Dataset Information

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.

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.

SUBMITTER: Harter PN

PROVIDER: S-EPMC4437987 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Publications

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

Harter Patrick N PN Jennewein Lukas L Baumgarten Peter P Ilina Elena E Burger Michael C MC Thiepold Anna-Luisa AL Tichy Julia J Zörnig Martin M Senft Christian C Steinbach Joachim P JP Mittelbronn Michel M Ronellenfitsch Michael W MW

PloS one 20150519 5

<h4>Background</h4>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 ...[more]

PMID: 25993328

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Dataset Information

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

Background

Methods and findings

Conclusion

Publications

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

Similar Datasets

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