8-stem-cells---cancer--2012-oct-

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Flatworm (planarian) Newt MRL mice Nature. 2012 Sep 27;489(7417):561-5. doi: 10.1038/nature11499. Skin shedding and tissue regeneration in African spiny mice (Acomys).Seifert AW, Kiama SG, Seifert MG, Goheen JR, Palmer TM, Maden M.

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Stem Cell Development Adult stem/ progenitors functional maturation PCD (apoptosis) Differentiation Death Senescence ESCs chronological aging

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Stem Cells & Cancer Three tumor biology puzzles: Most tumors are of a clonal origin but tumor cells are heterogeneous. It is very difficult to establish stable tumor cell lines from tumors. Large numbers of established tumor cells have to be injected to re-initiate an orthotopic tumor in mice. Key reviews: Reya T et al. Stem cells, cancer, and cancer stem cells. Nature 414, 105-111, 2001. Dick JE. Stem cell concepts renew cancer research. Blood 112: 4793-4807, 2008. Visvader JE, and Lindeman GJ. Cancer Stem Cells: Current Status and Evolving Complexicities. Cell Stem Cell 10: 717-728, 2012. 4. Tang DG. Understanding cancer stem cell heterogeneity and plasticity. Cell Res, 22(3):457-472, 2012. 5. Magee JA, Piskounova E, & Morrison SJ. Cancer Stem Cells: Impact, Heterogeneity, and Uncertainty. Cancer Cell 21: 283-296, 2012. (Dean Tang, Basic Concepts of Tumor Biology, Oct 31, 2012)

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1. Characteristics & Definition 2. SC Identification 3. SC Niche & Plasticity 4. SCs & Cancer 5. Cancer Stem Cells (CSCs) Stem Cells & Cancer

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Rare Generally small - Normally localized in a ‘protected’ environment called NICHE , where they only occasionally divide. - But they possess HIGH PROLIFERATIVE POTENTIAL and can give rise to large clones of progeny in vitro or in vivo following injury or appropriate stimulation. - Possess the ability to SELF-RENEW (i.e., asymmetric or symmetric cell division) - Can generate (i.e., DIFFERENTIATE into) one or multiple or all cell types (uni-, oligo-, multi-, pluri-, or toti-potent). Stem Cells

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Committed progenitor cells symmetric SC renewal asymmetric cell division (ACD) symmetric SC commitment (differentiation) SC Tang, Cell Res . 2012

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LT-SC ST-SC Late progenitors Differen- tiated cells Differen- tiating cells Early progenitors Proliferation Differentiation Transformation probability Self-renewal Niche Commitment Differentiation Expansion Tang, Cell Res . 2012 Cell lineage development: Self-renewal, proliferation, & differentiation

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Mouse ESCs were generated early 1980s by Evans and Martin. mES cells are cultured on mouse fibroblast feeders (irradiated or mitomycin C-treated) together with LIF. .mES cells are widely used in gene targeting. Human ES (hES) cells were first created by Jim Thomson (Uni. Wisconsin) in 1998. hES cells were initially cultured also on mouse fibroblast feeders but without LIF. Now they can be maintained in defined medium with high bFGF (100 ng/ml), activin, and some other factors. Embryonic Stem Cells (ESCs)

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Leftover or dead-end IVF embryos (PGD) How can hES cells be derived?

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16-cell morula

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Primitive ectoderm Trophectoderm Primitive Endoderm A. Nagy

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ES cells A. Nagy

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TS cells A. Nagy

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A. Nagy

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A. Nagy

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A. Nagy

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heart pancreas testis liver brain kidney A. Nagy

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Derived from umbilical cord Primarily blood stem cells Also contain mesenchymal stem cells that can differentiate into bone, cartilage, heart muscle, brain, liver tissue etc. *CB-SC could be stimulated to differentiate into neuron, endothelial cell, and insulin-producing cells Cord Blood Stem Cells (CB-SC) Germline Stem Cells (GSC) Other ‘embryonic’ SCs

