11 28 2006 Oncology Angiogenesis Notes

Angiogenesis, 陳炯東, Nov.2006

陳炯東 Chiung-Tong Chen, Ph.D. Biotechnology and Pharmaceutical Research National Health Research Institutes Phone: 02-2653-4401 Ext. 35711

November 28, 2006

TUMOR ANGIOGENESIS AND METASTASIS Steps of Tumor Metastasis Proliferation of primary tumor Vascular genesis Vascular invasion Transport into the blood stream Arrest in the capillary circulation of target organs Adhesion of tumor cells Extravasation Implant in the microenvironment Metastasis growth

Dr. Judah Folkman: Cancer cells implanted in vascular sites in animals grew rapidly and formed large tumors. Cancer cells implanted in avascular sites were unable to form tumor masses more than 1-2 mm in size. Hypothesis: Angiogenesis is obligatory for tumor growth.

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Angiogenesis, 陳炯東, Nov.2006

Diseases in Which Angiogenesis Plays a Role in Pathogenesis Inflammatory diseases

arthritis, chronic inflammations, psoriasis, inflammatory bowel diseases, endometriosis. Neoplasms breast, bladder, colon, liver, lung, prostate, testes, pancreas, kidney, uterine-cervix, glioblastoma, hemangioblastoma, melanoma, neuroblastoma, Kaposi’s sarcoma, leukemia, myelofibrosis, polycythemia vera and multiple myeloma. Ocular diseases age-related macular degeneration, proliferative retinopathy (diabetic). Developmental disorders bowel atresia, vascular malformations, hemingiomas and unilateral facial atrophy. Other diseases gastroduodenal ulcer, atherosclerotic plaques, dysfunction associated with physiological angiogenic processes, such as ovulation, repair of menstruating uterus and development of the placenta.

ANGIOGENESIS: A multistep, tightly controlled physiological process involving tissue repair and/or remodeling such as wound healing, placental development etc. Tissue repair and remodeling involves continuous feedback and interaction between endothelial cells and the extracellular matrix (ECM). Vascular remodeling is accomplished by targeted apoptosis and proliferation, deposition of matrix and its stabilization, and organization by enzymatic cross-linking and proteolysis. Angiogenesis involves an initial localized breakdown of the basement membrane in the parent vessel that is mediated by proteases.

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Endothelial cells migrate into the perivascular space and adjoining matrix, and form a capillary sprout.

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Elongate by further endothelial migration at the tip, and proliferation at the base, to replace the migrated cells. Remodeling occurs, and these cords anastamose to form a loop, basement membrane is laid out, and a new vessel is formed.

The complex sequential process of angiogenesis Step 1. Endothelial cell activation 2. Basement membrane and extracellular matrix degradation 3. New capillary-tubes formation 4. Vascular lumen and deposition of basement membrane 5. Linkage to pre-existing vessels and formation of new capillary loops and of the intratumoural vascular network Angiogenic cascade: activation and resolution phases. Activation: sequential basement membrane degradation, endothelial cell migration and invasion of the surrounding extracellular matrix, endothelial cell proliferation, and capillary lumen formation. Resolution: the maturation and stabilization of the newly formed microvasculature by investment of the vessel with pericytes and subsequent inhibition of endothelial proliferation, basement membrane reconstitution, and junctional complex formation. The resolution phase is often incomplete, resulting in tumor microvessels that are highly irregular and tortuous with only partial endothelial linings and basement membranes; arterio-venous shunts and blind ends are common. Role of Balanced Proteolysis in Angiogenesis Migratory path formation and remodeling: Basement membrane degradation Controlled ECM degradation involved during migration/invasion of endothelial and inflammatory cells into matrix Anastamoses and capillary lumen/tube formation Release of cytokines: Release of bound basic FGF and VEGF Activation of TGF-β from latent to active form Degradation products with angiogenesis modulating capability: Angiostatin (plasminogen) Collagen derived peptides Endostatin (collagen XVIII) Fibrin and fibronectin fragments 16-kDa fragment of prolactin

