Journal of Neuro-Oncology

, Volume 85, Issue 3, pp 231–240 | Cite as

Nonspecific immunotherapy with intratumoral lipopolysaccharide and zymosan A but not GM-CSF leads to an effective anti-tumor response in subcutaneous RG-2 gliomas

  • Christopher L. Mariani
  • Didier Rajon
  • Francis J. Bova
  • Wolfgang J. Streit
Lab Investigation-Human/Animal Tissue



Nonspecific stimulation of cells of the immune system may be useful in generating an anti-tumor response for a variety of cancers and may work synergistically with currently available cytotoxic therapies. In this study we examined the response of syngeneic rat gliomas to treatment with several nonspecific stimulators of dendritic cells and macrophages alone or in combination with radiation therapy.

Experimental design

RG-2 gliomas were implanted subcutaneously and treated with intratumoral (IT) injections of the toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and zymosan A (ZymA) and the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF). Combination treatment with IT LPS and single-fraction external beam radiotherapy (EBRT) was also evaluated.


Treatment with IT LPS and ZymA delayed tumor growth compared to saline controls. Multiple doses of both substances were superior to single doses, and led to complete tumor regression in 71% (LPS) and 50% (ZymA) of animals. GM-CSF showed no anti-tumor effects in this study. Combinations of IT LPS and EBRT appeared to have a synergistic effect in delaying tumor growth. Rechallenge studies and IT LPS treatment of RG-2 tumors in nude rats suggested the importance of T cells in this treatment paradigm.


Direct IT treatment with the TLR ligands LPS and ZymA are effective in generating an anti-tumor response. These treatments may synergize with cytotoxic therapies such as EBRT, and appear to require T cells for a successful outcome.


Glioblastoma multiforme Rat Brain tumor Radiation therapy Toll-like receptor Denditic cells Macrophages 


