title: Immunohistochemical Assessment of Phosphorylated mTORC1-Pathway Proteins in Human Brain Tumors.
abstract: BACKGROUND
  Current pathological diagnostics include the analysis of (epi-)genetic alterations as well as oncogenic pathways. Deregulated mammalian target of rapamycin complex 1 (mTORC1) signaling has been implicated in a variety of cancers including malignant gliomas and is considered a promising target in cancer treatment. Monitoring of mTORC1 activity before and during inhibitor therapy is essential. The aim of our study is to provide a recommendation and report on pitfalls in the use of phospho-specific antibodies against mTORC1-targets phospho-RPS6 (Ser235/236; Ser240/244) and phospho-4EBP1 (Thr37/46) in formalin fixed, paraffin embedded material.
  
  
  RESULTS
  Primary, established cell lines and brain tumor tissue from routine diagnostics were assessed by immunocyto-, immunohistochemistry, immunofluorescent stainings and immunoblotting. For validation of results, immunoblotting experiments were performed. mTORC-pathway activation was pharmacologically inhibited by torin2 and rapamycin. Torin2 treatment led to a strong reduction of signal intensity and frequency of all tested antibodies. In contrast phospho-4EBP1 did not show considerable reduction in staining intensity after rapamycin treatment, while immunocytochemistry with both phospho-RPS6-specific antibodies showed a reduced signal compared to controls. Staining intensity of both phospho-RPS6-specific antibodies did not show considerable decrease in stability in a timeline from 0-230 minutes without tissue fixation, however we observed a strong decrease of staining intensity in phospho-4EBP1 after 30 minutes. Detection of phospho-signals was strongly dependent on tissue size and fixation gradient. mTORC1-signaling was significantly induced in glioblastomas although not restricted to cancer cells but also detectable in non-neoplastic cells.
  
  
  CONCLUSIONS
  Here we provide a recommendation for phospho-specific immunohistochemistry for patient-orientated therapy decisions and monitoring treatment response.
authors.affiliation: Edinger 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.
authors.forename: Patrick N
authors.initials: PN
authors.lastname: Harter
authors.affiliation: Edinger Institute, Institute of Neurology, University of Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Lukas
authors.initials: L
authors.lastname: Jennewein
authors.affiliation: Edinger Institute, Institute of Neurology, University of Frankfurt am Main, Frankfurt am Main, Germany; Department of Neurosurgery, University of Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Baumgarten
authors.affiliation: Edinger Institute, Institute of Neurology, University of Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Ilina
authors.affiliation: German Cancer Consortium (DKTK), Heidelberg, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany; Senckenberg Institute of Neurooncology, University of Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Burger
authors.affiliation: Senckenberg Institute of Neurooncology, University of Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Anna-Luisa
authors.initials: AL
authors.lastname: Thiepold
authors.affiliation: Senckenberg Institute of Neurooncology, University of Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Tichy
authors.affiliation: Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Zörnig
authors.affiliation: German Cancer Consortium (DKTK), Heidelberg, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany; Department of Neurosurgery, University of Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Senft
authors.affiliation: German Cancer Consortium (DKTK), Heidelberg, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany; Senckenberg Institute of Neurooncology, University of Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Joachim P
authors.initials: JP
authors.lastname: Steinbach
authors.affiliation: Edinger 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.
authors.forename: Michel
authors.initials: M
authors.lastname: Mittelbronn
authors.affiliation: German Cancer Consortium (DKTK), Heidelberg, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany; Senckenberg Institute of Neurooncology, University of Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Ronellenfitsch
mesh_terms: D001932:Brain Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases