Stem cells Rare cells with the ability to perpetuate themselves through self-renewal and to

generate mature cells of a particular tissue through differentiation.

Asymmetric and symmetric stem cell divisions: stem cell strategies

Mechanisms of asymmetric stem cell division

Symmetric stem cell division in the adult germline

Stem cell can facultatively use both symmetric and asymmetric division

Stem cells occur in many different somatic tissues and are important for their physiology

Jordan et al. 2006 NEJM 355:1253-61

Neural stem cells generate cells in the central nervous system (Panel A). Hematopoietic stem cells generate mature blood cells (Panel B). Mammary stem cells generate breast tissue (Panel C).

HSCs can be subdivided into long-term self-renewing HSCs, short-term self-renewing HSCs and multipotent progenitors (red arrows indicate self-renewal). They give rise to common lymphoid progenitors (CLPs; the precursors of all lymphoid cells) and common myeloid progenitors (CMPs; the precursors of all myeloid cells). Both CMPs/GMPs (granulocyte macrophage precursors) and CLPs can give rise to all known mouse dendritic cells. ErP, erythrocyte precursor; MEP, megakaryocyte erythrocyte precursor; MkP, megakaryocyte precursor; NK, natural killer.

Development of Hematopoietic Stem Cells

Stem Cells

Multipotent Progenitors

Oligolineage Progenitors

Mature Cells

Reya et al. 2001 Nature 414:105-111

In vivo transfer to irradiated recipient mice

Recapitulation of the haematopoietic system in the recipient mouse

Positive and

negative s

election Peripheral Blood, Bone Marrow

or Cord Blood-derived cells

CD34+ cells

rare HSC in progenitor cell background

Origin of the Theory of Cancer Stem Cells

Only a small subset of cancer cells is capable of extensive proliferation

Liquid Tumors In vitro colony forming assays:

- 1 in 10,000 to 1 in 100 mouse myeloma cells obtained from ascites away from normal hematopoietic cells were able to form colonies

In vivo transplantation assays: - Only 1-4% of transplanted leukaemic cells could form spleen colonies

Solid Tumors - A large number of cells are required to grow tumors in xenograft models - 1 in 1,000 to 1 in 5,000 lung cancer, neuroblastoma cells, ovarian cancer cells, or breast cancer cells can form colonies in soft agar or in vivo

Two models for tumor heterogeneity and propagation

Normal cellular hierarchy

Clonal evolution model: all undifferentiated cell have similar

tumorigenic capacity

Cancer stem cell model model: only CSC can generate tumor, based on

its self-renewal properties and enormous proliferative potential

Both models of tumor maintenance may underlie tumorigenesis

Early in situ lesion low heterogeneity

Advanced lesion high heterogeneity

cancer stem cells

proliferating tumor cells

mutation

asymmetrycal division

symmetrycal division

senescent/dying cancer cells

Tumor formation according to the CSC hypothesis. A mutated stem cell can expand by symmetrical and asymmetrical division, giving rise to daughter stem cells and progenitor cells, which in turn generate other tumor cells without self-renewal capability. Proliferating tumorigenic cells are the target of additional mutations that eventually result in tumor progression. As CSC divide and mutate, the tumor can become more heterogeneous, although rapidly dividing CSC derivatives are likely to be positively selected.

Defects in regulation of the switch between symmetric and asymmetric divisions can be deleterious

Sublethally irradiated NOD/SCID Mice

FACS Cell Sorter

Cancer Cells (ex: Leukaemia cells)

Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell

Prospective identification of tumorigenic breast cancer cells.

FACS Cell Sorter

Solid Tumor Single Cell Suspension CD24 Expression

CD

44 E

xpre

ssio

n

Mince (small pieces)

Surgical Implantation

Prospective isolation of human CSC from freshly dissociated tumors

Cancerstemcellsfeatures:exvivoandinvivoassaysforCSCs

Abilitytopropagateindenitely.

Giverisetotheirdieren>atedprogeny

Resemble,uponinjec>oninvivo,thetumoroforigin

Reciprocalinterac>onsbetweentheCSCanditsmicroenvironmentorniche

Tumor tissue Dissociation

Brain Tumor Stem Cell Culture

Xenografts

Differentiation

GFAP Neu-N

MRI

H&E

H&E H&E

DICT IF

Glioblastoma stem cells

Nature. 2007; 445(7123):111-5.

Cell Death and Differentiation. 2008; 15: 504-514.

Colon and lung cancer stem cells have been identified for the first time at ISS

Cancer Stem Cells

Cancer stem cells have been isolated from solid tumors by surface marker sorting (i.e. CD133) or by specific culture conditions that allow CSC-containing tumor spheres to growth.

