Advertisement

Chaperones pp 307-320 | Cite as

Immunohistochemical and Flow Cytometric Analysis of Intracellular and Membrane-Bound Hsp70, as a Putative Biomarker of Glioblastoma Multiforme, Using the cmHsp70.1 Monoclonal Antibody

  • Stefan Stangl
  • Gemma A. Foulds
  • Helena Fellinger
  • Geoffrey J. Pilkington
  • A. Graham Pockley
  • Gabriele MulthoffEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1709)

Abstract

The major stress-inducible 70 kDa heat shock (stress) protein 70 (Hsp70) is frequently overexpressed in highly aggressive tumor cells and thus might serve as a tumor-specific biomarker of aggressive disease. We have previously shown that, in contrast to normal cells, tumor cells present Hsp70 on their plasma membrane. In order to elucidate the role of intracellular and membrane-bound Hsp70 as a potential tumor biomarker in glioblastoma multiforme, herein, we describe protocols for the staining of cytosolic Hsp70 in tumor formalin fixed paraffin-embedded (FFPE) sections using immunohistochemistry, and for plasma membrane-bound Hsp70 by multi-parametric flow cytometry using the cmHsp70.1 monoclonal antibody (mAb).

Key words

Intracellular and membrane-bound heat shock protein 70 (Hsp70) Glioblastoma multiforme Formalin fixed paraffin-embedded (FFPE) sections Immunohistochemistry Flow cytometry Hsp70 antibody epitope 

Notes

Acknowledgments

Gabriele Multhoff is supported by grants of the Deutsche Forschungsgemeinschaft (SFB824/2; DFG INST95/980-1 FUGG, INST411/37-1 FUGG), the DFG Cluster of Excellence: Munich-Centre for Advanced Photonics (MAP), the Bundesministerium für Forschung und Technologie (BMBF Innovative Therapies, 01GU0823; Kompetenzverbund Strahlenforschung 02NUK038A), EU-CELLEUROPE (315963) and the German Cancer Consortium Radiation Oncology Group, Munich (DKTK-ROG).

The John van Geest Cancer Research Centre is supported by funding from the John and Lucille van Geest Foundation, Nottingham Trent University, the European Regional Development Fund (ERDF) via the Healthcare and Bioscience iNET, as delivered by Medilink East Midlands, UK and the Headcase Cancer Trust.

The University of Portsmouth Brain Tumour Research Centre is core funded by Brain Tumour Research.

