Abstract
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.
Similar content being viewed by others
References
Lindquist S, Craig EA (1988) The heat-shock proteins. Annu Rev Genet 22:631–677
Kampinga HH, Hageman J, Vos MJ et al (2009) Guidelines for the nomenclature of the human heat shock proteins. Cell Stress Chaperones 14:105–111
Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381:571–579
Farkas B, Hantschel M, Magyarlaki M et al (2003) Heat shock protein 70 membrane expression and melanoma-associated marker phenotype in primary and metastatic melanoma. Melanoma Res 13:147–152
Gehrmann M, Pfister K, Hutzler P et al (2002) Effects of antineoplastic agents on cytoplasmic and membrane-bound heat shock protein 70 (Hsp70) levels. Biol Chem 383:1715–1725
Ciocca DR, Arrigo AP, Calderwood SK (2013) Heat shock proteins and heat shock factor 1 in carcinogenesis and tumor development: an update. Arch Toxicol 87:19–48
Multhoff G, Botzler C, Wiesnet M et al (1995) A stress-inducible 72-kDa heat-shock protein (HSP72) is expressed on the surface of human tumor cells, but not on normal cells. Int J Cancer 61:272–279
Stangl S, Gehrmann M, Riegger J et al (2011) Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody. Proc Natl Acad Sci USA 108:733–738
Stupp R, Mason WP, van den Bent MJ et al (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996
Ohgaki H, Kleihues P (2007) Genetic pathways to primary and secondary glioblastoma. Am J Pathol 170:1445–1453
Ohgaki H, Kleihues P (2013) The definition of primary and secondary glioblastoma. Clin Cancer Res 19:764–772
Louis DN, Perry A, Reifenberger G et al (2016) The 2016 World Health Organization classification of tumors of the central nervous system: a summary. Acta Neuropathol 131:803–820
Yan H, Parsons DW, Jin G et al (2009) IDH1 and IDH2 mutations in gliomas. N Engl J Med 360:765–773
Louis DN, Ohgaki H, Wiestler OD et al (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:97–109
Macdonald DR, Cascino TL, Schold SC Jr, Cairncross JG (1990) Response criteria for phase II studies of supratentorial malignant glioma. J Clin Oncol 8:1277–1280
Wiedorn KH, Olert J, Stacy RA et al (2002) HOPE—a new fixing technique enables preservation and extraction of high molecular weight DNA and RNA of >20 kb from paraffin-embedded tissues. Hepes-Glutamic acid buffer mediated organic solvent protection effect. Pathol Res Pract 198:735–740
Miebach S, Grau S, Hummel V et al (2006) Isolation and culture of microvascular endothelial cells from gliomas of different WHO grades. J Neurooncol 76:39–48
Qiang L, Yang Y, Ma YJ et al (2009) Isolation and characterization of cancer stem like cells in human glioblastoma cell lines. Cancer Lett 279:13–21
Stangl S, Themelis G, Friedrich L et al (2011) Detection of irradiation-induced, membrane heat shock protein 70 (Hsp70) in mouse tumors using Hsp70 Fab fragment. Radiother Oncol 99:313–316
Gehrmann M, Liebisch G, Schmitz G et al (2008) Tumor-specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3. PLoS ONE 3:e1925
Hermisson M, Strik H, Rieger J et al (2000) Expression and functional activity of heat shock proteins in human glioblastoma multiforme. Neurology 54:1357–1365
Beaman GM, Dennison SR, Chatfield LK, Phoenix DA (2014) Reliability of HSP70 (HSPA) expression as a prognostic marker in glioma. Mol Cell Biochem 393:301–307
Zhang WL, Tsuneishi S, Nakamura H (2001) Induction of heat shock proteins and its effects on glial differentiation in rat C6 glioblastoma cells. Kobe J Med Sci 47:77–95
Jaattela M, Wissing D, Kokholm K et al (1998) Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases. EMBO J 17:6124–6134
Murakami N, Kuhnel A, Schmid TE et al (2015) Role of membrane Hsp70 in radiation sensitivity of tumor cells. Radiat Oncol 10:149
