Transcript Cancer Biology Angiogenesis and Metastasi

Angiogenesis
Proangiogenic
- Angiogenic growth factors
VEGF, FGF, TNF, HGF, IGF,TGF, Proliferin
Anti angiogenetic - Thrombospondin -1, 2
Angiostatin
Endostatin
Platelet factor 4
Angiogenesis
Local degradation of basement membrane
Directional migration
invasion
Endothelial cell proliferation
Capillary tube morphogenesis
Coalescence of capillaries
Vascular pruning
What Is Metastasis?
The ability of cancer cells to:
1. Penetrate into
 Lymphatic.
 Blood vessels.
2. Circulate through bloodstream.
4. Invade and grow in normal tissues elsewhere.
It is the ability to spread to other tissues and
organs that makes cancer a potentially life
threatening disease.
So what makes metastasis possible for a
Cancerous tumor?
1. Cancer cells invade surounding
tissues and vessels
Blood vessel
2. Cancer cells are transported
by the circulatory system to
distant sites.
3. Cancer cells
reinvade and grow
at new location
Metastasis Requires Angiogenesis
Growth of a new network of blood vessels
What Is Tumor Angiogenesis?
Tumor angiogenesis
Proliferation of a network of
blood vessels that penetrates
into cancerous growths.
Function
Supplying nutrients and oxygen
and removing waste products.
Mechanism
Cance cells releas molecules
that send signals to surrounding
normal host tissue.
This signaling activates certain
genes in the host tissue that, in
turn, make proteins to encourage
growth of new blood vessels.
Normal Angiogenesis: vasculogenesis
Normal Angiogenesis in Adults
New blood vessels
form in the lining
of the uterus during
the menstrual cycle.
Repair or regeneration
of tissue during wound healing.
Angiogenesis and Vascular Endothelial Cells
The walls of blood vessels are
formed by vascular endothelial
cells .
These cells rarely divide, doing
so only about once every 3 years
on average.
When the situation requires it,
angiogenesis can stimulate them
to divide.
Angiogenesis and
Regulatory Proteins
High inhibitors Rare cell
Low activators= division
No angiogenesis
Low inhibitors Frequent cell
High activators =division
Angiogenesis
Angiogenesis and Cancer
The dilatation theory
Before the 1960s
Angiogenesis theory
Without Angiogenesis, Tumor Growth Stops
With Angiogenesis, Tumor Growth
Proceeds
In another experiment designed to find
out whether cancer growth can
continue when angiogenesis occurs,
researchers compared the behavior of
cancer cells in two regions of the same
organ. Both locations in the eye had
nutrients available, but only one could
support angiogenesis. Scientists found
that the same starting injection of
cancer cells grew to 1-2mm in
diameter and then stopped in the
region without nearby blood vessels,
but grew well beyond 2 mm when
placed in the area where angiogenesis
was possible. With angiogenesis,
tumor growth continued
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What Prompts Angiogenesis?
In an experiment designed to find
out whether molecules from the
cancer cells or from the
surrounding host tissues are
responsible for starting
angiogenesis, scientists implanted
cancer cells in a chamber
bounded by a membrane with
pores too small for the cells to
exit. Under these conditions,
angiogenesis still began in the
region surrounding the implant.
Small activator molecules
produced by the cancer cells must
have passed out of the chamber
and signaled angiogenesis in the
surrounding tissue
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Activators of Angiogenesis
Once researchers knew that cancer
cells can release molecules to activate
the process of angiogenesis, the
challenge became to find and study
these angiogenesis-stimulating
molecules in animal and human
tumors.
