Post on 02-May-2015
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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 self-renewal e nell’oncogenesi
• Ipotesi: le cellule tumorali -capaci di self-renewal- 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 self-renewal 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
1
3
2
Stem cells self renew--?immortal non-stem cells have finite life span
Rules of Normal Tissue Growth
1
3
2
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
Traditional View of Tumor Growth
Non-stem tumor model: every cell in a tumor should initiate a new tumor
Experiments showed that very rare cells in a tumorcan 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 TumorsIn 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)
Adult stem cell = undifferentiated
Transit amplifying cell
Tumor stem cell = tumorigenic
Tumor growth is similar to normal tissue growth
Normal differentiated cell
Non-tumorigenic cell
Normal Tissues Tumor
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
StemCells
MultipotentProgenitors
OligolineageProgenitors
MatureCells
Reya et al. 2001 Nature 414:105-111
CD34+CD38- CD4+
CD4+
CD35+
CD36+
CD8+
CD8+
CD20+CD34-CD38+
CD34-CD38-
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(Non-obese diabetic/severe combined immunodeficiency)
Sublethally irradiated NOD/SCID Mice
FACS Cell Sorter
Cancer Cellsex: Leukaemia cells CD38 Expression
CD
34 E
xpre
ssio
n
Leukaemia stem cells exist in human acute myeloid leukaemia (AML)
Leukaemic
blastsfrom
AML patients
CD34+/ CD38-
CD34+/ CD38+
LEUKAEMIA
NO LEUKAEMIA
John Dick and Dominique Bonnet
NOD/ SCIDmice
Leukaemia is arranged as a hierarchy similar to normal haematopoiesis
B-cell T-cell Erythrocyte Platelet Monocyte Granulocyte
CD34+/ CD38-
lymphoidprogenitor
myeloidprogenitor
HSC
NORMALLEUKAEMIA
Leukaemogenicevents
Bulk leukaemiacells (CD34+/CD38- and other cells)
Block terminaldifferentiation
John Dick and Dominique Bonnet
Le cellule staminali tumorali come meccanismo di mantenimento del tumore
1. 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
2. Reinoculo di queste cellule in topi NOD/SCID ed analisi della loro capacitá leuchemogenica
3. 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)
Tumor stem cells
Non-tumorigenic cells
Therapeutic predictions of tumor stem cell model
tumor grows back
tumor degenerates
Therapeutic predictions of tumor stem cell model
rapid growing cells killed
kill stem cells
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 modelsin vitro models (ex vivo )
• Cultured cell from human gliomas:D456MGD54MG
• 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+
• Glioma xenograft D456MG:
in vivo CD133+ enrichment after radiation
→enriched CD133+ population 48h after radiation (3-5x)
in vitro CD133+ enrichment after radiation
• Cultures from human glioma xenograft (D54MG):
→48h after radiation: 3x enrichment
• Patient glioblastoma samples:
Irradiation effects at molecular level
Early DNA damage checkpoint responses (phosphorylation) checked before treatment and after 1h.Higher amount of phosphorylated proteins in CD133+.
Early DNA damage checkpoint responses:
CD133+ subpopulation has
cancer stem cell properties
in vivo tumorigenic potentialof purified CD133+ tumor cells
D456MG CD133- (2 x 106) formed small tumors in 2 out of 5 xenotransplanted in immunocompromised mice.
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.
in vitroirradiation
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
Promyelocytes
Chr 15
Chr 17
t(15;17)
Acute Promyelocytic Leukemia (APL)
Myeloid differentiation
Monoblast
Leukemogenesis is a multi-stage process
Leu
kem
ia-f
ree
surv
ival
(%
)
Pre-leukemia
At the pre-leukemic stage, hematopoiesis is apparently normal
Molecular mechanism ofPML-RAR action
From DeThe and Chen
RA
DNMT/HMTs
tumor grows back
PML-RAR degradation
APL Tumor Recurrence
LICs
Bulk Cells
ATRA
ATRA acts on bulk APL cells, and on 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
tumor grows back
rapid growing cells killed
An assay to measure LICs
Leukemic Cells (Ly5.2)
Drug treatment
Harvest leukemiccells (Ly5.2+)
treated/untreated
(Ly5.1+)
Transplant in Limiting Dilutions
BulkLIC
Vehicle
Treatment
No Effect
LIC Expansion
LIC Reduction
(Ly5.1+))
An assay to measure LICs
BulkLIC
Vehicle
Treatment
No Effect
LIC Expansion
LIC Reduction
Leukemic Cells (Ly5.2)
Drug treatment
Harvest leukemiccells (Ly5.2+)
treated/untreated
(Ly5.1+)
Transplant in Limiting Dilutions
(Ly5.1+)
ATRA treatment reduces LIC frequency ≈ 100 fold
VPA spares LICs
Vehicle VPA
LIC Frequency 2.5x10^4 3.9x10^4
Limiting Dilution
Short-term inhibition of multiple HDACs with SAHA tackles LICs but does not prolong survival
Survival
Vehicle SAHA
LIC Frequency 2.5x10^4 2.3x10^6
LIC assay
Leukemia Tumor Recurrence
VPA
SAHA
LICs
Bulk Cells
ATRA
In Summary…
?
Eradication of APL by ATRA-SAHA-VPA
No leukemic cells detectable
Self-renewal Assay in NOD/SCID MiceFor solid tumors: surgical orthotopic implantation (SOI)
FACS Cell Sorter
Solid TumorSingle CellSuspension
Mince (small pieces)
Surgical Implantation
CD24 Expression
CD
44 E
xpre
ssio
n
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 moleculesB38.1 – breast/ovarian cancer-specific markerESA – epithelial specific antigen
Al-Hajj (2003) PNAS 100, 3983
Transplantation into the cleared fat pad of syngenic mice:
• WT mammospheres form a normal breast tissue
• ErbB2 mammospheres form tumors
1. Are Clonal in origin
2. Grow serially (self-renewal)
3. Contain SCs
Biological characterization of WT and ErbB2 Mammospheres
decrease in number during passages (limited lifespan)
Analysis of the replicative potential of Normal and Tumor mammospheres:
(serial growth)WT
increase in number during passages (near-immortal)
ErbB2
R2: 0,98 502%
decrease
101
105
103
10-1
WT
R2: 0,99 64%
decrease
ErbB2 : fixed increase at every passage (502%)
WT : fixed decrease at every passage (64%)
(exponential curves)
ErbB2
Stem Cell divisions permit generation of more SCs (‘self-renewal’) and
production of cells that differentiate
1. Asymmetric cell division
Pr.
SC
SC
Each SC divides to generate one daughter with SC fate and one that differentiates (progenitror)
Mechanisms:
1. Asymmetric localizzation of cell polarity (PINS and aPKC) and cell fate determinants (Numb and Prospero)
2. Asymmetric placement of daughter cells relative to the stem cell nicheThis strategy leaves stem cells
unable to expand in number
2. Symmetric cell division
Each SC divides to generate daughter cellsthat are destined to acquire the same fate
SC
SC
Pr.
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).
ErbB2
Asymmetric10,3%
Symmetric78,2%
Uncertain11,5%
WT
Asymmetric59,5%Symmetric
7,2%
Uncertain33,3%
Increased frequency of Symmetric Divisions in tumor cells (ErbB2) vs WT cells
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”