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Immunohistochemical Assessment of Phosphorylated mTORC1-Pathway Proteins in Human Brain Tumors

Patrick N Harter1, Lukas Jennewein2, Peter Baumgarten3

  • 1Edinger Institute, Institute of Neurology, University of Frankfurt am Main, Frankfurt am Main, Germany; German Cancer Consortium (DKTK), Heidelberg, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany.

Plos One
|May 21, 2015
PubMed
Abstract

Insights

This study evaluates phospho-specific antibodies for monitoring mammalian target of rapamycin complex 1 (mTORC1) signaling in cancer. Recommendations are provided for reliable immunohistochemistry in guiding patient therapy and treatment response.

Area of Science:

  • Oncology
  • Molecular Biology
  • Pathology

Background:

  • Deregulated mammalian target of rapamycin complex 1 (mTORC1) signaling is implicated in various cancers, including malignant gliomas.
  • Monitoring mTORC1 activity is crucial for guiding and assessing targeted cancer therapies.
  • Phospho-specific antibodies targeting mTORC1 pathway components are essential tools for this monitoring.

Purpose of the Study:

  • To provide recommendations for the use of phospho-specific antibodies against phospho-RPS6 and phospho-4EBP1 in formalin-fixed, paraffin-embedded tissues.
  • To identify and report potential pitfalls associated with these immunohistochemical techniques.
  • To establish reliable methods for monitoring mTORC1 activity in cancer diagnostics and treatment.

Main Methods:

  • Immunocytochemistry, immunohistochemistry, immunofluorescence, and immunoblotting were employed.
  • Experiments utilized primary cell lines and routine brain tumor diagnostics.
  • Pharmacological inhibition of mTORC1 signaling with torin2 and rapamycin was performed to validate antibody performance.

Main Results:

  • Torin2 treatment significantly reduced signal intensity for all tested antibodies.
  • Phospho-4EBP1 showed reduced staining after rapamycin treatment, unlike phospho-RPS6.
  • Staining intensity was dependent on tissue size and fixation, with phospho-4EBP1 stability decreasing after 30 minutes without fixation.
  • mTORC1 signaling was notably induced in glioblastomas, affecting both cancer and non-neoplastic cells.

Conclusions:

  • Established protocols for phospho-specific immunohistochemistry can aid in patient-oriented therapy decisions.
  • The study provides critical insights into the reliable application of phospho-specific antibodies for monitoring mTORC1 pathway activity.
  • Recommendations are offered to optimize immunohistochemistry for clinical use in cancer treatment monitoring.

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