TY  - JOUR
A1  - Harter, Patrick Nikolaus
A1  - Jennewein, Lukas
A1  - Baumgarten, Peter
A1  - Ilina, Elena
A1  - Burger, Michael Christian
A1  - Thiepold, Anna-Luisa
A1  - Tichy, Julia Frederike
A1  - Zörnig, Martin
A1  - Senft, Christian
A1  - Steinbach, Joachim Peter
A1  - Mittelbronn, Michel Guy André
A1  - Ronellenfitsch, Michael Wilfried
T1  - Immunohistochemical assessment of phosphorylated mTORC1-pathway proteins in human brain tumors
T2  - PLoS One
N2  - 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.
Methods and findings: 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.
Conclusion: Here we provide a recommendation for phospho-specific immunohistochemistry for patient-orientated therapy decisions and monitoring treatment response.
Y1  - 2015
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/37992
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-379929
SN  - 1932-6203
N1  - Copyright: © 2015 Harter et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
VL  - 10
IS  - (5): e0127123
SP  - 1
EP  - 20
PB  - PLoS
CY  - Lawrence, Kan.
ER  -