Cancer Stem Cells and Metastasis

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Cancer Stem Cells and Metastasis

• Maria M. (Marj) Peña, PhD
• Dept. of Biological Sciences
• Center for Colon Cancer Research
• University of South Carolina

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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
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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 growth
• Differentiation into phenotypically diverse
  mature cell types
• 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

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Development of Hematopoietic Stem Cells
Liquid Organ
Stem Cells
Multipotent Progenitors
Oligolineage Progenitors
Mature 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.
Differentiation
Self Renewal
Reya et al. 2001 Nature 414105-111
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How do we identify Hematopoietic Stem Cells
CD34- CD38
CD20
CD8
CD8
CD34 CD38-
CD4
CD34- CD38-
CD4
CD36
CD35
Reya et al. 2001 Nature 414105-111
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Anti-A Antibody
Anti-B Antibody
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Florescence Activated Cell Sorting (FACS)
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Self-renewal Assay Irradiated Mice
FACS Cell Sorter
Bone Marrow Cells
Lethally irradiated Mice
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How do we identify Hematopoietic Stem Cells
CD34- CD38
CD20
CD8
CD8
CD34 CD38-
CD34- CD38-
CD4
CD4
CD36
CD35
Reya et al. 2001 Nature 414105-111
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The anatomy of the small intestinal epithelium
Differentiation
Self Renewal
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)
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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
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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).
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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 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 cells can form colonies
in soft agar or in vivo
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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.

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Self renewal and differentiation are random. All
cells have equal but low probability of extensive
proliferation. Only cells with self renewal
capacity can sustain tumor growth.
Distinct classes of cells exist within a tumor.
Only a small definable subset, the cancer stem
cells can initiate tumor growth.
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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.

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Thymidylate synthase
Chu E. et al., Cancer Chemother Pharmacol (2003)
52 (Suppl 1) S80-S89
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Thymidylate Synthase Inhibitors
Raltitrexed
5-FU
Longley, DB et al., Nature Reviews Cancer (2003)
3330-338
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Which cells in the hierarchy are cancer stem
cells?
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Self-renewal Assay in NOD/SCID Mice (Non-obese
diabetic/severe combined immunodeficiency)
FACS Cell Sorter
Cancer Cells ex Leukaemia cells
Sublethally irradiated NOD/SCID Mice
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Hierarchies in normal and leukemic human
hematopoietic cells
Human hematopoietic cells are organized in a
hierarchy that is sustained by a small population
of self-renewing hematopoietic stem 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
15494-501
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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.
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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-RARa acute
promyelocytic leukaemia (APML)77 MRP8-BCRablXBCL-2
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Self-renewal Assay in NOD/SCID Mice For solid
tumors surgical orthotopic implantation (SOI)
FACS Cell Sorter
Single Cell Suspension
Solid Tumor
Mince (small pieces)
Surgical
Implantation
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Stem Cells in the Nervous System
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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.
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Brain tumor stem cells were identified by
intracranial transplantation of CD133 cells
into adult NOD/SCID mouse forebrain.
Singh et al. 2004 Nature 432 396-401
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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
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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.

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Metastasis

• 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

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Metastasis is a multi-step processMetastatic
cell Decathlon champion

• Vascularization of primary tumor
• Tumor grows through the synthesis and secretion
  of pro-angiogenic factors by the tumor and
  surrounding stroma
• 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

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Metastatic Cancer Cells Migratory Cancer Stem
Cells

• Cells arrest in the capillary beds of distant
  organs
• Extravasation into distant organ
• Survival and proliferation in target organ
• Depends on multiple interactions (cross-talk)
  between tumor cell and organ microenvironment

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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 specific
• 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

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Principles of the Seed and Soil Hypothesis

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

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Tumors are biologically heterogeneous and contain
subpopulations of cells with different
angiogenic, invasive, and metastatic properties.
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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. 200511544-55
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Noninvasive BLI to monitor the development of
osteolytic metastases from the same mouse
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Verification of macroscopic and microscopic
metastases by fluorescence histology
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SCPs exhibit different abilities to metastasize
to bone
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SCPs demonstrate different abilities to
metastasize to the lung
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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.
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SCPs from MDA-MB-231 cells have a poor-prognosis
gene expression signature
Minn, A. J. et al. J. Clin. Invest. 200511544-55
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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/collag
enase, promotes osteolysis by cleaving
a specific peptide bond in the collagen of
bone matrix OPN osteopontin (consistently
overexpressed in metastatic cells)
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Directing metastasis to the Bone
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Genetic determinants for metastasis to the bone
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The outcome of metastasis depends on multiple
interactions (cross-talk) between the
metastatic subpopulation in the primary tumor and
the host organ microenvironment.
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Summary The Metastatic Process
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FUTURE DIRECTIONS

• 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

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Wnt Signaling Pathway
Fodde, R et al., Nat Rev Cancer (2001) 157-67