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
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
 Development of Hematopoietic Stem Cells
Liquid Stem Multipotent Oligolineage Mature
Organ 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
Differentiation
progenitors (CMPs; the
precursors of all myeloid Self
cells). Both CMPs/GMPs
(granulocyte macrophage Renewal
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

How do we identify Hematopoietic Stem Cells
Stem Multipotent Oligolineage Mature
Cells Progenitors Progenitors Cells

CD34- CD20+
CD38+
CD8+

CD8+

CD34+
CD38-
CD34- CD4+
CD38-
CD4+

CD36+

CD35+

Reya et al. 2001 Nature 414:105-111

Anti-A Antibody
Anti-B Antibody
Florescence Activated Cell Sorting (FACS)
 Self-renewal Assay Irradiated Mice

CD34 Expression
FACS Cell
Sorter
Bone Marrow Cells
CD38 Expression

Lethally irradiated Mice

How do we identify Hematopoietic Stem Cells
Stem Multipotent Oligolineage Mature
Cells Progenitors Progenitors Cells

CD34- CD20+
CD38+
CD8+

CD8+

CD34+
CD38- CD34- CD4+
CD38-
CD4+

CD36+

CD35+

Reya et al. 2001 Nature 414:105-111

 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)
 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
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
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?
 Self-renewal Assay in NOD/SCID Mice
(Non-obese diabetic/severe combined immunodeficiency)

CD34 Expression
FACS Cell
Sorter
Cancer Cells
ex: Leukaemia cells CD38 Expression

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
 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.
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
 Self-renewal Assay in NOD/SCID Mice
For solid tumors: surgical orthotopic implantation (SOI)

CD44 Expression
FACS Cell
Sorter
Single Cell
Solid Tumor
Suspension CD24 Expression

Mince Surgical
(small
Implantation
pieces)
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+ 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.
 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
 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 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
• 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

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 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
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,
extravasation 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 microenvironment.
 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
 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
Wnt Signaling Pathway

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