References

  1. 1.
    Burton EC, Prados MD (2000) Malignant gliomas. Curr Treat Options in Oncol 1(5):459–468CrossRefGoogle Scholar
  2. 2.
    Nieder C, Grosu AL, Molls M (2000) A comparison of treatment results for recurrent malignant gliomas. Cancer Treat Rev 26(6):397–409CrossRefPubMedGoogle Scholar
  3. 3.
    Dazzi C, Cariello A, Giannini M, Del Duca M, Giovanis P, Fiorentini G, Leoni M, Rosti G, Turci D, Tienghi A, Vertogen B, Zumaglini F, De Giorgi U, Marangolo M (2000) A sequential chemo-radiotherapeutic treatment for patients with malignant gliomas: a phase II pilot study. Anticancer Res 20(1B):515–518PubMedGoogle Scholar
  4. 4.
    Barrié M, Couprie C, Dufour H, Figarella-Branger D, Muracciole X, Hoang-Xuan K, Braguer D, Martin PM, Peragut JC, Grisoli F, Chinot O (2005) Temozolomide in combination with BCNU before and after radiotherapy in patients with inoperable newly diagnosed glioblastoma multiforme. Ann Oncol: Off J Eur Soc Med Oncol/ESMO 16(7):1177–1184CrossRefGoogle Scholar
  5. 5.
    Combs SE, Gutwein S, Thilmann C, Debus J, Schulz-Ertner D (2005) Reirradiation of recurrent WHO grade III astrocytomas using fractionated stereotactic radiotherapy (FSRT). Strahlentherapie und Onkologie: Organ der Deutschen Rontgengesellschaft [et al] 181(12):768–773CrossRefGoogle Scholar
  6. 6.
    Combs SE, Thilmann C, Edler L, Debus J, Schulz-Ertner D (2005) Efficacy of fractionated stereotactic reirradiation in recurrent gliomas: long-term results in 172 patients treated in a single institution. J Clin Oncol: Off J Am Soc Clin Oncol 23(34):8863–8869CrossRefGoogle Scholar
  7. 7.
    Combs SE, Wagner J, Bischof M, Welzel T, Wagner F, Debus J, Schulz-Ertner D (2008) Postoperative treatment of primary glioblastoma multiforme with radiation and concomitant temozolomide in elderly patients. Int J Radiat Oncol Biol Phys 70(4):987–992CrossRefPubMedGoogle Scholar
  8. 8.
    Combs SE, Wagner J, Bischof M, Welzel T, Edler L, Rausch R, Wagner F, Zabel-du Bois A, Debus J, Schulz-Ertner D (2008) Radiochemotherapy in patients with primary glioblastoma comparing two temozolomide dose regimens. Int J Radiat Oncol Biol Phys 71(4):999–1005CrossRefPubMedGoogle Scholar
  9. 9.
    Frenay M, Lebrun C, Lonjon M, Bondiau PY, Chatel M (2000) Up-front chemotherapy with fotemustine (F)/cisplatin (CDDP)/etoposide (VP16) regimen in the treatment of 33 non-removable glioblastomas. Eur J Cancer 36(8):1026–1031CrossRefPubMedGoogle Scholar
  10. 10.
    Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO, European Organisation for R, Treatment of Cancer Brain T, Radiotherapy G, National Cancer Institute of Canada Clinical Trials G (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352(10):987–996CrossRefPubMedGoogle Scholar
  11. 11.
    Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K, Hau P, Brandes AA, Gijtenbeek J, Marosi C, Vecht CJ, Mokhtari K, Wesseling P, Villa S, Eisenhauer E, Gorlia T, Weller M, Lacombe D, Cairncross JG, Mirimanoff RO (2009) Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 10(5):459–466CrossRefPubMedGoogle Scholar
  12. 12.
    Multhoff G, Botzler C, Wiesnet M, Eissner G, Issels R (1995) CD3 large granular lymphocytes recognize a heat-inducible immunogenic determinant associated with the 72-kD heat shock protein on human sarcoma cells. Blood 86:1374–1382PubMedGoogle Scholar
  13. 13.
    Multhoff G, Botzler C, Wiesnet M, Muller E, Meier T, Wilmanns W, Issels RD (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 61:272–279CrossRefPubMedGoogle Scholar
  14. 14.
    Stangl S, Gehrmann M, Riegger J, Kuhs K, Riederer I, Sievert W, Hube K, Mocikat R, Dressel R, Kremmer E, Pockley AG, Friedrich L, Vigh L, Skerra A, Multhoff G (2011) Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody. Proc Natl Acad Sci U S A 108(2):733–738CrossRefPubMedGoogle Scholar
  15. 15.
    Gehrmann M, Stangl S, Foulds GA, Oellinger R, Breuninger S, Rad R, Pockley AG, Multhoff G (2014) Tumor imaging and targeting potential of an hsp70-derived 14-mer peptide. PLoS One 9(8):e105344CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Gehrmann M, Pfister K, Hutzler P, Gastpar R, Margulis B, Multhoff G (2002) Effects of antineoplastic agents on cytoplasmic and membrane-bound heat shock protein 70 (Hsp70) levels. Biol Chem 383:1715–1725PubMedGoogle Scholar
  17. 17.
    Gehrmann M, Stangl S, Kirschner A, Foulds GA, Sievert W, Doss BT, Walch A, Pockley AG, Multhoff G (2012) Immunotherapeutic targeting of membrane hsp70-expressing tumors using recombinant human granzyme B. PLoS One 7(7):e41341CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Pfister K, Radons J, Busch R, Tidball JG, Pfeifer M, Freitag L, Feldmann HJ, Milani V, Issels R, Multhoff G (2007) Patient survival by Hsp70 membrane phenotype: association with different routes of metastasis. Cancer 110:926–935CrossRefPubMedGoogle Scholar
  19. 19.
