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Stem Cells and Cancer

VIEWS: 3 PAGES: 36

									Stem Cells and Cancer


               Paul Clark
   Stem Cell Training Program Fellow
   Brain Tumor Research Laboratory
   B i T        R       hL b
  Department of Neurological Surgery
                   Outline

• What is a stem cell?
  What is a stem cell?
• How does stem cell theory apply to cancer?
• How can cancer stem cell hypothesis be 
                         ll h   h i b
  applied to development of novel therapies?
                   What is a stem cell?
• Self renewal: Able to reproduce without becoming
  Self‐renewal:  Able to reproduce without becoming 
    specialized, characterized by cell division in which 
    differentiation is blocked in at least one daughter cell
• Potency: Ability to differentiate to multiple types of 
    specialized cells under proper stimulation

• Pluripotent: Form all tissues 
  of adult body (embryonic 
           ll
  stem cell, ESC))                                          Embryonic stem cell


• Multipotent:  Form all cells of                              Adult stem cell

  a single tissue of the body 
  (adult stem cells)

                                                                 Differentiated




                                                            www.wikipedia.org
        Stem cells recapitulate tissue in vivo




                                                                        Cartilage/ Bone                  Intestine
                 Feeder cells                                            (mesoderm)                    (endoderm)




   ESC                                 ESC
                                                                           Neural                      Intestine
                                                                         (ectoderm)




Gertow et al., “Isolation of Human Embryonic Stem Cell‐Derived Teratomas for the Assessment of Pluripotency”, Curr. 
Protoc. Stem Cell Biol. 3:1B.4.1‐1B.4.29, 2007. 
Adult stem cells can be isolated from many tissues
     Clinical challenge of malignant brain tumors
                                                           Supratentorial Tumors
                                                               Astrocytoma
                                                               Oligodendroglioma
                                                               Ependymoma
                                                               Craniopharyngioma
                                                               Germinoma
                                                               Choroid Plexus Tumors
                                                               Meningioma

                                                             Infratentorial Tumors
                                                                      y
                                                                Astrocytoma
                                                                Ependymoma
                                                                Medulloblastoma
                                                                Atypical Teratoid
                                                                Meningioma
                                                                      g



•   Estimated 44,685 new cases of primary brain tumors to be diagnosed in
         (CBTRUS
    2007 (CBTRUS, 2006)
    – In children, brain tumors most common solid tumor (ACS, 2006)
Gliomas:
Astrocytoma
Oligodendroglioma
Ependymoma

Glioma grading (I-IV):
Nuclear atypia
Mitosis
V    l     d th li l    lif ti
Vascular endothelial proliferation
Necrosis

Grade IV Astrocytoma:
Glioblastoma multiforme (GBM)
Despite advances in surgery, imaging,
              chemotherapies
radiation and chemotherapies…
      Overall survival for GBM remains poor


    100
    90                                    RT           TMZ/RT
    80                 Median OS, mo: 12.1             14.6 p<0.001
                       2-yr survival: 10%              26% p<0.001
    70
    60                 PFS , mo:         5.0           7.2 P<0.001
%                      (no central review)
    50
    40
    30
                                                         TMZ/RT
    20
    10                                         RT
     0
          0   6   12   18            24        30           36          42
                            months




                                                    Stupp et al., NEJM (2005)
  Cancer arises from clonal evolution




• Cancer is a stochastic (random) process (?)
 Not every cancer cell can initiate disease
• Serial dilution of human cancer specimens
  – ≈ 1 in 250,000 cells are “cancer initiating”
                  leukemia,           cancer,       cancer, etc…
  – Originally in leukemia also brain cancer breast cancer etc
       Cancer initiating cell displayed stem cell properties

                                                            Cancer Stem Cells!!




•   CD34 = hematopoietic stem cell marker
•   CD34+ leukemia also differentiated to leukemic blasts
Clonal Evolution vs. Cancer Stem Cells
                         Cancer Stem Cell /
Clonal Evolution          Hierarchy Model
Tumor hierarchy and clonal evolution




                                Zhou, et al. Nature 2009
                       What is a CSC?
•   Cancer stem/initiating cells
     – Small subpopulation of cells within tumors ( 1%) highly efficient
                  p p                               (~ %) g y
       in tumor initiation
     – Display stem-like characteristics of self-renewal, enhanced
       proliferation, and multipotent differentiation
       p            ,           p

•   GBM cancer stem cells (CSCs)
     – Originally identified in Dirks lab (Singh et al., 2003 and 2004)
                                                    al
     – Enrichment for CD133+ expression (cell surface marker) or
       functional isolation as neurospheres in vitro
                      GBM CSC isolation




    W k1
    Week 1        250 µm
                  250

                                             100 µm
                            4‐6 weeks
                                                                       250 µm
                                                       Post‐chop

•    GBM specimens obtained from OR
      – Chopped into 100 μm cubes
      – Pl d i              i    l       d        h     develop i 4 6
        Placed into suspension culture and neurospheres d l in 4-6 weeks k
      – Passaged by chopping into 100 µm cubes to preserve stem cell niche
      – Successfully isolated 4 GBM CSC lines (BT22, BT12.1, BT33, BT44)
GBM CSC express stem cell markers and are 
             multipotent

A       Stem Cell Markers                       B           Multipotency
A1                      A2                      B1                       B2



                                       100 µm                   100 µm                   100 µm
GBM CSC                 NSC                     GBM CSC                  NSC
               100 µm


