Cancer Immunology, Immunotherapy

, Volume 64, Issue 3, pp 299–309 | Cite as

Glioblastoma exosomes and IGF-1R/AS-ODN are immunogenic stimuli in a translational research immunotherapy paradigm

  • Larry A. Harshyne
  • Kirsten M. Hooper
  • Edward G. Andrews
  • Brian J. Nasca
  • Lawrence C. Kenyon
  • David W. Andrews
  • D. Craig Hooper
Original Article


Glioblastomas are primary intracranial tumors for which there is no cure. Patients receiving standard of care, chemotherapy and irradiation, survive approximately 15 months prompting studies of alternative therapies including vaccination. In a pilot study, a vaccine consisting of Lucite diffusion chambers containing irradiated autologous tumor cells pre-treated with an antisense oligodeoxynucleotide (AS-ODN) directed against the insulin-like growth factor type 1 receptor was found to elicit positive clinical responses in 8/12 patients when implanted in the rectus sheath for 24 h. Our preliminary observations supported an immune response, and we have since reopened a second Phase 1 trial to assess this possibility among other exploratory objectives. The current study makes use of a murine glioma model and samples from glioblastoma patients in this second Phase 1 trial to investigate this novel therapeutic intervention more thoroughly. Implantation of the chamber-based vaccine protected mice from tumor challenge, and we posit this occurred through the release of immunostimulatory AS-ODN and antigen-bearing exosomes. Exosomes secreted by glioblastoma cultures are immunogenic, eliciting and binding antibodies present in the sera of immunized mice. Similarly, exosomes released by human glioblastoma cells bear antigens recognized by the sera of 6/12 patients with recurrent glioblastomas. These results suggest that the release of AS-ODN together with selective release of exosomes from glioblastoma cells implanted in chambers may drive the therapeutic effect seen in the pilot vaccine trial.


