Skip to main content
SpringerLink
Log in

Analysis of Three Properties of Newcastle Disease Virus for Fighting Cancer: Tumor-Selective Replication, Antitumor Cytotoxicity, and Immunostimulation

  • Protocol
  • First Online:
Oncolytic Viruses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 797))

Abstract

Newcastle disease virus (NDV), a bird paramyxovirus, is an antitumor agent which has shown benefits to cancer patients. Its antineoplastic efficacy appears to be associated with three properties of the virus:

  1. 1.

    Selective replication in tumor cells. This feature can be studied at the RNA level, for example by RT-PCR, and at the protein level by immunochemistry.

  2. 2.

    Oncolytic properties (of some strains). The use of cultures of tumor cell lines represents a selective model to study direct viral oncolysis at the cellular level. The capacity of NDV to lyse tumor cells can be analyzed in vitro using cytotoxic assays based on the WST1 chemical reagent. The endoplasmic reticulum stress, which is induced by infection with the oncolytic NDV strain MTH-68/H and which plays an important role in the viral oncolytic effects, can be analyzed by Western blotting using specific monoclonal antibodies. Such stress appears as a key component of NDV cytotoxicity.

  3. 3.

    Immunostimulatory capacity. We describe an in vitro test called “Tumor Neutralisation Assay” which allows the analysis of bystander antitumor immune effects induced in human peripheral blood mononuclear cells by NDV. There are two variants, one for oncolytic NDV strains and the other one for nonlytic NDV strains.

NDV may use several mechanisms to exert its tumor-killing action: direct cytotoxicity against cancer cells but also nonspecific as well as active-specific antitumor immune responses from the host organism. All the methods described here allow to evaluate the different oncolytic and immunostimulatory capacities of various strains of NDV. They are crucial to harness optimal antitumor activity by appropriate combinations of virus strains and application regimens.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

Abbreviations

α-SMA:

α-Smooth muscle actin

A490:

Absorbance at 490 nm

ATV-NDV:

Autologous tumor vaccine with NDV

BSA:

Bovin serum albumin

cDNA:

Complementary DNA

CT:

Threshold cycle

ELISA:

Enzyme-linked immunosorbent assay

ELISPOT:

Enzyme-linked immunosorbent spot

ER:

Endoplasmic reticulum

HN:

Hemagglutinin protein of NDV

HSC:

Hepatic stellate cell

HU:

Hemagglutination unit

IFN:

Interferon

IRF-3:

Interferon regulatory factor 3

IRF-7:

Interferon regulatory factor 7

MOI:

Multiplicity of infection

NDV:

Newcastle disease virus

pAb:

Polyclonal antibodies

PBMC:

Peripheral blood mononuclear cell

PBS:

Phosphate-buffered saline

PCR:

Polymerase chain reaction

PHA:

Phytohemagglutinin

RIG-I:

Retinoic acid-inducible gene I

rpm:

Rotation per minute

RPNI:

Roswell park memorial institute medium

RT-PCR:

Real-time PCR

SDS:

Sodium dodecyl sulfate

STAT:

Signal transducers and activation of transcription

Tet:

Tetracycline

TGI:

Tumor growth inhibition

TNA:

Tumor-neutralization assay

UV:

Ultraviolet

References

  1. Cassel WA, Garrett RE. Newcastle disease virus as an antineoplastic agent. Cancer 1965; 18:863–868.

    CAS  PubMed  Google Scholar 

  2. Csatary LK. Viruses in the treatment of cancer. Lancet 1971; ii:825.

    Google Scholar 

  3. Alexander DJ, Allan WH. Newcastle disease virus pathotypes. Avian Path. 1974; 3(4): 269–278.

    CAS  Google Scholar 

  4. Lorence RM, Roberts MS, Groene WS, Rabin H. Replication-competent, oncolytic Newcastle disease virus for cancer therapy. In: Hern aiz Driever P, Rabkin SD, eds. Replication-competent viruses for cancer therapy. Monographs in virology, Vol. 22. Basel, Switzerland: Karger, 2001:160–82.

    Google Scholar 

  5. Pecora AL, Rizvi N, Cohen GI, Meropol NJ, Sterman D, Marshall JL, Goldberg S, Gross P, O’Neil JD, Groene WS, Roberts MS, Rabin H, Bamat MK, Lorence RM. Phase I trial of intravenous administration of PV701, an oncolytic virus, in patients with advanced solid cancers. J. Clin. Oncol. 2002; 20:2251–66.

    CAS  PubMed  Google Scholar 

  6. Cassel WA, Murray DR, Olkowski ZL. Newcastle disease virus oncolysate in the management of stage III malignant melanoma. In: Sinkovics JG, Horvath JC, eds. Viral therapy of human cancer. New York: Marcel Dekker, 2005; 577–689.

