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

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).

Jordan et al. 2006 NEJM 355:1253-61

Development of Hematopoietic Stem Cells

Stem
Cells

Multipotent
Progenitors

Oligolineage
Progenitors

Mature
Cells

HSCs can be subdivided

into long-term self-renewing
HSCs,
short-term
selfrenewing
HSCs
and
multipotent progenitors (red
arrows
indicate
selfrenewal). 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.

Reya et al. 2001 Nature 414:105-111

CD34+ cells

Peripheral Blood, Bone Marrow

or Cord Blood-derived cells

tive

ga
d ne
n
a
ive
Posit

rare HSC
in progenitor cell
background

tion
c
e
l
se

In vivo transfer to
irradiated recipient mice

Recapitulation of the
haematopoietic system in
the recipient mouse

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

cancer
stem cells
proliferating
tumor cells
mutation
asymmetrycal
division

symmetrycal
division

Advanced lesion
high heterogeneity

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

Human acute myeloid leukemia is organized as a hierarchy

that originates from a primitive hematopoietic cell

FACS Cell
Sorter
Cancer Cells
(ex: Leukaemia cells)

Sublethally irradiated NOD/SCID Mice

FACS Cell
Sorter
Solid Tumor

Mince
(small
pieces)

Single Cell
Suspension

Surgical
Implantation

CD44 Expression

Prospective identification of tumorigenic breast cancer cells.

CD24 Expression

Prospective isolation of human CSC from freshly

dissociated tumors

Cancerstemcellsfeatures:exvivoandinvivoassaysforCSCs
Abilitytopropagateindenitely.

Giverisetotheirdieren>atedprogeny

Resemble,uponinjec>oninvivo,thetumoroforigin

Reciprocalinterac>onsbetweentheCSCanditsmicroenvironment
orniche

Glioblastoma
stem cells

MRI

Tumor tissue
Dissociation

H&E

Brain Tumor Stem

Cell Culture

IF
Differentiation

GFAP

Xenografts

Neu-N

H&E

H&E

DICT

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

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

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

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.
Surgical biopsy (colon cancer)

Culture in
serum free medium,
containing EGF and
FGF2

CD133

Sorting

Tumor Sphere

Xenograft

Conventional chemotherapy is unable to kill colon

cancer stem cells
Stem cells

CSC-based xenografts

Cell viability (%)

Control
Oxaliplatin

drug

1.6
drug

1.2

In vitro

100
80
60
40
20
0
5-FU

0.8
0.4
0
0

Weeks

10

12

CD133+ cells (%)

Tumour size (cm )

2.4

Differentiated cells

10

Irinitecan Oxaliplatin

In vivo

8
6
4
2
0
Untreated

5-FU

Irinotecan Oxaliplatin

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.
Experimental Design
Tumor
dissociation

Tumor sphere

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.

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.

Lung cancer stem cells are resistant to conventional chemotherapy

The 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.
Etoposide
100

100
80
60
40
20
0

40

40

35

20

LCNEC
Cell viability (%)

80
60
40
20

100
80
60
40

AC

Etoposide 10g/ml
Paclitaxel 30 ng/ml

spheres

differentiated

30
25
20
15
10
5

20

Cisplatin 5g/ml
Gemcitabine 250 m

60

100

Paclitaxel

80

SCLC
Cell viability (%)

Gemcitabine

Cell death (%)

Cisplatin

Cell viability (%)

Cell viability (%)

Control

SCC

NT

CIS

ETO

PACLI

GEMC

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.

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

Therapeutic implications of Cancer Stem Cells

conventional
therapy

drug
resistance

surviving CSCs

bulk tumor

tumor relapse

proliferating
tumoral cell
self-renewing
cancer stem cell

CSC-oriented
therapy

bulk tumor

degeneration

non-self renewing
tumor cells

tumor
eradication

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
Variable

Progression-Free Survival

Hazard Ratio
(95% CI)

P
Value

Hazard Ratio
(95% CI)

P
Value

CSC generation (vs. no CSC generation)

4.03 (1.82-8.93)

0.0006

4.45 (2.03-9.78)

0.0002

Symptom duration 3 months (vs > 3 months)

2.98 (1.25-7.09)

0.0133

1.89 (0.89-4.01)

0.0956

Partial surgery (vs. complete surgery)

3.09 (1.43-6.69)

0.0042

3.40 (1.58-7.31)