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1. Characteristics & Definition 2. SC Identification 3. SC Niche & Plasticity 4. SCs & Cancer 5. Cancer Stem Cells (CSCs) Stem Cells & Cancer

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Functional Assays of Stem Cells (Candidate) Stem Cells Stem Cells in situ (Xeno)Transplantation Lineage tracing

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How to identify and characterize (adult) stem cells? Marker analysis Label-retaining cells (LRC): Pulse-chase exper. Clonal/clonogenic assays Functional analysis: Side population (SP) assay Functional analysis: Aldefluor assay Cell size-based enrichment Genetic marking & lineage tracing

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Passegué, Emmanuelle et al. (2003) Proc. Natl. Acad. Sci. USA 100, 11842-11849 Hematopoietic stem/progenitor cell lineages (~1:5,000 or 0.02%; lifetime self-renewal) (~1:1,000 or 0.1%; self-renewal for 8 wks) (No self-renewal) Lin - Sca1 + ckit + CD150 + CD48 - (20%-50% such mouse BM cells are SCs) Till JE & McCulloch EA. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res 14, 213-222, 1961. Lin - CD34 + CD38 - CD45RA - Thy1 + Rho lo CD49f + (Notta F…..Dick JE. Science 333, 218-221, 2011)

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(Nestin) (GFAP) (Pax6) (A2B5) (NG2) (MBP) (NeuM) (Mash-1) (PDGFR a )

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Sue Fischer

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How to identify and characterize (adult) stem cells? Marker analysis Label-retaining cells (LRC): Pulse-chase exper. Clonal/clonogenic assays Functional analysis: Side population (SP) assay Functional analysis: Aldefluor assay Cell size-based enrichment Genetic marking & lineage tracing

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Till JE & McCulloch EA. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res 14, 213-222, 1961 . [The earliest report in which putative stem cells were identified by their ability to retain labeled radionucleotides for long period of time] Cotsarelis G, Cheng SZ, Dong G, Sun TT & Lavker RM. Existence of slow-cycling libmal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells. Cell 57, 201-209, 1989. Cotsarelis G, Sun TT, & Lavker RM. Label-retaining cell reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61, 1329-1337, 1990.

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LRCs in the Bulge & BM ARE Stem Cells Tumbar T et al., Defining the epithelial stem cell niche in skin. Science 303, 359-363, 2004. Blanpain, C., et al., Self-renewal, multipotency, and the existence of two cell populations in an epithelial stem cell niche. Cell 118, 635-648, 2004. Fuchs et al., Cell 116, 769, 2004 Fuchs E: The tortoise and the hair: Slow-cycling cells in the stem cell race. Cell 137, 811-819, 2009. Fuchs E & Horsley V. Ferreting out stem cells from their niches. Nat Cell Biology 13: 513-518, 2011. Wilson A et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 135, 1118-1129, 2008. Foudi A et al. Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells. Nat. Biotechnol. 27, 84-90, 2009.

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Not All Stem Cells Are Slow-Cycling Fuchs E & Horsley V. Ferreting out stem cells from their niches. Nat Cell Biology 13: 513-518, 2011. Li L & Clevers H. Co-existence of quiescent and active adult stem cells in mammals. 327, 542-545, 2010. ‘…. and even for the ones that do, approximately only 5-6 divisions of the label-retaining stem cell or its progeny can be monitored after a pulse-chase before the label has diluted out to the point where it can no longer be traced’. Not All Slow-Cycling Cells Are Stem Cells

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How to identify and characterize (adult) stem cells? Marker analysis Label-retaining cells (LRC): Pulse-chase exper. Clonal/clonogenic assays Functional analysis: Side population (SP) assays Functional analysis: Aldefluor assay Cell size-based enrichment Genetic marking & lineage tracing