    Google Scholar 

  7. Schirrmacher V, Fournier P. Newcastle Disease Virus : a promising vector for viral therapy of cancer. In: Harrington KJ, Pandha HS and Vile RG, eds. Viral therapy of cancer, vol 542. Wiley Publishers, 2008:171–186.

    Google Scholar 

  8. Reichard KW, Lorence RM, Cascino CJ, Peeples ME, Walter RJ, Fernando MB, Reyes HM, Greager JA. Newcastle disease virus selectively kills human tumor cells. J. Surg. Res. 1992; 52(5): 448–453.

    CAS  PubMed  Google Scholar 

  9. Fiola C, Peeters B, Fournier P, Arnold A, Bucur M, Schirrmacher V. Tumor-selective replication of Newcastle Disease Virus: association with defects of tumor cells defence. Int. J. Cancer 2006; 119(2): 328–38.

    CAS  PubMed  Google Scholar 

  10. Li YL, Wu J, Wei D, Zhang DW, Feng H, Chen ZN, Bian H. Newcastle disease virus represses the activation of human hepatic stellate cells and reverses the development of hepatic fibrosis in mice. Liver Int. 2009; 29(4):593–602.

    PubMed  Google Scholar 

  11. Sinkovics JG, Horvath JC. Newcastle disease virus (NDV): brief history of its oncolytic strains. J. Clin. Virol. 2000; 16:1–15.

    CAS  PubMed  Google Scholar 

  12. Apostolidis L, Schirrmacher V, Fournier P. Host mediated anti-tumor effect of oncolytic Newcastle disease virus after locoregional application. Int. J. Oncol. 2007; 31(5):1009–19.

    CAS  PubMed  Google Scholar 

  13. Fábián Z, Csatary CM, Szeberényi J, Csatary LK. p53-independent endoplasmic reticulum stress-mediated cytotoxicity of a Newcastle disease virus strain in tumor cell lines. J. Virol. 2007; 81(6):2817–30.

    PubMed  PubMed Central  Google Scholar 

  14. Schirrmacher V, Haas C, Bonifer R, Ahlert T, Gerhards R, Ertel C. Human tumor cell modification by virus infection: an efficient and safe way to produce cancer vaccine with pleiotropic immune stimulatory properties when using Newcastle Disease Virus. Gene Ther. 1999; 6: 63–73.

    CAS  PubMed  Google Scholar 

  15. Czeglédi A, Wehmann E, Lomniczi B. On the origins and relationships of Newcastle disease virus vaccine strains Hertfordshire and Mukteswar, and virulent strain Herts’33. Avian Pathol. 2003; 32, 271–276.

    PubMed  Google Scholar 

  16. Haas C, Lulei M, Fournier P, Arnold A, Schirrmacher V. T-cell triggering by CD3- and CD28-binding molecules linked to a human virus-modified tumor cell vaccine. Vaccine. 2005; 23(19):2439–53.

    CAS  PubMed  Google Scholar 

  17. Bian H, Fournier P, Moormann R, Peeters B, Schirrmacher V. Selective gene transfer in vitro to tumor cells via recombinant Newcastle disease virus. Cancer Gene Ther. 2005; 12:295–303.

    CAS  PubMed  Google Scholar 

  18. Van Meir EG, Polverini PJ, Chazin VR, Su Huang HJ, de Tribolet N, Cavenee WK. Release of an inhibitor of angiogenesis upon induction of wild type p53 expression in glioblastoma cells. Nat. Genet. 1994; 8(2):171–6.

    PubMed  Google Scholar 

  19. Van Meir EG, Kikuchi T, Tada M, Li H, Diserens AC, Wojcik BE, Huang HJ, Friedmann T, de Tribolet N, Cavenee WK. Analysis of the p53 gene and its expression in human glioblastoma cells. Cancer Res. 1994; 54(3):649–52.

    PubMed  Google Scholar 

  20. Xu C, Bailly-Maitre B, Reed JC. Endoplasmic reticulum stress: cell life and death decisions. J. Clin. Invest. 2005; 115: 2656–2664.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Horvath CM. Weapons of STAT destruction. Interferon evasion by paramyxovirus V protein. Eur. J. Biochem. 2004; 271:4621–8.

    CAS  PubMed  Google Scholar 

  22. Wilden H, Fournier P, Zawatzky R, Schirrmacher V. Expression of RIG-I, IRF3, IFN-beta and IRF7 determines resistance or susceptibility of cells to infection by Newcastle Disease Virus. Int. J. Oncol. 2009; 34(4):971–82.

    CAS  PubMed  Google Scholar 

  23. Csatary LK, Gosztonyi G, Szeberenyi J, Fabian Z, Liszka V, Bodey B, Csatary CM. MTH-68/H oncolytic viral treatment in human high-grade gliomas. J. Neurooncol. 2004; 67: 83–93.

