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Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies.

Iqbal U, Albaghdadi H, Luo Y, Arbabi M, Desvaux C, Veres T, Stanimirovic D, Abulrob A - Br. J. Cancer (2010)

Bottom Line: In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour.Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe₃O₄ NPs selectively in GBM vessels.Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biological Sciences, National Research Council of Canada, 1200 Montreal Road, Ottawa, Ontario, Canada K1A 0R6.

ABSTRACT

Background: Insulin-like growth factor-binding protein 7 (IGFBP7) is an abundant, selective and accessible biomarker of glioblastoma multiforme (GBM) tumour vessels. In this study, an anti-IGFBP7 single-domain antibody (sdAb) was developed to target GBM vessels for molecular imaging applications.

Methods: Human GBM was modelled in mice by intracranial implantation of U87MG.EGFRvIII cells. An anti-IGFBP7 sdAb, isolated from an immune llama library by panning, was assessed in vitro for its binding affinity using surface plasmon resonance and by ex vivo immunobinding on mouse and human GBM tissue. Tumour targeting by Cy5.5-labelled anti-IGFBP7 sdAb as well as by anti-IGFBP7 sdAb conjugated to PEGylated Fe₃O₄ nanoparticles (NPs)-Cy5.5 were assessed in U87MG.EGFRvIII tumour-bearing mice in vivo using optical imaging and in brain sections using fluorescent microscopy.

Results: Surface plasmon resonance analyses revealed a medium affinity (K(D)=40-50 nM) binding of the anti-IGFBP7 sdAb to the purified antigen. The anti-IGFBP7 sdAb also selectively bound to both mouse and human GBM vessels, but not normal brain vessels in tissue sections. In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour. Similarly, the anti-IGFBP7 sdAb-functionalised PEGylated Fe₃O₄ NP-Cy5.5 demonstrated enhanced tumour signal compared with non-targeted NPs. Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe₃O₄ NPs selectively in GBM vessels.

Conclusions: Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.

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Related in: MedlinePlus

Representative immunofluorescence images demonstrating IGFBP7 immunoreactivity detected with the anti-IGFBP7 sdAb 4.43 in tissue sections of the (A) mouse orthotopic GBM, (B) contralateral healthy mouse brain and (C) human GBM. IGFBP7 immunoreactivity is shown in red (middle panels); the staining for the endothelium-specific markers, CD31 and UEA1, for mouse and human tissues, respectively, is shown in green (right panels) and cell nuclei stained with DAPI are shown in blue in overlay images (left panels).
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fig2: Representative immunofluorescence images demonstrating IGFBP7 immunoreactivity detected with the anti-IGFBP7 sdAb 4.43 in tissue sections of the (A) mouse orthotopic GBM, (B) contralateral healthy mouse brain and (C) human GBM. IGFBP7 immunoreactivity is shown in red (middle panels); the staining for the endothelium-specific markers, CD31 and UEA1, for mouse and human tissues, respectively, is shown in green (right panels) and cell nuclei stained with DAPI are shown in blue in overlay images (left panels).

Mentions: The ability of anti-IGFBP7 sdAb to detect IGFBP7 expression in mouse and human GBM tissue was evaluated by immunofluorescence (Figure 2). In brain sections of GBM-bearing mice, IGFBP7 immunoreactivity detected by anti-IGFBP7 sdAb (clone 4.43) was enhanced and detected specifically in the tumours (Figure 2A), but not in normal brain (Figure 2B), and colocalised with the immunoreactivity against the vascular antigen, CD31 (Figure 2A). No IGFBP7 expression was observed in non-tumour brain vessels or in non-vascular cells. In surgically removed human GBM tissue, IGFBP7 immunoreactivity (Figure 2C, middle panel) colocalised with highly abnormal tumour vasculature stained with Ulex europeaus agglutinin I (UEA I) (Figure 2C, right panel), shown previously to selectively bind to carbohydrates of human brain vessels (Holthofer et al, 1982). These results indicate that anti-IGFBP7 sdAb detects its target, IGFBP7, in both mouse and human GBM tissue sections ex vivo. This analysis confirms our previous observation of a selective vascular upregulation of IGFBP7 in human GBM (Pen et al, 2007) and demonstrates that mouse vessels ingrowing into orthotopically implanted human GBM (U87MG-EGFRvIII) are selectively induced by the tumour microenvironment to overexpress IGFBP7. This mouse model is thus suitable for in vivo assessment of tumour targeting/imaging using anti-IGFBP7 sdAb.


Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies.