A3                      A4                      B3                       B4



                                                                50 µm    βIII-tubulin:
                        CD133
               100 µm                                                                    50 µm
CD133                                  100 µm   βIII-tubulin:
                                                DAPI                     DAPI


A5                       A6                     B5                       B6



               50 µm                   100 µm                   50 µm                    50 µm
Nestin :DAPI            Nestin :DAPI            GFAP                     GFAP
    GBM CSC initiate tumors at high efficiency




                       10-14      post-injection
• GBM tumor initiation 10 14 wks post injection
• Serial passage for 2° and 3° tumors
GBM CSC xenograft resembles patient tumor




                                  Singh, et al. Nature 2004
  GBM CSC genetically similar to patient tumor


                                               •      NBE = GBM CSC
                                               •      S = Serum cultured
                                                      glioma
                                               •      NSC = neural stem
                                                      cell




• Traditional glioma cell lines in serum rapidly diverge from
  patient/parental tumor
                                                   Lee et al., Cancer Cell, 2006
GBM CSC infiltrate surrounding brain
 U87 glioma cell line
 U87 glioma cell line         vs.
                              vs    GBM CSC
                                    GBM CSC




                        H&E                     H&E




                  hNestin                     hNestin
                     CSC vs Glioma Lines

                                   GBM CSC                   Glioma Cell Lines

                            Induce to become glial and       Do not respond to
Differentiation Potential
                                neuronal lineages           differentiation stimuli

                               Extensive infiltration
   Tumor Histology                                         Fail to show infiltration
                             Phenocopy human GBM

Global Gene Expression        Similar to human GBM       Different from primary tumor

                                                             Additional genomic
                             Same as parental tumor
       Genotype                                            alterations not found in
                             regardless of passages
                                                                parental tumor
       GBM CSCs are radiation resistant




• Preferential DNA repair activation by GBM CSCs
                                               Bao et al., Nature, 2006
GBM CSCs are chemotherapy resistant




                            Liu et al., Molec. Cancer, 2006
Cancer stem cells must be stopped!!
           …but how?
Shift in treatment paradigms




                           Zhou, et al. Nature 2009
Selective CSC elimination by HER2 blockade 

                 •   Breast cancer initiating cells / CSCs
                     overexpress HER2
                 •   Treatment with trastuzumab (Herceptin,
                     HER2 blocking antibody) prevents self-
                     renewal of CSCs




                                     Magnifico et al., Clin. Cancer Res., 2009
Differentiation therapy with BMPs




                          Piccirillo, et al. Nature 2006
     Targeting the leukemic stem cell niche




•   CD44 important for leukemic stem cell homing to niche
•   Blocking antibody prevents engraftment and leukemia in mice



                                                       Jin et al., Nat. Med., 2006
    Limitations of Cancer Stem Cell hypothesis

•   Not all cancers are driven by stem-like cells
     • Clonality in many types of cancers has been demonstrated
•   Cancer stem cells need not be rare




•   Multiple cancer stem cell populations within cancer
     • Dynamically evolving by clonal evolution
•   Cancer stem cells likely to vary among patients
Personalized cancer stem cell treatment for GBM
                  Conclusions

• Many cancers, such as GBM, are not
  homogenous
• Cancer stem / initiating cells may drive tumor
  initiation progression, recurrence and treatment
  initiation, progression recurrence,
  resistance
• Develop novel pe so a ed, ta geted t e ap es
    e e op o e personalized, targeted therapies
  directed to eradicate the cancer stem cells
     Open questions in CSC biology

• What is the distribution and location of CSCs in
  GBMs?
• Developing CSC-specific diagnostic and
  therapeutic strategies (i.e. identify CSCs)
• Screen for candidate CSC-specific genes




             NSC      CSC     Tumor
      Open questions in CSC biology

• Cell cycle regulators (p53, Rb, MDM2, CDK, Ink4a/ARF)
• Cell growth/developmental signaling pathways (
       g             p          g   gp                 ,
                                             y (EGFR, Akt, ,
  PTEN, PI3K)
• Developmental signaling pathways (Hedgehog, Notch, Wnt,
  BMP)
• Tumor angiogenesis (PDGF, FGF, VEGF)
• Neural developmental genes (musashi, sox, forkhead, zic)
• DNA repair / DNA methylation genes
• Novel CSC-specific genes may participate in oncogenesis and
  be viable therapeutic targets
             Acknowledgments
Brain tumor lab              Neurosurgery Department
   Daniel Treisman              Haviryaji Kalluri
             p
   Frank Hospod                   g           g
                                Raghu Vemuganti and lab members
   Sathyapriya Ezhilan          Doug Kintner, Dandan Sun Lab
   Michael Zorniak              Rao Adibhatla and lab members
   Sasha Rackman
                             Svendsen Lab
   Michael Jones
                                Erin McMillan
   Odaine Gordon
                                Dhruv Sareen
 Gordana Raca – State
 Laboratory of Hygiene,      Zhang Lab
 UW Madison

Funding
F di
   Stem Cell Training Program at UW-Madison (NIH)
   UW School of Medicine and Public Health
   UW Graduate School
   UW Department of Neurological Surgery
              CD133 Profile




• I j i
  Injection of 100 CD133+ cells produce a 
             f 100 CD133 ll         d
  phenotypic copy of parent tumor
• Injection of 105 CD133‐ cells engrafted but did 
  not cause a tumor

                                    Singh, et al. Nature 2004

								
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