Targeted Proteomics to Assess the Response to Anti-Angiogenic Treatment in Human Glioblastoma (GBM)

Glioblastoma (GBM) is a highly aggressive primary brain tumor with dismal outcome for affected patients. Because of the significant neo-angiogenesis exhibited by GBMs, anti-angiogenic therapies have been intensively evaluated during the past years. Recent clinical studies were however disappointing, although a subpopulation of patients may benefit from such treatment. We have previously shown that anti-angiogenic targeting in GBM increases hypoxia and leads to a metabolic adaptation toward glycolysis, suggesting that combination treatments also targeting the glycolytic phenotype may be effective in GBM patients. The aim of this study was to identify marker proteins that are altered by treatment and may serve as a short term readout of anti-angiogenic therapy. Ultimately such proteins could be tested as markers of efficacy able to identify patient subpopulations responsive to the treatment. We applied a proteomics approach based on selected reaction monitoring (SRM) to precisely quantify targeted protein candidates, selected from pathways related to metabolism, apoptosis and angiogenesis. The workflow was developed in the context of patient-derived intracranial GBM xenografts developed in rodents and ensured the specific identification of human tumor versus rodent stroma-derived proteins. Quality control experiments were applied to assess sample heterogeneity and reproducibility of SRM assays at different levels. The data demonstrate that tumor specific proteins can be precisely quantified within complex biological samples, reliably identifying small concentration differences induced by the treatment. In line with previous work, we identified decreased levels of TCA cycle enzymes, including isocitrate dehydrogenase, whereas malectin, calnexin, and lactate dehydrogenase A were augmented after treatment. We propose the most responsive proteins of our subset as potential novel biomarkers to assess treatment response after anti-angiogenic therapy that warrant future analysis in clinical GBM samples. Glioblastoma (GBM) is a highly aggressive primary brain tumor with dismal outcome for affected patients. Because of the significant neo-angiogenesis exhibited by GBMs, anti-angiogenic therapies have been intensively evaluated during the past years. Recent clinical studies were however disappointing, although a subpopulation of patients may benefit from such treatment. We have previously shown that anti-angiogenic targeting in GBM increases hypoxia and leads to a metabolic adaptation toward glycolysis, suggesting that combination treatments also targeting the glycolytic phenotype may be effective in GBM patients. The aim of this study was to identify marker proteins that are altered by treatment and may serve as a short term readout of anti-angiogenic therapy. Ultimately such proteins could be tested as markers of efficacy able to identify patient subpopulations responsive to the treatment. We applied a proteomics approach based on selected reaction monitoring (SRM) to precisely quantify targeted protein candidates, selected from pathways related to metabolism, apoptosis and angiogenesis. The workflow was developed in the context of patient-derived intracranial GBM xenografts developed in rodents and ensured the specific identification of human tumor versus rodent stroma-derived proteins. Quality control experiments were applied to assess sample heterogeneity and reproducibility of SRM assays at different levels. The data demonstrate that tumor specific proteins can be precisely quantified within complex biological samples, reliably identifying small concentration differences induced by the treatment. In line with previous work, we identified decreased levels of TCA cycle enzymes, including isocitrate dehydrogenase, whereas malectin, calnexin, and lactate dehydrogenase A were augmented after treatment. We propose the most responsive proteins of our subset as potential novel biomarkers to assess treatment response after anti-angiogenic therapy that warrant future analysis in clinical GBM samples. In the context of glioblastoma (GBM) 1The abbreviations used are:GBMGlioblastomaIDHIsocitrate dehydrogenaseMRMMultiple Reaction MonitoringSILStable Isotope LabeledSRMSelected Reaction MonitoringVEGFVascular Endothelial Growth FactorMRImagnetic resonance imagingTCAtricarboxylic acid cycleERendoplasmic reticulum., the quest for effective biomarkers is vital given that GBM is the most aggressive primary brain tumor in adults and no curative treatment is currently available (1.Jayaram S. Gupta M.K. Polisetty R.V. Cho W.C. Sirdeshmukh R. Towards developing biomarkers for glioblastoma multiforme: a proteomics view.Expert Rev. Proteomics. 