Journal of Neuro-Oncology

, Volume 119, Issue 2, pp 243–251 | Cite as

Expression of Hedgehog ligand and signal transduction components in mutually distinct isocitrate dehydrogenase mutant glioma cells supports a role for paracrine signaling

  • Sunday A. Abiria
  • Thomas V. Williams
  • Alexander L. Munden
  • Vandana K. Grover
  • Ato Wallace
  • Christopher J. Lundberg
  • J. Gerardo Valadez
  • Michael K. Cooper
Laboratory Investigation

Abstract

Hedgehog (Hh) signaling regulates the growth of malignant gliomas by a ligand-dependent mechanism. The cellular source of Sonic Hh ligand and mode of signaling have not been clearly defined due to the lack of methods to definitively identify neoplastic cells in glioma specimens. Using an antibody specific for mutant isocitrate dehydrogenase protein expression to identify glioma cells, we demonstrate that Sonic Hh ligand and the pathway components Patched1 (PTCH1) and GLI1 are expressed in neoplastic cells. Further, Sonic Hh ligand and its transcriptional targets, PTCH1 and GLI1, are expressed in mutually distinct populations of neoplastic cells. These findings support a paracrine mode of intratumoral Hh signaling in malignant gliomas.

Keywords

Glioma Hedgehog signaling pathway Isocitrate dehydrogenase 

Supplementary material

11060_2014_1481_MOESM1_ESM.eps (3.1 mb)
Fig. 1Comparable IDH1 R132H staining with chromogenic and fluorescence detection. Paraffin sections of an IDH1 R132H mutant anaplastic oligodendroglioma (12262 AO) and a wild type glioblastoma (15589 GBM) were stained with anti-IDH1 R132H antibody and then processed for detection by immunohistochemistry (A and B) or immunofluorescence (C and D) (EPS 3206 kb)
11060_2014_1481_MOESM2_ESM.eps (2.7 mb)
Fig. 2Specificity of SHH staining. An astrocytoma (13396 A) was stained with anti-Shh antibody (A) or with secondary antibody alone (B). In other control experiments, anti-Shh antibody was preincubated with a Shh blocking peptide (C) or with a control blocking peptide (D). Immunostaining was detected using Alexa Fluor 555 (red) and nuclei were counterstained with Hoechst dye (blue) (EPS 2774 kb)
11060_2014_1481_MOESM3_ESM.eps (3.2 mb)
Fig. 3SHH expression in IDH1 R132H-mutant glioma cells. Paraffin sections of IDH1 mutant gliomas were immunostained for IDH1 R132H (green) and SHH (red) and nuclei were counterstained with Hoechst dye (blue). (A) SHH expression was detected in IDH1 R132H-positive cells in 11 of 12 glioma specimens, and the percentage of double-positive cells in each high-powered field (points on the graph) varied within and among each specimen. (B-I) Colocalization of SHH and IDH1 R132H immunofluorescence staining in a cytosolic expression pattern in an anaplastic oligodendroglioma (12262 AO) (B-E and I), oligodendroglioma (12784 O), astrocytoma (13396 A), and oligodendroglioma (16772 O) (EPS 3258 kb)
11060_2014_1481_MOESM4_ESM.eps (9.9 mb)
Fig. 4Validation of two commercially available anti-Gli1 antibodies. Epilepsy and glioma specimens were stained for GLI1 using a goat anti-Gli1 antibody (C-18, sc-6152) from Santa Cruz Biotechnology (A and B), or a mouse anti-Gli1 antibody (2643S) from Cell Signaling Technologies and then for SHH (C and D). Inset demonstrates SHH (brown) and GLI1 (purple) staining in separate cells in a glioma specimen (EPS 10122 kb)
11060_2014_1481_MOESM5_ESM.eps (6.7 mb)
Fig. 5Validation of chromogenic multiplex in situ hybridization for SHH and GLI1. Glioma paraffin sections were evaluated for background signal following hybridization with probes for the bacterial gene DapB transcript (A) and for expression of transcripts from housekeeping human genes POLR2A and PPIB (B). Compared to the negative (A) and positive (B) controls, GLI1 (green) and SHH (red) transcripts were detected at moderate levels (C) (EPS 6836 kb)

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Sunday A. Abiria
    • 2
  • Thomas V. Williams
    • 2
  • Alexander L. Munden
    • 2
  • Vandana K. Grover
    • 2
  • Ato Wallace
    • 3
  • Christopher J. Lundberg
    • 2
  • J. Gerardo Valadez
    • 2
  • Michael K. Cooper
    • 1
    • 2
    • 4
  1. 1.Veterans AffairsTVHSNashvilleUSA
  2. 2.Department of NeurologyVanderbilt University Medical CenterNashvilleUSA
  3. 3.Vanderbilt University School of MedicineVanderbilt University Medical CenterNashvilleUSA
  4. 4.Vanderbilt Ingram Cancer CenterVanderbilt University Medical CenterNashvilleUSA

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