Antisense Exosome Antibody Diffusion chamber GL261 Glioblastoma 



Antisense oligodeoxynucleotide


Basic fibroblast growth factor


3,3′-dioctadecyloxacarbocyanine perchlorate


Draining lymph nodes


Epidermal growth factor


Fetal bovine serum




Granulocyte macrophage colony stimulating factor


Insulin-like growth factor type 1 receptor PBS




Median fluorescence intensity


Myeloid dendritic cell


Peripheral blood mononuclear cells


Phosphate buffered saline


Plasmacytoid dendritic cell


T helper type 2


  1. 1.
    Andrews DW, Resnicoff M, Flanders AE et al (2001) Results of a pilot study involving the use of an antisense oligodeoxynucleotide directed against the insulin-like growth factor type I receptor in malignant astrocytomas. J Clin Oncol 19:2189–2200PubMedGoogle Scholar
  2. 2.
    Baserga R, Reiss K, Alder H, Pietrzkowski Z, Surmacz E (1992) Inhibition of cell cycle progression by antisense oligodeoxynucleotides. Ann N Y Acad Sci 660:64–69CrossRefPubMedGoogle Scholar
  3. 3.
    Hernandez-Sanchez C, Blakesley V, Kalebic T, Helman L, LeRoith D (1995) The role of the tyrosine kinase domain of the insulin-like growth factor-I receptor in intracellular signaling, cellular proliferation, and tumorigenesis. J Biol Chem 270:29176–29181CrossRefPubMedGoogle Scholar
  4. 4.
    Resnicoff M, Abraham D, Yutanawiboonchai W et al (1995) The insulin-like growth factor I receptor protects tumor cells from apoptosis in vivo. Cancer Res 55:2463–2469PubMedGoogle Scholar
  5. 5.
    Bauer S, Kirschning CJ, Hacker H et al (2001) Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci U S A 98:9237–9242CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Iho S, Yamamoto T, Takahashi T, Yamamoto S (1999) Oligodeoxynucleotides containing palindrome sequences with internal 5′-CpG-3′ act directly on human NK and activated T cells to induce IFN-gamma production in vitro. J Immunol 163:3642–3652PubMedGoogle Scholar
  7. 7.
    Kobayashi N, Hong C, Klinman DM, Shirota H (2013) Oligodeoxynucleotides expressing polyguanosine motifs promote antitumor activity through the upregulation of IL-2. J Immunol 190:1882–1889CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Krieg AM, Yi AK, Matson S et al (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374:546–549CrossRefPubMedGoogle Scholar
  9. 9.
    Pasare C, Medzhitov R (2003) Toll pathway-dependent blockade of CD4+ CD25+ T cell-mediated suppression by dendritic cells. Science 299:1033–1036CrossRefPubMedGoogle Scholar
  10. 10.
    Stein CA, Subasinghe C, Shinozuka K, Cohen JS (1988) Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res 16:3209–3221CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Théry C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569–579PubMedGoogle Scholar
  12. 12.
    Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C (1987) Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem 262:9412–9420PubMedGoogle Scholar
  13. 13.
    Théry C, Boussac M, Véron P et al (2001) Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol 166:7309–7318CrossRefPubMedGoogle Scholar
  14. 14.
    Raposo G, Nijman HW, Stoorvogel W et al (1996) B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183:1161–1172CrossRefPubMedGoogle Scholar
  15. 15.
    Wolfers J, Lozier A, Raposo G et al (2001) Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med 7:297–303CrossRefPubMedGoogle Scholar
  16. 16.
    Clayton A, Court J, Navabi H et al (2001) Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods 247:163–174CrossRefPubMedGoogle Scholar
  17. 17.
    Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C (2005) Exosomal-like vesicles are present in human blood plasma. Int Immunol 17:879–887CrossRefPubMedGoogle Scholar
  18. 18.
    Graner MW, Alzate O, Dechkovskaia AM et al (2009) Proteomic and immunologic analyses of brain tumor exosomes. FASEB J 23:1541–1557CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G (2002) The biogenesis and functions of exosomes. Traffic 3:321–330CrossRefPubMedGoogle Scholar
  20. 20.
    Théry C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. Chapter 3: Unit 3.22. doi: 10.1002/0471143030.cb0322s30
  21. 21.
    Faure J, Lachenal G, Court M et al (2006) Exosomes are released by cultured cortical neurones. Mol Cell Neurosci 31:642–648CrossRefPubMedGoogle Scholar
  22. 22.
    Kramer-Albers EM, Bretz N, Tenzer S, Winterstein C (2007) Oligodendrocytes secrete exosomes containing major myelin and stress-protective proteins: trophic support for axons? Proteomics Clin Appl 1:1446–1461CrossRefPubMedGoogle Scholar
