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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

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slide1

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.

Recent key reviews:

1. Reya T et al. Stem cells, cancer, and cancer stem cells. Nature 414, 105-111, 2001.

2. Visvader JE, and Lindeman GJ. Cancer stem cells in solid tumours: accumulating evidence

and unresolved questions. Nat Rev Cancer 8: 755-768, 2008.

3. Dick JE. Stem cell concepts renew cancer research. Blood 112: 4793-4807, 2008.

4. Shackleton M, Quintana E, Fearon ER, and Morrison SJ. (2009). Heterogeneity in cancer:

cancer stem cells versus clonal evolution.Cell 138, 822-829, 2009.

(Dean Tang, Basic Concepts of Tumor Biology, Sept. 22, 2010)

slide2

Stem Cells & Cancer

1. Characteristics & Definition

2. Types & (Adult) SC Identification

3. SC Niche & Plasticity

4. SCs & Cancer

5. Cancer Stem Cells (CSCs)

slide3

Flatworm

(planarian)

Newt

MRL mice

slide4

Stem cell development

Terminal

differentiation

Progenitors/

Precursor cells

Senescence

Death (PCD)

Stem cells

slide5

Stem Cells

  • 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).
slide7

SC Development: Self-renewal, proliferation, differentiation

Differentiation

Transformation

targets

Self-renewal

?

LT-SC

ST-SC

Late

progenitors

Differen-

tiating cells

Differen-

tiated cells

Early

progenitors

Proliferation

Niche

Commitment

Differentiation

slide8

Embryonic Stem Cells (ESCs)

  • 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.
slide9

How can hES cells be derived?

  • Leftover or dead-end IVF embryos (PGD)
slide11

Primitive ectoderm

Trophectoderm

Primitive Endoderm

A. Nagy

slide12

ES cells

A. Nagy

slide13

TS cells

A. Nagy

slide17

heart

pancreas

testis

liver

brain

kidney

A. Nagy

slide19

Other ‘embryonic’ SCs

Germline Stem Cells (GSC)

Cord Blood Stem Cells (CB-SC)

  • 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
slide20

How to identify and characterize

(adult) stem cells?

Marker analysis

Clonal/clonogenic assays

Side population (SP): BCRP or ABCG2

Label-retaining cells (LRC)

Aldefluor assay (Aldh1 expression)

Cell size-based enrichment

Genetic marking

slide21

Hematopoietic and 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)

Passegué, Emmanuelle et al. (2003) Proc. Natl. Acad. Sci. USA 100, 11842-11849

slide22

(Nestin)

(PDGFR)

(Mash-1)

(NeuM)

(Pax6)

(A2B5)

(GFAP)

(MBP)

(NG2)

slide24

How to identify and characterize

(adult) stem cells?

Marker analysis

Clonal/clonogenic assays

Side population (SP): BCRP or ABCG2

Label-retaining cells (LRC)

Aldefluor assay (Aldh1 expression)

Cell size-based enrichment

Genetic marking

slide25

Anchorage-

independ.

survival

Plating efficiency

Clonogenic

‘In-gel’ assays

(plate cells at

low density)

Prolif. potential

Prolif.

Clonal

*Plate cells at

clonal density

(50-100 cells/well

in 6-well plate

or 10-cm dish

or T25 flask)

‘On-gel’ assays

(plate at low density)

*Plate single cells

into 96-well plates

(or using flow sorting)

- limiting dilution

Gels: Agar

Agarose

Methylcellulose

Matrigel

Poly-HEMA

fibroblasts

Spheres

(sphere-formation

assays)

Colonies

(colony-formation

assays)

Holoclone

Mero- or paraclone

A colony/sphere: a 3-D structure

Efficiency (%)

Colony/sphere size (cell number)

Colony/sphere development (tracking)

Immunostaining/tumor exp.

CLONAL vs CLONOGENIC ASSAYS

A clone: a two-dimensional structure

Cloning efficiency (CE; %)

Clonal size (cell number/clone)

Clonal development (tracking)

Clone types

mixing experiments to demonstrate the clonality of clones spheres
Mixing Experiments to Demonstrate the Clonality of Clones/Spheres

DU145:DU145 GFP (1:1) Clonal Assay

phase

DU145:DU145 GFP (1:1) MC

GFP

DU145 RFP:DU145 GFP (1:1) MC

slide27

How to identify and characterize

(adult) stem cells?

Marker analysis

Clonal/clonogenic assays

Side population (SP): BCRP or ABCG2

Label-retaining cells (LRC)

Aldefluor assay (Aldh1 expression)

Cell size-based enrichment

Genetic marking

slide28

Identification of

HSC by SP

Zhou et al.,

Nature Med

7, 1028, 2001

slide29

How to identify and characterize

(adult) stem cells?

