Transcript Document

Cellule staminali e cellule
staminali tumorali
Quali cellule sono responsabili per la
crescita del tumore?
Cellule staminali
• Sono cellule che hanno la capacitá di perpetuarsi
indefinitamente (“self-renewal”)
• Attraverso il differenziamento, esse danno vita
alle cellule “mature”
• Le cellule differenziate originano dalle cellule
staminali del medesimo compartimento
• Plasticitá delle cellule staminali: apparentemente,
le cellule staminali di un tessuto possono dare
origine anche a cellule mature di altri tessuti
Cellule staminali e cellule
tumorali
•Il tumore è costituito da cellule con
una capacità di self-renewal indefinita
•La comprensione dei meccanismi di
self-renewal delle cellule staminali
puó aiutarci a comprendere il tumore
Pathway coinvolti nel selfrenewal e nell’oncogenesi
• Ipotesi: le cellule tumorali -capaci di selfrenewal- utilizzano la “machinery” presente
nelle cellule staminali
• Dimostrazione indiretta di tale ipotesi é il
fatto che diversi pathway associati
all’oncogenesi sono stati coinvolti nel selfrenewal delle cellule staminali
I pathway di Notch, Shh, Wnt
• Notch: l’attivazione di questo pathway é associata
ad un aumento del pool delle cellule staminali
• Shh: popolazioni arricchite di cellule staminali
umane rispondono in vitro a Shh con un
aumentato self-renewal
• Wnt: la sua attivazione espande il pool di cellule
staminali, mentre la sua soppressione inibisce la
proliferazione delle cellule staminali
Rules of Normal Tissue Growth
1
2
3
Stem cells self renew--?immortal
non-stem cells have finite life span
Traditional View of Tumor Growth
1
2
3
Rules:
1.) Tumors are clonal – starts in a single cell
2.) All tumor cells have infinite lifespan
3.) All tumor cells divide symmetrically
30 cell divisions = 1 billion cells = 1 cm tumor
Non-stem tumor model:
every cell in a tumor should initiate a new tumor
Experiments showed that very rare cells in a tumor
can transplant a new tumor: Tumor Stem Cells
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 could 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 cell from cell lines can
form colonies in soft agar or in vivo (fewer with 10 tumor cells)
Tumor growth is similar to normal tissue growth
Normal Tissues
Adult stem cell = undifferentiated
Transit amplifying cell
Normal differentiated cell
Tumor
Tumor stem cell = tumorigenic
Non-tumorigenic cell
Cellule staminali tumorali:
organogenesi aberrante
• Il tumore puó essere immaginato come un organo
aberrante originato da una cellula trasformata che ha
acquisito la capacitá di proliferare indefinitamente
attraverso varie mutazioni
• La popolazione tumorale é eterogenea, e spesso contiene
cellule a diversi stadi di differenziamento (seppure
anomali): data la clonalitá dei tumori, questo dato implica
che la progenie delle cellule tumorali si diversifica
(“differenzia”)
Evidenze per la presenza di
cellule staminali tumorali
Hematopoietic Stem Cells
Stem
Cells
Multipotent
Progenitors
Oligolineage
Progenitors
CD34CD38+
Mature
Cells
CD20+
CD8+
CD8+
CD34+
CD38-
CD34CD38-
CD4+
CD4+
CD36+
CD35+
Reya et al. 2001 Nature 414:105-111
Cellule staminali ematopoietiche
• Le cellule caratterizzate con maggiore precisione,
grazie ad esperimenti di ripopolamento di topi
letalmente irradiati e ricostituiti con popolazioni
cellulari altamente purificate a partire dal midollo
osseo
• Le cellule staminali (0.05% delle cellule totali del
midollo) danno origine ai progenitori
ematopoietici che perdono il loro potenziale di
self-renewal
Self-renewal Assay in NOD/SCID Mice
FACS Cell
Sorter
Cancer Cells
ex: Leukaemia cells
Sublethally irradiated NOD/SCID Mice
CD34 Expression
(Non-obese diabetic/severe combined immunodeficiency)
CD38 Expression
Leukaemia stem cells exist in human acute myeloid
leukaemia (AML)
CD34+/ CD38-
Leukaemic
blasts
from
AML
patients
CD34+/ CD38+
N
O
D
/
S
C
I
D
m
i
c
e
LEUKAEMIA
NO LEUKAEMIA
John Dick and Dominique Bonnet
Leukaemia is arranged as a hierarchy similar to normal
haematopoiesis
LEUKAEMIA
NORMAL
CD34+/ CD38-
HSC
Leukaemogenic
events
lymphoid
progenitor
myeloid
progenitor
Bulk leukaemia
cells (CD34+/CD38- and other cells)
Block terminal
differentiation
John Dick and Dominique Bonnet
B-cell
T-cell
Erythrocyte Platelet
Monocyte Granulocyte
Le cellule staminali tumorali come
meccanismo di mantenimento del tumore
1.