Multhoff G, Botzler C, Jennen L et al (1997) Heat shock protein 72 on tumor cells: a recognition structure for natural killer cells. J Immunol 158:4341–4350
Multhoff G, Botzler C, Wiesnet M et al (1995) CD3-large granular lymphocytes recognize a heat-inducible immunogenic determinant associated with the 72-kD heat shock protein on human sarcoma cells. Blood 86:1374–1382
Gunther S, Ostheimer C, Stangl S et al (2015) Correlation of Hsp70 serum levels with gross tumor volume and composition of lymphocyte subpopulations in patients with squamous cell and adeno non-small cell lung cancer. Front Immunol 6:556
Bayer C, Liebhardt ME, Schmid TE et al (2014) Validation of heat shock protein 70 as a tumor-specific biomarker for monitoring the outcome of radiation therapy in tumor mouse models. Int J Radiat Oncol Biol Phys 88:694–700
Lee H, Palm J, Grimes SM, Ji HP (2015) The Cancer Genome Atlas Clinical Explorer: a web and mobile interface for identifying clinical-genomic driver associations. Genome Med 7:112
Ceccarelli M, Barthel FP, Malta TM et al (2016) Molecular profiling reveals biologically discrete subsets and pathways of progression in diffuse glioma. Cell 164:550–563
Chen R, Nishimura MC, Bumbaca SM et al (2010) A hierarchy of self-renewing tumor-initiating cell types in glioblastoma. Cancer Cell 17:362–375
Kleinjung T, Arndt O, Feldmann HJ et al (2003) Heat shock protein 70 (Hsp70) membrane expression on head-and-neck cancer biopsy—a target for natural killer (NK) cells. Int J Radiat Oncol Biol Phys 57:820–826
Gastpar R, Gehrmann M, Bausero MA et al (2005) Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells. Cancer Res 65:5238–5247
Vega VL, Rodriguez-Silva M, Frey T et al (2008) Hsp70 translocates into the plasma membrane after stress and is released into the extracellular environment in a membrane-associated form that activates macrophages. J Immunol 180:4299–4307
Shevtsov MA, Kim AV, Samochernych KA et al (2014) Pilot study of intratumoral injection of recombinant heat shock protein 70 in the treatment of malignant brain tumors in children. Onco Targets Ther 7:1071–1081
Shevtsov MA, Pozdnyakov AV, Mikhrina AL et al (2014) Effective immunotherapy of rat glioblastoma with prolonged intratumoral delivery of exogenous heat shock protein Hsp70. Int J Cancer 135:2118–2128
Yeh CH, Tseng R, Zhang Z et al (2009) Circulating heat shock protein 70 and progression in patients with chronic myeloid leukemia. Leuk Res 33:212–217
Mock A, Warta R, Geisenberger C et al (2015) Printed peptide arrays identify prognostic TNC serumantibodies in glioblastoma patients. Oncotarget 6:13579–13590
Motaln H, Koren A, Gruden K et al (2015) Heterogeneous glioblastoma cell cross-talk promotes phenotype alterations and enhanced drug resistance. Oncotarget 6:40998–41017
Breuninger S, Erl J, Knape C et al (2014) Quantitative analysis of liposomal Heat Shock Protein 70 (Hsp70) in the blood of tumor patients using a novel LipHsp70 ELISA. J Clin Cell Immunol 5:264. doi:10.4172/2155-9899.1000264
Acknowledgements
We thank Stefanie Lange and Sabrina Lakotta for technical support of the experiments.
Funding
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).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
JCT served on the advisory board of Roche, MerckSerono, Celldex and received speakers honoraria from Roche, MerckSerono, BrainLab and Siemens.
Ethical approval
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.
Electronic supplementary material
Below is the link to the electronic supplementary material.
11060_2017_2600_MOESM1_ESM.tif
Suppl. Fig. 1 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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11060-017-2600-z