Cancer Stem Cells Cancer Stem Cells Maria M Marj Peña PhD by hedongchenchen


									 Cancer Stem Cells

   Maria M. (Marj) Peña, PhD
  Dept. of Biological Sciences
Center for Colon Cancer Research
  University of South Carolina
             Normal stem cells
 Rare cells within organs with the ability to self-
renew and give rise to all types of cells within the
         organ to drive organogenesis

             Cancer stem cells

 Rare cells within tumors with the ability to self-
renew and give rise to the phenotypically diverse
  tumor cell population to drive tumorigenesis
Properties shared by normal stem cells and
             cancer stem cells

• Assymetric Division:
  – Self renewal
     • Tissue-specific normal stem cells must self-renew throughout
       the lifetime of the animal to maintain specific organs
     • Cancer stem cells undergo self-renewal to maintain tumor
  – Differentiation into phenotypically diverse mature cell
     • Give rise to a heterogeneous population of cells that
       compose the organ or the tumor but lack the ability for
       unlimited proliferation (hierarchical arrangement of cells)
• Regulated by similar pathways
  – Pathways that regulate self-renewal in normal stem
    cells are dys-regulated in cancer stem cells
            Development of Hematopoietic Stem Cells
                                    Stem    Multipotent   Oligolineage             Mature
                                    Cells   Progenitors   Progenitors              Cells

HSCs can be subdivided into
long-term self-renewing HSCs,
short-term self-renewing HSCs
and multipotent progenitors
(red arrows indicate self-
renewal). They give rise to
common lymphoid progenitors
(CLPs; the precursors of all
lymphoid cells) and common
myeloid progenitors (CMPs;
the precursors of all myeloid
cells). Both CMPs/GMPs
(granulocyte macrophage
precursors) and CLPs can
give rise to all known mouse
dendritic cells. ErP, erythrocyte
precursor; MEP,
megakaryocyte erythrocyte
precursor; MkP,
megakaryocyte precursor; NK,
natural killer.

                                                          Reya et al. 2001 Nature 414:105-111
     The anatomy of the small intestinal epithelium

The epithelium is shaped into crypts and villi (left). The lineage scheme (right) depicts the stem cell, the
transit-amplifying cells, and the two differentiated branches. The right branch constitutes the enterocyte
lineage; the left is the secretory lineage. Relative positions along the crypt-villus axis correspond to the
schematic graph of the crypt in the center.
                                                         F. Radtke et al., Science 307, 1904 -1909 (2005)
                                 Adult intestinal homeostasis

Schematic representation and section of the crypt-villus unit in the mature small intestine. Proliferative cells reside in the crypts,
while differentiated cells occupy the villus. Crypt progenitors migrate up (red arrow) the crypt-villus axis before shedding into the
lumen. The process of epithelial renewal takes 3-6 d and is ensured by a small number of asymmetrically dividing stem cells at the
bottom of the crypts. Wnt signaling in the adult intestine promotes proliferation of progenitor or transit-amplifying (TA) cells, as well
as commitment toward secretory lineages. Wnt signaling may also drive terminal differentiation of certain secretory lineages.
Although it is commonly believed that Wnt signaling may promote proliferation and/or differentiation of intestinal stem cells, there is
no evidence that formally proves this (see arrows with question marks). In panel A, black arrowheads indicate Ki67 positive transit-
amplifying cells, while white arrowheads indicate the Paneth cell compartment.
                                                                         Alex Gregorieff et al. Genes Dev. 2005; 19: 877-890
  Pathways involved in self-renewal that are deregulated in cancer cells

Wnt, Shh, and Notch pathways have been shown to contribute to the self-renewal of stem cells and/or progenitors in a variety
of organs, including the haematopoietic and nervous systems. When dysregulated, these pathways can contribute to
oncogenesis. Mutations of these pathways have been associated with a number of human tumours, including colon
carcinoma and epidermal tumours (Wnt), medulloblastoma and basal cell carcinoma (Shh), and T-cell leukaemias (Notch).
    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
         away from normal hematopoietic cells were able to form colonies
In vivo transplantation assays:
         - Only 1-4% of transplanted leukaemic cells could form spleen
Solid Tumors
       - A large number of cells are required to grow tumors in xenograft
       - 1 in 1,000 to 1 in 5,000 lung cancer, neuroblastoma cells, ovarian
       cancer cells, or breast cancer cells can form colonies in soft agar or
       in vivo
Two General Models for Cancer Heterogeneity

1.   All cancer cells are potential cancer stem
     cells but have a low probability of
     proliferation in clonogenic assays

2.   Only a small definable subset of cancer cells
     are cancer stem cells that have the ability to
     proliferate indefinitely.
Self renewal and differentiation are random.    Distinct classes of cells exist within a
All cells have equal but low probability of     tumor. Only a small definable subset,
extensive proliferation. Only cells with self   the cancer stem cells can initiate tumor
renewal capacity can sustain tumor growth.      growth.
Therapeutic implications of Cancer Stem Cells

