Summary

Title

HLA peptides (LC-MSMS) induced by Decitabine in Glioblastoma cell lines

Description

Treatment of cancer cells with anti-cancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumor’s gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anti-cancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine (Decitabine) has limited anti-tumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug.

Sample Processing Protocol

Drug treatment regimen: Cells were counted and plated in new 150 mm Petri dishes one day prior to the treatment. The following day, the media were removed and replaced with fresh medium with or without 1 μM of 5'-Aza-2'-deoxycytidine (Decitabine, Enco diagnostics, AdooQ bioscience). After incubation with Decitabine for 72 hours, the cells were harvested, counted and prepared for FACS, HLA peptidomics, proteomics analysis or for RNA sequencing. Affinity purification of HLA molecules: HLA class I molecules were purified from three biological replicates for each Glioblastoma cell line. Each replica with about 5*108 cells was lysed with 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 Protease Inhibitors Cocktail (Sigma), 1 mM PMSF and 1% octyl-β-D glucopyranoside (Sigma) in PBS at 4°C for 1 hour. The cell extracts were cleared by centrifugation for 45 minutes at 18,000 rpm, 4°C. The recovered HLA class I molecules were immunoaffinity purified using the W6/32 mAb bound to Amino-Link beads (Thermo Scientific) . The HLA molecules with their bound peptides were eluted from the affinity column with five column volumes of 0.1 N acetic acid. The eluted HLA class I proteins and the released peptides were loaded on disposable C18 columns (Harvard Apparatus) and the peptides fraction was recovered with 30% acetonitrile in 0.1% TFA. The peptide fractions were dried using vacuum centrifugation, reconstituted in 100 μl of 0.1% TFA, reloaded on Stage-Tips , eluted with 80% ACN, dried and reconstituted with 0.1% formic acid for μLC-MS-MS analysis. Identification of the HLA peptides: The HLA peptides were resolved by capillary chromatography with 75µ ID laser-pulled capillaries, self-packed with 3.5µ Reprosil-Aqua C18 (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Electrospray tandem mass spectrometry was performed with Q-Exactive plus mass spectrometers (Thermo-Fisher Scientific). Two approaches for proteome analysis were employed in this study, the first was in-solution tryptic digest followed by resolution of the peptides by long (3 hours) one dimensional reversed phase capillary chromatography and tandem mass spectrometry and the other was based on in-gel proteolysis of 5 gel slices from each lane, followed by two hours μLC-MS-MS of the tryptic peptides from each gel slice, as in (70). 30 µg of total proteins were used for the in-gel digest and μLC-MS-MS and 2.5 µg of proteins were used for the in-solution digest and μLC-MS-MS analysis.

Data Processing Protocol

Identification of the HLA peptides: The MS data was analyzed by the MaxQuant computational proteomics platform version 1.5.0.25, searching with the Andromeda search engine, with 5% FDR. Peptide identifications were based on the human section of the Uniprot database (http://www.uniprot.org/) of July 2015 containing 69693 entries. The proteins were identified with MaxQuant (version 1.5.0.25) using the same human section of the Uniprot database as for the HLA peptidome described above, and searched with the Andromeda search engine, with 1% FDR.

Contact

Bracha Shraibman, Department of Biology, Technion- Israel Institute of Technology
Arie Admon, Department of Biology, Technion - Israel Institute of Technology, Haifa, Israel ( lab head )

Submission Date

14/06/2016

Publication Date

18/07/2016

Instrument

Q Exactive

Software

Not available

Experiment Type

Shotgun proteomics

Publication

    Shraibman B, Melamed Kadosh D, Barnea E, Admon A. HLA peptides derived from tumor antigens induced by inhibition of DNA methylation for development of drug-facilitated immunotherapy. Mol Cell Proteomics. 2016 Jul 13. pii: mcp.M116.060350 PubMed: 27412690