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Angiogenesis, 陳炯東, Nov.2006

Molecules w/ Proangiogenic Activity Angiogenin Fibroblast growth factors (acidic and basic) Heparin Hepatocyte growth factor Insulin-like growth factors Interleukin-3 Interleukin-8 Leptin Placental growth factor Platelet-activating factor Platelet-derived endothelial cell growth factor Platelet derived growth factor Pleiotropin Proliferin Transforming growth factor-α Transforming growth factor-β Tumor necrosis factor-α Vascular endothelial growth factor

Molecules w/ Antiangiogenesis Activity Angiostatin Endostatin Interferon-α Tissue inhibitors of matrix metalloproteinases (TIMPs) Platelet factor 4 Somatostatin Thrombospondin

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Rationale of Targeting Angiogenesis 1. Angiogenesis required for growth and metastasis of many tumors 2. Angiogenesis prompted by paracrine stimuli (eg. VEGF) 3. Endothelial cells genetically stable than cancer cells 4. Potential for therapeutic versatility

Criteria for Antiangiogenic Activity • Differential cytotoxicity • Alters endothelial cell function • Critical mechanistic effects • Inhibition of angiogenesis in vivo

Differential cytotoxicity: Toxic to endothelial cells at doses less than those needed for a toxic effect on cancer cells Alters endothelial cell function: The agent interferes with endothelial cell function without causing endothelial cell death Critical mechanistic effects: The agent interferes with a specific portion of the angiogenic cascade Inhibition of angiogenesis in vivo: The agent shows in vivo evidence of antiangiogenic activity

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Potential Mechanisms of Resistance to Antiangiogenic Chemotherapy • Protective tumor microenvironment • Chemotherapy-induced tumor hypoxia stimulates angiogenesis • Regrowth independent of angiogenesis • Ineffective pharmacokinetics Protective tumor microenvironment : Tumor-associated endothelial cells are affected by the tumor microenvironment Chemotherapy-induced tumor hypoxia stimulates angiogenesis : Cytotoxic chemotherapy induced tumor necrosis and hypoxia stimulate VEGF production Regrowth independent of angiogenesis : Tumor regrowth is not dependent on angiogenesis Ineffective pharmacokinetics : Antiangiogenic activity requires different drug doses and schedules than those required for optimal cytotoxicity

Development of a new inhibitor of angiogenesis Pre-Clinical Stage • Chemical purification or synthesis of the agent • Evaluation of the angiosuppressive effect in vitro assays endothelial cell proliferation and migration endothelial cell invasiveness endothelial cell capillary-like tube formation in vivo assays chicken chorioallantoic membrane (CAM assay) Matrigel® implantation corneal neovascularization (micropocket assay) primary tumor growth metastasis models experimental tumor xenografts in nude mice • Pharmacokinetics and metabolism 9

Angiogenesis, 陳炯東, Nov.2006

PRECLINICAL ANGIOGENESIS MODELS In vitro models Proliferation assay: Endothelial cell counts, thymidine incorporation, cell cycle perturbation, apoptosis or immunohistochemical staining. The most basic, simple, rapid and inexpensive method. Endothelial cell migration, chemotaxis or chemoinvasion: (Transwell or Boyden chamber)

Tube formation: Activated endothelial cells in culture form cords and tubes in a process thought to recapitulate early capillary development. Tubule formation in two dimensions on tissue culture dishes or in three dimensions into a collagen matrix, Matrigel®, a simulated basement membrane, or a fibrin clot.

Rat aorta tube formation: Outgrowth of microvessels containing endothelial cells confirmed by staining for factor VIII, CD34 and CD31.