Tumor Sphere

Xenograft

Sorting

Surgical biopsy (colon cancer)

Culture in serum free medium, containing EGF and

FGF2

CD

133

Conventional chemotherapy is unable to kill colon cancer stem cells

Stem cells Differentiated cells

Untreated 5-FU Irinotecan Oxaliplatin

CD

133+

cel

ls (%

) C

ell v

iabi

lity

(%)

0 20 40 60 80

100

5-FU Irinitecan Oxaliplatin

0 2 4 6 8

10

In vitro

In vivo

Control

Oxaliplatin

drug

0

0.4

0.8

1.2

1.6

2

2.4

4 6 8 10 12

Weeks

Tum

our s

ize

(cm

3 )

drug

0

CSC-based xenografts

Our approach to isolate lung cancer stem cells from primary tumors

In our laboratory, human CSC were isolated based on their ability to survive in serum-free conditions and to proliferate as cellular aggregates, so called tumor spheres.

This experimental strategy represents the best approach so far to obtain unlimited expansion of the tumorigenic cancer cell population from primary tumors, providing a powerful tool to allow extensive studies on these cells.

Tumor samples are obtained from consainting patients.Tissue dissociation is carried out by mechanical disgregation and enzymatic digestion with collagenase II 1.5 mg/ml and DNaseI 20g/ml. Recovered cells were cultured at clonal density in serum-free medium and supplemented with 20 g/ml EGF and 10 g/ml bFGF. Non-treated flasks for tissue culture were used to reduce cell adherence and support growth as tumor spheres.

Tumor dissociation Tumor sphere

Experimental Design

Lung cancer stem cells are resistant to conventional chemotherapyThe establishment of exponentially growing lung CSC cultures may allow the direct evaluation of the cytotoxicity of antineoplastic agents on the cells responsible for tumor growth and spreading, which represented the optimal cellular target for successful therapies.

Lung CSC were more resistant to chemotherapeutic drugs than differentiated cells in line with the poor therapeutic effect of conventional chemotherapy on lung cancer patients.

Cisplatin 5g/ml Etoposide 10g/mlGemcitabine 250 m Paclitaxel 30 ng/ml

LCNEC

AC SCC

Cel

l via

bilit

y (%

)

Cel

l via

bilit

y (%

) C

ell v

iabi

lity

(%)

Cel

l via

bilit

y (%

)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0 20 40 60 80

100

Control Cisplatin Etoposide Gemcitabine Paclitaxel

SCLC

0

5

10

15

20

25

30

35

40

NT CIS ETO PACLI GEMC

spheres differentiated

Cell d

eath

(%)

Models of tumor drug resistence

Conventional model: rare cells with genetic alterations that confer multidrug resistance (MDR) form a drug resistant clone (yellow). Following chemotherapy, these cells survive and proliferate, forming a recurrent tumour

Cancer-stemcell model: tumours contain a small population of tumour stem cells (red) and their differentiated offspring, which are committed to a particular lineage (blue). Following chemotherapy, the committed cells are killed, but the stem cells, which express drug transporters, survive. These cells repopulate the tumour

Acquired resistance stem-cell model: the tumour stem cells (red), which express drug transporters, survive the therapy, whereas the committed but variably differentiated cells are killed. Mutation(s) in the surviving tumour stem cells (yellow) and their descendants (purple) can arise conferring a drug-resistant phenotype

Intrinsic resistance model: both the stem cells (yellow) and the variably differentiated cells (purple) are inherently drug resistant, so therapies have little or no effect, resulting in tumour growth.

Cancer stem cells display enhanced resistance to radiation

drug resistance

conventional therapy

bulk tumor surviving CSCs tumor relapse

bulk tumor non-self renewing

tumor cells tumor

eradication

CSC-oriented therapy degeneration

proliferating tumoral cell

self-renewing cancer stem cell

Therapeutic implications of Cancer Stem Cells

Cancer stem cells Cancer stem cells are responsible for tumor cell development, maintenance and spreading

Tumor relapse results from residual cancer stem cells surviving the therapeutic treatment

Cancer stem cells should represent the primary target for new therapeutic strategies aimed at tumor eradication

Cancer stem cell analysis may provide considerable information for prognostic study and patient stratification

Options Characterization: immunoistochemistry screening, mRNA and protein analysis

Functional assays: in vitro drug screening, in vivo drug validation,

Results of analyses with the Cox Proportional-Hazard Models Overall Survival Progression-Free Survival

Variable Hazard Ratio (95% CI)

P Value

Hazard Ratio (95% CI)