 • Most therapies fail to consider the difference in drug sensitivities of cancer stem cells
   compared to their non-tumorigenic progeny.
 • Most therapies target rapidly proliferating non-tumorigenic cells and spare the
   relatively quiescent cancer stem cells.
  Thymidylate synthase

Chu E. et al., Cancer Chemother Pharmacol (2003) 52 (Suppl 1) S80-S89
       Thymidylate Synthase Inhibitors

Raltitrexed   5-FU

                     Longley, DB et al., Nature Reviews Cancer (2003) 3:330-338
Which cells in the hierarchy are cancer stem cells?
Hematopoietic and Progenitor Cell Lines

      Passegue, Emmanuelle et al. (2003) Proc. Natl. Acad. Sci. USA 100, 11842-11849
        Self-renewal Assay in NOD/SCID Mice
     (Non-obese diabetic/severe combined immunodeficiency)

                         FACS Cell
    Cancer Cells
(ex: Leukaemia cells

      Sublethally irradiated NOD/SCID Mice
Human hematopoietic cells are
organized in a hierarchy that is
                                         Hierarchies in normal and leukemic human
sustained by a small population of
self-renewing hematopoietic stem
                                                     hematopoietic cells
cells (HSCs). HSCs give rise to
progressively more lineage-
restricted, differentiated progenitors
with reduced self-renewal capacity
(LTC-ICs, long-term culture-initiating
cells; CFU, colony-forming units),
which in turn produce functionally
mature blood cells.

Disruption of pathways regulating
self-renewal and differentiation
through the acquisition of
transforming mutations generates
leukemic stem cells (LSCs) capable
of sustaining growth of the leukemic
clone in vivo. LSCs possess an
altered differentiation program, as
demonstrated by aberrant
expression of some cell-surface
markers (indicated in blue) and give
rise to an aberrant developmental
hierarchy that retains aspects of its
normal counterpart.

 In vivo reconstitution assays using
immune-deficient mouse recipients
enable detection of HSCs and LSCs
as SCID-repopulating cells (SRCs)
and SCID leukemia-initiating cells
(SL-ICs), respectively.
                                                  Wang and Dick 2005 Trends in Cell Biology 15:494-501
Hematopoietic Cancer
Stem Cells

   Acute myeloid leukemia
   (AML) – CD34+ CD38-

              Leukaemic Mouse Models:
              chronic myelomonocytic leukaemia (CMML)   MRP8-BCL-2
              acute myeloid leukaemia (AML)             MRP8-BCL2Xlpr/lpr
              chronic myeloid leukaemia (CML)/Blast     MRP8-PML-RARα
              acute promyelocytic leukaemia (APML)77    MRP8-BCRablXBCL-2
          The importance of self-renewal in leukemic initiation and progression.

Self-renewal is a key property of both normal and leukemic stem cells. Fewer mutagenic changes are required to transform stem
cells in which the self-renewal machinery is already active (a), as compared with committed progenitors in which self-renewal must
be activated ectopically (b). In addition, self-renewing stem cells are long-lived; thus, there is an increased chance for genetic
changes to accumulate in individual stem cells in comparison with more mature, short-lived progenitors. If a committed progenitor
with limited life span acquires a genetic mutation that does not confer increased self-renewal (c), that cell will likely die or undergo
terminal differentiation before enough mutations occur to propagate a full leukemogenic program.
              Self-renewal Assay in NOD/SCID Mice
       For solid tumors: surgical orthotopic implantation (SOI)

                                                  CD44 Expression
                                  FACS Cell
                   Single Cell
Solid Tumor
                   Suspension                                       CD24 Expression

  Mince       Surgical
Stem Cells in the Nervous System
    Brain Tumor Stem Cells: CD133+
    CD133 – neuronal stem cell marker

    Brain tumor stem cells were identified from human brain tumor samples by in
    vitro neurosphere assays normally used to isolate normal neural stem cells

GFAP = glial fibrillary acidic protein    Singh et. al 2003 Cancer Research 63: 5821-5828.
Brain tumor stem cells were identified by intracranial transplantation of
CD133+ cells into adult NOD/SCID mouse forebrain.


              CD133+                           CD133-

                                                    Singh et al. 2004 Nature 432: 396-401
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, Muhammad et al. (2003) Proc. Natl. Acad. Sci. USA 100, 3983-3988
                 FUTURE DIRECTIONS

- Need to characterize cancer stem cells at the single cell level

- Understand the genetic and biochemical mechanisms that
  control the self-renewal phenotype, assymetric subdivision,
  and the role of the stem cell niche in regulating the biological
  properties of both normal and cancer stem cells.

- Characterize the response of cancer stem cells to
  chemotherapeutic regimens

- Develop therapeutic strategies to target cancer stem cells to
  prevent tumor recurrence.
• Process by which a tumor cell leaves the
  primary tumor, travel to a distant site via the
  circulatory system and then establishes a
  secondary tumor.