0.0018

Age < 50 years (vs. 50)

3.80 (1.44-10.04)

0.0071

2.42 (1.04-5.63)

0.0403

MGMT status negative (vs. positive)

1.08 (0.52-2.22)

0.8369

1.03 (0.52-2.05)

0.9231

p53 status positive (vs. negative)

1.24 (0.54-2.84)

0.6161

0.86 (0.39-1.88)

0.7045

CD133/Ki67 positive (vs. negative)

4.87 (1.94-12.22)

0.0007

6.64 (2.65-16.64)

<.0001

Symptom duration 3 months (vs > 3 months)

4.95 (2.05-11.96)

0.0004

3.46 (1.56-7.67)

0.0022

Partial surgery (vs. complete surgery)

3.21 (1.45-7.10)

0.0041

3.80 (1.73-8.30)

0.0008

Age < 50 years (vs. 50)

1.95 (0.76-5.03)

0.1674

1.35 (0.58-3.13)

0.4840

MGMT status negative (vs. positive)

1.06 (0.51-2.18)

0.8808

0.92 (0.45-1.85)

0.8080

p53 status positive (vs. negative)

1.06 (0.47-2.39)

0.8925

0.86 (0.40-1.86)

0.7064

CD133 2 percent (vs < 2 percent)

1.81 (0.91-3.59)

0.0883

1.79 (0.9-3.56)

0.097

Ki67 expression

1.04 (1.0-1.09)

0.0674

1.06 (1.02-1.10)

0.0061

Symptom duration 3 months (vs > 3 months)

3.44 (1.43-8.30)

0.0060

2.57 (1.19-5.52)

0.0158

Partial surgery (vs. complete surgery)

3.23 (1.49-6.99)

0.0029

3.19 (1.48-6.93)

0.0033

Age < 50 years (vs. 50)

2.53 (0.98-6.53)

0.0545

1.59 (0.66-3.81)

0.2965

MGMT status negative (vs. positive)

1.13 (0.54-2.37)

0.7467

1.35 (0.65-2.82)

0.4185

p53 status positive (vs. negative)

0.99 (0.44-2.22)

0.9796

0.96 (0.42-2.16)

0.957

Cancer stem cell generation correlates with worse prognosis

Probability of Overall Survival (%)

100
90
80
70
60
50

No CSC generation

40
30

P=0.0002

20

CSC generation

10
0

12

18
Months

24

30

Colon cancer stem cell characterization

Mutational profiling
Ras BRAF
G12V V600E PTEN PI3KCA TP53 Smad4 MSH6 MLH1
ctsc11 +/+

+/+

+/+

E542K +/+nfR361C +/+

+/+

ctsc12 +/+

+/+

+/+

E542K +/+nfR361C +/+

+/+

ctsc18 G12V +/+

+/+

+/+

down

ctsc85 +/+

+/+

down +/+

+/+nfR361C +/+

+/+

ctsc26 +/+

+/+

+/+

+/+nf+/+

down

+/+

+/fs +/+

+/+

+/+

Available Resources
Diagnosis and
tissue banking

Tumor
database

H&E

Tumor
dissociation
Stem cell culture
and expansion

Cancer stem cell

banking

Transcriptional profiling
High throughput
microRNA expression

NMuc

HT29

HCT116

CTSC12

CTSC26

CTSC85

CTSC18

CTSC11

CTSC220

MSI2
SFRP5 (11,12)
MSI1
LGR4
LGR5 (85)
NANOG
KLF4
POU5F1
LIN28
KLF5
PROM1
CD44
Entero-endocrine
FABP2
CHGA
CHGB
REG1A
SPON1
Paneth
DEFA6
DEFA5
NELL2
SERPINI1
Enterocyte
TFF3
MUC3B
MUC20
MUC1
MUC6
MUC12

Experimental design for reverse phase-based high

throughput proteomic analysis
Stem cell
Culture

Tumor sphere
DIC

Differentiation

Absence of
Growth Factors
Protein Lysate

Arrayer
Nitrocellulose coated slides

Antibody-based
phosphoprotein detection

Data Analysis

Experimental protocol
Compounds with
known mechanism unknown mechanism

RPPA
In vitro drug testing

Resistant

Sensitive

Patients
1 2 3 4 5 6 7 8 9

Pathway inhibitors

In vivo drug testing

<5%
45-55%
>80%