TY - JOUR
T1 - Immunohistochemical assessment of phosphorylated mTORC1-pathway proteins in human brain tumors
AU - Harter, Patrick N.
AU - Jennewein, Lukas
AU - Baumgarten, Peter
AU - Ilina, Elena
AU - Burger, Michael C.
AU - Thiepold, Anna Luisa
AU - Tichy, Julia
AU - Zörnig, Martin
AU - Senft, Christian
AU - Steinbach, Joachim P.
AU - Mittelbronn, Michel
AU - Ronellenfitsch, Michael W.
N1 - Publisher 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.
PY - 2015/5/19
Y1 - 2015/5/19
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 nonneoplastic cells. Conclusion: Here we provide a recommendation for phospho-specific immunohistochemistry for patientorientated therapy decisions and monitoring treatment response.
AB - 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 nonneoplastic cells. Conclusion: Here we provide a recommendation for phospho-specific immunohistochemistry for patientorientated therapy decisions and monitoring treatment response.
UR - http://www.scopus.com/inward/record.url?scp=84930654995&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0127123
DO - 10.1371/journal.pone.0127123
M3 - Article
C2 - 25993328
AN - SCOPUS:84930654995
SN - 1932-6203
VL - 10
JO - PLoS ONE
JF - PLoS ONE
IS - 5
M1 - e0127123
ER -