Background

[PubMed]

Angiogenesis, the development of new vasculature from pre-existing blood vessels (for details see Carmeliet and Jain (1)), is essential for the development, maintenance, progression, and metastasis of neoplastic tumors (2). Therefore, angiogenesis is considered to be the hallmark of cancerous tumors, and early detection of this process can facilitate the initiation of treatment and management of the disease (3). Although there are several known pro-angiogenic biomarkers, among these only vascular endothelial growth factor receptor 2 (VEGFR-2) (4), α_Vβ₃ integrin (5), and endoglin (6) are well characterized and are overexpressed in many cancerous tumors, such as those of the breast, ovaries, and the pancreas (3). The VEGFR-2 mediates its effects through a family of receptor tyrosine kinases that promote the mitogenesis, survival, differentiation, migration, and vascular permeability of endothelial cells (7). The α_Vβ₃ integrins are heterodimeric cell adhesion molecules that can bind several different endogenous ligands such as fibronectin, von Willebrand factor, fibrinogen, etc., and assist with the survival and migration of cancerous cells, which increases the invasive potential of these cells, and promotes angiogenesis in tumors (8). Endoglin (CD105) is a co-receptor of the transforming growth factor beta (TGF-β) and co-modulates the different activities, including angiogenesis, of the activated TGF-β receptor (9).

Little information is available regarding the expression of the different angiogenic markers during the progression of a tumor from a small size to a larger size. Noninvasive visualization of angiogenic markers that are overexpressed during initial stages of the neoplasia can facilitate early detection and treatment of the disease (3). For this, Deshpande et al. developed a series of microbubble (MB; perfluorocarbon gas enclosed within spherical lipid shells harboring streptavidin moieties to bind biotinylated monoclonal antibodies (mAb) directed toward specific targets) based contrast agents that were coated with specific antibodies targeted to α_Vβ₃ integrin VEGFR 2 (MB_vegfr2), (MB_integrin), and endoglin (MB_endoglin), respectively (3). The targeted MBs were then used with ultrasound imaging to determine the expression of the angiogenic biomarkers during the growth of human ovarian (SKOV3 cells), human breast (MDA-MB-361 cells), and human pancreatic (MiaPaCa2 cells) cell line xenograft tumors in mice. This chapter describes the studies performed with MB_vegfr2. Studies performed with MB_endoglin and MB_integrin are described in separate chapters of MICAD (www.micad.nih.gov) (10, 11).

Synthesis

[PubMed]

The synthesis of MB_vegfr2 has been described in detail by Deshpande et al. (3). Briefly, the MBs were obtained as a freeze-dried preparation from a commercial source and reconstituted in 1 mL sterile 0.9% sodium chloride. Subsequently, 5 × 10⁷ of the reconstituted MBs were incubated with 5 μg biotinylated anti-mouse VEGFR 2 mAb for 10 min at room temperature to obtain MB_vegfr2. A similar procedure was used to obtain MB_endoglin and MB_integrin. For use as controls, another batch of MBs was linked to biotinylated rabbit immunoglobulin G antibodies (MB_control). The procedure used to remove excess mAb, if any, from the final preparations was not reported. The average number of antibody molecules bound per square micrometer of the MBs of each preparation was reported to be ~7,600 as determined with fluorescence-activated cell sorter analysis (3).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

A parallel plate flow chamber cell-binding assay with SVR cells (the high expression of VEGFR 2 and the other angiogenic biomarkers by these cells was confirmed with immunofluorescence staining) revealed that, compared to MB_control, a significantly higher (P = 0.003) amount of MB_vegfr2 bound to these cells (3). Little or no binding of these MBs was observed with control cells (4T1 cells) that did not express any of the angiogenic biomarkers. An overall significantly positive correlation (ρ = 0.83; P = 0.042) was observed between the number of MBs that attached to the SVR cells and the expression of VEGFR 2 by the cells. Pre-incubation of the SVR cells with the rabbit anti-mouse VEGFR 2 mAb was reported to block the binding of MB_vegfr2.

An ex vivo immunofluorescence study of the tumor sections showed that VEGFR 2 in the cells was colocalized with CD31, a biomarker specific for endothelial cells (3). This indicated that the in vivo ultrasound signal obtained from the cancerous lesions was indeed generated by the MB_vegfr2 bound to the VEGFR 2 receptors expressed by the cells in the tumors. In addition, a good correlation (ρ = 0.63) was evident between the in vivo ultrasound imaging signal and the ex vivo expression level of VEGFR 2 in the tumors (P = 0.05) (3).

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

Supplemental Information

[Disclaimers]

No Supplemental Information is currently available.

References

1.

Carmeliet P., Jain R.K. Molecular mechanisms and clinical applications of angiogenesis. Nature. 2011;473(7347):298–307. [PMC free article: PMC4049445] [PubMed: 21593862]

2.

De Bock K., Mazzone M., Carmeliet P. Antiangiogenic therapy, hypoxia, and metastasis: risky liaisons, or not? Nat Rev Clin Oncol. 2011;8(7):393–404. [PubMed: 21629216]

3.

Deshpande N., Ren Y., Foygel K., Rosenberg J., Willmann J.K. Tumor angiogenic marker expression levels during tumor growth: longitudinal assessment with molecularly targeted microbubbles and US imaging. Radiology. 2011;258(3):804–11. [PMC free article: PMC3042640] [PubMed: 21339349]

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Shibuya M., Claesson-Welsh L. Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis. Exp Cell Res. 2006;312(5):549–60. [PubMed: 16336962]

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Cai W., Chen X. Anti-angiogenic cancer therapy based on integrin alphavbeta3 antagonism. Anticancer Agents Med Chem. 2006;6(5):407–28. [PubMed: 17017851]

6.

Nassiri F., Cusimano M.D., Scheithauer B.W., Rotondo F., Fazio A., Yousef G.M., Syro L.V., Kovacs K., Lloyd R.V. Endoglin (CD105): a review of its role in angiogenesis and tumor diagnosis, progression and therapy. Anticancer Res. 2011;31(6):2283–90. [PubMed: 21737653]

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Koch S., Tugues S., Li X., Gualandi L., Claesson-Welsh L. Signal transduction by vascular endothelial growth factor receptors. Biochem J. 2011;437(2):169–83. [PubMed: 21711246]

8.

Wu G., Wang X., Deng G., Wu L., Ju S., Teng G., Yao Y., Liu N. Novel peptide targeting integrin alphavbeta3-rich tumor cells by magnetic resonance imaging. J Magn Reson Imaging. 2011;34(2):395–402. [PubMed: 21780231]

9.

Perez-Gomez E., Del Castillo G., Juan Francisco S., Lopez-Novoa J.M., Bernabeu C., Quintanilla M. The role of the TGF-beta coreceptor endoglin in cancer. ScientificWorldJournal. 2010;10:2367–84. [PMC free article: PMC5763795] [PubMed: 21170488]

10.

Chopra, A., Microbubbles coated with biotinylated rabbit anti-mouse endoglin monoclonal antibody. Molecular Imaging and Contrast agent Database (MICAD) [database online]. National Library of Medicine, NCBI, Bethesda, MD, USA. Available from www​.micad.nih.gov, 2004 -to current. [PubMed: 21977535]

11.

Chopra, A., Microbubbles coated with biotinylated rabbit anti-mouse alphaV beta3 integrin monoclonal antibody. Molecular Imaging and Contrast agent Database (MICAD) [database online]. National Library of Medicine, NCBI, Bethesda, MD, USA. Available from www​.micad.nih.gov, 2004 -to current.