Identification of T cell target antigens in glioblastoma stem-like cells using an integrated proteomics-based approach in patient specimens


Glioblastoma (GBM) is a highly aggressive brain tumor and still remains incurable. Among others, an immature subpopulation of self-renewing and therapy-resistant tumor cells—often referred to as glioblastoma stem-like cells (GSCs)—has been shown to contribute to disease recurrence. To target these cells personalized immunotherapy has gained a lot of interest, e.g. by reactivating pre-existing anti-tumor immune responses against GSC antigens. To identify T cell targets commonly presented by GSCs and their differentiated counterpart, we used a proteomics-based separation of GSC proteins in combination with a T cell activation assay. Altogether, 713 proteins were identified by LC–ESI–MS/MS mass spectrometry. After a thorough filtering process, 32 proteins were chosen for further analyses. Immunogenicity of corresponding peptides was tested ex vivo. A considerable number of these antigens induced T cell responses in GBM patients but not in healthy donors. Moreover, most of them were overexpressed in primary GBM and also highly expressed in recurrent GBM tissues. Interestingly, expression of the most frequent T cell target antigens could also be confirmed in quiescent, slow-cycling GSCs isolated in high purity by the DEPArray technology. Finally, for a subset of these T cell target antigens, an association between expression levels and higher T cell infiltration as well as an increased expression of positive immune modulators was observed. In summary, we identified novel immunogenic proteins, which frequently induce tumor-specific T cell responses in GBM patients and were also detected in vitro in therapy-resistant quiescent, slow-cycling GSCs. Stable expression of these T cell targets in primary and recurrent GBM support their suitability for future clinical use.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. 1.

    Ahmed N, Salsman VS, Kew Y, Shaffer D, Powell S, Zhang YJ et al (2010) HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors. Clin Cancer Res 16:474–485. doi:10.1158/1078-0432.CCR-09-1322

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SAJR, Behjati S, Biankin AV et al (2013) Signatures of mutational processes in human cancer. Nature 500:415–421. doi:10.1038/nature12477

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Aretz S, Krohne TU, Kammerer K, Warnken U, Hotz-Wagenblatt A, Bergmann M et al (2013) In-depth mass spectrometric mapping of the human vitreous proteome. Proteome Sci 11:22. doi:10.1186/1477-5956-11-22

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Azad TD, Razavi S-M, Jin B, Lee K, Li G (2015) Glioblastoma antigen discovery–foundations for immunotherapy. J Neurooncol 123:347–358. doi:10.1007/s11060-015-1836-8

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB et al (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444:756–760. doi:10.1038/nature05236

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Beckhove P, Warta R, Lemke B, Stoycheva D, Momburg F, Schnölzer M et al (2010) Rapid T cell–based identification of human tumor tissue antigens by automated two-dimensional protein fractionation. J Clin Invest 120:2230–2242. doi:10.1172/JCI37646

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Ben-Baruch A (2006) The multifaceted roles of chemokines in malignancy. Cancer Metastasis Rev 25:357–371. doi:10.1007/s10555-006-9003-5

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Bonertz A, Weitz J, Pietsch D-HK, Rahbari NN, Schlude C, Ge Y et al (2009) Antigen-specific Tregs control T cell responses against a limited repertoire of tumor antigens in patients with colorectal carcinoma. J Clin Invest 119:3311–3321. doi:10.1172/JCI39608

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Brown CE, Starr R, Aguilar B, Shami AF, Martinez C, D’Apuzzo M et al (2012) Stem-like tumor-initiating cells isolated from IL13Ralpha2 expressing gliomas are targeted and killed by IL13-zetakine-redirected T Cells. Clin Cancer Res 18:2199–2209. doi:10.1158/1078-0432.CCR-11-1669

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Butler MW, Fukui T, Salit J, Shaykhiev R, Mezey JG, Hackett NR et al (2011) Modulation of cystatin A expression in human airway epithelium related to genotype, smoking, COPD, and lung cancer. Cancer Res 71:2572–2581. doi:10.1158/0008-5472.CAN-10-2046

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Campa MJ, Wang MZ, Howard B, Fitzgerald MC, Patz EF (2003) Protein expression profiling identifies macrophage migration inhibitory factor and cyclophilin a as potential molecular targets in non-small cell lung cancer. Cancer Res 63:1652–1656

    CAS  PubMed  Google Scholar 

  12. 12.

    Campos B, Gal Z, Baader A, Schneider T, Sliwinski C, Gassel K et al (2014) Aberrant self-renewal and quiescence contribute to the aggressiveness of glioblastoma. J Pathol 234:23–33. doi:10.1002/path.4366

    Article  PubMed  Google Scholar 

  13. 13.