P Value

CSC generation (vs. no CSC generation) Symptom duration 3 months (vs > 3 months) Partial surgery (vs. complete surgery) Age < 50 years (vs. 50) MGMT status negative (vs. positive) p53 status positive (vs. negative)

4.03 (1.82-8.93) 2.98 (1.25-7.09) 3.09 (1.43-6.69)

3.80 (1.44-10.04) 1.08 (0.52-2.22) 1.24 (0.54-2.84)

0.0006 0.0133 0.0042 0.0071 0.8369 0.6161

4.45 (2.03-9.78) 1.89 (0.89-4.01) 3.40 (1.58-7.31) 2.42 (1.04-5.63) 1.03 (0.52-2.05) 0.86 (0.39-1.88)

0.0002 0.0956 0.0018 0.0403 0.9231 0.7045

CD133/Ki67 positive (vs. negative) Symptom duration 3 months (vs > 3 months) Partial surgery (vs. complete surgery) Age < 50 years (vs. 50) MGMT status negative (vs. positive) p53 status positive (vs. negative)

4.87 (1.94-12.22) 4.95 (2.05-11.96) 3.21 (1.45-7.10) 1.95 (0.76-5.03) 1.06 (0.51-2.18) 1.06 (0.47-2.39)

0.0007 0.0004 0.0041 0.1674 0.8808 0.8925

6.64 (2.65-16.64) 3.46 (1.56-7.67) 3.80 (1.73-8.30) 1.35 (0.58-3.13) 0.92 (0.45-1.85) 0.86 (0.40-1.86)

3 months) Partial surgery (vs. complete surgery) Age < 50 years (vs. 50) MGMT status negative (vs. positive) p53 status positive (vs. negative)

1.81 (0.91-3.59) 1.04 (1.0-1.09)

3.44 (1.43-8.30) 3.23 (1.49-6.99) 2.53 (0.98-6.53) 1.13 (0.54-2.37) 0.99 (0.44-2.22)

0.0883 0.0674 0.0060 0.0029 0.0545 0.7467 0.9796

1.79 (0.9-3.56) 1.06 (1.02-1.10) 2.57 (1.19-5.52) 3.19 (1.48-6.93) 1.59 (0.66-3.81) 1.35 (0.65-2.82) 0.96 (0.42-2.16)

0.097 0.0061 0.0158 0.0033 0.2965 0.4185 0.957

P=0.0002

0 6 12 18 24 30 0

10 20 30 40 50 60 70 80 90

100

CSC generation

No CSC generation

Months

Prob

abili

ty o

f Ove

rall

Surv

ival

(%)

Cancer stem cell generation correlates with worse prognosis

Colon cancer stem cell characterization

Mutational profiling RasG12V

BRAFV600E PTEN PI3KCA TP53 Smad4 MSH6 MLH1

ctsc11 +/+ +/+ +/+ E542K +/+nfR361C +/+ +/+

ctsc12 +/+ +/+ +/+ E542K +/+nfR361C +/+ +/+

ctsc18 G12V +/+ +/+ +/+ +/fs +/+ +/+ down

ctsc85 +/+ +/+ down +/+ +/+nfR361C +/+ +/+

ctsc26 +/+ +/+ +/+ +/+ +/+nf+/+ +/+ down

Cancer stem cell banking

Tumor dissociation

Stem cell culture and expansion

Tumor database

H&E

Diagnosis and tissue banking

Available Resources

High throughput microRNA expression

Transcriptional profiling

Entero-endocrine FABP2 CHGA CHGB

REG1A SPON1 Paneth DEFA6 DEFA5 NELL2

SERPINI1 Enterocyte

TFF3 MUC3B MUC20 MUC1 MUC6

MUC12

CTS

C11

MSI2 SFRP5 (11,12)

MSI1 LGR4

LGR5 (85) NANOG

KLF4 POU5F1

LIN28 KLF5

PROM1 CD44

CTS

C18

CTS

C85

CTS

C26

CTS

C12

HC

T116

HT2

9

NM

uc

CTS

C22

0

Stem cell Culture

Differentiation

Tumor sphere

DIC

Absence of Growth Factors

Protein Lysate

Data Analysis

Arrayer

Nitrocellulose coated slides Antibody-based

phosphoprotein detection

Experimental design for reverse phase-based high throughput proteomic analysis

Compounds withunknown mechanismknown mechanism

Experimental protocol

In vitro drug testing

Resistant Sensitive

In vivo drug testing 1 2 3 4 5 6 7 8 9

80% 45-55%

Path

way

inhi

bito

rs

Patients

RPPA