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E Rheinwald JG & Green H . Serial cultivation of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell 6, 331-343, 1975. Sun TT Cell 9, 511-521, 1976 Nature 269, 489-493, 1977 Cell 14, 469-476, 1978 Fuchs E Cell 19, 1033-1042, 1980 Cell 25, 617-625, 1981 Barrandon Y PNAS 82, 5390-4, 1985 Cell 50, 1131-1137, 1987 Rice RH Cell 11, 417-422, 1977 Cell 18, 681-694, 1979 Watt FM JCB 90, 738-742, 1981

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CLONAL vs CLONOGENIC ASSAYS Clonal *Plate cells at clonal density (50-100 cells/well in 6-well plate or 10-cm dish or T25 flask) *Plate single cells into 96-well plates (or using flow sorting) - limiting dilution Holoclone Mero- or paraclone Cloning efficiency (CE; %) Clonal size (cell number/clone) Clonal development (tracking) Clone types A clone: a two-dimensional structure Plating efficiency Prolif. potential Clonogenic ‘In-gel’ assays (plate cells at low density) ‘On-gel’ assays (plate at low density) Efficiency (%) Colony/sphere size (cell number) Colony/sphere development (tracking) Immunostaining/tumor exp. A colony/sphere: a 3-D structure Colonies (colony-formation assays) Anchorage- independ. survival Prolif. Spheres (sphere-formation assays) Gels: Agar Agarose Methylcellulose Matrigel Poly-HEMA fibroblasts

Mixing Experiments to Demonstrate the Clonality of Clones/Spheres:

Mixing Experiments to Demonstrate the Clonality of Clones/Spheres DU145 RFP:DU145 GFP (1:1) MC DU145:DU145 GFP (1:1) MC phase GFP DU145:DU145 GFP (1:1) Clonal Assay Pastrana E, Silva-Vargas V, and Doetsch F. Eyes wide open: A critical review of sphere-formation as an assay of stem cells. Cell Stem Cell 8, 486-498, 2011

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How to identify and characterize (adult) stem cells? Marker analysis Label-retaining cells (LRC): Pulse-chase exper. Clonal/clonogenic assays Functional analysis: Side population (SP) assays Functional analysis: Aldefluor assay Cell size-based enrichment Genetic marking & lineage tracing

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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. Golebiewska A, Brons NH, Bjerkvig R, and Niclou SP. Critical appraisal of the side population assay in stem cell and cancer stem cell research. Cell Stem Cell 8, 136-147, 2011.

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Bcrp (ABCG2) is a major mediator of the SP phenotype Zhou et al., Nature Med 7, 1028, 2001

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How to identify and characterize (adult) stem cells? Marker analysis Label-retaining cells (LRC): Pulse-chase exper. Clonal/clonogenic assays Functional analysis: Side population (SP) assays Functional analysis: Aldefluor assay Cell size-based enrichment Genetic marking & lineage tracing

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Alison MR, Guppy NJ, Lim SML, & Nicholson LJ. Finding cancer stem cells: Are aldehyde dehydrogenases fit for purpose? J Pathol. 222, 335-344, 2010. Ma I & Allan AL. The role of human aldehyde dehydrogenase in normal and cancer stem cells. Stem Cell Rev and Report 7, 292-306, 2011. *ALDH1A1 and ALDH3A1: Seem to be the major isozymes mediating the Aldefluor phenotype and are preferentially expressed in SC. *ALDH superfamily: 19 putatively functional genes in 11 families and 4 subfamilies. ALDH superfamily of NAD(P) + -dependent enzyme catalyzes oxidations of aldehydes to carboxylic acids. Kastan MB et al. Direct demonstration of elevated aldehyde dehydrogenase in human hematopoietic progenitor cells. Blood 75, 1947-1960, 1990. Jones RJ et al., Assessment of aldehyde dehydrogenase in viable cells. Blood 85, 2742-46, 1995. Storms RW et al. Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity. PNAS 96, 9118-9123, 1999.