    Castriconi R, Daga A, Dondero A, Zona G, Poliani PL, Melotti A et al (2009) NK cells recognize and kill human glioblastoma cells with stem cell-like properties. J Immunol 182:3530–3539. doi:10.4049/jimmunol.0802845

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Celldex Therapeutics Inc. (2016) Data safety and monitoring board recommends Celldex’s Phase 3 Study of RINTEGA® (rindopepimut) in newly diagnosed glioblastoma be discontinued as it is unlikely to meet primary overall survival endpoint in patients with minimal residual disease. Accessed 26 Jan 2017

  15. 15.

    Cheever MA, Allison JP, Ferris AS, Finn OJ, Hastings BM, Hecht TT et al (2009) The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational research. Clin Cancer Res 15:5323–5337. doi:10.1158/1078-0432.CCR-09-0737

    Article  PubMed  Google Scholar 

  16. 16.

    Cheng S, Luo M, Ding C, Peng C, Lv Z, Tong R et al (2016) Downregulation of Peptidylprolyl isomerase A promotes cell death and enhances doxorubicin-induced apoptosis in hepatocellular carcinoma. Gene 591:236–244. doi:10.1016/j.gene.2016.07.020

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Chen J, McKay RM, Parada LF (2012) Malignant glioma: lessons from genomics, mouse models, and stem cells. Cell 149:36–47. doi:10.1016/j.cell.2012.03.009

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Dahlrot RH, Hermansen SK, Hansen S, Kristensen BW (2013) What is the clinical value of cancer stem cell markers in gliomas? Int J Clin Exp Pathol 6:334–348

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Di Tomaso T, Mazzoleni S, Wang E, Sovena G, Clavenna D, Franzin A et al (2010) Immunobiological characterization of cancer stem cells isolated from glioblastoma patients. Clin Cancer Res 16:800–813. doi:10.1158/1078-0432.CCR-09-2730

    Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Dufour JH, Dziejman M, Liu MT, Leung JH, Lane TE, Luster AD (2002) IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J Immunol 168:3195–3204

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Dunn GP, Dunn IF, Curry WT (2007) Focus on TILs: Prognostic significance of tumor infiltrating lymphocytes in human glioma. Cancer Immun 7:12

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Dutoit V, Herold-Mende C, Hilf N, Schoor O, Beckhove P, Bucher J et al (2012) Exploiting the glioblastoma peptidome to discover novel tumour-associated antigens for immunotherapy. Brain 135:1042–1054. doi:10.1093/brain/aws042

    Article  PubMed  Google Scholar 

  23. 23.

    Eyler CE, Rich JN (2008) Survival of the fittest: cancer stem cells in therapeutic resistance and angiogenesis. J Clin Oncol 26:2839–2845. doi:10.1200/JCO.2007.15.1829

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Foghsgaard L, Wissing D, Mauch D, Lademann U, Bastholm L, Boes M et al (2001) Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol 153:999–1010

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, de Vitis S et al (2004) Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64:7011–7021. doi:10.1158/0008-5472.CAN-04-1364

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Galluzzi L, Vacchelli E, Bravo-San Pedro J-M, Buque A, Senovilla L, Baracco EE et al (2014) Classification of current anticancer immunotherapies. Oncotarget 5:12472–12508. doi:10.18632/oncotarget.2998

    Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP et al (2015) Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med 372:2018–2028. doi:10.1056/NEJMoa1501824

    Article  PubMed  Google Scholar 

  28. 28.

    Ge Y, Domschke C, Stoiber N, Schott S, Heil J, Rom J et al (2012) Metronomic cyclophosphamide treatment in metastasized breast cancer patients: immunological effects and clinical outcome. Cancer Immunol Immunother 61:353–362. doi:10.1007/s00262-011-1106-3

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Ghosh JC, Siegelin MD, Dohi T, Altieri DC (2010) Heat shock protein 60 regulation of the mitochondrial permeability transition pore in tumor cells. Cancer Res 70:8988–8993. doi:10.1158/0008-5472.CAN-10-2225

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Ghosh JC, Dohi T, Kang BH, Altieri DC (2008) Hsp60 regulation of tumor cell apoptosis. J Biol Chem 283:5188–5194. doi:10.1074/jbc.M705904200

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Gilbert CA, Ross AH (2009) Cancer stem cells: cell culture, markers, and targets for new therapies. J Cell Biochem 108:1031–1038. doi:10.1002/jcb.22350