2.
3.
Isolamento di sub-popolazioni cellulari con marcatori di superficie
caratteristici delle SC normali (CD34+CD38-), o di cellule piú
differenziate, da blasti leucemici di pazienti affetti da varie forme di
leucemia mieloide acuta
Reinoculo di queste cellule in topi NOD/SCID ed analisi della loro
capacitá leuchemogenica
Mentre le cellule CD34+38- sono leuchemogeniche, quelle
CD34+CD38+ non possono trasferire la leucemia nell’animale
immunocompromesso
4. Le cellule tumorali non sono tutte
uguali, e le CSC sono responsabili del
mantenimento della massa tumorale
Evidenze da altri tumori
Nei tumori solidi si può osservare
sperimentalmente una simile struttura
gerarchica (i marker sono definiti in
maniera meno precisa)
Therapeutic predictions of tumor stem cell model
Tumor
stem cells
Non-tumorigenic
cells
Therapeutic predictions of tumor stem cell model
rapid growing
cells killed
kill
stem cells
tumor
grows back
tumor
degenerates
Therapeutic implications of Cancer Stem Cells
Hypothesis:
-Most therapies (chemotherapy and radiation) target rapidly
proliferating, non-tumorigenic cells and spare the relatively
quiescent cancer stem cells
-Cell surface pumps
-Cancer stem cells have greater invasive and migratory
properties and can home to specific tissue niches
Cancer stem cells sono più
resistenti alle terapie antitumorali
Experimental models
in vitro models (ex vivo )
• Cultured cell from human gliomas:
D456MG
D54MG
• Patient glioblastoma samples
in vivo models
• Human xenograft models in immunocompromised mice
Brain tumor stem cells: identified by intracranial transplantation of
CD133+ cells into adult NOD/SCID mouse forebrain.
CD133+
CD133+
CD133-
Singh et al. 2004 Nature 432: 396-401
Resistance to radiation:
→ given by CD133+
in vivo CD133+ enrichment after radiation
• Glioma xenograft D456MG:
→enriched CD133+ population 48h after radiation (3-5x)
in vitro CD133+ enrichment after radiation
• Cultures from human glioma xenograft (D54MG):
• Patient glioblastoma samples:
→48h after
radiation: 3x
enrichment
Irradiation effects at molecular level
Early DNA damage checkpoint responses:
Early DNA damage checkpoint responses (phosphorylation) checked before
treatment and after 1h.
Higher amount of phosphorylated proteins in CD133+.
CD133+ subpopulation has
cancer stem cell properties
in vivo tumorigenic potential
of purified CD133+ tumor cells
in vitro
irradiation
CD133+ cells (104) from patient sample or xenograft transplanted into
brains of immunocompromised mice.
Brain observed at appearence of neurological signs or after 8 weeks.
D456MG CD133- (2 x 106) formed small tumors in 2 out of 5
xenotransplanted in immunocompromised mice.
Domanda fondamentale
È sufficiente attaccare esclusivamente
le CSC?
Nessuno ha finora dimostrato che
l'incapacità di self-renewal delle CSC
sia sufficiente ad impedire lo
sviluppo di un tumore
Acute Promyelocytic Leukemia (APL)
Myeloid differentiation
Monoblast
Promyelocytes
PML
Chr 15
RAR
Chr 17
PML RAR
t(15;17)
Leukemia-free survival (%)
Leukemogenesis is a multi-stage process
Pre-leukemia
At the pre-leukemic stage, hematopoiesis is apparently norma
Molecular mechanism of
PML-RAR action
DNMT/HMTs
RA
From DeThe and C
ATRA acts on bulk APL cells, and on LICs
APL
ATRA
Tumor Recurrenc
tumor
grows back
PML-RAR degradation
Bulk Cells
LICs
Continuous treatment with HDACi is required
for prolonging survival of leukemic mice
Continuous treatment with HDACi is required
for prolonging survival of leukemic mice
rapid growing
cells killed
tumor
grows back
An assay to measure LICs
Bulk
LIC
(Ly5.1+))
Vehicle
Treatment
No Effect
Leukemic Cells (Ly5.2)
Drug treatment
Harvest leukemic
cells (Ly5.2+)
treated/untreated
LIC Expansion
Transplant in Limiting Dilutions
(Ly5.1+)
LIC Reduction
An assay to measure LICs
Bulk
LIC
(Ly5.1+)
Vehicle
Treatment
No Effect
Leukemic Cells (Ly5.2)
Drug treatment
Harvest leukemic
cells (Ly5.2+)
treated/untreated
LIC Expansion
Transplant in Limiting Dilutions
(Ly5.1+)
LIC Reduction
ATRA treatment reduces LIC frequency ≈ 100 fold
VPA spares LICs
Limiting Dilution
Vehicle
LIC Frequency
2.5x10^4
VPA
3.9x10^4
Short-term inhibition of multiple HDACs with SAHA
tackles LICs but does not prolong survival
Survival
LIC assay
LIC Frequency
Vehicle
SAHA
2.5x10^4
2.3x10^6
In Summary…
Leukemia
ATRA
Tumor Recurrenc
?
