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”