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Gonzalez-Galarza FF, Takeshita LYC, Santos EJM, Kempson F, Maia MHT, da Silva Andrea, Soares Luciana et al (2015) Allele frequency net 2015 update: new features for HLA epitopes, KIR and disease and HLA adverse drug reaction associations. Nucleic Acids Res 43:D784–D788. doi:10.1093/nar/gku1166

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674. doi:10.1016/j.cell.2011.02.013

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Hashimoto N, Tsuboi A, Kagawa N, Chiba Y, Izumoto S, Kinoshita M et al (2015) Wilms tumor 1 peptide vaccination combined with temozolomide against newly diagnosed glioblastoma: safety and impact on immunological response. Cancer Immunol Immunother 64:707–716. doi:10.1007/s00262-015-1674-8

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Hashimoto Y, Kim DJ, Adams JC (2011) The roles of fascins in health and disease. J Pathol 224:289–300. doi:10.1002/path.2894

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    He J, Liu Y, Lubman DM (2012) Targeting glioblastoma stem cells: cell surface markers. Curr Med Chem 19:6050–6055

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Herold-Mende C, Mueller MM, Bonsanto MM, Schmitt HP, Kunze S, Steiner HH (2002) Clinical impact and functional aspects of tenascin-C expression during glioma progression. Int J Cancer 98:362–369

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Higano CS, Small EJ, Schellhammer P, Yasothan U, Gubernick S, Kirkpatrick P et al (2010) Sipuleucel-T. Nat Rev Drug Discov 9:513–514. doi:10.1038/nrd3220

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Horn T, Grab J, Schusdziarra J, Schmid S, Maurer T, Nawroth R et al (2013) Antitumor T cell responses in bladder cancer are directed against a limited set of antigens and are modulated by regulatory T cells and routine treatment approaches. Int J Cancer 133:2145–2156. doi:10.1002/ijc.28233

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Huang DW, Sherman BT, Lempicki RA (2009) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37:1–13. doi:10.1093/nar/gkn923

    Article  Google Scholar 

  41. 41.

    Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57. doi:10.1038/nprot.2008.211

    CAS  Article  Google Scholar 

  42. 42.

    ImmunoCellular Therapeutics Ltd. (2013) ImmunoCellular Therapeutics Phase II Study Demonstrates That Glioblastoma Patients Live Longer Without Disease Progression When Treated With ICT-107. Accessed 26 Jan 2017

  43. 43.

    Jarboe JS, Johnson KR, Choi Y, Lonser RR, Park JK (2007) Expression of interleukin-13 receptor alpha2 in glioblastoma multiforme: implications for targeted therapies. Cancer Res 67:7983–7986. doi:10.1158/0008-5472.CAN-07-1493

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Ji J, Judkowski VA, Liu G, Wang H, Bunying A, Li Z et al (2014) Identification of novel human leukocyte antigen-A*0201-restricted, cytotoxic T lymphocyte epitopes on CD133 for cancer stem cell immunotherapy. Stem Cells Transl Med 3:356–364. doi:10.5966/sctm.2013-0135

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF et al (2010) Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med 363:411–422. doi:10.1056/NEJMoa1001294

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Keller BO, Sui J, Young AB, Whittal RM (2008) Interferences and contaminants encountered in modern mass spectrometry. Anal Chim Acta 627:71–81. doi:10.1016/j.aca.2008.04.043

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Kikuchi T, Akasaki Y, Abe T, Fukuda T, Saotome H, Ryan JL et al (2004) Vaccination of glioma patients with fusions of dendritic and glioma cells and recombinant human interleukin 12. J Immunother 27:452–459

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Kmiecik J, Zimmer J, Chekenya M (2014) Natural killer cells in intracranial neoplasms: presence and therapeutic efficacy against brain tumours. J Neurooncol 116:1–9. doi:10.1007/s11060-013-1265-5

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Koelsche C, Sahm F, Capper D, Reuss D, Sturm D, Jones DTW et al (2013) Distribution of TERT promoter mutations in pediatric and adult tumors of the nervous system. Acta Neuropathol 126:907–915. doi:10.1007/s00401-013-1195-5

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD et al (2015) PD-1 blockade in tumors with mismatch-repair deficiency. N Engl J Med 372:2509–2520. doi:10.1056/NEJMoa1500596

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Lee EY, Lee Z-H, Song YW (2009) CXCL10 and autoimmune diseases. Autoimmun Rev 8:379–383. doi:10.1016/j.autrev.2008.12.002

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Liau LM, Prins RM, Kiertscher SM, Odesa SK, Kremen TJ, Giovannone AJ et al (2005) Dendritic cell vaccination in glioblastoma patients induces systemic and intracranial T-cell responses modulated by the local central nervous system tumor microenvironment. Clin Cancer Res 11:5515–5525. doi:10.1158/1078-0432.CCR-05-0464

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Li M, Zhai Q, Bharadwaj U, Wang H, Li F, Fisher WE et al (2006) Cyclophilin A is overexpressed in human pancreatic cancer cells and stimulates cell proliferation through CD147. Cancer 106:2284–2294. doi:10.1002/cncr.21862

    CAS  Article  PubMed  Google Scholar 

  54. 54.