• Final and most devastating step of a malignancy

• Leading cause of death in cancer patients
   – Before mets tumors may be cured by surgery
   Metastasis is a multi-step process
      Metastatic cell = “Decathlon champion”

• Vascularization of primary tumor
  – Tumor grows through the synthesis and secretion of
    pro-angiogenic factors by the tumor and surrounding
• Invasion of the organ stroma through enhanced
  expression of enzymes (MMP9)
• Invasion of the lymphatic or vascular channels
  (may grow in these places)
• Tumors cells enter circulation
  – Must survive turbulence of circulation and evade both
    immune and non-immune mechanisms
• Cells arrest in the capillary beds of distant
• Extravasation into distant organ
• Survival and proliferation in target organ
  – Depends on multiple interactions (“cross-talk”)
    between tumor cell and organ

      Metastatic Cancer Cells =
     Migratory Cancer Stem Cells
            Metastasis is not random
• Seed and soil hypothesis
   – 1889: Stephen Paget analyzed autopsy records of 735 women
     with breast cancer
   – Metastasis to distant sites was not due to chance
   – Certain tumor cells (the “seed”) has an affinity for the milieu (the
     “soil”) of certain organs. Metastases resulted when the seed and
     soil were compatible
• Metastatic dissemination occurs by purely mechanical
  factors that are the result of the anatomical structure of
  the vascular system
   – 1929: J. Ewing
• Regional metastases can be attributed to anatomic and
  mechanical factors but distant organ metastases is
   – 1964: Sugarbaker
   – Lymphatic drainage to regional lymph nodes
   – Organ-specific metastases: breast, prostate, and lung cancer
     metastasize to the bone, while colorectal cancer metastasized to
     the liver and lymph nodes
 Principles of the Seed and Soil Hypothesis

1.   Tumors are biologically heterogeneous and contain
     subpopulations of cells with different angiogenic,
     invasive, and metastatic properties.
2.   Metastases is a selective process for cells that
     succeed in invasion, embolization, survival in the
     circulation, arrest in a distal capillary bed, extravastion
     into the distant organ, and survival and proliferation in
     the distant organ.
3.   The outcome of metastasis depends on multiple
     interactions (“cross-talk”) between the metastatic
     subpopulation in the primary tumor and the host organ
Tumors are biologically heterogeneous
  and contain subpopulations of cells
  with different angiogenic, invasive,
      and metastatic properties.
Organ Specific metastasis of Breast Cancer Cells

               MDA-MB-231 Breast Cancer Cell Line

                              Isolate Single Clonal Populations (SCPs)

  Introduce Luciferase Bioluminescent Marker and GFP Fluorescence Marker

               Introduce into Nude Mice by intracardiac Injection

                                         Minn, A. J. et al. J. Clin. Invest. 2005;115:44-55
Noninvasive BLI to monitor the development of
 osteolytic metastases from the same mouse
Verification of macroscopic and microscopic metastases by
                   fluorescence histology
SCPs exhibit different abilities to metastasize to bone
SCPs demonstrate different abilities
   to metastasize to the lung
 Metastases is a selective process for cells that
succeed in invasion, embolization, survival in the
    circulation, arrest in a distal capillary bed,
 extravastion into the distant organ, and survival
       and proliferation in the distant organ.
SCPs from MDA-MB-231 cells have a poor-prognosis
           gene expression signature

                        Minn, A. J. et al. J. Clin. Invest. 2005;115:44-55
              Genes that mediate metastasis to the Bone

CXCR4 – bone homing chemokine receptor
CTGF – connective tissue growth factor
IL-11 – activator of osteoclast differentiation (mediators of bone resorption in bone metastases)
MMP1 – matrix metalloproteinase/collagenase, promotes osteolysis by cleaving
         a specific peptide bond in the collagen of bone matrix
OPN – osteopontin (consistently overexpressed in metastatic cells)
Directing metastasis to the Bone
Genetic determinants for metastasis to the bone
 The outcome of metastasis depends on multiple
interactions (“cross-talk”) between the metastatic
 subpopulation in the primary tumor and the host
             organ microenvironment.
Summary: The Metastatic Process
• Understand the factors and mechanisms that lead to
  metastasis rather than study metastatic end points
   – What steps of metastasis provides good therapeutic targets?
   – Are the early steps clinically detectable and is the process a
     good biological target?
• Understand the “cross-talk’ between metastatic cells and
  target organs that establish metastases
   – What are the “messages”
   – What are the “messengers”
• Target the soil to prevent the growth of the seed
• Develop therapies to alleviate metastases while
  minimizing therapies that will subject the patient to
  unnecessary toxicities
Wnt Signaling Pathway

            Fodde, R et al., Nat Rev Cancer (2001) 1:57-67

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