    Multhoff G, Botzler C, Jennen L, Schmidt J, Ellwart J, Issels R (1997) Heat shock protein 72 on tumor cells: a recognition structure for natural killer cells. J Immunol 158:4341–4350PubMedGoogle Scholar
  20. 20.
    Multhoff G, Hightower LE (2011) Distinguishing integral and receptor-bound heat shock protein 70 (Hsp70) on the cell surface by Hsp70-specific antibodies. Cell Stress Chaperones 16(3):251–255CrossRefPubMedGoogle Scholar
  21. 21.
    Multhoff G, Pfister K, Gehrmann M, Hantschel M, Gross C, Hafner M, Hiddemann W (2001) A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity. Cell Stress Chaperones 6(4):337–344CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Multhoff G, Pfister K, Botzler C, Jordan A, Scholz R, Schmetzer H, Burgstahler R, Hiddemann W (2000) Adoptive transfer of human natural killer cells in mice with severe combined immunodeficiency inhibits growth of Hsp70-expressing tumors. Int J Cancer 88:791–797CrossRefPubMedGoogle Scholar
  23. 23.
    Gross C, Hansch D, Gastpar R, Multhoff G (2003) Interaction of heat shock protein 70 peptide with NK cells involves the NK receptor CD94. Biol Chem 384:267–279CrossRefPubMedGoogle Scholar
  24. 24.
    Gross C, Holler E, Stangl S, Dickinson A, Pockley AG, Asea AA, Mallappa N, Multhoff G (2008) An Hsp70 peptide initiates NK cell killing of leukemic blasts after stem cell transplantation. Leuk Res 32(4):527–534CrossRefPubMedGoogle Scholar
  25. 25.
    Gross C, Koelch W, DeMaio A, Arispe N, Multhoff G (2003) Cell surface-bound heat shock protein 70 (Hsp70) mediates perforin-independent apoptosis by specific binding and uptake of granzyme B. J Biol Chem 278(42):41173–41181CrossRefPubMedGoogle Scholar
  26. 26.
    Krause SW, Gastpar R, Andreesen R, Gross C, Ullrich H, Thonigs G, Pfister K, Multhoff G (2004) Treatment of colon and lung cancer patients with ex vivo heat shock protein 70-peptide-activated, autologous natural killer cells: a clinical phase I trial. Clin Cancer Res 10:3699–3707CrossRefPubMedGoogle Scholar
  27. 27.
    Specht HM, Ahrens N, Blankenstein C, Duell T, Fietkau R, Gaipl US, Gunther C, Gunther S, Habl G, Hautmann H, Hautmann M, Huber RM, Molls M, Offner R, Rodel C, Rodel F, Schutz M, Combs SE, Multhoff G (2015) Heat shock protein 70 (Hsp70) peptide activated natural killer (NK) cells for the treatment of patients with non-small cell lung cancer (NSCLC) after Radiochemotherapy (RCTx) - from preclinical studies to a clinical phase II trial. Front Immunol 6:162CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Gehrmann M, Doss BT, Wagner M, Zettlitz KA, Kontermann RE, Foulds G, Pockley AG, Multhoff G (2011) A novel expression and purification system for the production of enzymatic and biologically active human granzyme B. J Immunol Methods 371(1–2):8–17CrossRefPubMedGoogle Scholar
  29. 29.
    Milani V, Stangl S, Issels R, Gehrmann M, Wagner B, Hube K, Mayr D, Hiddemann W, Molls M, Multhoff G (2009) Anti-tumor activity of patient-derived NK cells after cell-based immunotherapy--a case report. J Transl Med 7:50CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Stangl S, Gehrmann M, Dressel R, Alves F, Dullin C, Themelis G, Ntziachristos V, Staeblein E, Walch A, Winkelmann I, Multhoff G (2010) In vivo imaging of CT26 mouse tumors by using cmHsp70.1 monoclonal antibody. J Cell Mol Med 15(4):874–887CrossRefPubMedCentralGoogle Scholar
  31. 31.
    Stangl S, Varga J, Freysoldt B, Trajkovic-Arsic M, Siveke JT, Greten FR, Ntziachristos V, Multhoff G (2014) Selective in vivo imaging of syngeneic, spontaneous, and xenograft tumors using a novel tumor cell-specific hsp70 peptide-based probe. Cancer Res 74(23):6903–6912CrossRefPubMedGoogle Scholar
  32. 32.
    Gehrmann MK, Kimm MA, Stangl S, Schmid TE, Noel PB, Rummeny EJ, Multhoff G (2015) Imaging of Hsp70-positive tumors with cmHsp70.1 antibody-conjugated gold nanoparticles. Int J Nanomedicine 10:5687–5700CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Zhang H, Liu R, Huang W (2007) A 14-mer peptide from HSP70 protein is the critical epitope which enhances NK activity against tumor cells in vivo. Immunol Investig 36(3):233–246CrossRefGoogle Scholar
  34. 34.
    Fouchaq B, Benaroudj N, Ebel C, Ladjimi MM (1999) Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70. Eur J Biochem 259(1–2):379–384CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Stefan Stangl
    • 1
  • Gemma A. Foulds
    • 2
  • Helena Fellinger
    • 1
  • Geoffrey J. Pilkington
    • 3
  • A. Graham Pockley
    • 1
    • 2
  • Gabriele Multhoff
    • 1
    • 4
    Email author
  1. 1.Department of Radiation Oncology, Klinikum Rechts der IsarTechnische Universität MünchenMunichGermany
  2. 2.John van Geest Cancer Research Centre, School of Science and TechnologyNottingham Trent UniversityNottinghamUK
  3. 3.Brain Tumour Research Centre, Institute of Biomedical and Biomolecular Sciences, School of Pharmacy and Biomedical SciencesUniversity of PortsmouthPortsmouthUK
  4. 4.CCG-Innate Immunity in Tumor BiologyHelmholtz Zentrum MünchenNeuherbergGermany

Personalised recommendations