Cellule staminali e cellule staminali tumorali

Presentazione sul tema: "Cellule staminali e cellule staminali tumorali"— Transcript della presentazione:

1 Cellule staminali e cellule staminali tumorali
Quali cellule sono responsabili per la crescita del tumore?

2 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

3 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

4 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

5 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

6

7 Rules of Normal Tissue Growth
1 2 3 Stem cells self renew--?immortal non-stem cells have finite life span

8 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

9 every cell in a tumor should initiate a new tumor
Non-stem tumor model: every cell in a tumor should initiate a new tumor

10 Experiments showed that very rare cells in a tumor
can transplant a new tumor: Tumor Stem Cells

11 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)

12 Tumor growth is similar to normal tissue growth
Normal Tissues Tumor Tumor stem cell = tumorigenic Adult stem cell = undifferentiated Transit amplifying cell Non-tumorigenic cell Normal differentiated cell

13 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”)

14 Evidenze per la presenza di cellule staminali tumorali

15 Hematopoietic Stem Cells
Multipotent Progenitors Oligolineage Mature CD34- CD38+ CD20+ CD8+ CD8+ CD34+ CD38- CD34- CD38- CD4+ CD4+ CD36+ CD35+ Reya et al Nature 414:

16 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

17 Self-renewal Assay in NOD/SCID Mice
(Non-obese diabetic/severe combined immunodeficiency) CD38 Expression CD34 Expression FACS Cell Sorter Cancer Cells ex: Leukaemia cells Sublethally irradiated NOD/SCID Mice

18 Leukaemia stem cells exist in human acute myeloid leukaemia (AML)
CD34+/ CD38- NOD/ SCID mice LEUKAEMIA Leukaemic blasts from AML patients CD34+/ CD38+ NO LEUKAEMIA John Dick and Dominique Bonnet

19 Leukaemia is arranged as a hierarchy similar to normal haematopoiesis
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

20 Le cellule staminali tumorali come meccanismo di mantenimento del tumore
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 CD sono leuchemogeniche, quelle CD34+CD38+ non possono trasferire la leucemia nell’animale immunocompromesso Le cellule tumorali non sono tutte uguali, e le CSC sono responsabili del mantenimento della massa tumorale

21 Evidenze da altri tumori
Nei tumori solidi si può osservare sperimentalmente una simile struttura gerarchica (i marker sono definiti in maniera meno precisa)

22 Therapeutic predictions of tumor stem cell model
stem cells Non-tumorigenic cells

23 Therapeutic predictions of tumor stem cell model
grows back rapid growing cells killed tumor degenerates kill stem cells

24 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

25 Cancer stem cells sono più resistenti alle terapie antitumorali

26 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

27 Brain tumor stem cells: identified by intracranial transplantation of CD133+ cells into adult NOD/SCID mouse forebrain. CD133+ CD133- Singh et al Nature 432:

28 Resistance to radiation: → given by CD133+

29 in vivo CD133+ enrichment after radiation
Glioma xenograft D456MG: Not induced by radiation itself →enriched CD133+ population 48h after radiation (3-5x)

30 in vitro CD133+ enrichment after radiation
Cultures from human glioma xenograft (D54MG): →48h after radiation: 3x enrichment Patient glioblastoma samples: Not induced by radiation itself

31 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+.

32 CD133+ subpopulation has cancer stem cell properties
Or proliferative ability

33 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. Minimum number of CD133+ for tumor initiation (8 weeks): No tumor detected with up to 2 x 106 CD133- (8 weeks) D456MG: pediatric xenograft D456MG CD133- (2 x 106) formed small tumors in 2 out of 5 xenotransplanted in immunocompromised mice.

34 È sufficiente attaccare esclusivamente le CSC?
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

35 Myeloid differentiation
Acute Promyelocytic Leukemia (APL) Myeloid differentiation Monoblast Promyelocytes Chr 15 Leukemia t(15;17) Chr 17

36 Leukemogenesis is a multi-stage process
Controls Leukemia-free survival (%) PML - RAR Pre-leukemia At the pre-leukemic stage, hematopoiesis is apparently normal

37 Molecular mechanism of PML-RAR action
DNMT/HMTs RA From DeThe and Chen

38 ATRA acts on bulk APL cells, and on LICs
tumor grows back PML-RAR degradation Tumor Recurrence LICs Bulk Cells

39 Continuous treatment with HDACi is required for prolonging survival of leukemic mice

40 Continuous treatment with HDACi is required for prolonging survival of leukemic mice
tumor grows back rapid growing cells killed