From such studies more than a dozen
different proteins, as well as several
smaller molecules, have been
meaning ”,angiogenic“ identified as
that they are released by tumors as
signals for angiogenesis. Among these
molecules, two proteins appear to be
the most important for sustaining
tumor growth: vascular endothelial
and basic )VEGF( growth factor
VEGF .)bFGF( fibroblast growth factor
and bFGF are produced by many
kinds of cancer cells and by certain
.types of normal cells, too
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The Angiogenesis Signaling
Cascade
VEGF and bFGF are first
synthesized inside tumor cells and
then secreted into the surrounding
tissue. When they encounter
endothelial cells, they bind to
specific proteins, called receptors,
sitting on the outer surface of the
cells. The binding of either VEGF
or bFGF to its appropriate
receptor activates a series of relay
proteins that transmits a signal
into the nucleus of the endothelial
cells. The nuclear signal ultimately
prompts a group of genes to make
products needed for new
.endothelial cell growth
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Endothelial Cell Activation
The activation of endothelial cells by
VEGF or bFGF sets in motion a series
of steps toward the creation of new
blood vessels. First, the activated
endothelial cells produce matrix
a special ,)metalloproteinases (MMPs
class of degradative enzymes. These
enzymes are then released from the
endothelial cells into the surrounding
tissue. The MMPs break down the
extracellular matrix—support material
that fills the spaces between cells and
is made of proteins and
polysaccharides. Breakdown of this
matrix permits the migration of
endothelial cells. As they migrate into
the surrounding tissues, activated
endothelial cells begin to divide. Soon
they organize into hollow tubes that
evolve gradually into a mature network
of blood vessels
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Inhibitors of Angiogenesis
Although many tumors produce angiogenic
molecules such as VEGF and bFGF, their
presence is not enough to begin blood
vessel growth. For angiogenesis to begin,
these activator molecules must overcome a
variety of angiogenesis inhibitors that
normally restrain blood vessel growth.
Almost a dozen naturally occurring proteins
can inhibit angiogenesis. Among this group
,angiostatin of molecules, proteins called
thrombospondin appear to and ,endostatin
be especially important. A finely tuned
balance, between the concentration of
angiogenesis inhibitors and of activators
such as VEGF and bFGF, determines
whether a tumor can induce the growth of
new blood vessels. To trigger angiogenesis,
the production of activators must increase
as the production of inhibitors decreases
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Angiogenesis Inhibitors and Metastasis
The discovery that angiogenesis inhibitors
such as endostatin can restrain the growth
of primary tumors raises the possibility that
such inhibitors might also be able to slow
tumor metastasis.
In one striking study, researchers injected
several kinds of mouse cancer cells beneath
the animals' skin and allowed the cells to
grow for about two weeks. The primary
tumors were then removed, and the animals
checked for several weeks. Typically, mice
developed about 50 visible tumors from
individual cancer cells that had spread to the
lungs prior to removal of the primary tumor.
But mice treated with angiostatin developed
an average of only 2-3 tumors in their lungs.
Inhibition of angiogenesis by angiostatin had
reduced the rate of spread (metastasis) by
about 20-fold
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Angiogenesis and Tumor Dormancy
It has been known for many years that
cancer cells originating in a primary
tumor can spread to another organ
and form tiny, microscopic tumor
masses (metastases) that can remain
dormant for years. A likely explanation
for this tumor dormancy is that no
angiogenesis occurred, so the small
tumor lacked the new blood vessels
needed for continued growth.
One possible reason for tumor
dormancy may be that some primary
tumors secrete the inhibitor angiostatin
into the bloodstream, which then
circulates throughout the body and
inhibits blood vessel growth at other
sites. This could prevent microscopic
metastases from growing into visible
tumors
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Cancer in Angiogenesis-Deficient Mice
Additional support for the idea that interfering with
the process of angiogenesis can restrain tumor
growth has come from genetic studies of mice.
Scientists have recently created strains of mice that
lack two genes, called Id1 and Id3, whose absence
hinders angiogenesis. When mouse breast cancer
cells are injected into such angiogenesis-deficient
mutant mice, there is a small period of tumor
growth, but the tumors regress completely after a
few weeks, and the mice remain healthy with no
signs of cancer. In contrast, normal mice injected
with the same breast cancer cells die of cancer
within a few weeks.
When lung cancer cells are injected into the same
strain of angiogenesis-deficient mutant mice, the
results are slightly different. The lung cancer cells
do develop into tumors in the mutant, but the
tumors grow more slowly than in normal mice and
fail to spread (metastasize) to other organs. As a
result, the mutant mice live much longer than
normal mice injected with the same kinds of lung
.cancer cells
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Angiogenesis Inhibitors in the •
Treatment of Human Cancer
Researchers are now asking if •
inhibiting angiogenesis can slow
down or prevent the growth and
spread of cancer cells in humans.
To answer this question, almost •
two dozen angiogenesis inhibitors
are currently being tested in
cancer patients. The inhibitors
being tested fall into several
different categories, depending on
their mechanism of action. Some
inhibit endothelial cells directly,
while others inhibit the
angiogenesis signaling cascade or
block the ability of endothelial cells
to break down the extracellular
matrix
Drugs That Inhibit Angiogenesis
Directly
One class of angiogenesis
inhibitors being tested in cancer
patients are molecules that
directly inhibit the growth of
endothelial cells. Included in this
category is endostatin, the
naturally occurring protein known
to inhibit tumor growth in animals.
