Article Text

Download PDFPDF

1463 The intra-tumoral spatial heterogeneity of T cell antigens in glioblastoma: An integrated multi-omics approach
  1. Marcel Wacker1,2,3,
  2. Gioele Medici4,5,
  3. Marissa Dubbelaar2,3,6,7,
  4. Jens Bauer2,3,6,
  5. Annika Nelde2,3,6,
  6. Friederike Hanssen7,
  7. Carolin Schwitalla7,
  8. Luca Regli4,
  9. Michael Weller5,
  10. Hans-Georg Rammensee2,3,8,
  11. Marian Christoph Neidert4,9,10 and
  12. Juliane S Walz2,3,6,11
  1. 1University and University Hospital Tübingen, Tübingen, Germany
  2. 2Institute for Cell Biology, Department of Immunology, University of Tübingen, Tübingen, Germany
  3. 3Cluster of Excellence iFIT (EXC2180) ‘Image-Guided and Functionally Instructed Tumor Therapies’, University of Tübingen, Tübingen, Germany
  4. 4Clinical Neuroscience Center and Department of Neurosurgery, University Hospital Zürich, University of Zürich, Zürich, Switzerland
  5. 5Laboratory of Molecular Neuro-Oncology, Department of Neurology and Brain Tumor Center, University Hospital Zürich, Zürich, Switzerland
  6. 6Department of Peptide-based Immunotherapy, University and University Hospital Tübingen, Tübingen, Germany
  7. 7Quantitative Biology Center (QBiC), University of Tübingen, Tübingen, Germany
  8. 8German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ) partner site Tübingen, Tübingen, Germany
  9. 9Department of Neurosurgery, Cantonal Hospital St. Gallen, St. Gallen, Switzerland
  10. 10Neuroscience Center Zürich (ZNZ), University of Zürich and ETH Zürich, Zürich, Switzerland
  11. 11Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK), Department of Internal Medicine, University Hospital Tübingen, Tübingen, Germany
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Glioblastoma is the most common primary malignant neoplasm of the central nervous system in adults. Current treatment options comprise maximal surgical resection followed by radiation and/or chemotherapy with temozolomide. These procedures are unable to eliminate all tumor cells, leading to disease recurrence and accounting for the poor prognosis. Glioblastoma is a highly infiltrative tumor with recurrence originating from the unresectable peritumoral infiltration zone (INF). Thus, novel treatment options specifically targeting tumor cells in the INF zone are needed to prevent relapse and enable long-lasting remission.

Methods In this work, we performed a multi-omics spatial analysis of the necrotic center (NEC), the gadolinium contrast-enhancing region (T1), and the INF zone integrating mass spectrometry-based immunopeptidome analysis with next-generation sequencing methods (whole exome and RNA sequencing) to assess immunologically relevant aspects of tumor heterogeneity. For the multi-omics analysis, HLA-restricted peptides and genetic material from 15 glioblastoma patients were analyzed from the three zones NEC, T1, INF. Additionally, adjacent benign (BEN) brain tissue was analyzed from four patients.

Results A total of 31,655 unique HLA class I and 10,071 unique HLA class II peptides were identified in 12 of the 15 glioblastoma patients. Comparative profiling of peptides from our study and a benign tissue database (HLA ligand atlas ( identified 38% of HLA class I and 70% of HLA class II peptides as tumor-associated antigens (TAAs) originating from either INF, T1 or NEC zones, respectively. Of these HLA class I TAAs, 17%, 19%, and 17% and of the HLA class II TAAs 16%, 19%, and 43%, were exclusively presented in the INF, T1, or NEC zones, respectively. Five INF-associated HLA class I ligands were frequently presented in 42% of glioblastoma immunopeptidomes, whereas nine INF-associated HLA class II ligands were presented in up to 62% of glioblastoma immunopeptidomes. Of note, the glioblastoma-associated proteins BAALC, NCAN, and SLC20A1A were identified among these INF-associated ligands. Furthermore, integrated RNA/DNA sequencing enabled a greater understanding of spatial tumor antigen presentation. It led to the identification of 1,287 and 923 predicted possible INF-specific HLA class I and HLA class II neoepitopes, respectively, which are derived from tumor-associated mutations. The immunogenicity of these peptides will be further validated in upcoming T cell assays of tumor-infiltrating lymphocytes.

Conclusions In summary, intra-tumoral regional heterogeneity of tumor antigens was identified, which could be used for specific immunotherapy approaches targeting the INF zone of glioblastoma.

Ethics Approval Written informed consent was obtained for all patients following the Declaration of Helsinki protocol and the local review board (Kantonale Ethikkomission Zürich; KEK-ZH-Nr.2015–0163)

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.