  23. 23.
    Potolicchio I, Carven GJ, Xu X et al (2005) Proteomic analysis of microglia-derived exosomes: metabolic role of the aminopeptidase CD13 in neuropeptide catabolism. J Immunol 175:2237–2243CrossRefPubMedGoogle Scholar
  24. 24.
    Zitvogel L, Regnault A, Lozier A et al (1998) Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes. Nat Med 4:594–600CrossRefPubMedGoogle Scholar
  25. 25.
    Février B, Raposo G (2004) Exosomes: endosomal-derived vesicles shipping extracellular messages. Curr Opin Cell Biol 16:415–421CrossRefPubMedGoogle Scholar
  26. 26.
    Al-Nedawi K, Meehan B, Micallef J et al (2008) Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol 10:619–624CrossRefPubMedGoogle Scholar
  27. 27.
    Skog J, Wurdinger T, van Rijn S et al (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 10:1470–1476CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Wieckowski E, Whiteside TL (2006) Human tumor-derived vs dendritic cell-derived exosomes have distinct biologic roles and molecular profiles. Immunol Res 36:247–254CrossRefPubMedGoogle Scholar
  29. 29.
    Akira S, Takeda K (2004) Toll-like receptor signalling. Nat Rev Immunol 4:499–511CrossRefPubMedGoogle Scholar
  30. 30.
    Andre F, Schartz NE, Movassagh M et al (2002) Malignant effusions and immunogenic tumour-derived exosomes. Lancet 360:295–305CrossRefPubMedGoogle Scholar
  31. 31.
    Chaput N, Schartz NE, André F et al (2004) Exosomes as potent cell-free peptide-based vaccine. II. Exosomes in CpG adjuvants efficiently prime naive Tc1 lymphocytes leading to tumor rejection. J Immunol 172:2137–2146CrossRefPubMedGoogle Scholar
  32. 32.
    Creagh EM, O’Neill LA (2006) TLRs, NLRs and RLRs: a trinity of pathogen sensors that co-operate in innate immunity. Trends Immunol 27:352–357CrossRefPubMedGoogle Scholar
  33. 33.
    Mignot G, Roux S, Thery C, Ségura E, Zitvogel L (2006) Prospects for exosomes in immunotherapy of cancer. J Cell Mol Med 10:376–388CrossRefPubMedGoogle Scholar
  34. 34.
    Morelli AE (2006) The immune regulatory effect of apoptotic cells and exosomes on dendritic cells: its impact on transplantation. Am J Transplant 6:254–261CrossRefPubMedGoogle Scholar
  35. 35.
    Peche H, Renaudin K, Beriou G, Merieau E, Amigorena S, Cuturi MC (2006) Induction of tolerance by exosomes and short-term immunosuppression in a fully MHC-mismatched rat cardiac allograft model. Am J Transpl 6:1541–1550CrossRefGoogle Scholar
  36. 36.
    Tang J, Flomenberg P, Harshyne L, Kenyon L, Andrews DW (2005) Glioblastoma patients exhibit circulating tumor-specific CD8+ T cells. Clin Cancer Res 11:5292–5299CrossRefPubMedGoogle Scholar
  37. 37.
    Abraham D, Rotman HL, Haberstroh HF et al (1995) Strongyloides stercoralis: protective immunity to third-stage larvae inBALB/cByJ mice. Exp Parasitol 80:297–307CrossRefPubMedGoogle Scholar
  38. 38.
    Tavernier J, Tuypens T, Verhee A et al (1995) Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist. Proc Natl Acad Sci USA 92:5194–5198CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Lee GR, Fields PE, Griffin TJ, Flavell RA (2003) Regulation of the Th2 cytokine locus by a locus control region. Immunity 19:145–153CrossRefPubMedGoogle Scholar
  40. 40.
    Rolink AG, Thalmann P, Kikuchi Y, Erdei A (1990) Characterization of the interleukin 5-reactive splenic B cell population. Eur J Immunol 20:1949–1956CrossRefPubMedGoogle Scholar
  41. 41.
    McHeyzer-Williams MG (1989) Combinations of interleukins 2, 4 and 5 regulate the secretion of murine immunoglobulin isotypes. Eur J Immunol 19:2025–2030CrossRefPubMedGoogle Scholar
  42. 42.
    Valenti R, Huber V, Iero M, Filipazzi P, Parmiani G, Rivoltini L (2007) Tumor-released microvesicles as vehicles of immunosuppression. Cancer Res 67:2912–2915CrossRefPubMedGoogle Scholar
  43. 43.
    Murphy KA, Erickson JR, Johnson CS et al (2014) CD8+ T cell-independent tumor regression induced by Fc-OX40L and therapeutic vaccination in a mouse model of glioma. J Immunol 192:224–233CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Larry A. Harshyne
    • 1
  • Kirsten M. Hooper
    • 1
  • Edward G. Andrews
    • 1
  • Brian J. Nasca
    • 1
  • Lawrence C. Kenyon
    • 2
  • David W. Andrews
    • 1
  • D. Craig Hooper
    • 1
    • 3
  1. 1.Department of Neurological SurgeryThomas Jefferson UniversityPhiladelphiaUSA
  2. 2.Department of PathologyThomas Jefferson UniversityPhiladelphiaUSA
  3. 3.Department of Cancer BiologyThomas Jefferson UniversityPhiladelphiaUSA

Personalised recommendations