Statistics of Cancer Death in Taiwan

Tumor Angiogenesis and Metastasis

Cancer has been the leading cause of death in the past 2 decades. Tumor Metastasis
37,222 patients died of cancer in Taiwan in 2005.
陳 炯 東 Cancer related death is increasing annually.
Chiung-Tong Chen, Ph.D. The spread or movement of cancer cells from the
2002 2004 2005 primary cancer site to another area of the body,
Lung 6,943; 7,153; 7,302 deaths which is the most deadly characteristics of cancer.
生物技術與藥物研究組 副研究員 Hepatic 6,846; 7,059; 7,108 deaths
Æ MALIGNANCY
實驗動物中心 主任 Colorectal 3,649; 3,898; 4,111 deaths
Gastric 2,433; 2,500; 2,490 deaths
國家衛生研究院 Oral 1,613; 1,993; 2,041 deaths
November 28th, 2006
Breast 1,203; 1,339; 1,439 deaths

Hematogenous Route & Lymphatic Route Tumor Metastasis

Types of Tumor Metastasis

This is the type in which the cancerous cells

of the disease float away from the
Hematogenous metastasis
cancerous tissue into the blood circulation
and migrate to another parts of the body.

This is the type in which the cancerous cells

of the disease flow to the outlying metastasis in
Lymphatic metastasis lymphatic knobs, circulate with the lymphatic fluid and
invade the lymphatic glands one by one, and
eventually enter into the veins through the left clavicle,
and disperse into the whole body.

Chambers et al, Nat. Rev Cancer 2:563-572, 2002 Nat. Rev. Cancer, Vol 2, 563-572 (2002)

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This is the type whereby the cancerous
Destructive metastasis
cells grow larger steadily and destruct adjoining
organs, tissues, e.g. invasion of metastasis
carcinoma of stomach into the abdominal cavity
to cause the type of carcinomatous peritonitis.
This is the type whereby cancerous cells
Contact metastasis contact the adjoining organs, e.g. metastasis
complication of carcinoma of stomach to neighboring
liver cancer.
The Seed-and-Soil hypothesis was challenged
by James Ewing in 1920s
Circulatory patterns between a primary tumor and
specific secondary organs were sufficient to account
for organ-specific metastasis.
66%, not all, accounted for by this blood flow hypothesis
Extracellular Matrix and Seed and Soil
Basement Membrane Structure
Stephen Paget, 1889, “The Lancet”
Tumor development, angiogenesis, and metastatic spread
depend on the ability of degradation of ECM and BM.
* The distribution of secondary tumor growth is
not a matter of chance.
* Which organ will suffer the disseminated
cancer?
* When a plant goes to seed, its seeds are
carried in all directions, but only congenital
soil will make it growth.
Annu. Rev. Biochem., 68:729-777 (1999)
Steps of Tumor Metastasis
Proliferation of primary tumor
Vascular neogenesis
Vascular invasion
Breast cancer frequently metastases to bone, liver, brain, lungs Disconnection of intercellular adhesions
Proteolysis of extracellular matrix
Migration in the extracellular matrix
Prostate cancer frequently metastases to bone
Transport into the blood stream
Immunological escape in the blood stream
Colon cancer frequently metastases to liver Arrest in the capillary circulation of target organs
Adhesion of tumor cells to endothelial cells
Extravasation from lymph and blood vessels
Infiltrating in the microenvironment
Metastasis growth
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1961 Tumor grow w/ blood vessels
To starve the tumor to death Angiogenesis Angiogenesis Angiogenesis
In vitro observations

1971 In vivo micropocket assay in rabbits • Formation of new blood vessels from pre-existing
vasculature ≥ a trillion (1,000,000,000,000) endothelial cells line the
1972 Successfully grew endothelial cells in inside of blood vessels and cover an area of
culture (w/ Gimbrone)
approximately 1000 m2 in a 70-kg adult.
1979 Tumor angiogenesis factors promote • Physiologic angiogenesis
growth of capillary endothelial cells – Reproduction, development, wound healing
(w/ Zetter) The turnover time of these normally quiescent cells can
– Intermittent, localized, tightly regulated
1989 No more questions about the importance – Vessels well-formed
exceed 1000 days.
of angiogenesis from clinicians or
pharmaceutical companies
• Pathologic angiogenesis During angiogenesis, capillary endothelial cells can
1999 Endostatin in clinical trials
– Cancer and other disease states
proliferate as rapidly as bone marrow cells, with a mean
($7 million/kg for the 1st batch)
– Sustained, localized turnover time of 5 days.
2006 81 (19 - 2003) related clinical trials as of today
– Vessels tortuous, leaky