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How to identify and characterize (adult) stem cells? Marker analysis Label-retaining cells (LRC): Pulse-chase exper. Clonal/clonogenic assays Functional analysis: Side population (SP) assays Functional analysis: Aldefluor assay Cell size-based enrichment Genetic marking & lineage tracing

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Fuchs E & Horsley V. Ferreting out stem cells from their niches. Nature Cell Biology 13: 513-518, 2011.

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Fuchs E & Horsley V. Ferreting out stem cells from their niches. Nature Cell Biology 13: 513-518, 2011. Liver J et al., Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56-62, 2007. Snippert HJ et al., Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lrg stem cells. Cell 143, 134-144, 2010.

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1. Characteristics & Definition 2. SC Identification 3. SC Niche & Plasticity 4. SCs & Cancer 5. Cancer Stem Cells (CSCs) Stem Cells & Cancer

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Stem Cell Niche in Hair Follicles: The Bulge Moore KA & Lemischka IR. Science 311, 1880-1885, 2006

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Bulge Stem Cells Tumbar et al., Science 303, 359-363, 2004; Fuchs et al., Cell 116, 769, 2004

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Stem Cell Niche in Small Intestine: The Crypt Moore KA & Lemischka IR. Science 311, 1880-1885, 2006 Barker N et al., Cell Stem Cell 11: 452-460, 2012.

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Stem Cell Niches in BM Moore KA & Lemischka IR. Science 311, 1880-1885, 2006 Naveiras O et al., Bone-marrow adipocytes as negative regulators of the hematopoietic microenvironment. Nature 460, 259, 2009. Mendez-Ferrer, S et al., Mesenchymal and hematopoietic stem cells form a unique bone marrow niche. Nature 466, 829-834, 2010.

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Stem cell lineage Differentiated cells Death (PCD) Senescence Stem cells Progenitors/ Precursor cells Other differ. c ell(s)

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*First report : Long-term cultured adult brain (stem) cells can reconstitute the whole blood in lethally irradiated mice (Bjornson et al., Science 283, 534-537, 1999 ). *Cells from skeletal muscle have hematopoietic potential (Jackson et al., PNAS 96, 14482-14486, 1999 ) and can also “differentiate” into many other cell types (Qu-Petersen, Z, et al., JCB 157, 851- 864, 2002). *CNS “SCs” can “differentiate” into muscle cells (Clarke et al., Science 288, 1660-1663, 2000 ; Galli et al., Nat. Neurosci 3, 986-991, 2000 ; Tsai and McKay, J. Neurosci 20, 3725-3735, 2000 ). *Vice versa, “SCs” from blood or bone marrow can “transdifferentiate” into muscle (Ferrari et al., Science 279, 1528-1530, 1998 ; Gussoni et al., Nature 401, 390-394, 1999 ), hepatocytes (Petersen et al., Science 284, 1168-1170, 1999 ; Lagasse et al., Nat Med 6, 1229-1234, 2000 ), cardiac myocytes (Orlic et al., Nature 410, 701-705, 2001 ), or neural cells (Mezey et al., Science 290, 1779-1782, 2000 ; Brazelton et al., Science 290, 1775-1779, 2000 ). *Bone marrow appears to contain two distinct SCs: the HSC and MSC. A single HSC could contribute to epithelia of multiple organs of endodermal and ectodermal origin (Krause et al., Cell 105 369-377, 2001 ). MSC, on the other hand, can adopt a wider range of fates (endothelial, liver, neural cells, and perhaps all cell types) (Pittenger et al., Science 284, 143-146, 1999 ; Schwartz et al., JCI 109, 1291-1302, 2002 ; Jiang et al., Nature 418, 41-49, 2002 ). *Pluripotent “SCs” have also been isolated from skin that can “differentiate” into neural cells, epithelial cells, and blood cells (Toma et al., Nat Cell Biol. 3, 778-784, 2001 ) *Highly purified adult rat hepatic oval “stem’ cells, which are capable of differentiating into hepatocytes and bile duct epithelium, can “trans-differentiate” into pancreatic endocrine hormone- producing cells when cultured in a high glucose environment (Yang et al., PNAS 99, 8078- 8083, 2002) “Transdifferentiation” of Stem Cells

