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Novel anti IGFBP2 single chain variable fragment inhibits glioma cell migration and invasion

Journal of Neuro-Oncology volume 123, pages 225–235 (2015)Cite this article

Abstract

Insulin like growth factor binding protein 2 (IGFBP2) is highly up regulated in glioblastoma (GBM) tissues and has been one of the prognostic indicators. There are compelling evidences suggesting important roles for IGFBP2 in glioma cell proliferation, migration and invasion. Extracellular IGFBP2 through its carboxy terminal arginine glycine aspartate (RGD) motif can bind to cell surface α5β1 integrins and activate pathways downstream to integrin signaling. This IGFBP2 activated integrin signaling is known to play a crucial role in IGFBP2 mediated invasion of glioma cells. Hence a molecular inhibitor of carboxy terminal domain of IGFBP2 which can inhibit IGFBP2-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of IGFBP2, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I (Library size 1.47 × 108) and Tomlinson J (Library size 1.37 × 108) using human recombinant IGFBP2. After screening we obtained three IGFBP2 specific binders out of which one scFv B7J showed better binding to IGFBP2 at its carboxy terminal domain, blocked IGFBP2-cell surface association, reduced activity of matrix metalloprotease 2 in the conditioned medium of glioma cells and inhibited IGFBP2 induced migration and invasion of glioma cells. We demonstrate for the first time that in vitro inhibition of extracellular IGFBP2 activity by using human scFv results in significant reduction of glioma cell migration and invasion. Therefore, the inhibition of IGFBP2 can serve as a potential therapeutic strategy in the management of GBM.

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Acknowledgments

We acknowledge Department of Biotechnology, Government of India for funding this study (Program support and Center of Excellence); University Grants Commission for a fellowship to SSP and infrastructure and Department of Science and Technology for infrastructure support (FIST).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

The experiments conducted for this manuscript comply with current laws of the country.

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Affiliations

  1. Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore, 560012, India

    Shilpa S. Patil, Reema Railkar, Rajan R. Dighe & Paturu Kondaiah

  2. NMR Research Centre, Indian Institute of Science, Bangalore, 560012, India

    Monalisa Swain & Hanudatta S. Atreya

Authors
  1. Shilpa S. Patil
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  2. Reema Railkar
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  3. Monalisa Swain
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Corresponding author

Correspondence to Paturu Kondaiah.

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Fig S1

Purification and characterization of recombinant human IGFBP2 a Map of IGFBP2 cloning vector pET 15 b b Surface plasmon resonance analysis showing binding of recombinant 1–289h-IGFBP2 to hIGF-I and to c hIGF-II d Map of PIT2 scFv expression vector e A semi quantitative PCR showing 900 bp scFv amplicon from identified clones of E coli HB2151. Supplementary material 1 (TIFF 2723 kb)

Fig S2

a aligned amino acid sequences of identified scFv clones. Supplementary material 2 (TIFF 1910 kb)

Fig S3

Purification of soluble scFv by protein A affinity chromatography Protein A affinity purified scFvs; western blots showing scFv detected with anti c Myc antibody a purified anti BSA scFv b purified B7J scFv c purified F2J scFv d ELISA showing binding of purified scFvs to IGFBP2 at increasing concentrations e ELISA showing cross reactivity of anti IGFBP2 scFv (B7J) to BSA and cross reactivity of anti BSA scFv to IGFBP2. Supplementary material 3 (TIFF 1655 kb)

Fig S4

a Purified 190–289 hIGFBP2 (C domain), Western blot probed for IGFBP2 antibody b Western blot showing endogenous IGFBP2 levels across U251, LN229, A172, U87 glioma cell lines c Western blot showing IGFBP2 levels in U251 vector control clone and IGFBP2 knockdown clones 2 and 4 d, e, f Real time PCR analysis showing regulation of IGFBP2, MMP2 and CD24 m RNAs respectively in vector control clone and IGFBP2 knockdown clones 2 and 4 (***P value ≤ 0.0002, n = 3). Supplementary material 4 (TIFF 3079 kb)

Fig S5

a wound healing assay showing the rate of migration of U251 vector control clones and IGFBP2 knockdown clones 2 and 4 at 0, 8 and 24 h b Migration assay represented as percentage wound healing (***P value ≤ 0.0002, n = 3) c Matrigel invasion assay showing the invasiveness of U251 vector control clone and IGFBP2 knockdown clones 2 and 4.(***P value ≤ 0.0002, n = 3) d Gelatin zymography showing the MMP2 activity in conditioned medium of Vector control clone and IGFBP2 knockdown clones 2 and 4 of U251 cell line. Supplementary material 5 (TIFF 3333 kb)

Fig S6

a Bio-layer interferometry (BLI) showing the binding of IGF-II to the IGFBP2-B7J complex; IGF-II could not bind to IGFBP2-B7J complex b Western blot showing intracellular IGFBP2 levels upon treatment with B7J scFv for 24 h c Western blot showing the IGFBP2 protein in the conditioned medium of U87 cells after 24 h of IGFBP2 + B7J treatment; there was no detectable degradation of IGFBP2w. Supplementary material 6 (TIFF 6236 kb)

Supplementary material 7 (docx 12 kb)

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Patil, S.S., Railkar, R., Swain, M. et al. Novel anti IGFBP2 single chain variable fragment inhibits glioma cell migration and invasion. J Neurooncol 123, 225–235 (2015). https://doi.org/10.1007/s11060-015-1800-7

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  • DOI: https://doi.org/10.1007/s11060-015-1800-7

Keywords

  • IGFBP2
  • scFv
  • Glioma
  • Invasion