In vitro assays: Advantages: Lower cost and more rapid results; Controlled experimental conditions; Allowing each component of endothelial cell function to be studied independently. Limitations: Source of endothelial cells; Experimental technique and method of quantification make comparisons across studies difficult. Results are not always reproducible in different assays.

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In vivo Models Four distinct in vivo approaches of angiogenesis: (1) microcirculatory preparations (2) vascularization into matrix implants (3) tumor xenografts (4) genetic manipulation 1) Microcirculatory preparations a. Chick chorioallantoic membrane (CAM) assay Advantages: the least expensive and most commonly used Limitations: nonspecific inflammatory response generated by the implanted material; difficulty of precise quantification. b. Corneal micropocket assay: rabbit, rat, mouse Advantages: amenable to frequent observation, reliable model Limitations: technically demanding; expensive as a screening tool c. Dorsal skin chambers

d. Hamster cheek pouch

e. Subcutaneous air sac technique: rat, rabbit

f. Intradermal injection of tumor cells or angiogenic factors

2) Vascularization into a semi-permeable implant: microcapsules, discs, matrix, Matrigel®, alginate-tumor pellets Vascularization into a matrix implant forms the basis of several techniques including the commonly used disc angiogenesis system, Matrigel® and above listed.

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3) Orthotopic and subcutaneous tumor xenograft models Advantages: The most intact physiologic system Limitations: Significant intra- and inter-tumor variability - large sample size. Tumor angiogenesis is heterogeneous. Excision of the tumor required, not if a proper image system used. Major differences between orthotopic and heterotopic implants. Different strains of mice may have a 10-fold range of response.

4) Transgenic Animals A new approach is the creation of transgenic mouse models of tumor angiogenesis to investigate the angiogenic switch, its molecular controls, and prospects for preclinical therapeutic models.

Quantitative indices: Vascularization can be assessed histologically and morphometrically (vascular density) or biochemically (protein and hemoglobin content). Determination of Vascular Permeability

Estimation of Vessel Density

In vivo assays: Advantage: Limitation:

Physiologically relevant environment Time/labor consuming

Research goals for anti-angiogenics For targeting endothelial cells For chronic long-term dosing For very low toxicity To determine possible mechanism(s) of resistance To explore possibility of combinational therapy

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References: 1. Teicher, B.A., Antiangiogenic agents in cancer therapy, Humana Press Inc., New Jersey, USA, 1999. 2. Miller, K.D., Sweeney, C.J., and Sledge Jr., G.W., Redefining the target: Chemotherapeutics as antiangiogenics. J. Clin. Oncol. 19:1195-1206, 2001. 3. Li, W.W., Tumor Angiogenesis: Molecular Pathology, Therapeutic Targeting, and Imaging. Acad. Radiol., 7:800-811, 2000. 4. http://clinicaltrials.gov 5. Hoffmann, J., Schirner, M., Menrad, A., and Schneider, M.R., Animal Models for Determination of Anti-Angiogenic Drug Effects, In: ”Relevance of Tumor Models for Anticancer Drug Development.” Eds. by Fiebig, H.H. and Burger, A.M., Karger Press, Basel, Germany, 1999, vol. 54, pp 169-180. 6. Cooke, R., DR. FOLKMAN’S WAR: Angiogenesis and the Struggle to Defeat Cancer. Random House Inc., New York, USA, 2001. 7. J. Folkman. Angiogenesis-Dependent Diseases. Sem. Oncol. 28:536-542, 2001. 8. J. Folkman. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N. Engl. J. Med. 333: 1757-1763, 1995. 9. Croix et al. Genes expressed in human tumor endothelium. Science 289, 1197-1202,2000. 10. Chambers et al, Nature Rev Cancer 2:563-572, 2002. 11. Wang et al, ASSAY Drug Dev. Technol., 2(1):31-38, 2004. 12. Kuo et al, Cancer Res., 64:4621-4628, 2004.

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Li, W.W., Acad. Radiol. 7:800-811, 2000.

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