    Liu G, Yuan X, Zeng Z, Tunici P, Ng H, Abdulkadir IR et al (2006) Analysis of gene expression and chemoresistance of CD133 + cancer stem cells in glioblastoma. Mol Cancer 5:67. doi:10.1186/1476-4598-5-67

    Article  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Lohr J, Ratliff T, Huppertz A, Ge Y, Dictus C, Ahmadi R et al (2011) Effector T-cell infiltration positively impacts survival of glioblastoma patients and is impaired by tumor-derived TGF-beta. Clin Cancer Res 17:4296–4308. doi:10.1158/1078-0432.CCR-10-2557

    CAS  Article  PubMed  Google Scholar 

  56. 56.

    Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A et al (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:97–109. doi:10.1007/s00401-007-0243-4

    Article  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Lynch GW, Turville S, Carter B, Sloane AJ, Chan A, Muljadi N et al (2006) Marked differences in the structures and protein associations of lymphocyte and monocyte CD4: resolution of a novel CD4 isoform. Immunol Cell Biol 84:154–165. doi:10.1111/j.1440-1711.2005.01403.x

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    Mock A, Chiblak S, Herold-Mende C (2014) Lessons we learned from high-throughput and top-down systems biology analyses about glioma stem cells. Curr Pharm Des 20:66–72

    CAS  Article  PubMed  Google Scholar 

  59. 59.

    Mussunoor S, Murray GI (2008) The role of annexins in tumour development and progression. J Pathol 216:131–140. doi:10.1002/path.2400

    CAS  Article  PubMed  Google Scholar 

  60. 60.

    Okada H, Kalinski P, Ueda R, Hoji A, Kohanbash G, Donegan TE et al (2011) Induction of CD8 + T-cell responses against novel glioma-associated antigen peptides and clinical activity by vaccinations with {alpha}-type 1 polarized dendritic cells and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose in patients with recurrent malignant glioma. J Clin Oncol 29:330–336. doi:10.1200/JCO.2010.30.7744

    CAS  Article  PubMed  Google Scholar 

  61. 61.

    Okada H, Lieberman FS, Walter KA, Lunsford LD, Kondziolka DS, Bejjani GK et al (2007) Autologous glioma cell vaccine admixed with interleukin-4 gene transfected fibroblasts in the treatment of patients with malignant gliomas. J Transl Med 5:67. doi:10.1186/1479-5876-5-67

    Article  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Pallini R, Ricci-Vitiani L, Montano N, Mollinari C, Biffoni M, Cenci T et al (2011) Expression of the stem cell marker CD133 in recurrent glioblastoma and its value for prognosis. Cancer 117:162–174. doi:10.1002/cncr.25581

    Article  PubMed  Google Scholar 

  63. 63.

    Peraud A, Mondal S, Hawkins C, Mastronardi M, Bailey K, Rutka JT (2003) Expression of fascin, an actin-bundling protein, in astrocytomas of varying grades. Brain Tumor Pathol 20:53–58

    CAS  Article  PubMed  Google Scholar 

  64. 64.

    Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567. doi:10.1002/(SICI)1522-2683(19991201)20:18<3551:AID-ELPS3551>3.0.CO;2-2

    CAS  Article  PubMed  Google Scholar 

  65. 65.

    Perretti M, D’Acquisto F (2009) Annexin A1 and glucocorticoids as effectors of the resolution of inflammation. Nat Rev Immunol 9:62–70. doi:10.1038/nri2470

    CAS  Article  PubMed  Google Scholar 

  66. 66.

    Phuphanich S, Wheeler CJ, Rudnick JD, Mazer M, Wang H, Nuno MA et al (2013) Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma. Cancer Immunol Immunother 62:125–135. doi:10.1007/s00262-012-1319-0

    CAS  Article  PubMed  Google Scholar 

  67. 67.

    Powles T, Eder JP, Fine GD, Braiteh FS, Loriot Y, Cruz C et al (2014) MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature 515:558–562. doi:10.1038/nature13904

    CAS  Article  PubMed  Google Scholar 

  68. 68.