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De-differentiation: Cell-cycle re-entry *Many ‘post-mitotic’ cells such as hepatocytes, endothelial cells, and Schwann cells have long been known to retain proliferative (progenitor) potential. * Dedifferentiation is a genetically regulated process that may ensure a return path to the undifferentiated state when necessary (Katoh et al., PNAS 101, 7005, 2004 ). *Regeneration of male GSC by spermatogonial dedifferentiation in vivo (Brawley and Matunis, Science 304, 1331, 2004 ). *Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors (Nature 449, 473-477, 2007). *During Salamander limb regeneration, complete de-differentiation to a pluripotent state is not required – Progenitor cells in the blastema keep a memory of their tissue origin (Nature 460, 60-65, 2009). *Epigenetic reversion of post-implantation epiblast to pluripotent embryonic cells (Nature, 461, 1292-1295, 2009). *Evidence for cardiomyocyte renewal in humans (Bergmann O et al., Science 324, 98-102, 2009). (Cardiomyocytes turn over at an estimated rate of ~1% per year at age 20, declining to 0.4% per year at age 75. At age 50, 55% of human cardiomyocytes remain from birth while 45% were generated afterward. Over the first decade of life, cardiomyocytes often undergo a final round of DNA synthesis and nuclear division without cell division, resulting in ~25% of human cardiomyocytes being binucleated.) *Neuregulin 1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury (Bersell et al., Cell, 138, 257-270, 2009). *MafB/c-Maf deficiency enables self-renewal of differentiated functional macrophages (Aziz A, et al., Science 326, 867-871, 2009).

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Pancreatic b -cells: Interesting insulin-producing cells *Insulin-producing b -cells in adult mouse pancreas can self-duplicate during normal homeostasis as well as during injury (Dor et al., Nature 429, 41, 2004). *In vivo reprogramming of adult pancreatic exocrine cells to b cells using 3 TFs (Ngn3, Pdx1, and Mafa), suggesting a paradign for directing cell reprogramming without reversion to a pluripotent cell state (Zhou et al., Nature 455, 627-632, 2008). *In response to injury, a population of pancreatic progenitors can generate glucagon-expressing alpha cells that then transdifferentiate (with ectopic expression of Pax4) into beta cells (Collmbat et al, Cell 138, 449-462, 2009). *Conversion of adult pancreatic a -cells to b -cells after extreme b -cell loss (Nature 464, 1149-1154, 2010).

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NUCLEAR REPROGRAMMING CELL 126, 652-655, 2006

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Induced Pluripotent Cells (iPS cells) (Infection of somatic cells with 2-4 factors: Oct4, Sox2, Klf-4, Myc) Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006 Aug 25;126(4):663-76. Maherali N, ……., Hochedlinger K . Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell. 2007 Jun 7;1(1):55-70. Okita K, Ichisaka T, Yamanaka S . Generation of germline-competent induced pluripotent stem cells. Nature. 2007 Jul 19;448(7151):313-7. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872, 2007. Yu J, ……, Thomson JA . Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007 Dec 21;318(5858):1917-20. Hanna J, …. Jaenisch R. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin (Science, 318, 1920-1923, 2007). Nakagawa M, …… Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol. 2008 Jan;26(1):101-6. Park IH, …….. Daley GQ. Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 2008 Jan 10;451(7175):141-6. Kobayashi T et al., Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 142, 787-799, 2010. *Up to now: ~5,000 PubMed publications on iPS cells! *Yamanaka S: Elite and stochastic models for induced pluripotent stem cell generation. Nature 460: 49-52, 2009. *Yamanaka S & Blau HM. Nuclear reprogramming to a pluripotent state by three approaches. Nature 465, 704, 2010

Development and epigenetic (re)programming:

Development and epigenetic (re)programming Hochedlinger 2009, Development 136, 509-523