  1. 1.
    Akman F, Cooper RA, Sen M, Tanriver Y, Kentli S (2002) Validation of the Medical Research Council and a newly developed prognostic index in patients with malignant glioma: how useful are prognostic indices in routine clinical practice? J Neurooncol 59:39–47PubMedCrossRefGoogle Scholar
  2. 2.
    Basso U, Ermani M, Vastola F, Brandes AA (2002) Non-cytotoxic therapies for malignant gliomas. J Neurooncol 58:57–69PubMedCrossRefGoogle Scholar
  3. 3.
    Nieder C, Grosu AL, Molls M (2000) A comparison of treatment results for recurrent malignant gliomas. Cancer Treat Rev 26:397–409PubMedCrossRefGoogle Scholar
  4. 4.
    Badie B, Schartner JM (2000) Flow cytometric characterization of tumor-associated macrophages in experimental gliomas. Neurosurgery 46:957–961; discussion 961–952PubMedCrossRefGoogle Scholar
  5. 5.
    Graeber MB, Scheithauer BW, Kreutzberg GW (2002) Microglia in brain tumors. Glia 40:252–259PubMedCrossRefGoogle Scholar
  6. 6.
    Kielian T, van Rooijen N, Hickey WF (2002) MCP-1 expression in CNS-1 astrocytoma cells: implications for macrophage infiltration into tumors in vivo. J Neurooncol 56:1–12PubMedCrossRefGoogle Scholar
  7. 7.
    Morioka T, Baba T, Black KL, Streit WJ (1992) Immunophenotypic analysis of infiltrating leukocytes and microglia in an experimental rat glioma. Acta Neuropathol 83:590–597PubMedCrossRefGoogle Scholar
  8. 8.
    Rossi ML, Hughes JT, Esiri MM, Coakham HB, Brownell DB (1987) Immunohistological study of mononuclear cell infiltrate in malignant gliomas. Acta Neuropathol (Berl) 74:269–277CrossRefGoogle Scholar
  9. 9.
    Huettner C, Paulus W, Roggendorf W (1995) Messenger RNA expression of the immunosuppressive cytokine IL-10 in human gliomas. Am J Pathol 146:317–322PubMedGoogle Scholar
  10. 10.
    Merlo A, Juretic A, Zuber M, Filgueira L, Luscher U, Caetano V, Ulrich J, Gratzl O, Heberer M, Spagnoli GC (1993) Cytokine gene expression in primary brain tumours, metastases and meningiomas suggests specific transcription patterns. Eur J Cancer 29A:2118–2125PubMedCrossRefGoogle Scholar
  11. 11.
    Parney IF, Hao C, Petruk KC (2000) Glioma immunology and immunotherapy. Neurosurgery 46:778–791; discussion 791–772PubMedCrossRefGoogle Scholar
  12. 12.
    Schneider J, Hofman FM, Apuzzo ML, Hinton DR (1992) Cytokines and immunoregulatory molecules in malignant glial neoplasms. J Neurosurg 77:265–273PubMedGoogle Scholar
  13. 13.
    Weller M, Fontana A (1995) The failure of current immunotherapy for malignant glioma. Tumor-derived TGF-beta, T-cell apoptosis, and the immune privilege of the brain. Brain Res Brain Res Rev 21:128–151PubMedCrossRefGoogle Scholar
  14. 14.
    Wischhusen J, Jung G, Radovanovic I, Beier C, Steinbach JP, Rimner A, Huang H, Schulz JB, Ohgaki H, Aguzzi A, Rammensee HG, Weller M (2002) Identification of CD70-mediated apoptosis of immune effector cells as a novel immune escape pathway of human glioblastoma. Cancer Res 62:2592–2599PubMedGoogle Scholar
  15. 15.
    Giulian D, Baker TJ, Shih LC, Lachman LB (1986) Interleukin 1 of the central nervous system is produced by ameboid microglia. J Exp Med 164:594–604PubMedCrossRefGoogle Scholar
  16. 16.
    Sawada M, Kondo N, Suzumura A, Marunouchi T (1989) Production of tumor necrosis factor-alpha by microglia and astrocytes in culture. Brain Res 491:394–397PubMedCrossRefGoogle Scholar
  17. 17.
    Dobrovolskaia MA, Vogel SN (2002) Toll receptors, CD14, and macrophage activation and deactivation by LPS. Microbes Infect 4:903–914PubMedCrossRefGoogle Scholar
  18. 18.
    Williams MA, Kelsey SM, Newland AC (1999) GM-CSF and stimulation of monocyte/macrophage function in vivo relevance and in vitro observations. Eur J Cancer 35(Suppl 3):S18–S22PubMedCrossRefGoogle Scholar
  19. 19.
    Young SH, Ye J, Frazer DG, Shi X, Castranova V (2001) Molecular mechanism of tumor necrosis factor-alpha production in 1→3-beta-glucan (zymosan)-activated macrophages. J Biol Chem 276:20781–20787PubMedCrossRefGoogle Scholar
  20. 20.
    Miura T, Ohno N, Miura NN, Adachi Y, Shimada S, Yadomae T (1999) Antigen-specific response of murine immune system toward a yeast beta-glucan preparation, zymosan. FEMS Immunol Med Microbiol 24:131–139PubMedGoogle Scholar
  21. 21.
    Murata J, Ricciardi-Castagnoli P, Dessous L’Eglise Mange P, Martin F, Juillerat-Jeanneret L (1997) Microglial cells induce cytotoxic effects toward colon carcinoma cells: measurement of tumor cytotoxicity with a gamma-glutamyl transpeptidase assay. Int J Cancer 70:169–174PubMedCrossRefGoogle Scholar
  22. 22.
    Beutler B, Rietschel ET (2003) Innate immune sensing and its roots: the story of endotoxin. Nat Rev Immunol 3:169–176PubMedCrossRefGoogle Scholar
  23. 23.
    Berendt MJ, North RJ, Kirstein DP (1978) The immunological basis of endotoxin-induced tumor regression. Requirement for T-cell-mediated immunity. J Exp Med 148:1550–1559PubMedCrossRefGoogle Scholar
  24. 24.
    Goto S, Sakai S, Kera J, Suma Y, Soma GI, Takeuchi S (1996) Intradermal administration of lipopolysaccharide in treatment of human cancer. Cancer Immunol Immunother 42:255–261PubMedCrossRefGoogle Scholar
  25. 25.
    Otto F, Schmid P, Mackensen A, Wehr U, Seiz A, Braun M, Galanos C, Mertelsmann R, Engelhardt R (1996) Phase II trial of intravenous endotoxin in patients with colorectal and non-small cell lung cancer. Eur J Cancer 32A:1712–1718PubMedCrossRefGoogle Scholar
  26. 26.
    Shear MJ, Turner FC (1943) Chemical treatment of tumors. V. Isolation of the hemorrhage-producing fraction from Serratia marcescens (Bacillus prodigiosus) culture filtrate. J Natl Cancer Inst 4:81–97Google Scholar
  27. 27.
    Engelhardt R, Mackensen A, Galanos C, Andreesen R (1990) Biological response to intravenously administered endotoxin in patients with advanced cancer. J Biol Response Mod 9:480–491PubMedGoogle Scholar
  28. 28.
    Chicoine MR, Won EK, Zahner MC (2001) Intratumoral injection of lipopolysaccharide causes regression of subcutaneously implanted mouse glioblastoma multiforme. Neurosurgery 48:607–614; discussion 614–605PubMedCrossRefGoogle Scholar
  29. 29.
    Won EK, Zahner MC, Grant EA, Gore P, Chicoine MR (2003) Analysis of the antitumoral mechanisms of lipopolysaccharide against glioblastoma multiforme. Anticancer Drugs 14:457–466PubMedCrossRefGoogle Scholar
  30. 30.
    Akira S, Takeda K (2004) Toll-like receptor signalling. Nat Rev Immunol 4:499–511PubMedCrossRefGoogle Scholar
  31. 31.
    Pistoia V (1991) Granulocyte-macrophage colony stimulating factor (GM-CSF); sources, targets and mechanism of action. Leukemia 5(Suppl 1):114–118PubMedGoogle Scholar
  32. 32.
    Wallenfriedman MA, Conrad JA, DelaBarre L, Graupman PC, Lee G, Garwood M, Gregerson DS, Jean WC, Hall WA, Low WC (1999) Effects of continuous localized infusion of granulocyte-macrophage colony-stimulating factor and inoculations of irradiated glioma cells on tumor regression. J Neurosurg 90:1064–1071PubMedCrossRefGoogle Scholar
  33. 33.
    Dranoff G (2002) GM-CSF-based cancer vaccines. Immunol Rev 188:147–154PubMedCrossRefGoogle Scholar
  34. 34.
    Dranoff G (2003) GM-CSF-secreting melanoma vaccines. Oncogene 22:3188–3192PubMedCrossRefGoogle Scholar
  35. 35.
    Reinisch W, Holub M, Katz A, Herneth A, Lichtenberger C, Schoniger-Hekele M, Waldhoer T, Oberhuber G, Ferenci P, Gangl A, Mueller C (2002) Prospective pilot study of recombinant granulocyte-macrophage colony-stimulating factor and interferon-gamma in patients with inoperable hepatocellular carcinoma. J Immunother 25:489–499PubMedCrossRefGoogle Scholar
  36. 36.