    Prasad S, Gaedicke S, Machein M, Mittler G, Braun F, Hettich M et al (2015) Effective eradication of glioblastoma stem cells by local application of an AC133/CD133-specific T-cell-engaging antibody and CD8 T cells. Cancer Res 75:2166–2176. doi:10.1158/0008-5472.CAN-14-2415

    CAS  Article  PubMed  Google Scholar 

  69. 69.

    Rebhan M, Chalifa-Caspi V, Prilusky J, Lancet D (1997) GeneCards: integrating information about genes, proteins and diseases. Trends Genet 13:163

    CAS  Article  PubMed  Google Scholar 

  70. 70.

    Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414:105–111. doi:10.1038/35102167

    CAS  Article  PubMed  Google Scholar 

  71. 71.

    Rhen T, Cidlowski JA (2005) Antiinflammatory action of glucocorticoids–new mechanisms for old drugs. N Engl J Med 353:1711–1723. doi:10.1056/NEJMra050541

    CAS  Article  PubMed  Google Scholar 

  72. 72.

    Roma AA, Prayson RA (2005) Fascin expression in 90 patients with glioblastoma multiforme. Ann Diagn Pathol 9:307–311. doi:10.1016/j.anndiagpath.2005.07.005

    Article  PubMed  Google Scholar 

  73. 73.

    Sahm F, Schrimpf D, Jones DTW, Meyer J, Kratz A, Reuss D et al (2016) Next-generation sequencing in routine brain tumor diagnostics enables an integrated diagnosis and identifies actionable targets. Acta Neuropathol 131:903–910. doi:10.1007/s00401-015-1519-8

    CAS  Article  PubMed  Google Scholar 

  74. 74.

    Sampson JH, Heimberger AB, Archer GE, Aldape KD, Friedman AH, Friedman HS et al (2010) Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma. J Clin Oncol 28:4722–4729. doi:10.1200/JCO.2010.28.6963

    Article  PubMed  PubMed Central  Google Scholar 

  75. 75.

    Schuster J, Lai RK, Recht LD, Reardon DA, Paleologos NA, Groves MD et al (2015) A phase II, multicenter trial of rindopepimut (CDX-110) in newly diagnosed glioblastoma: the ACT III study. Neuro Oncol 17:854–861. doi:10.1093/neuonc/nou348

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  76. 76.

    Seder RA, Darrah PA, Roederer M (2008) T-cell quality in memory and protection: implications for vaccine design. Nat Rev Immunol 8:247–258. doi:10.1038/nri2274

    CAS  Article  PubMed  Google Scholar 

  77. 77.

    Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T et al (2004) Identification of human brain tumour initiating cells. Nature 432:396–401. doi:10.1038/nature03128

    CAS  Article  PubMed  Google Scholar 

  78. 78.

    Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J et al (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63:5821–5828

    CAS  PubMed  Google Scholar 

  79. 79.

    Snyder A, Makarov V, Merghoub T, Yuan J, Zaretsky JM, Desrichard A et al (2014) Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med 371:2189–2199. doi:10.1056/NEJMoa1406498

    Article  PubMed  PubMed Central  Google Scholar 

  80. 80.

    Sotiropoulou PA, Christodoulou MS, Silvani A, Herold-Mende C, Passarella D (2014) Chemical approaches to targeting drug resistance in cancer stem cells. Drug Discov Today 19:1547–1562. doi:10.1016/j.drudis.2014.05.002

    CAS  Article  PubMed  Google Scholar 

  81. 81.

    Steiner HH, Bonsanto MM, Beckhove P, Brysch M, Geletneky K, Ahmadi R et al (2004) Antitumor vaccination of patients with glioblastoma multiforme: a pilot study to assess feasibility, safety, and clinical benefit. J Clin Oncol 22:4272–4281. doi:10.1200/JCO.2004.09.038

    Article  PubMed  Google Scholar 

  82. 82.

    Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJB et al (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996. doi:10.1056/NEJMoa043330

    CAS  Article  PubMed  Google Scholar 

  83. 83.

    The UniProt Consortium (2015) UniProt: a hub for protein information. Nucleic Acids Res 43:D204–D212. doi:10.1093/nar/gku989

    Article  Google Scholar 

  84. 84.

    Uhlen M, Bjorling E, Agaton C, Szigyarto CA-K, Amini B, Andersen E et al (2005) A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol Cell Proteomics 4:1920–1932. doi:10.1074/mcp.M500279-MCP200

    CAS  Article  PubMed  Google Scholar 

  85. 85.