Marker analysis

Clonal/clonogenic assays

Side population (SP): BCRP or ABCG2

Label-retaining cells (LRC)

Aldefluor assay (Aldh1 expression)

Cell size-based enrichment

Genetic marking

slide30

LRCs in the Bulge ARE Stem Cells

Tumbar et al., Science 303, 359-363, 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.

slide31

How to identify and characterize

(adult) stem cells?

Marker analysis

Clonal/clonogenic assays

Side population (SP): BCRP or ABCG2

Label-retaining cells (LRC)

Aldefluor assay (Aldh1 expression)

Cell size-based enrichment

Genetic marking

slide32

Stem Cell Niche

The most important function of a stem cell

niche is to keep the stem cells quiescent and

from differentiating and simultaneously

maintain their “stemness” (i.e., the repertoire

of gene expression profiles characteristic

of stem cells).

slide33

Stem Cell Niche in Hair Follicles: The Bulge

Moore KA & Lemischka IR. Science 311, 1880-1885, 2006

slide34

Bulge Stem Cells

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

slide35

Stem Cell Niche in Small Intestine: The Crypt

Moore KA & Lemischka IR. Science 311, 1880-1885, 2006

slide36

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.

slide37

Stem cell lineage

Differentiated

cells

Progenitors/

Precursor cells

Other

cell(s)

Senescence

Death (PCD)

Stem cells

slide38

Adult Stem Cell Plasticity

Plasticity: the ability of SCs to

regenerate and trans-differentiate into

(many) other cell types (the cell type-

specific programming of apparently

committed primary progenitors is not

irrevocably fixed, but may be radically

re-specified in response to a single

transcriptional regulator. Heyworth C

et al., EMBO J. 21, 3770-3781, 2002).

**Transdifferentiation vs Dedifferenitation: Transdifferentiation refers to adult stem cells

directly differentiating into other cell lineages of cells; de-differentiation refers to somatic

stem/progenitor cells first reverting back to a more primitive state then differentiating into

a specific cell type.

slide40

“Transdifferentiation” of Stem Cells: Exciting!!!

*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)

slide41

Cell fusion as one mechanism of trans-differentiation: Not only

stem/progenitor cells but also terminally differentiated cells can fuse with other cells

Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle

(Matsuura K et al., JCB 167, 351, 2004)

slide42

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).

slide43

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).

slide44

CELL 126, 652-655, 2006

NUCLEAR REPROGRAMMING

slide45

Induced Pluripotent Cells (iPS cells)

(Infection of somatic cells with 2-4 factors:Sox2, Oct4, 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: >2000 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

Transcription factors and chromatin remodelers and key modulators of ‘potency’

slide47

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 multi-

potent 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.

slide48

Stem Cells & Tumorigenesis

(Terminal) differentiation

functional

maturation

Senescence

Perinatal stem/

progenitors

PCD

Death

Adult Stem/

Progenitor Cells

ESCs

Tumor cells

slide49

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?

slide50

Tumorigenecities of Orthotopically Implanted Prostate Cancer Cells

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

slide51

Nanog

CK5

PSA

CD57

AR

slide52

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

slide53

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.

slide54

A

C

Du145

B

D

LNCaP

Evidence for CSCs: Long-term cutured cancer cells have only

a minor subset of clone-initiating cells

a

slide56

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).

slide57

CSC studies in human solid tumors (2003 - 2008)

Tumor type Samples Marker Mice Transplantation Results Ref.

Breast cancer9 (1 primary; 8 met.) CD44+CD24-/loESA+ NOD/SCID mice mammary fat pad >50 fold enrichment 1

FACS pretreated with VP16 in tumorigenicity

Breast cancer4 xenotransplants ALDH+ NOD/SCID mice humanized mammary 500 ALDH+ cells generate T; 2

(from 2 primary; 2 met.) FACS fat pad 20 ALDH+CD44+CD24-Lin- cells

generate T

Brain tumors 7 primary tumors CD133+ (MACS) 6-8 wk NOD/SCID intracranial injection CD133+ more tumorigenic 3

Prostate cancer7 (4 primary, CD44+21hiCD133+ (MACS) no tumor experiments marker+ cells more clonogenic 4

1 benign, 2 LN mets) purified from long-term cultured cells

Colon cancer17 (6 primary, 10 liver CD133+ (double MACS) 8 wk NOD/SCID renal capsule 1 CSC/57,000 T. cells 5 & 1 retroperitoneal met.) irradiated 1 CSC/262 CD133+ cells

Colon cancer19 primary (5 Dukes A) CD133+ (FACS or MACS) SCID subcutaneous 3,000 CD133+ cells generate T 6 Colon cancer 21 primary CRC CD133+ 5-6 wk nude mice subcutaneous 2,500 CD133+ cells generate T 7

25 CD133+-derived spheres generate T

Colon cancer 2 primary, 6 xenografts EpCAMCD166+CD44+ 6-8 wk NOD/SCID subcutaneous 150 EpCAMCD166+CD44+ 8

(FACS) cells generate T

Pancreatic cancer10 (2 primary; 2 met.) CD44+CD24+ESA+ NOD/SCID subcutaneous+pancreas >100 fold enrichment 9

Pancreatic cancer11 (6 met.); sorting for 7 CD133+ (MACS) 8-12 wk nude mice pancreas 500 CD133+ cells generate T 10

L3.6pl metastatic line CD133+CXCR4+ (FACS)the CD133+CXCR4+ pop. mediates met.

