper. 3. Sphere assays 4. Clonal/clonogenic assays 5. Side population (SP) assay 6. Aldefluor assay 7. Cell size-based enrichment 8. Genetic marking & lineage tracing
Cancer stem cell in solid tumors Nature Reviews Cancer 8, 755-768 (October 2008)
4-Regulation of cancer stem cell fate? How tumoral stem cells differentiate or are maintained?
Decision making is based on processing of information in complex networks (stochasticity also matters!) Parliament BRAIN CELL
Computation of internal and external information to make good (or bad..) decision COMPUTING (stochasticity)
Regulation of cell differentiation
4-Regulation of cancer stem cell fate? How tumoral stem cells differentiate or are maintained? Influence of local cell-cell signaling: a key role for the Notch pathway
Notch signaling Evolutionary conserved pathway in Metazoan regulates Cellular identity Proliferation Differentiation Apoptosis Developmental processes of lateral inhibition and boundary induction. Notch proteins (4): transmembrane receptors Notch ligands: Delta-like (Dll1/3/4) and Jagged (Jag1/2) Notch targets: Hes 1or 5 and Hey families of bhlh trancriptional repressors
Inhibition of differentiation Notch Inhibition of differentiation Delta proteolyse proteolyse
Lateral inhibition and Notch Neuronal progenitor cell Multipotential neural stem cells Notch signalling cell Neuron Delta expressing cell
Intimate contact between glioblastoma stem cells and endothelial cells activate the Notch pathway in cancer stem cells to maintain them Cancer stem cell A Vascular Niche for Cancer Stem Cells Vessel Nature Reviews Cancer 7, 733-736 (October 2007) Endothelial cells create a stem cell niche in glioblastoma by providing NOTCH ligands that nurture selfrenewal of cancer stem-like Cancer Res. 2011 Sep 15;71(18):6061-72
4-Regulation of cancer stem cell fate? How tumoral stem cells differentiate or are maintained? Reduction of growth factors in the environment Neurospheres Differentiation Growth factor removal
4-Regulation of cancer stem cell fate? How tumoral stem cells differentiate or are maintained? Asymmetric division Example of asymmetric distribution of Numb (regulator of Notch pathway) Numb
Asymmetric division in glioblastoma stem cells
Evidence for asymmetric division in cancer stem cells
Evidence for asymmetric division in cancer stem cells Nat Cell Biol. 2014 Mar;16(3):212-4. doi: 10.1038/ncb2924. A microrna-operated switch of asymmetric-to-symmetric cancer stem cell divisions. Lerner RG, Petritsch C.
Alteration of asymmetric division may lead to accumulation of cancer stem cells Asymmetric division genes behave as tumor suppressors in Drosophila Numb Dlg (Disc large) Scrib Pins Mira Prospero Lethal giant larvae (Lgl) Aurora-A
5-Cancer Stem Cells and Treatment Resistance Normal stem cells are resistant to treatement: For instance hair loss and regrowth after chemotherapy
5.1 Cancer stem cells have a higher capacity to repair their DNA
5.2-Cancer stem cells are slow cycling cells or quiescent cells
5.3-Cancer stem cells express high levels of efflux pumps such as ATP cassette transporters (MDR1, BRCP1, ABCG2, ) which enables chemotherapy extrusion from the cells
Purification of stem cells by side population method Goodell MA et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 183, 1797-1806, 1996.
Zhou et al., Nature Med 7, 1028, 2001 Bcrp (ABCG2) is a major mediator of the SP phenotype
5.4-Cancer stem cells can transdifferentiate in a reversible manner into vascular cells Control Endothelial cells Notch Glioma derived pericytes Guichet et al, 2014, Stem Cells and Cell. 2013 Mar 28;153(1):139-52.
Current cancer treatment Similar to the weed and the mower problem? Very efficient at first but only transient?..
6-Strategies to eliminate cancer stem cells 1-Find new molecules tageting the cancer stem cell population 2-Force the terminal differentiation of cancer stem cells 3-Make the cancer stem cells more sensitive to currently used drugs
1-Find new molecules tageting the cancer stem cell population
Use inhibitors against canonical pathways used for cancer stem cell selfrenewal
2-Force the terminal differentiation of cancer stem cells
3-Make the cancer stem cells more sensitive to currently used drugs
Thanks for your attention