    Verhaak RGW, Hoadley KA, Purdom E, Wang V, Qi Y, Wilkerson MD et al (2010) Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17:98–110. doi:10.1016/j.ccr.2009.12.020

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  86. 86.

    Vita R, Overton JA, Greenbaum JA, Ponomarenko J, Clark JD, Cantrell JR et al (2015) The immune epitope database (IEDB) 3.0. Nucleic Acids Res 43:D405–D412. doi:10.1093/nar/gku938

    CAS  Article  PubMed  Google Scholar 

  87. 87.

    de Vleeschouwer S, Fieuws S, Rutkowski S, van Calenbergh F, van Loon J, Goffin J et al (2008) Postoperative adjuvant dendritic cell-based immunotherapy in patients with relapsed glioblastoma multiforme. Clin Cancer Res 14:3098–3104. doi:10.1158/1078-0432.CCR-07-4875

    Article  PubMed  Google Scholar 

  88. 88.

    Walter S, Weinschenk T, Stenzl A, Zdrojowy R, Pluzanska A, Szczylik C et al (2012) Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival. Nat Med 18:1254–1261. doi:10.1038/nm.2883

    CAS  Article  PubMed  Google Scholar 

  89. 89.

    Wheeler CJ, Black KL, Liu G, Mazer M, X-x Zhang, Pepkowitz S et al (2008) Vaccination elicits correlated immune and clinical responses in glioblastoma multiforme patients. Cancer Res 68:5955–5964. doi:10.1158/0008-5472.CAN-07-5973

    CAS  Article  PubMed  Google Scholar 

  90. 90.

    Wu A, Wei J, Kong L-Y, Wang Y, Priebe W, Qiao W et al (2010) Glioma cancer stem cells induce immunosuppressive macrophages/microglia. Neuro Oncol 12:1113–1125. doi:10.1093/neuonc/noq082

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  91. 91.

    Wu G, Diaz AK, Paugh BS, Rankin SL, Ju B, Li Y et al (2014) The genomic landscape of diffuse intrinsic pontine glioma and pediatric non-brainstem high-grade glioma. Nat Genet 46:444–450. doi:10.1038/ng.2938

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  92. 92.

    Xu Q, Liu G, Yuan X, Xu M, Wang H, Ji J et al (2009) Antigen-specific T-cell response from dendritic cell vaccination using cancer stem-like cell-associated antigens. Stem Cells 27:1734–1740. doi:10.1002/stem.102

    CAS  Article  PubMed  Google Scholar 

  93. 93.

    Yamanaka R, Homma J, Yajima N, Tsuchiya N, Sano M, Kobayashi T et al (2005) Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma: results of a clinical phase I/II trial. Clin Cancer Res 11:4160–4167. doi:10.1158/1078-0432.CCR-05-0120

    CAS  Article  PubMed  Google Scholar 

  94. 94.

    Yu JS, Wheeler CJ, Zeltzer PM, Ying H, Finger DN, Lee PK et al (2001) Vaccination of malignant glioma patients with peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial T-cell infiltration. Cancer Res 61:842–847

    CAS  PubMed  Google Scholar 

  95. 95.

    Zhou B-BS, Zhang H, Damelin M, Geles KG, Grindley JC, Dirks PB (2009) Tumour-initiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov 8:806–823. doi:10.1038/nrd2137

    CAS  Article  PubMed  Google Scholar 

  96. 96.

    Zhu X, Prasad S, Gaedicke S, Hettich M, Firat E, Niedermann G (2015) Patient-derived glioblastoma stem cells are killed by CD133-specific CAR T cells but induce the T cell aging marker CD57. Oncotarget 6:171–184. doi:10.18632/oncotarget.2767

    PubMed  Google Scholar 

Download references


We like to thank the Tissue Bank of the National Center for Tumor Diseases (NCT, Heidelberg, Germany) for providing us with tissue samples. We further thank Melanie Greibich, Mandy Barthel, Farzaneh Kashfi, Ilka Hearn, Hildegard Goeltzer, Axel Schoeffel, and Cinja Sackmann for excellent technical assistance.

Author information



Corresponding author

Correspondence to Christel Herold-Mende.

Ethics declarations


This project was supported by the Anni Hofmann Stiftung.