Head & Neck 25 primary (3 recurrences)CD44+Lin- (FACS) NOD/SCID & Rag2-/- subcutaneous 5,000 CD44+Lin- cells generate T 11

9 for sorting (4 primary+5 xenografts)only13/25 HNSCC samples gave tumors

Melanoma 7 (1 primary; 4 LN & ABCB5+ (MACS) NOD/SCID subcutaneous 1 MMIC/1 million bulk T cells 12

2 visceral met.) 1ary xeno: 1 MMIC/160,000 ABCB5+ cells

2ary xeno: 1 MMIC/120,000 ABCB5+ cells

Lung cancer 19 (18 primary; 1 met.) CD133+ (FACS) 4 wk SCID or nude subcutaneous 104 CD133+ cells generate T. 13

Liver cancer 28 primary (only 13 used) CD45-CD90+ (MACS) SCID intrahepatic CD45-CD90+ more tumorigenic 14

Al-Hajj M, et al. PNAS2003;100:3983-8.2. Ginestier C et al., Cell Stem Cell 2007;1:555-67.

Singh SK, et al. Nature 2004;432:396-401. 4. Collins AT, et al. Cancer Res 2005; 65: 10946-51.

O'Brien CA, et al. Nature 2007; 445:106-10. 6. Ricci-Vitiani L, et al. Nature 2007; 445:111-5.

Todaro M et al., Cell Stem Cell 2007; 1: 389-402. 8. Dalerba P et al., PNAS 2007; 104: 10158-63.

Li C et al., Cancer Res. 2007; 67:1030-7 10. Hermann PC et al., Cell Stem Cell 2007;1:313-32

Prince ME et al., PNAS 2007; 104: 973-8. 12. Schatton T, et al., Nature 2008; 451:345-9.

13. Eramo A et al., Cell Death Differ. 2008; 15: 504-14. 14. Yang ZF et al., Cancer Cell 2008; 13: 153-66.

slide58

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. Oncogene25, 1696-1708, 2006.

slide59

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.

slide60

The SP in LAPC-9 tumor is more tumorigenic

e

LAPC9-SP 100 2/8 (52 and 108 d)

1,000 3/4 (108 d)

1,500 3/3 (60 d)

LAPC9-NSP 1,500 0/8

150,000 0/7 (terminated in 9 mo.)

300,000 1/6 (92 d)

slide61

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.

slide62

Identification of putative CSCs by size selection

100

1000

Small

(<10 µm)

small

Large

(>30 µm)

large

slide63

TSS (+1)

ATG (164)

K5 promoter

Exon 1

718

116

Identification of putative CSCs by promoter tracking

Exon 2

-1187

slide64

Where did tumor cells come from?

Differentiation

Transformation

targets

?

Self-renewal

LT-SC

ST-SC

Late

progenitors

Differen-

tiating cells

Differen-

tiated cells

Early

progenitors

Proliferation

Niche

Commitment

Differentiation

slide65

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.

CancerCell. 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.

slide66

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 Set al.,Skin carcinoma arising from donor cells in a kidney transplant recipient. Cancer Res. 2005, 65:1755-60.

Kaplan RNet al.,VEGFR1-positive hematopoietic bone marrow

progenitors initiate the pre-metastatic niche. Nature. 2005, 438, 820-827.

Riggi Net al.,Development of Ewing’s sarcoma from primary bone marrow-derived mesenchymal progenitor cells. Cancer Res. 2005, 65:11459-11468.

Palapattu GSet al.,Epithelial architectural destruction is necessary for bone marrow derived cell contribution to regenerating prostate epithelium. J. Urol, 2006, 176:813-18.

slide67

Cancer Stem Cells & Treatment

Weissman, Nature 414, 105-111, 2001

slide68

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)

slide69

Using stem/progenitor cells to treat tumors:

*Using neural progenitor cells alone: these cells produce large amounts of TGFb

(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

slide70

‘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.

slide71

Rx

Nanog

CK5

PSA

CD57

AR

slide73

Nanog

CK5

PSA

CD57

AR

slide74

Rx

Rx

Nanog

CK5

PSA

CD57

AR