41 An assay to measure LICs Transplant in Limiting Dilutions
Bulk LIC Vehicle Treatment No Effect LIC Expansion LIC Reduction (Ly5.1+)) Leukemic Cells (Ly5.2) Drug treatment Harvest leukemic cells (Ly5.2+) treated/untreated Transplant in Limiting Dilutions (Ly5.1+)

42 An assay to measure LICs
Bulk LIC Vehicle Treatment No Effect LIC Expansion LIC Reduction Leukemic Cells (Ly5.2) Drug treatment Harvest leukemic cells (Ly5.2+) treated/untreated (Ly5.1+) Transplant in Limiting Dilutions ATRA treatment reduces LIC frequency ≈ 100 fold

43 VPA spares LICs Limiting Dilution Vehicle VPA LIC Frequency 2.5x10^4
Survival 4 weeks Limiting dilution 2 days harvest spl and limiting dilution

44 Short-term inhibition of multiple HDACs with SAHA tackles LICs but does not prolong survival
LIC assay Survival Survival 2 weeks leukemic splenomegaly and pb positive for 5.2 Limitin dilution after 2 days of treatment Vehicle SAHA LIC Frequency 2.5x10^4 2.3x10^6

45 In Summary… ATRA Leukemia Tumor Recurrence ? SAHA VPA LICs Bulk Cells

46 Eradication of APL by ATRA-SAHA-VPA
No leukemic cells detectable

47 Self-renewal Assay in NOD/SCID Mice
For solid tumors: surgical orthotopic implantation (SOI) CD24 Expression CD44 Expression FACS Cell Sorter Single Cell Suspension Solid Tumor Mince (small pieces) Surgical Implantation

48 CD 44 staining of breast cancer model
T. A. Ince 2001

49 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

50

51

52

53 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

54 WT ErbB2 Analysis of the replicative potential of
Normal and Tumor mammospheres: (serial growth) WT ErbB2 decrease in number during passages (limited lifespan) increase in number during passages (near-immortal) ErbB2 R2: 0,98 502% decrease 101 105 103 10-1 WT R2: 0,99 64% ErbB2 : fixed increase at every passage (502%) WT : fixed decrease at every passage (64%) (exponential curves)

55 Stem Cell divisions permit Asymmetric cell division
generation of more SCs (‘self-renewal’) and production of cells that differentiate Asymmetric cell division Mechanisms: Asymmetric localizzation of cell polarity (PINS and aPKC) and cell fate determinants (Numb and Prospero) Asymmetric placement of daughter cells relative to the stem cell niche Pr. SC SC Each SC divides to generate one daughter with SC fate and one that differentiates (progenitror) This strategy leaves stem cells unable to expand in number

56 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: Some mammalian SCs use conserved mechanism to divide asymmetrically; Mammalian SCs can expand in number during development (HSCs, Neural and Epidermal SCs) or after injury (neural SCs after stroke or HSCs after chemotherapy). SCs are defined by their abilities to generate more SCs (‘self-renewal’) and to produce cells that differentiate. One mechanism by which SCs accomplish these two tasks is asymmetric cell division, whereby each SC divides to generate one daughter with SC fate and one that differentiates. SCs, however, possess the ability to expand in number, as it occurs during development and in adulthood after injury or disease. This increase is not accounted by asymmetric divisions, in which only one daughter cell maintains SC identity. Recent findings in C.elegans and Drosophila indicate that SCs can also generate daughter cells that are destined to acquire the same fate (symmetric cell division). Limited data are available on the modes of division of mammalian SCs in vivo. Some mammalian SCs use conserved mechanisms to divide asymmetrically (asymmetric partitioning of cell polarity/fate determinants). As in invertebrates, however, they can expand in number during development (HSCs; neural and epidermal SCs) or after injury (neural SCs after stroke or HSCs after chemotherapy), suggesting that they can also divide symmetrically.

57 Increased frequency of Symmetric Divisions
in tumor cells (ErbB2) vs WT cells WT ErbB2 Uncertain Asymmetric 11,5% 10,3% Uncertain 33,3% Asymmetric Symmetric 59,5% 7,2% Symmetric 78,2%

58 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

59 Importanza del topo ricevente e delle condizioni sperimentali

60 I melanomi possono iniziare a partire da una singola cellula

61 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”