A unique feature in several non-CNS-tumors is the overexpression of heat shock protein 70 (Hsp70, HSPA1A) in the cytosol, but also its unusual plasma membrane expression and release. Although in gliomas, cytosolic Hsp70 levels are not associated with histological grading, the role of membrane bound and released Hsp70 is still completely unknown. Membrane bound as well as cytosolic Hsp70 can be detected in viable tumor cells with the monoclonal antibody (mAb) cmHsp70.1. Herein, we analysed membrane bound Hsp70 levels in primary and secondary gliomas of different grades and on isolated glioma subpopulations (endothelial cells, CD133-positive cells, primary cultures) by immunohistochemistry and flow cytometry using cmHsp70.1 mAb. Extracellular Hsp70 was determined by a commercial Hsp70 sandwich ELISA (R&D) in plasma samples of glioblastoma patients and healthy volunteers. We found an overexpression of Hsp70 in primary glioblastomas compared to low-grade, anaplastic, or secondary gliomas as determined by immunohistochemistry. Especially in flow cytometry, a strong plasma membrane Hsp70 expression was only observed in primary but not secondary glioblastomas. Within the heterogeneous tumor mass, CD133-positive tumor-initiating and primary glioblastoma cells showed a high membrane Hsp70 expression density, whereas endothelial cells, isolated from glioblastoma tissues only showed a weak staining pattern. Also in plasma samples, secreted Hsp70 protein was significantly increased in patients harbouring primary glioblastomas compared to those with secondary and low grade glioblastomas. Taken together, we show for the first time that cytosolic, membrane bound and extracellular Hsp70 is uniquely overexpressed in primary glioblastomas.
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We thank Stefanie Lange and Sabrina Lakotta for technical support of the experiments.
This study was supported by Grants of the SFB 824/B2, Deutsche Forschungsgemeinschaft (DFG), the EU ERA net bilateral INREMOS project on Systems Biology Tools development for cell Therapy and Drug Development/SYSTHER (Con. No.: 3211-06-000539) (2006–2011) funded by the German and Slovenian Federal Ministries of Education and Research and the “Familie Mehdorn Stiftung”. Peng Fu was supported by China Scholarship Council (No. 2009616010) and Ketai Guo was supported SYSTHER. The work of Gabriele Multhoff was supported by grants of the DFG (SFB824/2 B4; DFG INST/980-1FUGG, INST411/37-1FUGG), the DFG Cluster of Excellence: Munich-Centre for Advanced Photonics (MAP), the Bundesministerium für Forschung und Technologie (BMBF Innovative Therapies, 01GU0823; BMBF m4 Spitzencluster, 16EX1021C, 16GW0030; BMBF Kompetenzverbund Strahlenforschung 02NUK038A), European Union (EU-CELLEUROPE 315963) and the Wilhelm-Sander Stiftung (2012.078.1).
Conflict of interest
JCT served on the advisory board of Roche, MerckSerono, Celldex and received speakers honoraria from Roche, MerckSerono, BrainLab and Siemens.
The study was approved by the institutional review board of the University of Munich, Germany and have been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Informed consent was obtained from all patients.
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Kaplan-Meier curve for PFS and OS stratified for MGMT promoter status (a, b) and Hsp70 expression as determined by IHC (c, d) (TIF 299 KB)
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Thorsteinsdottir, J., Stangl, S., Fu, P. et al. Overexpression of cytosolic, plasma membrane bound and extracellular heat shock protein 70 (Hsp70) in primary glioblastomas. J Neurooncol 135, 443–452 (2017). https://doi.org/10.1007/s11060-017-2600-z