Complex sequential process of angiogenesis

Angiogenic Switch
Step
1. Endothelial cell activation Stimulators
VEGF, FGF, PDGF,
2. Basement membrane and extracellular matrix degradation PD-ECGF, EGF, HGF,
TGF-α, and others

3. New capillary-tubes formation +

Angiogenesis
4. Vascular lumen and deposition of basement membrane _
5. Linkage to pre-existing vessels and formation of new Inhibitors
capillary loops and of the intratumoural vascular network Thrombospondin, endostatin,
angiostatin, interferons, TIMPs, and
others

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 Tumor Angiogenesis Tumor Angiogenesis Tumor Angiogenesis
Tumor Tumor
Tumor CO2 ↑
Hypoxia ↑
COX–2 ↑
NO ↑
Oncogenes
Secretion of Endothelial cell (EC)
Angiogenic proliferation and migration Formation of
angiogenic new tumor
factors
factors vasculature
Proteolytic degradation of
the extracellular matrix
(ECM) Angiogenic
factors

Sprouting capillary

Tumor angiogenesis Tumor Growth and Metastasis

Depend on Angiogenesis
5. Intravasation
1. Secretion of Tumor • Preclinical evidence
angiogenic
factors
4. Appearance – Angiogenesis is essential for sustained tumor growth
of new tumor
vasculature – In animal models, inhibiting angiogenesis slows or stops
2. Proteolytic
destruction 3. Endothelial
tumor growth
of ECM cell proliferation – Inhibition of angiogenesis limits tumor cell access to
and migration
the circulation and prevents tumor growth at metastatic
sites
• Clinical evidence
– Vessel density in the tumor correlates with progression and
survival*†
Sprouting capillary
*Takahashi et al. Cancer Res. 1995.
ECM, extracellular matrix † Weidner et al. N Engl J Med. 1991.

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 Huamn endothelial Cell Migration Assay
Angiogenesis Models - in vitro model
Methods for evaluation
of angiogenesis activity
In vitro Endothelial cell proliferation, migration, chemotaxis
or chemoinvasion assay and tube formation

Ex vivo Rat aorta tube formation

In vivo Chicken chorioallantoic membrane assay

(dish SPF egg method)

Matrigel plug assay

Mouse corneal micro-pocket assay

Rat Aorta Tube Formation Assay Rat Aorta Tube Formation Assay
Endothelial Cell Migration Assay - ex vivo model (3D)
- ex vivo model (3D)
- in vitro model

w/o VEGF w/ VEGF, 10 ng/ml A B

SD rats
C D

Wang et al, ASSAY Drug Dev. Technol., 2(1):31-38, 2004.

Nicosia & Ottinetti, In Vitro Cell Biol. 1990; Lab. Investigation 1990.

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Rat Aorta Tube Formation Assay Rat Aorta Tube Formation Assay Rat Aorta Tube Formation Assay
- ex vivo model (3D) - ex vivo model (3D) - ex vivo model (3D)
MTT staining MTT staining
Day 1 Day 5 MTS reaction Control Taxol 0.1 µM

MTT staining
Day 2 Day 6

Taxol 10 µM Taxol 0.01 µM

Image
Optical Density Analysis

Day 3 Day 7 Data

Analysis
Taxol 1 µM Taxol 0.001 µM
Area/OD
Day 4 Day 8
insoluble formazan

Wang et al, ASSAY Drug Dev. Technol. 2(1):31-38, 2004. Wang et al, ASSAY Drug Dev. Technol. 2(1):31-38, 2004.

