Dataset Information

Identification of proteomic markers in head and neck cancer using MALDI-MS Imaging, LC-MS/MS and IHC

ABSTRACT: Purpose The heterogeneity of squamous cell carcinoma tissue complicates diagnosis and an individualized therapy to a great extent. Therefore it is of utmost importance to spatially characterize this tissue heterogeneity and to identify appropriate biomarkers. Matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) can analyze spatially resolved tissue biopsies on a molecular level. Experimental design MALDI MSI of snap frozen tissues as well as of formalin-fixed and paraffin-embedded (FFPE) tissue samples from patients with head and neck cancer (HNC) were used to analyze m/z values localized in tumor and non-tumor regions. Peptide identification was performed by using liquid chromatography (LC)-MS/MS and immunohistochemistry (IHC). Results In both FFPE and frozen tissue specimen seven characteristic masses of the tumor epithelial region were found identified. Using LC-MS/MS, the peaks were identified as Vimentin, Keratin type II, Nucleolin, Heat shock protein 90, Prelamin-A/C, Junction plakoglobin and PGAM1. Finally, Vimentin, Nucleolin and PGAM1 were verified with IHC. Conclusions and Clinical Relevance The combination of MALDI MSI, LC-MS/MS and subsequent IHC is an appropriate tool to characterize the molecular heterogeneity of tissue as well as to identify new representative biomarkers for a more individualized therapy.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Head, Neck

DISEASE(S): Head And Neck Cancer

SUBMITTER: Thomas Krüger

LAB HEAD: Olaf Kniemeyer

PROVIDER: PXD009144 | Pride | 2018-11-14

REPOSITORIES: Pride

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Publications

Identification of Proteomic Markers in Head and Neck Cancer Using MALDI-MS Imaging, LC-MS/MS, and Immunohistochemistry.

Hoffmann Franziska F Umbreit Claudia C Krüger Thomas T Pelzel Daniela D Ernst Günther G Kniemeyer Olaf O Guntinas-Lichius Orlando O Berndt Alexander A von Eggeling Ferdinand F

Proteomics. Clinical applications 20181126 1

<h4>Purpose</h4>The heterogeneity of squamous cell carcinoma tissue greatly complicates diagnosis and individualized therapy. Therefore, characterizing the heterogeneity of tissue spatially and identifying appropriate biomarkers is crucial. MALDI-MS imaging (MSI) is capable of analyzing spatially resolved tissue biopsies on a molecular level.<h4>Experimental design</h4>MALDI-MSI is used on snap frozen and formalin-fixed and paraffin-embedded (FFPE) tissue samples from patients with head and neck ...[more]

PMID: 30411850

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Project description:An integrative functional genomics of multiple forms of data is vital for discovering molecular drivers of cancer development and progression. Here, we present an integrated genomic strategy utilizing DNA methylation and transcriptome profile data to discover epigenetically regulated genes implicated in cancer development and invasive progression. More specifically, this analysis identified Fibromodulin (FMOD) as a glioblastoma (GBM) upregulated gene due to the loss of promoter methylation. Secreted FMOD promotes glioma cell migration through its ability to induce filamentous actin stress fiber formation. Treatment with Cytochalasin D, an actin polymerization inhibitor, significantly reduced the FMOD induced glioma cell migration. siRNA and small molecule inhibitor-based studies identified that FMOD-induced glioma cell migration is dependent on Integrin-FAK-Src-Rho-ROCK signaling pathway. FMOD lacking C terminus LRR11 domain (FMOD), which does not bind collagen type I, failed to induce integrin and promote glioma cell migration. Further, FMOD-induced integrin activation and migration was abrogated by a 9-mer wild type peptide from the FMOD C-terminus. However, the same peptide with mutation in two residues essential for FMOD interaction with collagen type I failed to compete with FMOD, thus signifying the importance of collagen type I-FMOD interaction in integrin activation. ChIP-PCR experiments revealed that TGF-Ã1 regulates FMOD expression through epigenetic remodeling of FMOD promoter that involved demethylation and gain of active histone marks with a simultaneous loss of DNMT3A and EZH2 occupancy, but enrichment of SMAD2 and CBP. FMOD silencing inhibited the TGF-Ã1 mediated glioma cell migration significantly. In univariate and multivariate cox regression analysis, both FMOD promoter methylation and transcript levels predicted prognosis in GBM. Thus, the present study identified several epigenetically regulated alterations responsible for cancer development and progression. Specifically, we found that secreted FMOD as an important regulator of glioma cell migration downstream of TGF-Ã1 pathway and forms a potential basis for therapeutic intervention in GBM. We used DNA isolated from 17 diffuse astrocytoma, 16 anaplastic astrocytoma, 36 glioblastoma and 9 control brain tissue for the array. P53 status: All cases were subjected to histopathology and the diagnosis of GBM was confirmed. Paraffin sections (4 Î¼m) from the tissues were collected on silane-coated slides, and the protein expression of p53 was assessed by immunohistochemistry (IHC). Antigen retrieval was done by heat treatment at 850W in citrate buffer or at 700W. After the initial processing steps, sections were incubated overnight with primary antibody for p53 (Mouse Monoclonal DO-7; Biogenex, USA)at 4Â°C with 1: 200 dilution. MIB: Paraffin blocks of all the cases were retrieved, and histological features were reviewed on freshly cut and stained (hematoxylin and eosin) sections. After confirming the diagnosis of glioblastoma, 4 Âµm thick sections were collected on silane-coated slides, and IHC was performed using MIB-1 (anti Ki-67 monoclonal BGX 297, diluted to 1:30) EGFR status: Paraffin blocks of all the cases were retrieved, and histological features were reviewed on freshly cut and stained (hematoxylin and eosin) sections. After confirming the diagnosis of glioblastoma, 4 Âµm thick sections were collected on silane-coated slides, and IHC was performed using EGFR (monoclonal E-30, diluted to 1:50) GFAP status: Paraffin blocks of all the cases were retrieved, and histological features were reviewed on freshly cut and stained (hematoxylin and eosin) sections. After confirming the diagnosis of glioblastoma, 4 Âµm thick sections were collected on silane-coated slides, and IHC was performed using glial fibrillary acidic protein (GFAP, monoclonal GA-5, diluted to 1:100).

Dataset Information

Identification of proteomic markers in head and neck cancer using MALDI-MS Imaging, LC-MS/MS and IHC

Publications

Identification of Proteomic Markers in Head and Neck Cancer Using MALDI-MS Imaging, LC-MS/MS, and Immunohistochemistry.

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