Another drug, combretastatin A4,
causes growing endothelial cells
to commit suicide (apoptosis).
Other drugs, which interact with a
molecule called integrin, also can
promote the destruction of
proliferating endothelial cells
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Old Drug With a New •
Use
Another interesting drug •
is thalidomide, a sedative
used in the 1950s that
was subsequently taken
off the market because it
caused birth defects
when taken by pregnant
women. Although this
drug clearly would not be
suitable for pregnant
women, its ability to
prevent endothelial cells
from forming new blood
vessels might make it
useful in treating non.pregnant cancer patients
Drugs That Block the
Angiogenesis Signaling
Cascade
A second group of angiogenesis
inhibitors being tested in human
clinical trials are molecules that
interfere with steps in the
angiogenesis signaling cascade.
Included in this category are antiVEGF antibodies that block the
VEGF receptor from binding
growth factor. Another agent,
interferon-alpha, is a naturally
occurring protein that inhibits the
production of bFGF and VEGF,
preventing these growth factors
from starting the signaling
cascade.
•Also, several synthetic drugs
capable of interfering with
endothelial cell receptors are
being tested in cancer patients .
On to Clinical Trials
Researchers have answered many
questions about angiogenesis, but
many questions still remain. Scientists
do not know whether using
angiogenesis inhibitors to treat cancer
will trigger unknown side effects, how
long treatment will need to last, or
whether tumor cells will find alternative
routes to vascularization. To answer
such questions, human clinical trials
are currently under way.
For an updated list of ongoing and
currently planned clinical trials
involving angiogenesis inhibitors,
including phone numbers for obtaining
additional information, refer to the
National Cancer Institute CancerTrial
™ Web site, which has a section
Angiogenesis Inhibitors in devoted to
.Clinical Trials
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Tumor angiogenesis
Tumorgrowth
Hypoxia
Growth factors
Vessels sprouting
Tumor growth
Rheumatoid
Arthritis
Blindness
Stroke
Cancer
AIDS
complications
EXCESSIVE
Psoriasis
INSUFFICIENT
Infertility
Heart Disease
Scleroderma
Ulcers
Tumorangiogenesis
Stimulators vs. Inhibitors
„ANGIOGENIC SWITCH“
Prevascular phase
Vascular phase
Proliferation - Progression - Metastases - Survival
Mast cells
Macrophages
Recruitment
activation
Tumor
Lytic enzymes
Extra cellular matrix
Blood vessel
Angiogenic factors
Hypoxia in Angiogenesis
Carmeliet, Nature, 2000
Vascular endothelial growth
factor
Four isoforms: 121, 165, 189, 206
VEGF 121: diffusible
VEGF 165: cell surface
Two receptors: Flt-1, KDR
Endothelial cell proliferation and
migration
Extracellular matrix degeneration
Vascular permeability (VPF)
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Vascular Morphogenesis
Ligands and EC selective RTKs
Vascular Endothelial Growth Factor
VEGF-R2 (Flk 1/KDR): angioblast/EC proliferation
VEGF-R1 (Flt 1): capillary tube formation
Angiopoietin-1 / Angiopoietin-2 (-)
Tie2: vessel maturation, periendothelial recruitment
Tie1: intercapillary hemodynamic balance
Distinct phenotype – complimentary function
Pathological Vascular Growth
Carmeliet, Nat Med, 2000
Cascade of Vessel Formation
Vasodilatation 
Vascular permeability 
Extravasation of plasma proteins
EC detachement and migration 
EC proliferation and sprouting 
Lumen formation
Endothelial survival and differentiation
Remodeling of endothelial network
Cascade of Vessels Formation
ANGIOPLAST
Regression
Extracellular matrix
Physical barrier
Production of angiogenic factors
Reservoir of inactive growth factos (bFGF)
Macrophages: secrete GFs
Mediation of EC binding (integrins)
EC migration, signalling, apoptosis
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VEGF/bFGF - MMP – matrix degradation
Interactions in
Tumorangiogenesis
Jones, BJUI, 1999
Cancer Cells & Tumor Vessels
• Cancer cells occupy 4%
of vascular surface
• Tumor intra-vasation in 2
days
• Shedding of 106 cells / day
/ gram
Angiogenesis in Bladder cancer
MVD = prognostic indicator in TCC
MVD + p53 = aggressive subset identified
bFGF higher in metastatic pts.
