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

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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.

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Acknowledgments

This material is based upon work supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development through grant 1 I01 BX000744-01. The contents do not represent the views of the Department of Veterans Affairs or the United States Government. We are particularly indebted to patients at Vanderbilt University Medical Center who provided invaluable research material for the Molecular Neurosurgical Tissue Bank. We thank those who established and maintain the Tissue Bank, Reid C. Thompson MD (principal investigator), Cherryl Kinnard RN (research nurse) and Larry A. Pierce MS (manager). Histological services were performed, in part, by the Vanderbilt University Medical Center (VUMC) Translational Pathology Shared Resource (supported by award 5P30 CA068485 to the Vanderbilt-Ingram Cancer Center). We thank Li-Chong Wang PhD and Ruby Hsu PhD at Advanced Cell Diagnostics for expertise and assistance with in situ hybridization studies.

Conflict of interest

The authors of this study declare no conflict of interest.

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Authors and Affiliations

  1. Veterans Affairs, TVHS, Nashville, TN, 37212, USA

    Michael K. Cooper

  2. Department of Neurology, Vanderbilt University Medical Center, MRB III, Rm. 6160, 465 21st Avenue South, Nashville, TN, 37232, USA

    Sunday A. Abiria, Thomas V. Williams, Alexander L. Munden, Vandana K. Grover, Christopher J. Lundberg, J. Gerardo Valadez & Michael K. Cooper

  3. Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Nashville, TN, 37232, USA

    Ato Wallace

  4. Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, 37232, USA

    Michael K. Cooper

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  1. Sunday A. Abiria
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  2. Thomas V. Williams
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  4. Vandana K. Grover
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  5. Ato Wallace
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  7. J. Gerardo Valadez
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Corresponding author

Correspondence to Michael K. Cooper.

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Sunday A. Abiria, Thomas V. Williams and Alexander L. Munden have contributed equally to this work.

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Fig. 1

Comparable 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)

Fig. 2

Specificity 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)

Fig. 3

SHH 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)

Fig. 4

Validation 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)

Fig. 5

Validation 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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Abiria, S.A., Williams, T.V., Munden, A.L. et al. Expression of Hedgehog ligand and signal transduction components in mutually distinct isocitrate dehydrogenase mutant glioma cells supports a role for paracrine signaling. J Neurooncol 119, 243–251 (2014). https://doi.org/10.1007/s11060-014-1481-7

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