Rat Aorta Tube Formation Assay Rat Aorta Tube Formation Assay (3-D)
- ex vivo model (3D) Rat Aorta Tube Formation Assay
1.5 - ex vivo model (3D)
suramin 2-methoxyestradiol
Absorbance, OD490nm
1.2

Two tetrazolium used in cell proliferation assays 0.9

0.6 100 100

Angiogenic Activity
0.3 80

Angiogenic Activity
insoluble formazan 80
A

(% of control)

(% of control)
0.0
0 8 16 24 32 40 48
60
MTS incubation, hr 60
40
MTS assay CD31 staining 20 40

0
soluble formazan 20
-20
0
0.1 10 1000 0.001 0.1 10

Concentration (µM) Concentration (µM)

Marshall et al, Growth Regul. 5(2):69-84, 1995
Wang et al, ASSAY Drug Dev. Technol., 2(1):31-38, 2004. Wang et al, ASSAY Drug Dev. Technol., 2(1):31-38, 2004.

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 Chick embryo chorioallantoic membrane (CAM) assay
In Vivo Models
Chick embryo chorioallantoic membrane (CAM) assay
• Chicken embryo chorioallantoic membrane (CAM) assay
(dish SPF egg method)
• Advantage: proper for massive screening
• Disadvantage: lack of metabolic activation disc w/ the
potential differences from human testing agent
• Subcutaneous Matrigel plug with angiogenic factors
• Advantage: physiologically relevant, ease of implementation
• Disadvantage: local inflammation

• Mouse corneal micro-pocket assay

• Advantage: physiologically relevant
• Disadvantage: surgery needed; local inflammation; time/labor consuming

• Tumor xenograft orthotopic and subcutaneous models

• Advantage: pathologically relevant
• Disadvantage: time/labor consuming; expensive

CAM assay Matrigel Plug Assay

(mouse) Corneal Micro-pocket Assay (Mouse)

(O'Reilly et al., Cell, 1994)

P: bFGF pallet

Nishikawa et al, Eur. J. Pharmacol. 539(3):151-7, 2006

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 Subcutaneous xenograft tumor model
HepG2 DLD-1
Rationale of Targeting Angiogenesis
• Angiogenesis required for growth and metastasis of many
tumors
• Angiogenesis prompted by paracrine stimuli (eg. VEGF)
• Endothelial cells genetically stable than cancer cells
• Potential for therapeutic versatility
Anti-Tumoral Effects Against Human Tumor Xenografts Orthotopic Liver Tumor Model
PLC/PRF/5-GFP Orthotopic implant
KB cervical cancer, 1x106 SC
Subcutaneous implant
Kuo et al, Cancer Res., 64:4621-4628, 2004.
Goals of basic research for anti-angiogenics Strategies to design anti-angiogenesis agents
• For targeting endothelial cells (tumor specific)
• Block factors that stimulate angiogenesis (eg. VEGF)
• For chronic long-term dosing
• Block mediators for stimulating angiogenesis (eg. VEGFR)
• For very low toxicity
• Block the ability of the endothelial cells or matrix proteases to break
down the surrounding matrix
• To determine possible mechanism(s) of resistance
• Block the action of integrin, a molecule on the endothelial cell surface
• To explore possibility of combinational therapy
• Ιnhibit normal/tumor endothelial cells directly
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 Summary of VEGF functions Activation of VEGF receptor signaling VEGF Expression Correlates With
VEGF-A
PlGF
• VEGF is a key angiogenic regulator directly implicated in VEGF-A
VEGF-C
VEGF-D
VEGF-C
VEGF-D
Colon Cancer Metastasis
– endothelial cell proliferation VEGF-B
100 13/14 • Tumor specimens obtained
– endothelial cell migration
80 from 62 patients were stained
– endothelial cell protease expression

Metastases (%)
with an anti-VEGF antibody.

Patients With
60
– endothelial cell adhesion For each patient group with
Blood vascular 40 10/29
– capillary tube formation different levels of staining
endothelial cell
– vessel maturation/pericyte recruitment VEGFR-1 VEGFR-2 VEGFR-3 Lymphatic 20 intensity (measured on a 0–
vascular 1/19
endothelial 0
3+ scale), the percentage of
cell 0 and 1+ 2+ 3+
metastatic disease was
• It also acts as Proliferation, vascular permeability, migration, survival Intensity of VEGF Staining
calculated.
– a survival factor for newly formed vasculature
PlGF, placental growth factor
– Vascular Permeability Factor VEGFR, vascular endothelial growth factor receptor
*Takahashi et al. Cancer Res. 1995.