Additional information

C. Rapp, R. Warta, A. Abdollahi, P. Beckhove and C. Herold-Mende contributed equally to the work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Immunohistochemistry and multicolor immunofluorescent stainings:

Two consecutive tissue sections, cytospins or wells of adherent cells on one slide allowed the concurrent staining of antibodies and the respective control. All primary antibodies were diluted in Antibody Diluent (Dako, Hamburg, Germany) and incubated for 1 h. All incubation steps were performed at room temperature. After the application of first and secondary antibodies as well as after the incubation with the ABC reagent, three washing steps with PBS containing 0.05 % Tween (Sigma-Aldrich, Taufkirchen, Germany) were performed. For immunohistochemical stainings, appropriate biotinylated secondary antibodies of the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, USA) were diluted (1:200) with serum (1:66) in DPBS and incubated for 30 minutes. In the next step, the ABC reagent was prepared 30 min. before the sections were incubated with peroxidase substrate solution until the desired staining intensity. The duration of incubation was based on the isotype control staining. After stopping incubation with water, nuclei were counterstained with hematoxylin. Finally, all slides were mounted with Elvanol (Roth, Karlsruhe, Germany). For all stainings, appropriate concentration and species of isotype controls were used (PPTX 107 kb)

Supplementary Fig. 1

Characterization of glioblastoma stem-like cells. a, c Phase-contrast images of glioblastoma stem-like cells (GSCs) from NCH663 and NCH711d growing as neurospheres (scale bar: 200 µm), while b, d expression of the stem cell marker CD133 was determined by flow cytometry (grey outline: isotype control, red outline: CD133 staining). e, f Immunofluorescence analysis of stem cell markers Nestin (green) and CD133 (red) as well as g, h the lineage markers GFAP (purple), MBP (red), and ßIII-tubulin (green) stained on cytospins (scale bar: 50 µM). i mRNA expression analysis of stem cell markers POU5F1, ID1, BMI1, FABP7, and SOX2 in undifferentiated and ATRA-treated GSCs from NCH663 and NCH711d. Expression levels were assessed by qPCR and normalized against the housekeeping gene GAPDH. Stem cell marker expression decreased markedly upon ATRA-induced differentiation (JPEG 5199 kb)


Supplementary Fig. 2 Expression of stem cell and differentiation markers before and after differentiation of glioblastoma stem-like cells. Percentage of positive GSCs expressing a stem cell markers Nestin and CD133 or b lineage markers GFAP, MBP, and ßIII-tubulin before and after ATRA-induced differentiation. Three representative areas of immunofluorescence stainings were quantitatively evaluated. Significant differences are indicated by asterisks (p < 0.05 *, p < 0.01 **, p < 0.001 ***). Error bars show the standard error of the mean (SEM) of triplicates (JPEG 2365 kb)


Supplementary Fig. 3 Tumorigenicity of glioblastoma stem-like cells. Anti-human Nuclei and Ki67 staining of an exemplary tumor-bearing mouse brain, derived from a NCH663 or b NCH711d GSCs, after xenotransplantation of 1x105 GSCs (dashed scale bar: 500 µm, insert: 20 µM). Mice were sacrificed upon occurrence of neurological symptoms (NCH663: symptom-free until the 8th week; NCH711d: symptom-free until the 14th week) or after 20 weeks at the latest (JPEG 4283 kb)


Supplementary Fig. 4 Characterization of ATRA-treated glioblastoma stem-like cells. a, b Phase-contrast images of adherently growing ATRA-treated GSCs from NCH663 and NCH711d (scale bar: 200 µm) c ATRA-treated NCH663 and d NCH711d cells were stained by immunofluorescence for the expression of the differentiation markers GFAP (purple), MBP (red), and ßIII-tubulin (green). e, f ATRA-treated cells were further stained for Nestin and CD133. Scale bar: 50 µM (JPEG 2787 kb)


Supplementary Fig. 5 IFN-γ ELISpot assays of 1st and 2nd PF2D dimension fractions. 1st (left) and 2nd PF2D dimension fractions (right) of a differentiated NCH663 GSCs, b undifferentiated NCH711d GSCs, and c differentiated NCH711d GSCs are shown. Fractions of the 1st PF2D dimension triggering higher T cell responses than the control (PBMC lysate) were further fractionated in the 2nd PF2D dimension (marked in black). 2nd PF2D fractions which had shown a significantly higher immune response than the control were analyzed by LC-ESI-MS/MS to identify protein contents (marked in black). Significant differences are indicated by asterisks (p < 0.05 *, p < 0.01 **, p < 0.001 ***). Abbreviations: GSCs = glioblastoma stem-like cells (JPEG 1154 kb)