2014; 11: 621-639Crossref PubMed Scopus (28) Google Scholar). GBM is characterized by extensive invasion into the brain parenchyma, a high proliferation rate, neo-angiogenesis and significant cellular and molecular heterogeneity. Current treatment involves neurosurgery, radiotherapy and chemotherapy, yet the median life expectancy of affected patients is less than fifteen months. Recent efforts have focused on targeting the vascular endothelial growth factor (VEGF) system which is critical for tumor angiogenesis, however GBM quickly develop escape mechanisms leading to tumor progression (2.Arrillaga-Romany I. Reardon D.A. Wen P.Y. Current status of antiangiogenic therapies for glioblastomas.Expert Opin. Investig. Drugs. 2014; 23: 199-210Crossref PubMed Scopus (27) Google Scholar, 3.Plate K.H. Scholz A. Dumont D.J. Tumor angiogenesis and anti-angiogenic therapy in malignant gliomas revisited.Acta Neuropathol. 2012; 124: 763-775Crossref PubMed Scopus (171) Google Scholar). Previous work from our group demonstrated that GBMs adapt to anti-VEGF treatment via a metabolic switch in tumor cells toward increased glycolysis (4.Keunen O. Johansson M. Oudin A. Sanzey M. Rahim S.A. Fack F. Thorsen F. Taxt T. Bartos M. Jirik R. Miletic H. Wang J. Stieber D. Stuhr L. Moen I. Rygh C.B. Bjerkvig R. Niclou S.P. Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma.Proc. PubMed Scopus Google Scholar, F. H. O. A. M. O. A. Stuhr L. Miletic H. Stieber D. Rygh C.B. M. L. Niclou S.P. Bjerkvig R. treatment metabolic adaptation toward in Neuropathol. PubMed Scopus Google Scholar). was by increased hypoxia and tumor cell with no on tumor growth (4.Keunen O. Johansson M. Oudin A. Sanzey M. Rahim S.A. Fack F. Thorsen F. Taxt T. Bartos M. Jirik R. Miletic H. Wang J. Stieber D. Stuhr L. Moen I. Rygh C.B. Bjerkvig R. Niclou S.P. Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma.Proc. PubMed Scopus Google Scholar). In with clinical the of a targeting in GBM patients although progression to be no on was T. for J. 2014; PubMed Scopus Google Scholar, H. A. S. M. R. D. Stieber M. A of for J. 2014; PubMed Scopus Google Scholar). The of such studies are by the that anti-angiogenic blood used to treatment Reardon D.A. Wen P.Y. in the of glioblastoma in the of antiangiogenic and therapy PubMed Scopus Google Scholar, O. Taxt T. R. M. T. Bjerkvig R. Niclou S.P. Thorsen F. of gliomas in the context of cellular and molecular Rev. 2014; PubMed Scopus Google Scholar). is a for molecular biomarkers to treatment response to anti-angiogenic Glioblastoma dehydrogenase Reaction Isotope Reaction Endothelial Growth resonance acid cycle proteomics R. and a proteomics PubMed Scopus Google Scholar, S. and proteomics PubMed Scopus Google is used in the of in in the context of including and The of the selected reaction monitoring (SRM) approach to proteomics the of in for identifying mechanisms and biomarkers of in Proteomics. PubMed Scopus Google Scholar, for protein Proteomics. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). SRM is a targeted proteomics approach that a and of previously selected marker S. reaction monitoring applied to PubMed Scopus Google Scholar, J. L. M. I. L. A. Wang L. A targeted for of biomarkers in PubMed Scopus Google Scholar). can be applied in a for potential biomarkers S.A. analysis of and proteins in by targeted PubMed Scopus Google Scholar, J. in protein toward clinical 2014; PubMed Scopus Google the of a of selected biomarkers of by the of in Because of high and also reaction monitoring is currently the in targeted proteomics PubMed Scopus Google Scholar, S. J. proteomics applied to PubMed Scopus Google Scholar). The aim of this study was to identify proteins that are altered by anti-angiogenic of tumor response in Ultimately such protein markers could be evaluated for as markers of efficacy that to from The study was focused on proteins that may significant differences in protein the metabolic switch exhibited during anti-angiogenic therapy. SRM workflow on a R. reaction monitoring for A PubMed Scopus Google was developed and in the context of GBM xenografts with in to in a a of proteins. We have previously shown that patient GBM xenografts developed in rodents human and a analysis of the tumor and (4.Keunen O. Johansson M. Oudin A. Sanzey M. Rahim S.A. Fack F. Thorsen F. Taxt T. Bartos M. Jirik R. Miletic H. Wang J. Stieber D. Stuhr L. Moen I. Rygh C.B. Bjerkvig R. Niclou S.P. Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma.Proc. PubMed Scopus Google Scholar, A. S. Stieber D. L. F. Bjerkvig R. Niclou S.P. in human glioblastoma is and brain endothelial PubMed Scopus Google Scholar, D. A. L. Oudin A. S. F. Bjerkvig R. L. Niclou S.P. are of with potential and Neuropathol. 