Chambers et al, Nat. Rev Cancer 2:563-572, 2002 Nat. Rev. Cancer, Vol 2, 563-572 (2002)
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Extracellular Matrix and Seed and Soil
This is the type whereby the cancerous
Destructive metastasis
cells grow larger steadily and destruct adjoining Basement Membrane Structure
organs, tissues, e.g. invasion of metastasis Stephen Paget, 1889, “The Lancet”
carcinoma of stomach into the abdominal cavity Tumor development, angiogenesis, and metastatic spread
to cause the type of carcinomatous peritonitis. depend on the ability of degradation of ECM and BM.
* The distribution of secondary tumor growth is
not a matter of chance.
66%, not all, accounted for by this blood flow hypothesis 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
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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
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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
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
SD rats
C D
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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
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
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
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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
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Subcutaneous xenograft tumor model Anti-Tumoral Effects Against Human Tumor Xenografts Orthotopic Liver Tumor Model
PLC/PRF/5-GFP Orthotopic implant
HepG2 DLD-1
Rationale of Targeting Angiogenesis Goals of basic research for anti-angiogenics Strategies to design anti-angiogenesis agents
• Angiogenesis required for growth and metastasis of many • For targeting endothelial cells (tumor specific)
tumors • Block factors that stimulate angiogenesis (eg. VEGF)
• For chronic long-term dosing
• Block mediators for stimulating angiogenesis (eg. VEGFR)
• Angiogenesis prompted by paracrine stimuli (eg. VEGF)
• For very low toxicity
• Block the ability of the endothelial cells or matrix proteases to break
• Endothelial cells genetically stable than cancer cells down the surrounding matrix
• To determine possible mechanism(s) of resistance
• Potential for therapeutic versatility • 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.
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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
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
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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
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.
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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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