    Salgia R, Lynch T, Skarin A, Lucca J, Lynch C, Jung K, Hodi FS, Jaklitsch M, Mentzer S, Swanson S, Lukanich J, Bueno R, Wain J, Mathisen D, Wright C, Fidias P, Donahue D, Clift S, Hardy S, Neuberg D, Mulligan R, Webb I, Sugarbaker D, Mihm M, Dranoff G (2003) Vaccination with irradiated autologous tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor augments antitumor immunity in some patients with metastatic non-small-cell lung carcinoma. J Clin Oncol 21:624–630PubMedCrossRefGoogle Scholar
  37. 37.
    Verra N, Jansen R, Groenewegen G, Mallo H, Kersten MJ, Bex A, Vyth-Dreese FA, Sein J, van de Kasteele W, Nooijen WJ, de Waal M, Horenblas S, de Gast GC (2003) Immunotherapy with concurrent subcutaneous GM-CSF, low-dose IL-2 and IFN-alpha in patients with progressive metastatic renal cell carcinoma. Br J Cancer 88:1346–1351PubMedCrossRefGoogle Scholar
  38. 38.
    Barth RF (1998) Rat brain tumor models in experimental neuro-oncology: the 9L, C6, T9, F98, RG2 (D74), RT-2 and CNS-1 gliomas. J Neurooncol 36:91–102PubMedCrossRefGoogle Scholar
  39. 39.
    Hoeller C, Jansen B, Heere-Ress E, Pustelnik T, Mossbacher U, Schlagbauer-Wadl H, Wolff K, Pehamberger H (2001) Perilesional injection of r-GM-CSF in patients with cutaneous melanoma metastases. J Invest Dermatol 117:371–374PubMedCrossRefGoogle Scholar
  40. 40.
    Pasare C, Medzhitov R (2003) Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 299:1033–1036PubMedCrossRefGoogle Scholar
  41. 41.
    Matzinger P (2002) The danger model: a renewed sense of self. Science 296:301–305PubMedCrossRefGoogle Scholar
  42. 42.
    Zeisberger E, Roth J (1998) Tolerance to pyrogens. Ann N Y Acad Sci 856:116–131PubMedCrossRefGoogle Scholar
  43. 43.
    Yang Y, Huang CT, Huang X, Pardoll DM (2004) Persistent Toll-like receptor signals are required for reversal of regulatory T cell-mediated CD8 tolerance. Nat Immunol 5:508–515PubMedCrossRefGoogle Scholar
  44. 44.
    Jiang H, Stewart CA, Fast DJ, Leu RW (1992) Tumor target-derived soluble factor synergizes with IFN-gamma and IL-2 to activate macrophages for tumor necrosis factor and nitric oxide production to mediate cytotoxicity of the same target. J Immunol 149:2137–2146PubMedGoogle Scholar
  45. 45.
    Berendt MJ, North RJ (1980) T-cell-mediated suppression of anti-tumor immunity. An explanation for progressive growth of an immunogenic tumor. J Exp Med 151:69–80PubMedCrossRefGoogle Scholar
  46. 46.
    Mason KA, Ariga H, Neal R, Valdecanas D, Hunter N, Krieg AM, Whisnant JK, Milas L (2005) Targeting toll-like receptor 9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy. Clin Cancer Res 11:361–369PubMedGoogle Scholar
  47. 47.
    Milas L, Mason KA, Ariga H, Hunter N, Neal R, Valdecanas D, Krieg AM, Whisnant JK (2004) CpG oligodeoxynucleotide enhances tumor response to radiation. Cancer Res 64:5074–5077PubMedCrossRefGoogle Scholar
  48. 48.
    Lake RA, Robinson BW (2005) Immunotherapy and chemotherapy—a practical partnership. Nat Rev Cancer 5:397–405PubMedCrossRefGoogle Scholar
  49. 49.
    Ford AL, Goodsall AL, Hickey WF, Sedgwick JD (1995) Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared. J Immunol 154:4309–4321PubMedGoogle Scholar
  50. 50.
    Carpentier AF, Xie J, Mokhtari K, Delattre JY (2000) Successful treatment of intracranial gliomas in rat by oligodeoxynucleotides containing CpG motifs. Clin Cancer Res 6:2469–2473PubMedGoogle Scholar
  51. 51.
    Bowles AP Jr, Perkins E (1999) Long-term remission of malignant brain tumors after intracranial infection: a report of four cases. Neurosurgery 44:636–642; discussion 642–633PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Christopher L. Mariani
    • 1
    • 3
  • Didier Rajon
    • 2
  • Francis J. Bova
    • 2
  • Wolfgang J. Streit
    • 1
  1. 1.Department of Neuroscience, McKnight Brain Institute, College of MedicineUniversity of FloridaGainesvilleUSA
  2. 2.Department of Neurosurgery, McKnight Brain Institute, College of MedicineUniversity of FloridaGainesvilleUSA
  3. 3.Department of Clinical Sciences, College of Veterinary MedicineNorth Carolina State UniversityRaleighUSA

Personalised recommendations