Approaches to VEGF Signal Inhibition Approaches to VEGF signal inhibition

Inhibition of VEGF signaling 1. Ligand sequestration: Ligands
mAbs, soluble receptors
VEGF-A
Anti-VEGF antibodies VEGF-B
Monoclonal
VEGF-C
antibody
VEGF-D
• Inhibiting VEGF signaling
– inhibits growth of new tumor vessels
2. Receptor blocking: p85
– decreases vascular density, diameter and permeability
mAbs
– may induce regression of recently developed tumor microvessels
PLCγ GRB2
• Therapeutic inhibition of tumor angiogenesis should be effective in a Blood vascular
broad range of solid malignancies 3. Tyrosine kinase endothelial cell Lymphatic
inhibition: TKIs vascular
endothelial
• Target tissue is in direct contact with blood, facilitating drug delivery 5. Inhibition of downstream cell
signaling events VEGFR-1
VEGFR-2
6. Transcription VEGFR-3
4. VEGFR antisense factor inhibition
oligonucleotide Endothelial cell

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 Approaches to VEGF signal inhibition Approaches to VEGF signal inhibition
Ligands Ligands VEGFR tyrosine kinase inhibitors
VEGF-A VEGFR-TKIs VEGF-A
Anti-VEGFR antibodies VEGF-B VEGF-B
Monoclonal VEGF-C
VEGF-C
antibodies VEGF-D
VEGF-D • Preclinical studies have evaluated the potency, selectivity,
Lymphatic anti-angiogenic and antitumor activity of VEGFR tyrosine
Blood vascular vascular
endothelial
kinase inhibitors (VEGFR-TKIs)
endothelial cell
cell

Blood vascular
• A VEGFR-TKI from AstraZeneca is currently in Phase II/III
endothelial cell Lymphatic trials in NSCLC and CRC.
vascular
VEGFR-TKI
endothelial VEGFR-1 VEGFR-2 VEGFR-3

VEGFR-1
cell Inhibition X X X X
VEGFR-2
VEGFR-3 Angiogenesis Lymphangiogenesis

VEGFR-TKI, vascular endothelial growth factor receptor-tyrosine kinase inhibitor

Treatment options are limited for NSCLC response rates decrease with
Lung cancer
advanced lung cancer subsequent regimens of chemotherapy
• Lung cancer is the leading cause of cancer death,
with approximately 1.1 million deaths/year worldwide Objective 25
• Cytotoxic chemotherapy is often a palliative treatment with response 20.9
– ~80% of lung cancers are of the non-small-cell histology
effectiveness for a limited period rate (%) 20
– the majority of patients have locally advanced or 16.3
metastatic disease • With additional cytotoxic treatment, the
15
– chemotherapy is the mainstay of treatment potential for toxicity often outweighs any possible benefits
• Symptoms of advanced disease are often severe, debilitating and • Response rates are known to decrease with subsequent regimens 10
difficult to manage of chemotherapy
5 2.3 2.3
• Median survival, from the start of the last chemotherapy treatment, is – 5.5% of patients survive for 1 year following 3rd- or
0.0
short for patients with advanced lung cancer (4 months) 4th-line chemotherapy 0
1st 2nd 3rd 4th Last

Ferlay et al 2000; Cersosimo 2002; Massarelli et al 2003 Massarelli et al 2003 Line of therapy Massarelli et al 2003

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 3rd- and 4th-line chemotherapy Activation of the EGFR:
is of limited benefit in NSCLC a pivotal driver of malignancy
Survival 1.0 Ligand
probability 0.9 Median survival 4 months
1-year survival 5.5% EGFR
0.8
0.7
The epidermal growth factor receptor
EGFR-TK
0.6
0.5
(EGFR) signalling pathway and the PI3-K pY
pY
pY GRB2
SOS
RAS RAF
0.4 mechanism of action of gefitinib PTEN Akt
STAT3

0.3 MEK
0.2 (IRESSA) Gene transcription
Cell-cycle progression MAPK
0.1 PP
DNA Myc Cyclin D1
0.0 Proliferation / JunFos
Survival
maturation Myc Cyclin D1 (anti-apoptosis)
0 6 12 18 24
Months after start of 3rd- or 4th-line therapy
Chemotherapy / radiotherapy / Metastasis
n=43 Massarelli et al 2003 hormonal resistance Angiogenesis