    CAS  PubMed  Google Scholar 

  24. Csatary LK, Moss RW, Beuth J, Torocsik B, Szeberenyi J, Bakacs T. Beneficial treatment of patients with advanced cancer using a Newcastle disease virus vaccine (MTH-68/H). Anticancer Res. 1999; 19:635–638.

    CAS  PubMed  Google Scholar 

  25. Bai L, Koopmann J, Fiola C, Fournier P and Schirrmacher V. Dendritic cells pulsed with viral oncolysates potently stimulate autologous T cells from cancer patients. Int. J. Oncol. 2002; 21:685–694.

    CAS  PubMed  Google Scholar 

  26. Bian H, Wilden H, Fournier P, Peeters B and Schirrmacher V. In vivo efficacy of systemic tumor targeting of a viral RNA vector with oncolytic properties using a bispecific adapter protein. Int. J. Oncol.29: 1359–1369, 2006.

    CAS  PubMed  Google Scholar 

  27. Phuangsab A, Lorence RM, Reichard KW, Peeples ME, Walter RJ. Newcastle disease virus therapy of human tumor xenografts: antitumor effects of local or systemic administration. Cancer Lett. 2001; 172:27–36.

    CAS  PubMed  Google Scholar 

  28. Schirrmacher V, Griesbach A and Ahlert T. Antitumor effects of Newcastle disease virus in vivo: Local versus systemic effects. Int. J. Oncol. 2001; 18:945–952.

    CAS  PubMed  Google Scholar 

  29. Batliwalla FM, Bateman BA, Serrano D, Murray D, Macphail S, Maino VC, Ansel JC, Gregersen PK, Armstrong CA. A 15-year follow-up of AJCC stage III malignant melanoma patients treated postsurgically with Newcastle disease virus (NDV) oncolysate and determination of alterations in the CD8 T cell repertoire. Mol. Med. 1998; 4(12):783–94.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Washburn B, Schirrmacher V. Human tumor cell infection by Newcastle disease virus leads to upregulation of HLA and cell adhesion molecules and to induction of interferons, chemokines and finally apoptosis. Int. J. Oncol. 2002; 21:85–93.

    CAS  PubMed  Google Scholar 

  31. Matzinger P. The danger model: a renewed sense of self. Science 2002; 296:301–5.

    CAS  PubMed  Google Scholar 

  32. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NK-k B by Toll-like receptor 3. Nature 2001; 413:732–8.

    CAS  PubMed  Google Scholar 

  33. Fournier P, Zeng J, Schirrmacher V. Two ways to induce innate immune responses in human PBMCs. Paracrine stimulation of IFN-α responses by viral protein or dsRNA. Int. J. Oncol. 2003; 23:673–80.

    CAS  PubMed  Google Scholar 

  34. Termeer CC, Schirrmacher V, Brocker EB, Becker JC. Newcastle Disease Virus infection induces a B7-1/B7-2 independent T cell-costimulatory activity in human melanoma cells. Cancer Gene Ther. 2000; 7:316–23.

    CAS  PubMed  Google Scholar 

  35. Zeng J, Fournier P, Schirrmacher V. Induction of interferon-α and tumor necrosis factor-related apoptosis-inducing ligand in human blood mononuclear cells by hemagglutinin-neuraminidase but not F protein of Newcastle Disease Virus. Virology 2002; 297:19–30.

    CAS  PubMed  Google Scholar 

  36. Luster AD. The role of chemokines in linking innate and adaptive immunity. Curr. Opin. Immunol. 2002; 14:129–35.

    CAS  PubMed  Google Scholar 

  37. Washburn B, Weigand MA, Grosse-Wilde A, Janke M, Stahl H, Rieser E, Sprick MR, Schirrmacher V, Walczak H. TNF-related apoptosis-inducing ligand mediates tumoricidal activity of human monocytes stimulated by Newcastle disease virus. J. Immunol. 2003; 170:1814–21.

    CAS  PubMed  Google Scholar 

  38. Haas C, Lulei M, Fournier P, Arnold A, Schirrmacher V. A tumor vaccine containing anti-CD3 and anti-CD28 bispecific antibodies triggers strong and durable antitumor activity in human lymphocytes. Int. J. Cancer. 2006; 118(3):658–67.

    CAS  PubMed  Google Scholar 

  39. Ahlert T, Sauerbrei W, Bastert G, Ruhland S, Bartik B, Simiantonaki N, Schumacher J, Hacker B, Schumacher M, Schirrmacher V. Tumor cell number and viability as quality and efficacy parameters of autologous virus modified cancer vaccines. J. Clin. Oncol. 1997; 15(4):1354–66.