SAHA
VPA
Bulk Cells
LICs
Eradication of APL by ATRA-SAHA-VPA
No leukemic cells
detectable
Self-renewal Assay in NOD/SCID Mice
FACS Cell
Sorter
Solid Tumor
Mince
(small
pieces)
Single Cell
Suspension
Surgical
Implantation
CD44 Expression
For solid tumors: surgical orthotopic implantation (SOI)
CD24 Expression
CD 44 staining of breast cancer model
T. A. Ince 2001
Breast Cancer Stem Cells: CD44+ CD24low Lin- B38.1+ ESA+
CD44 and CD24 – adhesion molecules
B38.1 – breast/ovarian cancer-specific marker
ESA – epithelial specific antigen
Al-Hajj (2003) PNAS 100, 3983
Biological characterization of
WT and ErbB2 Mammospheres
1. Are Clonal in origin
2. Grow serially (self-renewal)
3. Contain SCs
Transplantation into the cleared fat pad of syngenic
mice:
•
WT mammospheres form a normal breast tissue
•
ErbB2 mammospheres form tumors
cell n.
wt (FVB)
100000
5/5
10000
11/11
1000
6/6
ErbB2
4/4
2/2
6/6
Analysis of the replicative potential of
Normal and Tumor mammospheres:
(serial growth)
WT
WT
12
6
6
0
0
1
2
3
4
5
45000
160
30000
80
15000
0
6
0
1
passages
cumulative
sphere number
12
ErbB2
240
sphere number
18
cumulative
sphere number
sphere number
18
ErbB2
2
3
4
5
6
passages
decrease in number during
passages (limited lifespan)
1,E+05
105
increase in number during
passages (near-immortal)
ErbB2
cumulative sphere number
1,E+04
R2: 0,98
502%
decrease
1,E+03
103
1,E+02
ErbB2 : fixed increase at every
passage (502%)
(exponential curves)
1,E+01
101
R2: 0,99
64%
decrease
1,E+00
1,E-01
10-1
WT
1,E-02
1
3
passages
5
WT : fixed decrease at every
passage (64%)
Stem Cell divisions permit
generation of more SCs (‘self-renewal’) and
production of cells that differentiate
1. Asymmetric cell division
Pr.
SC
Mechanisms:
1. Asymmetric localizzation of
cell polarity (PINS and aPKC)
and cell fate determinants
(Numb and Prospero)
SC
2. Asymmetric placement of
daughter cells relative to
the stem cell niche
Each SC divides
to generate one daughter
with SC fate and one that
differentiates
This strategy leaves stem cells
(progenitror)
unable to expand in number
2. Symmetric cell division
Pr.
Each SC divides to
generate daughter cells
that are destined
to acquire the same fate
SC
SC
SC
SC
Limited data available on the modes of division of
mammalian SCs:
1. Some mammalian SCs use conserved mechanism to divide
asymmetrically;
2. Mammalian SCs can expand in number during development (HSCs,
Neural and Epidermal SCs) or after injury (neural SCs after stroke
or HSCs after chemotherapy).
Increased frequency of Symmetric Divisions
in tumor cells (ErbB2) vs WT cells
ErbB2
WT
Uncertain Asymmetric
10,3%
11,5%
Uncertain
33,3%
Symmetric
7,2%
Asymmetric
59,5%
Symmetric
78,2%
Nuovi risultati e incertezze
• I dati di maggiore rilevanza a supporto della teoria
delle CSC derivano da xenotrapianti di cellule
tumorali umane in topi immunocompromessi
• Molto recentemente è apparso un lavoro molto
importante sulla caratterizzazione delle CSC nel
melanoma, dove emerge che:
– almeno in questo tumore, il numero di cellule con
caratteristiche di CSC è altissimo (se si accettano
alcune assunzioni, si arriva quasi al 100% delle cellule):
se tutte le cellule sono CSC, le CSC non esistono
– i protocolli sperimentali per gli xenotrapianti possono
influenzare l’attecchimento di determinate
sottopopolazioni
Importanza del topo ricevente e
delle condizioni sperimentali
I melanomi possono iniziare a
partire da una singola cellula
Ci sono modelli
complementari/alternativi?
• Plasticità fenotipica: non c’è una vera e propria
gerarchia (staminale->non-staminale), ma diversi
stati cellulari determinati dalle condizioni
“ambientali” (microambiente e segnali)
– La stessa cellula può assumere reversibilmente
morfologia diversa, espressione di diversi pattern
trascrizionali e non di mutazioni irreversibili,
manifestando nei suoi diversi fenotipi una maggiore o
minore propensione alla “staminalità”