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Nicholas CR & Kriegstein AR. Cell reprogramming gets direct. Nature 463, 1031-1032, 2010. Vierbuchen T et al., Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035-1041, 2010 ( Ascl1, Brn2, and Myt1l ). Ieda M, et al., Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell 142, 375-386, 2010 ( Gata4, Mef2c, and Tbx5 ). Bonfanti P, et al., Microenvironmental reprogramming of thymic epithelial cells to skin multipotent stem cells. Nature 466, 978-982, 2010. Bussard KM, et al., Reprogramming human cancer cells in the mouse mammary gland. Cancer Res 70, 6336-6343, 2010. Direct Reprogramming and Lineage Conversion of Cells

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1. Characteristics & Definition 2. SC Identification 3. SC Niche & Plasticity 4. SCs & Cancer 5. Cancer Stem Cells (CSCs) Stem Cells & Cancer

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Perinatal stem/ progenitors functional maturation PCD Stem Cells & Tumorigenesis (Terminal) differentiation Adult Stem/ Progenitor Cells Death Senescence ESCs Tumor cells

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Several fundamental tumor biology questions Why is it so difficult to establish tumor cell lines from established tumors or even metastases? 2. Why tens or hundreds of thousands of established tumor cells have to be injected to initiate an orthotopic tumor? 3. Tumors are clonal (i.e., all tumors were initially derived from ‘going bad’ of one cell) but why is the tumor itself heterogeneous?

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Cell type Cell# injected Incidence Latency (days) Du145 1,000 0/4 10,000 0/4 100,000 1/4 103 500,000 3/5 53, 53, 59 LAPC4 100 0/4 1,000 0/4 10,000 0/4 100,000 0/4 500,000 3/6 43, 43, 48 LAPC9 100 0/3 1,000 0/9 10,000 4/8 46, 53, 75, 75 100,000 6/9 32, 42, 42, 45, 62, 69 1,000,000 4/4 48, 56, 56, 69 Tumorigenecities of Orthotopically Implanted Prostate Cancer Cells

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PSA AR CD57 CK5 Nanog

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Several fundamental tumor biology questions Why is it so difficult to establish tumor cell lines from tumors or even metastases? 2. Why tens or hundreds of thousands of established tumor cells have to be injected to initiate an orthotopic tumor? 3. Tumors are clonal (i.e., all tumors were initially derived from ‘going bad’ of one cell) but why is the tumor itself heterogeneous (i.e., comprising multiple cell types)? These questions can be potentially explained by the presence of stem-like cells in the tumor, i.e., tumor (or cancer) stem cells

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Cancer stem cells (CSC): Tumorigenic cells Hewitt, HB. Studies of the quantitative transplantation of mouse sarcoma. Brit. J. Cancer. 7, 367- 383, 1953 (~0.01% of the tumor cells are tumor stem cells; limiting dilution method). Bruce, W.R & van der Gaag, H. A quantitative assay for the number of murine lymphoma cells capable of proliferation in vivo. Nature 199, 79-80, 1963 (~0.8% total cells forming spleen colonies). Wodinsky, I., Swiniarski, J., and Kensler, CJ. Spleen colony studies of leukemia L1210. I. Growth kinetics of lymphocytic L1210 cells in vivo as determined by spleen colony assay. Cancer Chemother. Rep . 51, 415-421, 1967 (1-3% total cells forming spleen colonies). Bergsahel, DE & Valeriote FA. Growth characteristics of a mouse plasma cell tumor. Cancer Res . 28, 2187-2196, 1968 (<4.4% cells are tumor stem cells). Park CH, Bergsagel DE, and McCulloch, EA. Mouse myeloma tumor stem cells: a primary cell culture assay. JNCI 46, 411-422, 1971 (0.7 - 1.2% clonogenic, tumor stem cells). Hamburger, A.W. and Salmon SE. Primary bioassay of human tumor stem cells. Science 197, 461-463, 1977 0.001 - 0.1% myeloma cells forming colonies in soft agar). Fidler, IJ and Kripke, ML. Metastasis results from preexisting variant cells within a malignant tumor. Science 197, 893-895, 1977. Salmon, SE, Hamburger, A.W., Soehnlen, B., Durie, B.G.M., Alberts, D.S., and Moon, T.E.. Quantitation of differential sensitivity of human -tumor stem cells to anticancer drugs. N. Eng. J. Med . 298, 1321 -1327, 1978. Sell, S., and Pierce, G.B. Maturation arrest of stem cell cell differentiation is a common pathway for the cellular origin of teratocarcinomas and epithelial cancers. Lab. Invest . 70, 6-22, 1994. Trott, KR. Tumor stem cells: the biological concept and its application in cancer treatment. Radiother. Oncol. 30, 1-5, 1994.

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Evidence for CSCs: Long-term cutured cancer cells have only a minor subset of clone-initiating cells A C B D Du145 LNCaP

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Only 0.01 -0.1% of the actutely purified tumor cells have the sphere-forming abilities

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Leukemic stem cells (LSC): Classic examples of CSC Lapidot T, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367, 645-648, 1994. Blair et al., Blood 89, 3104-3112, 1997 Bonnet, D., & Dick, J.E. Nature Med . 3, 730-737, 1997 Most of the leukemic cells are unable to proliferate extensively and only a small, defined subset of cells was consistently clonogenic. LSCs for human AML were identified prospectively and purified as [Thy1-, CD34+, CD38-] cells from various patient samples and they represent 0.2 - 1% of the total. The LSCs are the only cells capable of transferring AML from human patient to NOD/SCID mice and are referred to as SCID leukemia -initiating cells (SL-IC).

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Identification of CSC in Solid Tumors by Markers Al-Hajj, M et al., Prospective identification of tumorigenic breast cancer cells. PNAS 100, 3983-3988, 2003. Hemmati, H.D et al., Cancerous stem cells can arise from pediatric brain tumors. PNAS 100, 15178-15183, 2003 (using neural SC markers). Galli R et al., Isolation and characterization of tumorigenic, stem-like neural precursors from human glioma. Cancer Res. 64, 7011, 2004. Singh SK et al. Identification of human brain tumour initiating cells. Nature 432: 396-401, 2004 (using CD133 as a marker) . Patrawala L, et al. Highly purified CD44 + prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells. Oncogene 25, 1696-1708 , 2006.

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Identification of CSCs by SP Kondo T, et al. Persistence of a small population of cancer stem-like cells in the C6 rat glioma cell line. PNAS 101: 781-786, 2004. Hirschmann-Jax C, et al. A distinct "side population" of cells with high drug efflux capacity in human tumor cells. PNAS 101: 14228, 2004. Patrawala, L., et al. Side population (SP) is enriched in tumorigenic, stem-like cancer cells whereas ABCG2 + and ABCG2 - cancer cells are similarly tumorigenic. Cancer Res. 65, 6207, 2005. Haraguchi N, et al. Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells 24, 506-13, 2006. Chiba T, et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology 44:240-51, 2006. Szotek PP, et al., Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness. PNAS 103:11154-9, 2006.

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Identification of putative CSCs by quiescence Pece S, et al., Biological and molecular heterogeneity of breast cancer correlates with their cancer Stem cell content. Cell 140, 62-73, 2010.

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-1187 Exon 1 TSS (+1) ATG (164) K5 promoter 116 718 Exon 2 Identification of putative CSCs by promoter tracking

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Identification of CSCs by lineage tracing Chen J et al., Nature . 2012 Aug 23;488(7412):522-6. A restricted cell population propagates glioblastoma growth after chemotherapy. Schepers AG et al. Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science 337: 730-735, 2012. Driessens G et al . Defining the mode of tumor growth by clonal analysis. Nature 488: 527-530, 2012.