Iqbal U, Albaghdadi H, Luo Y, Arbabi M, Desvaux C, Veres T, Stanimirovic D, Abulrob A - Br. J. Cancer (2010)

Representative immunofluorescence images demonstrating IGFBP7 immunoreactivity detected with the anti-IGFBP7 sdAb 4.43 in tissue sections of the (A) mouse orthotopic GBM, (B) contralateral healthy mouse brain and (C) human GBM. IGFBP7 immunoreactivity is shown in red (middle panels); the staining for the endothelium-specific markers, CD31 and UEA1, for mouse and human tissues, respectively, is shown in green (right panels) and cell nuclei stained with DAPI are shown in blue in overlay images (left panels).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990581&req=5

fig2: Representative immunofluorescence images demonstrating IGFBP7 immunoreactivity detected with the anti-IGFBP7 sdAb 4.43 in tissue sections of the (A) mouse orthotopic GBM, (B) contralateral healthy mouse brain and (C) human GBM. IGFBP7 immunoreactivity is shown in red (middle panels); the staining for the endothelium-specific markers, CD31 and UEA1, for mouse and human tissues, respectively, is shown in green (right panels) and cell nuclei stained with DAPI are shown in blue in overlay images (left panels).
Mentions: The ability of anti-IGFBP7 sdAb to detect IGFBP7 expression in mouse and human GBM tissue was evaluated by immunofluorescence (Figure 2). In brain sections of GBM-bearing mice, IGFBP7 immunoreactivity detected by anti-IGFBP7 sdAb (clone 4.43) was enhanced and detected specifically in the tumours (Figure 2A), but not in normal brain (Figure 2B), and colocalised with the immunoreactivity against the vascular antigen, CD31 (Figure 2A). No IGFBP7 expression was observed in non-tumour brain vessels or in non-vascular cells. In surgically removed human GBM tissue, IGFBP7 immunoreactivity (Figure 2C, middle panel) colocalised with highly abnormal tumour vasculature stained with Ulex europeaus agglutinin I (UEA I) (Figure 2C, right panel), shown previously to selectively bind to carbohydrates of human brain vessels (Holthofer et al, 1982). These results indicate that anti-IGFBP7 sdAb detects its target, IGFBP7, in both mouse and human GBM tissue sections ex vivo. This analysis confirms our previous observation of a selective vascular upregulation of IGFBP7 in human GBM (Pen et al, 2007) and demonstrates that mouse vessels ingrowing into orthotopically implanted human GBM (U87MG-EGFRvIII) are selectively induced by the tumour microenvironment to overexpress IGFBP7. This mouse model is thus suitable for in vivo assessment of tumour targeting/imaging using anti-IGFBP7 sdAb.

Bottom Line: In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour.Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe₃O₄ NPs selectively in GBM vessels.Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biological Sciences, National Research Council of Canada, 1200 Montreal Road, Ottawa, Ontario, Canada K1A 0R6.

ABSTRACT

Background: Insulin-like growth factor-binding protein 7 (IGFBP7) is an abundant, selective and accessible biomarker of glioblastoma multiforme (GBM) tumour vessels. In this study, an anti-IGFBP7 single-domain antibody (sdAb) was developed to target GBM vessels for molecular imaging applications.

Methods: Human GBM was modelled in mice by intracranial implantation of U87MG.EGFRvIII cells. An anti-IGFBP7 sdAb, isolated from an immune llama library by panning, was assessed in vitro for its binding affinity using surface plasmon resonance and by ex vivo immunobinding on mouse and human GBM tissue. Tumour targeting by Cy5.5-labelled anti-IGFBP7 sdAb as well as by anti-IGFBP7 sdAb conjugated to PEGylated Fe₃O₄ nanoparticles (NPs)-Cy5.5 were assessed in U87MG.EGFRvIII tumour-bearing mice in vivo using optical imaging and in brain sections using fluorescent microscopy.

Results: Surface plasmon resonance analyses revealed a medium affinity (K(D)=40-50 nM) binding of the anti-IGFBP7 sdAb to the purified antigen. The anti-IGFBP7 sdAb also selectively bound to both mouse and human GBM vessels, but not normal brain vessels in tissue sections. In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour. Similarly, the anti-IGFBP7 sdAb-functionalised PEGylated Fe₃O₄ NP-Cy5.5 demonstrated enhanced tumour signal compared with non-targeted NPs. Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe₃O₄ NPs selectively in GBM vessels.

Conclusions: Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.

Show MeSH
Related in: MedlinePlus