VEGF, TGF- higher in TCC
Urinary VEGF, aFGF higher in TCC pts.
VEGF mRNA ~ recurrence + progression
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Tumorangiogenesis
and LN-Metastases
T2-4 bladder cancer, n=41
Cx, pelvic LN dissection
MVD (FVIII-RA), 200x
neg. LN (n=27): 56.2 MV (SD 29.5)
pos. LN (n=14): 138.1 MV (SD 37.9)
p<0.0001
Angiogenesis in Prostate cancer
MVD higher in PCa
MVD ~ stage (superior to grade, preop. PSA)
MVD predictor for metastasis after Px
TURP-MVD ~ failure of radiotherapy
Biopsy-MVD (+ GS, PSA): extraprostatic ext.
Urine-bFGF: BPH > PCa > control
Serum-bFGF: PCa > BPH
VEGF: ?
PSA = antiangiogenic
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Microscopic Tumor Extension
Capsular penetration
Seminal vesicle extension
Pelvic lymph node metastases
Pathologic upstaging: 11%-60%
Sensitive markers: serum, urine, imaging?
Tumorangiogenesis and PCa
Microvessel density ~ Pathologic stage
Organ confined tumor vs. non organ confined tumor
n=31, retropubic Px,
CD31, 200x, 0.74 mm2
Stage
Mean Range
Organ confined (n=23) 49.7
23-97
Positive margins (n=5) 81.6 54-104
N1 (n=3)
87.2 65-111
MVD in Prostatic Carcinoma
CD31, 200x
Urine VEGF and T-Stage in PCa
VEGF-Elisa (R&D Systems), TNM
n=14 (64.6, ± 7.5 yrs.)
T2: n=6, Urine VEGF = 191 pg/ml (± 75)
T3: n=8, Urine VEGF = 309 pg/ml (± 163)
p=0.04
T2a/T2b and T3a/T3b: n.s.
Angiogenic Topography in PCa
PCa, rad Px, n=60 (64 ± 6 yrs)
MVD endothelial antigen CD34
VEGF165 monoclonal Ab
7,9 mm2 area, serial sections
Distribution and topography CD34+VEGF165
Topography divided in 4 categories:
Identical, intersecting, adjacent, no contact
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Angiogenic Topography in PCa
CD34
VEGF
Angiogenic Topography in PCa
MVD between four groups n.s. •
VEGF165-expression and highest MVD •
- identical: 19 (31.6%)
- intersecting: 18 (30%)
- adjacent: 11 (18.3%)
- no contact: 12 (20%)
Close topographical relation in 80% •
Angiogenesis and NE
PCa with high NE differentiation: poor •
prognosis
NE and Neovascularisation? •
PCa, rad Px, n=102 (65.2 ± 6.6 yrs) •
Chromogranin A, CD34, 200x, morphometry •
NE: scattered cells •
small clusters (<10 cells)
large clusters (>10 cells)
Angiogenesis and NE
Scattered cells
14.6 ± 2.2
Small cluster
82.8 ± 16.3
Chromogranin A, 200x
Large cluster
291.3 ± 40.1
Angiogenesis and NE
G Ia / Ib / IIa, Gleason 2-6, n=36 •
G IIb / IIIa / IIIb, Gleason 7-10, n=66 •
Low grade PCa: 33.8 ± 6.7 NE cells •
High grade PCa: 90.5 ± 16.4 NE cells •
(p=0.028)
Angiogenesis and NE
pT2
n=36
1p<0.05
pT3
n=58
pT4
n=6
49.6
NE tumor ± 10.63
cells
73.3
±
15.23
201.8
±
86.61,2
2.56
MVD ± 0.183
2.79
±
0.163
1.33
±
0.411,2
vs. pT2 tumors, 2p<0.05 vs. pT3 tumors, 3p<0.05 vs. pT4 tumors
Angiogenesis and NE
Low grade PCa, high NE •
CD34: 2.4% ± 0.26
High grade PCa, high NE •
CD34: 3.3% ± 0.26
(p=0.026)
Angiogenesis, NE
and Proliferation
S-VEGF: high angiogenic potential •
Ki-67: proliferation •
Neuroendocrine differentiation (NE) •
Correlation of S-VEGF and Ki-67 in •
neuroendocrine differentiated PCa?