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LT-SC ST-SC Late progenitors Differen- tiated cells Differen- tiating cells Early progenitors Proliferation Differentiation Transformation probability Self-renewal Niche Commitment Differentiation Expansion Tang, Cell Res . 2012 Where did tumor cells come from?

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Progenitor or differentiated cells as transformation targets or CSCs Passegue, E., et al. Normal and leukemic hematopoiesis: are leukemias a stem cell disorder or a reacquisition of stem cell characteristics? Proc. Natl. Acad. Sci. USA. 100 (Suppl 1): 11842-11849, 2003. Huntly, B.J., et al. MOZ-TIF2, but not BCR-ABL, confers properties of leukemic stem cells to committed murine hematopoietic progenitors. Cancer Cell. 6: 587-596, 2004. Jamieson, C.H., et al. Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML. NEJM . 351: 657-667, 2004. Krivtsov, AV et al., Transformation from committed progenitor to leukemia stem cells initiated by MLL-AF-9. Nature 442, 818-822, 2006. McCormack MP, et al. The Lmo2 oncogene initiates leukemia in mice by inducing thymocyte self-renewal. Science 327, 879-883, 2010.

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Where do tumors REALLY come from? Houghton J et al., Gastric cancer originating from bone marrow-derived cells. Science. 2004, 306: 1568-71. Direkze NC, et al., Bone marrow contribution to tumor-associated myofibroblasts and fibroblasts. Cancer Res. 2004, 64: 8492-5. Aractingi S et al., Skin carcinoma arising from donor cells in a kidney transplant recipient. Cancer Res. 2005, 65:1755-60. Kaplan RN et al., VEGFR1-positive hematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature. 2005, 438, 820-827. Riggi N et al., Development of Ewing’s sarcoma from primary bone marrow-derived mesenchymal progenitor cells. Cancer Res. 2005, 65:11459-11468. Palapattu GS et al., Epithelial architectural destruction is necessary for bone marrow derived cell contribution to regenerating prostate epithelium. J. Urol, 2006, 176:813-18.

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Cancer Stem Cells & Treatment Weissman, Nature 414, 105-111, 2001

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How to specifically target CSCs? *Identify functional CSC molecules (e.g., CD44, c-KIT, Bmi-1, Nanog)  Knock down these molecules to inhibit CSC properties (e.g., Jeter et al., 2009; Levina V et al., Elimination of human lung CSCs through targeting of the stem cell factor-c-kit autocrine signaling loop. Cancer Res. 70, 338-346, 2010). *Identify CSC-specific cell-surface markers  Develop antibody or prodrug based therapeutics *Take advantage of differential signaling requirement between normal and cancer SC (Yilmaz OH et al., Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. 2006 441:475-82 )

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Using stem/progenitor cells to treat tumors: *Using neural progenitor cells alone: these cells produce large amounts of TGF b (Staflin et al., Cancer Res. 64, 5347-5354, 2004) *Using neural progenitor cells to deliver cytokines or cytotoxic genes or products --- Benedetti et al., Nat. Med. 6, 447-450, 2000 --- Aboody et al., PNAS 97, 12846-12851, 2000 --- Herrlinger et al., Mol. Ther. 1, 347-357, 2000 --- Ehtesham et al., Cancer Res. 62, 5657-5663, 2002 --- Ehtesham et al., Cancer Res. 62, 7170-7174, 2002 --- Barresi et al., Cancer Gene Ther. 10, 396-402, 2003 *Using IL23-expressing BM-derived neural stem-like cells to attack glioma cells --- Yuan X et al., Cancer Res. 66, 2630-2638, 2006 *Using hMSC to attack Kaposi’s sarcoma --- Khakoo AY et al., JEM. 203, 1235-1247, 2006

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‘Reprogramming’ the microenvironment of CSC to treat tumors: Kulesa P et al. Reprogramming metastatic melanoma cells to assume a neural crest cell-like phenotype in an embryonic microenvironment. PNAS 103, 3752-3757, 2006. Topczewska JM., et al., Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness. Nature Med. 12, 925-932, 2006.

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