2014; PubMed Scopus Google Scholar, J. Miletic H. H. S. M. Bjerkvig R. A brain from human glioblastoma PubMed Scopus Google Scholar, M. S. O. Wang J. Miletic H. Bjerkvig R. Niclou S.P. proteomics increased metabolic and cellular in with glioblastoma Proteomics. PubMed Scopus Google Scholar). the to control as as the of studies T. R. A with analysis identification of novel biomarkers for human 2012; 11: PubMed Scopus Google Scholar). The demonstrate the of SRM to precisely quantify small in protein concentration after treatment. We the of data and of the of proteins within complex biological concentration in of candidates, we proteins and identified responsive to anti-angiogenic treatment. We propose malectin, calnexin, lactate dehydrogenase A and isocitrate dehydrogenase as novel response markers to anti-angiogenic therapy. Glioblastoma were at the of the in at the in from patients given and of patient tumor was by the for of and by the of the in GBM xenografts were in A. S. Stieber D. L. F. Bjerkvig R. Niclou S.P. in human glioblastoma is and brain endothelial PubMed Scopus Google in (4.Keunen O. Johansson M. Oudin A. Sanzey M. Rahim S.A. Fack F. Thorsen F. Taxt T. Bartos M. Jirik R. Miletic H. Wang J. Stieber D. Stuhr L. Moen I. Rygh C.B. Bjerkvig R. Niclou S.P. Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma.Proc. PubMed Scopus Google Scholar, F. H. O. A. M. O. A. Stuhr L. Miletic H. Stieber D. Rygh C.B. M. L. Niclou S.P. Bjerkvig R. treatment metabolic adaptation toward in Neuropathol. PubMed Scopus Google as previously tumor from patients and were for a of tumor were into the in in Tumor was by and were into control and treatment from the treatment group of whereas of for the the of the the were the were and into the at the tumor was for the was in for protein The of the and the were in with the on and the and were by the for tumor and was with in a was into the by of of with and were at the the and in a at during sample and at of for were were at during to cell and were to and at treatment. of to the proteins were D. 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A SRM workflow is in the context of intracranial GBM xenografts developed in into the of in the the of the and the of the The of was and by the of a developed in our Niclou S.P. A for targeted studies from different 2014; PubMed Scopus Google Scholar). the and available related of a given protein in a of as as the for within a such as in the of the that the proteins are to be tumor and from the The of protein including for the of and (4.Keunen O. Johansson M. Oudin A. Sanzey M. Rahim S.A. Fack F. Thorsen F. Taxt T. Bartos M. Jirik R. Miletic H. Wang J. Stieber D. Stuhr L. Moen I. Rygh C.B. Bjerkvig R. Niclou S.P. Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma.Proc. PubMed Scopus Google Scholar, M. S. O. Wang J. Miletic H. Bjerkvig R. Niclou S.P. proteomics increased metabolic and cellular in with glioblastoma Proteomics. PubMed Scopus Google from previous data M. Rahim S.A. Oudin A. A. T. L. Bjerkvig R. A. 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A. 2012; PubMed Scopus Google of the proteins in GBM xenografts after treatment. are for the proteins affected in and the GBM xenografts of the heterogeneity in of the protein within GBM GBM xenografts of the heterogeneity in of the protein within GBM that are after dehydrogenase A acid that are after dehydrogenase, that are after that are after that are after that are after dehydrogenase that are after that are after that are after that are after that are after brain that are after that are after that are after dehydrogenase that are after that are after that are after dehydrogenase of the heterogeneity in of the protein within GBM that are after in a the SRM we the of and the most responsive proteins of our subset by different GBM we were able to the of and the of treatment as shown in The were to the SRM versus by versus by that the SRM is and to based the proteins potential novel markers of GBM treatment response to anti-angiogenic is to our the targeted proteomics study in GBM the identification of biomarkers in response to anti-angiogenic treatment. on our previous studies the tumor escape mechanisms to (4.Keunen O. Johansson M. Oudin A. Sanzey M. Rahim S.A. Fack F. Thorsen F. Taxt T. Bartos M. Jirik R. Miletic H. Wang J. Stieber D. Stuhr L. Moen I. Rygh C.B. Bjerkvig R. Niclou S.P. Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma.Proc. PubMed Scopus Google Scholar, F. H. O. A. M. O. A. Stuhr L. Miletic H. Stieber D. Rygh C.B. M. L. Niclou S.P. Bjerkvig R. treatment metabolic adaptation toward in Neuropathol. PubMed Scopus Google we and SRM workflow for complex which of small protein in response to treatment. In with our previous work, we identified decreased levels of metabolic related to whereas a of the glycolytic for lactate was to be induced by the treatment. We the of and calnexin, in and which have been previously to anti-angiogenic treatment. The and reproducibility of the data was by which the of and the identified proteins potential novel biomarkers to assess treatment response to anti-angiogenic and of the in clinical GBM is In of the heterogeneity within complex biological samples, our the of data and protein for SRM We protein can be and into in In the context of previous studies were focused on the analysis of human Gupta M.K. R. Polisetty R.V. Sirdeshmukh R. with altered levels in from glioblastoma patients as by 2012; PubMed Scopus Google S. R. D. S. J. R. A. S. of in human glioblastoma 2012; PubMed Scopus Google S. T. A. J. analysis of glioblastoma by a PubMed Scopus Google Scholar, R.V. R. Gupta M.K. S. A. Sirdeshmukh R. analysis of glioblastoma altered and protein of Proteomics. 2012; PubMed Scopus Google and in most human GBM cell R. A. D. of proteins in human glioblastoma cell and PubMed Scopus Google Scholar, S. H. J. proteomics of cells and tumor cells as a potential PubMed Scopus Google Scholar, of in PubMed Scopus Google Scholar, D. S. M. A. H. S. H. J. S. L. and 2012; PubMed Scopus Google Scholar). A study targeted proteins by SRM in the of GBM cell different of the growth factor D. S. D. analysis of growth factor on proteins by glioblastoma Proteomics. 2014; PubMed Scopus (28) Google Scholar). no study a which is to in protein levels. We small significant which are with a highly and SRM the of to small protein concentration in complex biological samples. the of was in tumor cells also in the that was of the specific SRM workflow the workflow to human GBM developed in a rodent was on the of different within the workflow for specific and the of protein in human tumor versus cells been in developed in and in this Niclou S.P. A for targeted studies from different 2014; PubMed Scopus Google Scholar). We have previously applied analysis in GBM samples, the was a in the data analysis M. S. O. Wang J. Miletic H. Bjerkvig R. Niclou S.P. proteomics increased metabolic and cellular in with glioblastoma Proteomics. PubMed Scopus Google Scholar, Rahim S.A. I. K.H. Bjerkvig R. Niclou S.P. based data analysis differences in a brain PubMed Scopus Google Scholar). the of human and rodent proteins within the The that in were treatment is in with our previous studies of tumor hypoxia and increased the glycolytic metabolic switch is by levels of dehydrogenase and although was the for lactate to S. T. in the glycolytic of a PubMed Scopus Google was in we a in the that is with tumor specific and glycolysis S. a of the metabolic system in tumor J. PubMed Scopus Google Scholar). The of in glycolysis is however less and data that the of and in may be than A. H. in A Proteomics. 2014; Scholar, H. M. for a in to during PubMed Scopus Google Scholar, Gupta S. and 2014; PubMed Scopus Google Scholar). proteins which were after treatment we including the most increased proteins and calnexin, whereas and were is a protein in the and been to be in the of protein T. M. T. T. a novel protein of the and a in the of protein PubMed Scopus Google Scholar). in whereas and to molecular in the and control of in the A. M. protein and control in the PubMed Scopus Google Scholar). was shown that is induced by and to of proteins the control is R. T. M. in a control in the PubMed Scopus Google Scholar). the of malectin, calnexin, and is a of hypoxia induced in response to to be is to that and may be affected by anti-angiogenic suggesting that could serve as response A study that high levels of are with tumor control and patient after treatment F. M. M. A. A. M. D. L. O. of with response and in patients with for 2014; PubMed Scopus Google Scholar). was in our protein however be to levels by SRM and response in our GBM In this to the of from tumor In future the of SRM proteins could be to potential markers related to anti-angiogenic growth factor also markers of and endothelial cells M. M. S. J. T. Wen P.Y. a tumor and in glioblastoma 11: PubMed Scopus Google Scholar, M. J. L. J. M. J. in after antiangiogenic therapy is with patients with PubMed Scopus Google Scholar). The of a of proteins anti-angiogenic therapy may the for based patient the identification of molecular biomarkers to response and to in GBM is a high clinical and clinical studies with anti-angiogenic aim to patient and effective combination therapies with Reardon D.A. M. therapy for status and future 2014; PubMed Scopus Google Scholar). In we and highly SRM workflow for tumor specific protein differences in complex biological samples, and novel protein that are in response to anti-angiogenic treatment in The of in clinical GBM and as potential markers of efficacy be with