Supplementary Fig. 6

Filtering process to select the most interesting potential T cell target antigens. Mass spectrometry analyses of 2D PF2D fractions identified 713 proteins, which passed the following filtering process: First, false positive peptide identifications were reduced by applying cutoffs for the protein sequence coverage (> 10 %) and protein matches (> 5) as well as common contaminations resulting in 332 proteins. Additionally, literature and publicly available databases such as UniProt, GeneCards, and PubMed of the National Center for Biotechnology Information were used to characterize all proteins regarding their known function. Proteins (i) already described in the context of tumor diseases, (ii) involved in tumor-related signaling pathways such as cell cycle control, cell proliferation, angiogenesis, apoptosis, or invasion, and (iii) proteins with immunomodulatory effects were selected revealing 201 proteins which were further assessed regarding protein expression using The Human Protein Atlas. Proteins with a strong and homogenous expression in normal tissues (especially in the brain) were excluded. Based on this approach, 32 proteins were chosen for further characterization (JPEG 665 kb)


Supplementary Fig. 7 Epitope prediction of immunogenic epitopes. a The Immune Epitope Database (IEDB) was used to predict the most immunogenic epitope of selected proteins for the HLA alleles HLA-A*01:01, HLA-A*02:01, HLA-A*24:02, HLA-A*03:01, HLA-B*07:02. Epitopes were selected based on the calculation of a low HLA IC50 value (< 500 nm) (grey line) combined with a high product of the number of epitopes within the respective sequence and the number of HLA types (inverse, black line). Selection of long peptide sequences is exemplarily shown for HSPA5 (red box). b Amino acid sequence for HSPA5 containing the selected epitope marked in red (JPEG 996 kb)


Supplementary Fig. 8 Validation of synthesized peptides in the patients of origin. All selected peptides were validated for immunogenicity with peripheral autologous blood of the patients of origin by IFN-γ ELISpot assays. All peptides showing significantly higher T cell responses relative to the control (IgG) were further validated (marked in grey). Significant differences are indicated by asterisks (p < 0.05 *, p < 0.01 **, p < 0.001 ***). Error bars show the standard error of the mean (SEM) of triplicates (JPEG 708 kb)


Supplementary Fig. 9 Validation of immunogenicity of candidate proteins in additional GBM patients and healthy donors. Peptides corresponding to 11 potential TAAs identified by PF2D analysis were tested for immunogenicity in an independent cohort of GBM patients (n = 28) as well as in healthy donors (n = 22) by IFN-γ ELISpot analyses. GBM patients showed a significantly higher immune response against peptides than healthy donors. Asterisks indicate significant differences (***, p < 0.001) (JPEG 563 kb)


Supplementary Fig. 10 mRNA expression of immunogenic T cell target antigens in GSCs before and after ATRA-induced differentiation. mRNA expression of HSPD1, PPIA, FSCN1, ANXA1, and CSTA was analyzed in undifferentiated and differentiated GSCs (n = 9) by microarray analyses. Data of NCH663 and NCH711d are represented by a clear circle. Abbreviations: diff. = differentiated; undiff. = undifferentiated (JPEG 646 kb)


Supplementary Fig. 11 Survival analysis of CSTA. IDH1-wt pGBM cases retrieved from the TCGA microarray data set were used to calculate a Cox proportional hazard model. Results of a the univariate analysis (n = 356) and b the multivariate analysis (n = 259) are shown. All significant covariates of the univariate model were included into the multivariate model. P-values were calculated by log-rank test (p < 0.05 *, p < 0.01 **, p < 0.001 ***) (JPEG 648 kb)


Supplementary Fig. 12 Survival analysis of CSTA combined with CD8 (A) and TGFB1 (B). 356 IDH1-wt pGBM cases were used to calculate a Cox proportional hazard model. Groups which did not show significant differences in a and c were summarized in one group for a better visualization (b, d) (JPEG 986 kb)

Supplementary Fig. 13

Association of CSTA expression and cytokines. a TCGA microarray data of 357 IDH1-wt pGBM cases were median-grouped for the expression of CXCL10, TNF-α (TNF), and TGF-β1 (TGFB1) while CSTA expression was analyzed in the respective groups. b Correlation between CSTA and the respective immunomodulators. Significant correlations are indicated by asterisks (p < 0.05 *, p < 0.01 **, p < 0.001 ***) (JPEG 983 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rapp, C., Warta, R., Stamova, S. et al. Identification of T cell target antigens in glioblastoma stem-like cells using an integrated proteomics-based approach in patient specimens. Acta Neuropathol 134, 297–316 (2017).

Download citation


  • IDH1-wt glioblastoma
  • T cell target antigen repertoire
  • Quiescent stem-like cells
  • Heterogeneity
  • Plasticity