Immunohistochemical Assessment of Phosphorylated mTORC1-Pathway Proteins in Human Brain Tumors.
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 can...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2015-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4437987?pdf=render |
| _version_ | 1811269882864467968 |
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| author | Patrick N Harter Lukas Jennewein Peter Baumgarten Elena Ilina Michael C Burger Anna-Luisa Thiepold Julia Tichy Martin Zörnig Christian Senft Joachim P Steinbach Michel Mittelbronn Michael W Ronellenfitsch |
| author_facet | Patrick N Harter Lukas Jennewein Peter Baumgarten Elena Ilina Michael C Burger Anna-Luisa Thiepold Julia Tichy Martin Zörnig Christian Senft Joachim P Steinbach Michel Mittelbronn Michael W Ronellenfitsch |
| author_sort | Patrick N Harter |
| collection | DOAJ |
| description | 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.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.Here we provide a recommendation for phospho-specific immunohistochemistry for patient-orientated therapy decisions and monitoring treatment response. |
| first_indexed | 2024-04-12T21:50:58Z |
| format | Article |
| id | doaj.art-9d545b75c567496b8ede51c49eb4ed0f |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-12T21:50:58Z |
| publishDate | 2015-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-9d545b75c567496b8ede51c49eb4ed0f2022-12-22T03:15:29ZengPublic Library of Science (PLoS)PLoS ONE1932-62032015-01-01105e012712310.1371/journal.pone.0127123Immunohistochemical Assessment of Phosphorylated mTORC1-Pathway Proteins in Human Brain Tumors.Patrick N HarterLukas JenneweinPeter BaumgartenElena IlinaMichael C BurgerAnna-Luisa ThiepoldJulia TichyMartin ZörnigChristian SenftJoachim P SteinbachMichel MittelbronnMichael W RonellenfitschCurrent 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.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.Here we provide a recommendation for phospho-specific immunohistochemistry for patient-orientated therapy decisions and monitoring treatment response.http://europepmc.org/articles/PMC4437987?pdf=render |
| spellingShingle | Patrick N Harter Lukas Jennewein Peter Baumgarten Elena Ilina Michael C Burger Anna-Luisa Thiepold Julia Tichy Martin Zörnig Christian Senft Joachim P Steinbach Michel Mittelbronn Michael W Ronellenfitsch Immunohistochemical Assessment of Phosphorylated mTORC1-Pathway Proteins in Human Brain Tumors. PLoS ONE |
| title | Immunohistochemical Assessment of Phosphorylated mTORC1-Pathway Proteins in Human Brain Tumors. |
| title_full | Immunohistochemical Assessment of Phosphorylated mTORC1-Pathway Proteins in Human Brain Tumors. |
| title_fullStr | Immunohistochemical Assessment of Phosphorylated mTORC1-Pathway Proteins in Human Brain Tumors. |
| title_full_unstemmed | Immunohistochemical Assessment of Phosphorylated mTORC1-Pathway Proteins in Human Brain Tumors. |
| title_short | Immunohistochemical Assessment of Phosphorylated mTORC1-Pathway Proteins in Human Brain Tumors. |
| title_sort | immunohistochemical assessment of phosphorylated mtorc1 pathway proteins in human brain tumors |
| url | http://europepmc.org/articles/PMC4437987?pdf=render |
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