Angiogenesis, NE
and Proliferation
PCa, rad Px, T2b-3a, n=9, ( 65.8 yrs)
S-VEGF (quantitative ELISA)
Chromogranin A
Ki-67 (MIB-1-Ab)
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Expression, topography •
Angiogenesis, NE
and Proliferation
S-VEGF correlates with Ki-67 in areas of •
PCa with high NE differentiation.
(p=0.0228, r=0.67)
S-VEGF does not correlate with Ki-67 in •
areas with little NE differentiation.
(p=0.2110)
Vascular Morphogenesis
Microvessel Maturity
Newly formed immature vessels
vs.
Established and mature vessels
Coating of periendothelial cells:
Pericytes, smooth muscle cells, myocardial cells
Anti-angiogenic therapy:
MVD , change of ratio immature/mature vessels
Vulnerability of uncoated ECs
SMC-EC Interaction
ECs initiate angiogenesis •
Periendothelial cells for vascular maturation •
Vascular myogenesis / mural cells
Vessel stability
Inhibition of EC proliferation
Production of extracellular matrix
Viscoelastic and vasomotor properties
Specialized functions of ECs
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Angioarchitecture
HMW-MAA (MAb 225.28S)•
Staining restricted to pericytes•
Staining „clusters of cellular processes“•
Pericytes aquire HMW-MAA•
HMW-MAA pos. pericytes = proliferation?•
HMW-MAA in PCa?•
Antibody supplied by S. Ferrone, Roswell Park Cancer Insitute, USA
Angioarchitecture HMW-MAA
MAb 225.28S, 40x
Human Plasminogen - Kringle 5
K5
K1-4=Angiostatin™
Kringle 5 in Urine
of Cancer Patients
Breast cancer
Lung cancer
Rheumatoid arthritis
Prostate cancer
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Band reacts only with K5 Ab, not K1-3 or K4 Abs
K5 has a molecular mass of 10459 kDa
HMVEC bFGF/VEGF Stimulated Migration
Control
(15 ng/ml bFGF/VEGF)
400 pM K5
(15 ng/ml bFGF/VEGF)
36 Cells Migrated
2 Cells Migrated
40x magnification
Migration Inhibition of HMVEC Cells
by K5 with Various Activators
None
K5 (400 pM)
Endothelial Cell Migration
120
100
80
60
40
20
0
None
bFGF
aFGF
VEGF PDGF
TGF-
IL-8 HGF/SF
Pharmacokinetic MRI-Model
Gd
Gd
k21
Gd
Gd
Gd
Gd
extracellular
space
Amplitude A
Pharmacokinetic MRI-Model
k21
Amplitude A - Signal intensity - Vessel density
k21 - Exchangerate - Vessel density / Vessel permeability
Pharmacokinetic MRI-Model
MR-Spectroscopy in PCa
200
100
O2
0
min
0
1,5
3
4,5
6
0
1,5
3
4,5
6 min
200
100
0
Change of signal intensity [%]
MR-Spectroscopy in PCa
PCa, before inhalation
PCa, after inhalation
MR-Spectroscopy in PCa
Change of signal
intensity in ROI
Angiogenic Surroundings
Tumorproliferation / Apoptosis
Microvessel sprouting
Recruitment of periendothelial cells
Angioarchitecture
Neuroendocrine differentiation
Hormonal influence (Androgens)
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Paracrine pathway of angiogenic 
stimulation?
Tumor microenvironment?
Antiangiogenesis?
VEGF and Androgens in PCa
Androgens induce VEGF in hormone-sensitive tissues
VEGF induces EC proliferation
Hormonal ablation in androgen-sensitive tumors
Withdrawal of tumor cell VEGF:
Apoptosis of ECs AND tumor cells surrounding ECs
Regression of tumor mass
Problem: Recurrence due to hormone refractory variants
Antiangiogenic Trials
Natl Cancer Inst Database, 8/99
Tumorangiogenesis and
Cancer:
Relevance?
Diagnostic:
Screening (Serum, Urine)
Staging (Expression of GF, MVD, Imaging)
Prognosis
Therapeutic:
Antiangiogenesis (30 cancer drugs on trial NCI)
Market: $ 3 billion by 2005 (FT Pharmaceuticals)