IRESSA EGFR inhibition by IRESSA mTOR Inhibition Decreases Angiogenesis

Ligand
• IRESSA is an orally active • mTOR regulates HIF-1α
EGFR tyrosine kinase inhibitor EGFR and HIF-2α expression
(TKI) • HIF-1 and HIF-2 are
transcription factors for
• IRESSA is the first approved hypoxic stress-related
EGFR-TK
EGFR-TKI for the treatment of genes
advanced NSCLC EGFR-TKI • HIF-1α/2α are normally
degraded by VHL protein
• The mechanism of action of
• HIF-1 and HIF-2
IRESSA is distinct from condition the tumor to
Proliferation Inhibition of
cytotoxic chemotherapy adapt to growth under
apoptosis
hypoxic conditions and
Invasion Metastasis promote angiogenesis and
Angiogenesis metastasis
HIF = hypoxia-inducible factor; VHL = von Hippel-Lindau protein.

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mTOR - Tumor Cell Growth and Angiogenesis Angiogenesis Inhibitors and mTOR Inhibitors Development of a new inhibitor of angiogenesis
- Pre-Clinical Stage
May Act Synergistically
Endothelial cell • Chemical purification or synthesis of the agent – CMC/GMP production
Growth factors Smooth muscle cell (pericyte) Primary orthotopic (ear) B16 melanoma
• Pharmacokinetics and Metabolism/Toxicology/Safety Pharmacology
Cancer cell 8 Vehicle, n = 6
1 mg/kg mTOR inhibitor, po q24h, n = 6
PI3-K

PI3-K
100 mg/kg VEGFR inhibitor, po q24h, n = 6
1 mg/kg mTOR inhibitor + VEGFR inhibitor, n = 6
• Evaluation of the angiosuppressive effects – Pharmacological Evaluations
PTEN
Akt/ PI3-K
6
5 mg/kg mTOR inhibitor + VEGFR inhibitor, n = 6
in vitro assays

Fractional Tumor Volume

PKB

endothelial cell proliferation and migration

Akt/

(V/Vo, mean ± SEM)

PKB
TSC2 TSC1 Akt/
PKB

mTOR mTOR
endothelial cell invasiveness
4
mTOR
endothelial cell capillary-like tube formation
Protein production

HIF-1α
in vivo assays
VHL
HIF-2α 2 * *P < .05 vs vehicle controls chicken chorioallantoic membrane (CAM assay)
* and single agents.
Cell growth
and proliferation Angiogenic
Cell growth Matrigel® plug assay
growth factors
and proliferation
0 corneal vascularization (micropocket assay)
7 14 21
Cell growth
and proliferation Days Post Tumor Cell Injection primary tumor growth metastasis models
experimental tumor xenografts in mice
*O’Reilly et al. Proc Am Assoc Cancer Res. 2005;46:715. Abstract 3038.

Pharmacological Indices for Anti-angiogenics

Challenges in Clinical Development Clinical Trial Design
• Inhibition of pre-existing vasculature and of vascularization of tumors
• Dose selection • Clinical end points — Phase II
• Mechanism of action and identification of the specific step(s) of
angiogenesis inhibited – MTD may not be relevant. – Response rate: absence of effect may not mean lack of activity
– Optimal biologic effective dose may be preferable if – Stable disease, time to progression, biologic response, clinical
• Definition of the activity-related factors for the anti-angiogenics determination possible benefit

• Angiosuppressive activity of the serum of the treated animals (yes/no)

• Demonstration of clinical benefit • Combination therapy
• Chemotherapy
• Development of tissue and serum surrogate of predictive markers of the – Disease stabilization rather than objective response
– Phase III: AvastinTM (bevacizumab), w/ 5-FU/leucovorin/CPT-11, first-line for
antiangiogenic activity metastatic colorectal cancer, increase survival and FDA approved.
• Tumor target selection • Radiotherapy
• In vivo pharmacodynamic (dose & time vs effects) monitoring of the • Other novel agents
effects induced by the agent on tumor vascularization
• Reliable measurement of in vivo biological activities

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 Animal Pharmacology Group

陳 炯 東
ctchen@nhri.org.tw
http://lac.nhri.org.tw ; http://www.nhri.org.tw
02-2653-4401 ext. 35711

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