A Quantitative High-Throughput Endothelial Cell Migration Assay

Abstract

By combining the use of BD Biosciences Fluoro Blok ™ membrane-based Boyden chambers with the Cellomics HCS Array Scan, a more sensitive method formeasuring cellmigration has been developed. This assay is based on counting nuclei ofmigrated cells on the bottomof the filter rather than conventional approaches, which usemeasurement of totalwell fluorescence. This cellmigration assay provides approximately 10-fold increased signal/background compared to conventional approaches and can be used to assess the effects of growth factors on endothelial cell migration and to screen chemical compounds for inhibitory effects on growth factor–mediated endothelial cell migration.

Keywords

human umbilical vein endothelial cells (HUVEC)vascular endothelial growth factor (VEGF)automated cell migration (ACM)

References

Stupack DG , Cho SY , Klemke RL : Molecular signaling mechanisms of cell migration and invasion. Immunol Res 2000;21:83-88. Mastyugin et al.

Cockerill GW , Gamble JR , Vadas MA : Angiogenesis: models and modulators. Int Rev Cytol 1995;159:113-160.

Fan TP , Polverini PJ : In vivo models of angiogenesis. In Bicknell R , Lewis CE , Ferrara N (eds): Tumor Angiogenesis. Oxford, UK: Oxford University Press, 1997:5-18.

Jain RK , Schlenger K , Hockel M , Yuan F : Quantitative angiogenesis assays: progress and problems. Nat Med 1997;3:1203-1208.

Gavrieli Y , Sherman Y , Ben-Sasson SA : Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119:493-501.

Parent CA , Devreotes PN : Acell's sense of direction. Science 1999;284:765-770.

Montesano R , Pepper MS , Vassalli JD , Orci L : Modulation of angiogenesis in vitro. EXS 1992;61:129-136.

Jin T , Zhang N , Long Y , Parent CA , Devreotes PN : Localization of theGprotein betagamma complex in living cells during chemotaxis. Science 2000;287:1034-1036.

Servant G , Weiner OD , Herzmark P , Balla T , Sedat JW , Bourne HR : Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science 2000;287:1037-1040.

10.

Boyden S : The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes. J Exp Med 1962;115:453-466.

11.

Goldberger A , Septak M : BD Biosciences Discovery Labware. Technical Bulletin 428, 1998. Retrieved from http://www.bdbiosciences.com/discovery_labware/technical_resources/pdf/tb428.pdf

12.

Maliakal JC : Quantitative high throughput endothelial cell migration and invasion assay system. Methods Enzymol 2002;352:175-182.

13.

Bold G , Altmann KH , Frei J , et al: New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis. J Med Chem 2000;43:2310-2323.

14.

Drevs J , Muller-Driver R , Wittig C , et al: PTK787/ZK222584, a specific vascular endothelial growth factor-receptor tyrosine kinase inhibitor, affects the anatomy of the tumor vascular bed and the functional vascular properties as detected by dynamic enhanced magnetic resonance imaging. Cancer Res 2002;62:4015-4022.

15.

Solorzano CC , Baker CH , Bruns CJ , et al: Inhibition of growth andmetastasis of human pancreatic cancer growing in nude mice by PTK787/ZK222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases. Cancer Biother Radiopharm 2001;16:359-370.

16.

Drevs J , Hofmann I , Hugenschmidt H , et al: Effects of PTK787/ZK222584, a specific inhibitor of vascular endothelial growth factor receptor tyrosine kinases, on primary tumor, metastasis, vessel density, and blood flowin amurine renal cell carcinoma model. Cancer Res 2000;60:4819-4824.

17.

Wood JM , Bold G , Buchdunger E , et al: PTK787/ZK222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res 2002;60:2178-2189.

18.

Wood JM : Inhibition of vascular endothelial growth factor (VEGF) as a novel approach for cancer therapy. Medicina (B Aires) 2000;60(suppl 2):41-47.

19.

Manley PW , Furet P , Bold G , et al: Anthranilic acid amides: a novel class of antiangiogenicVEGF receptor kinase inhibitors. J Med Chem 2002;45:5687-5693.

20.

Esser S , Wolburg K , Wolburg H , Breier G , Kurzchalia T , Risau W : Vascular endothelial growth factor induces endothelial fenestrations in vitro. J Cell Biol 1998;140:947-959.

21.

Millauer B , Wizigmann-Voos S , Schnurch H , et al: High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 1993;72:835-846.

22.

Fong GH , Rossant J , Gertsenstein M , Breitman ML : Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 1995;376:66-70.

23.

Shalaby F , Ho J , Stanford WL , et al: A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell 1997;89:981-990.

24.

Neufeld G , Cohen T , Gengrinovitch S , Poltorak Z : Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999;13:9-22.

25.

Veikkola T , Karkkainen M , Claesson-Welsh L , Alitalo K : Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res 2000;60:203-212.

26.

Joukov V , Pajusola K , Kaipainen A , et al: Anovel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) andKDR(VEGFR-2) receptor tyrosine kinases. EMBO J 1996;15:290-298.

27.

Makinen T , Jussila L , Veikkola T , et al: Inhibition of lymphangiogenesis with resulting lymphedema in transgenicmice expressing solubleVEGF receptor-3. Nat Med 2001;7:199-205.

28.

Zhang JH , Chung TDY , Oldenburg KR : Asimple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67-73.