    CAS  PubMed  Google Scholar 

  40. Steiner HH, Bonsanto MM, Beckhove P, Brysch M, Geletneky K, Ahmadi R, Schuele-Freyer R, Kremer P, Ranaie G, Matejic D, Bauer H, Kiessling M, Kunze S, Schirrmacher V, Herold-Mende C. Anti-tumor vaccination of patients with glioblastoma multiforme: a pilot study to assess: Feasibility, safety and clinical benefit. J. Clin. Oncol 2004; 22 (21):4272–4281.

    PubMed  Google Scholar 

  41. Karcher J, Dyckhoff G, Beckhove P, Reisser C, Brysch M, Ziouta Y, Helmke BH, Weidauer H, Schirrmacher V, Herold-Mende C. Antitumor vaccination in patients with head and neck squamous cell carcinomas with autologous virus-modified tumor cells. Cancer Res. 2004; 64(21):8057–61.

    CAS  PubMed  Google Scholar 

  42. Schulze T, Kemmner W, Weitz J, Wernecke KD, Schirrmacher V, Schlag PM. Efficiency of adjuvant active specific immunization with Newcastle disease virus modified tumor cells in colorectal cancer patients following resection of liver metastases: results of a prospective randomized trial. Cancer Immunol. Immunother. 2009; 58(1):61–9.

    CAS  PubMed  Google Scholar 

  43. Schirrmacher V. Clinical trials of antitumor vaccination with an autologous tumor cell vaccine modified by virus infection: Improvement of patient survival based on improved anti-tumor immune memory. Cancer Immunol. Immunother. 2005; 54(6): 587–598.

    CAS  PubMed  Google Scholar 

  44. Aigner M, Janke M, Lulei M, Beckhove P, Fournier P, Schirrmacher V. An effective tumor vaccine optimized for costimulation via bispecific and trispecific fusion proteins. Int. J. Oncol. 2008; 32(4):777–89.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors are grateful to many colleagues at the German Cancer Research Center (DKFZ, Heidelberg, Germany), at the Department of Medical Biology, Medical School, University of Pécs, Pécs, Hungary, at the United Cancer Research Institute, Alexandria, Virginia, and at Cell Engineering Research Centre and Department of Cell Biology, Fourth Military Medical University, Xi’an, China, but also all the clinicians worldwide who have been implicated in clinical trials testing NDV as therapeutic agent. The authors would like to thank Drs. L.K. Csatary, Zs. Fábián, and M. Pap for their contributions in various phases of the research work on oncolytic MTH-68/H supported by the United Cancer Research Institute, Alexandria, Virginia, USA.

The aim of all these studies at the DKFZ was to acquire the basic scientific knowledge necessary for the development of cancer immunovirotherapies using NDV. The authors would like to thank previous colleagues and especially Claudia Haas and Leonidas Apostolidis for their contribution, respectively, in the development of the in vitro tumor-neutralization assays based on nonlytic and lytic NDV strains. We would also like to thank Annette Arnold for her excellent technical help throughout the project.

This work was supported by many grants over the last 10 years. We thank the IOZK Köln (www.iozk.de) for support and for clinical application of NOV to cancer patients. At the time when this manuscript is written, P.F. is supported by the Christian Berndt-Stiftung, Rostock, Germany.

Author information

Authors and Affiliations

  1. German Cancer Research Center(DKFZ), Heidelberg, Germany

    Philippe Fournier

  2. Fourth Military Medical University, Xian, China

    Huijie Bian

  3. University of Pécs Medical School, Pécs, Hungary

    József Szeberényi

  4. Tumor Immunology Program, German Cancer Research Center (DKFZ), Heidelberg, Germany

    Volker Schirrmacher

Authors
  1. Philippe Fournier
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Huijie Bian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. József Szeberényi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Volker Schirrmacher
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Volker Schirrmacher .

Editor information

Editors and Affiliations

  1. Jennerex Biotherapeutics, Inc., Market Street 1, San Francisco, 94105, California, USA

    David H. Kirn

  2. School of Medicine, Dept. Pathology & Immunology, Washington University, S. Euclid Ave. 660, St.Louis, 63110, Missouri, USA

    Ta-Chiang Liu

  3. Hillman Cancer Center, University of Pittsburgh, Centre Avenue 5117, Pittsburgh, 15213, Pennsylvania, USA

    Stephen H. Thorne

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Fournier, P., Bian, H., Szeberényi, J., Schirrmacher, V. (2012). Analysis of Three Properties of Newcastle Disease Virus for Fighting Cancer: Tumor-Selective Replication, Antitumor Cytotoxicity, and Immunostimulation. In: Kirn, D., Liu, TC., Thorne, S. (eds) Oncolytic Viruses. Methods in Molecular Biology, vol 797. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-61779-340-0_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-340-0_13

  • Published:

  • Publisher Name: Humana, Totowa, NJ

  • Print ISBN: 978-1-61779-339-4

  • Online ISBN: 978-1-61779-340-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation