Breast Cancer Research and Treatment

, Volume 107, Issue 2, pp 211–223 | Cite as

Prophylactic IL-12 treatment reduces postoperative metastasis: mediation by increased numbers but not cytotoxicity of NK cells

  • Yossi Schwartz
  • Roi Avraham
  • Marganit Benish
  • Ella Rosenne
  • Shamgar Ben-Eliyahu
Preclinical Study


Despite a promising potential, interleukin-12 immunotherapy has yielded limited clinical success while causing perilous toxicities. Here we study a context in which IL-12 may prove clinically beneficial—the removal of the primary tumor, when cell-mediated immunity (CMI) may eradicate minimal residual disease (MRD), but is inhibited by postoperative immunosuppression, potentially leading to enhanced malignant progression. F344 rats were preoperatively treated with IL-12 and inoculated postoperatively with syngeneic MADB106 tumor cells. An optimal regimen of eight-day sustained exposure to IL-12 was developed (1 μg/rat/day), which caused mild side effects, increased baseline resistance to experimental MADB106 metastasis, and abolished the promotion of metastasis by laparotomy and other immunosuppressive paradigms. Depletion of NK cells indicated their major role in controlling MADB106 metastasis in naïve and IL-12 treated rats. Studying NK cytotoxicity, we found that IL-12 did not potentiate activity per NK cell, nor protected it from suppression by surgery. However, IL-12 increased the numbers of NK cells in the circulation and marginating pulmonary pool of naïve and operated rats, and correspondingly increased total NK activity in these compartments. Therefore, this study indicates anti-tumor effects of IL-12 based on increased numbers of strategically located NK cells, and advocates a prophylactic approach against the potential metastasis-promoting effects of surgery.


Active immunotherapy Surgery Cellular immunity Cytokines IL-12 



cell-mediated immunity





This research was supported by NIH/NCI grant CA73056 and by a grant from the Israel Science Foundation to Dr. S. Ben-Eliyahu.


  1. 1.
    Brunda MJ, Luistro L, Warrier RR et al (1993) Antitumor and antimetastatic activity of interleukin 12 against murine tumors. J Exp Med 178:1223–1230PubMedCrossRefGoogle Scholar
  2. 2.
    Boggio K, Di Carlo E, Rovero S et al (2000) Ability of systemic interleukin-12 to hamper progressive stages of mammary carcinogenesis in HER2/neu transgenic mice. Cancer Res 60:359–364PubMedGoogle Scholar
  3. 3.
    Smyth MJ, Taniguchi M, Street SE (2000) The anti-tumor activity of IL-12: mechanisms of innate immunity that are model and dose dependent. J Immunol 165:2665–2670PubMedGoogle Scholar
  4. 4.
    Kobayashi T, Shiiba K, Satoh M et al (2002) Interleukin-12 administration is more effective for preventing metastasis than for inhibiting primary established tumors in a murine model of spontaneous hepatic metastasis. Surg Today 32:236–242PubMedCrossRefGoogle Scholar
  5. 5.
    Zou JP, Yamamoto N, Fujii T et al (1995) Systemic administration of rIL-12 induces complete tumor regression and protective immunity: response is correlated with a striking reversal of suppressed IFN-gamma production by anti-tumor T cells. Int Immunol 7:1135–1145PubMedCrossRefGoogle Scholar
  6. 6.
    Nastala CL, Edington HD, McKinney TG et al (1994) Recombinant IL-12 administration induces tumor regression in association with IFN-gamma production. J Immunol 153:1697–1706PubMedGoogle Scholar
  7. 7.
    Colombo MP, Trinchieri G (2002) Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev 13:155–168PubMedCrossRefGoogle Scholar
  8. 8.
    Caminschi I, Venetsanakos E, Leong CC et al (1998) Interleukin-12 induces an effective antitumor response in malignant mesothelioma. Am J Respir Cell Mol Biol 19:738–746PubMedGoogle Scholar
  9. 9.
    Cui J, Shin T, Kawano T et al (1997) Requirement for Valpha14 NKT cells in IL-12-mediated rejection of tumors. Science 278:1623–1626PubMedCrossRefGoogle Scholar
  10. 10.
    Hurteau JA, Blessing JA, DeCesare SL et al (2001) Evaluation of recombinant human interleukin-12 in patients with recurrent or refractory ovarian cancer: a gynecologic oncology group study. Gynecol Oncol 82:7–10PubMedCrossRefGoogle Scholar
  11. 11.
    Atkins MB, Robertson MJ, Gordon M et al (1997) Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clin Cancer Res 3:409–417PubMedGoogle Scholar
  12. 12.
    Portielje JE, Kruit WH, Schuler M et al (1999) Phase I study of subcutaneously administered recombinant human interleukin 12 in patients with advanced renal cell cancer. Clin Cancer Res 5:3983–3989PubMedGoogle Scholar
  13. 13.
    Leonard JP, Sherman ML, Fisher GL et al (1997) Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood 90:2541–2548PubMedGoogle Scholar
  14. 14.
    Rook AH, Wood GS, Yoo EK et al (1999) Interleukin-12 therapy of cutaneous T-cell lymphoma induces lesion regression and cytotoxic T-cell responses. Blood 94:902–908PubMedGoogle Scholar
  15. 15.
    Shakhar G, Ben-Eliyahu S (2003) Potential prophylactic measures against postoperative immunosuppression: could they reduce recurrence rates in oncological patients? Ann Surg Oncol 10:972–992PubMedCrossRefGoogle Scholar
  16. 16.
    Brittenden J (1996) Natural killer cells and cancer. Cancer 77:1226–1243PubMedCrossRefGoogle Scholar
  17. 17.
    Ben-Eliyahu S, Page GG, Yirmiya R et al (1999) Evidence that stress and surgical interventions promote tumor development by suppressing natural killer cell activity. Int J Cancer 80:880–888PubMedCrossRefGoogle Scholar
  18. 18.
    Dithmar SA, Rusciano DA, Armstrong CA et al (1999) Depletion of NK cell activity results in growth of hepatic micrometastases in a murine ocular melanoma model. Curr Eye Res 19:426–431PubMedCrossRefGoogle Scholar
  19. 19.
    Smyth MJ, Godfrey DI, Trapani JA (2001) A fresh look at tumor immunosurveillance and immunotherapy. Nat Immunol 2:293–299PubMedCrossRefGoogle Scholar
  20. 20.
    Taketomi A, Shimada M, Shirabe K et al (1998) Natural killer cell activity in patients with hepatocellular carcinoma: a new prognostic indicator after hepatectomy. Cancer 83:58–63PubMedCrossRefGoogle Scholar
  21. 21.
    Schantz SP, Brown BW, Lira E et al (1987) Evidence for the role of natural immunity in the control of metastatic spread of head and neck cancer. Cancer Immunol Immunother 25:141–148PubMedCrossRefGoogle Scholar
  22. 22.
    Ben-Eliyahu S, Page GG, Yirmiya R et al (1996) Acute alcohol intoxication suppresses natural killer cell activity and promotes tumor metastasis. Nat Med 2:457–460PubMedCrossRefGoogle Scholar
  23. 23.
    Ben-Eliyahu S, Page GG (1992) In vivo assessment of natural killer cell activity in rats. Prog Neuroendocrinemmunol 5:199–214Google Scholar
  24. 24.
    Barlozzari T, Leonhardt J, Wiltrout RH et al (1985) Direct evidence for the role of LGL in the inhibition of experimental tumor metastases. J Immunol 134:2783–2789PubMedGoogle Scholar
  25. 25.
    Shakhar G, Ben-Eliyahu S (1998) In vivo beta-adrenergic stimulation suppresses natural killer activity and compromises resistance to tumor metastasis in rats. J Immunol 160:3251–3258PubMedGoogle Scholar
  26. 26.
    Ben-Eliyahu S, Yirmiya R, Liebeskind JC et al (1991) Stress increases metastatic spread of a mammary tumor in rats: evidence for mediation by the immune system. Brain Behav Immun 5:193–205PubMedCrossRefGoogle Scholar
  27. 27.
    Melamed R, Rosenne E, Shakhar K et al (2005) Marginating pulmonary-NK activity and resistance to experimental tumor metastasis: suppression by surgery and the prophylactic use of a beta-adrenergic antagonist and a prostaglandin synthesis inhibitor. Brain Behav Immun 19:114–126PubMedCrossRefGoogle Scholar
  28. 28.
    Barlozzari T, Reynolds CW, Herberman RB (1983) In vivo role of natural killer cells: involvement of large granular lymphocytes in the clearance of tumor cells in anti-asialo GM1-treated rats. J Immunol 131:1024–1027PubMedGoogle Scholar
  29. 29.
    Bar-Yosef S, Melamed R, Page GG et al (2001) Attenuation of the tumor-promoting effect of surgery by spinal blockade in rats. Anesthesiology 94:1066–1073PubMedCrossRefGoogle Scholar
  30. 30.
    Dengler HG, Hengstmann JH (1976) Metabolism and pharmacokinetics of orciprenaline in various animal species and man. Arch Int Pharmacodyn Ther 223:71–87PubMedGoogle Scholar
  31. 31.
    Zuckerman L, Rimmerman N, Weiner I (2003) Latent inhibition in 35-day-old rats is not an “adult” latent inhibition: implications for neurodevelopmental models of schizophrenia. Psychopharmacology (Berl) 169:298–307CrossRefGoogle Scholar
  32. 32.
    Chambers WH, Brumfield AM, Hanley-Yanez K et al (1992) Functional heterogeneity between NKR-P1bright/Lycopersicon esculentum lectin (L.E.)bright and NKR-P1bright/L.E.dim subpopulations of rat natural killer cells. J Immunol 148:3658–3665PubMedGoogle Scholar
  33. 33.
    Chambers WH, Vujanovic NL, DeLeo AB et al (1989) Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokine-activated killer cells. J Exp Med 169:1373–1389PubMedCrossRefGoogle Scholar
  34. 34.
    Page GG, Ben-Eliyahu S, Liebeskind JC (1994) The role of LGL/NK cells in surgery-induced promotion of metastasis and its attenuation by morphine. Brain Behav Immun 8:241–250PubMedCrossRefGoogle Scholar
  35. 35.
    Ben-Eliyahu S, Page GG, Shakhar G et al (1996) Increased susceptibility to metastasis during pro-oestrus/oestrus in rats: possible role of oestradiol and natural killer cells. Br J Cancer 74:1900–1907PubMedGoogle Scholar
  36. 36.
    Fishkin RJ, Winslow JT (1997) Endotoxin-induced reduction of social investigation by mice: interaction with amphetamine and anti-inflammatory drugs. Psychopharmacology (Berl). 132:335–341CrossRefGoogle Scholar
  37. 37.
    Yirmiya R, Tio DL, Taylor AN (1996) Effects of fetal alcohol exposure on fever, sickness behavior, and pituitary-adrenal activation induced by interleukin-1 beta in young adult rats. Brain Behav Immun 10:205–220PubMedCrossRefGoogle Scholar
  38. 38.
    Yirmiya R, Pollak Y, Morag M et al (2000) Illness, cytokines, and depression. Ann NY Acad Sci 917:478–487PubMedCrossRefGoogle Scholar
  39. 39.
    Katafuchi T, Kondo T, Yasaka T et al (2003) Prolonged effects of polyriboinosinic:polyribocytidylic acid on spontaneous running wheel activity and brain interferon-alpha mRNA in rats: a model for immunologically induced fatigue. Neuroscience 120:837–845PubMedCrossRefGoogle Scholar
  40. 40.
    Ben-Eliyahu S, Shakhar G, Page GG et al (2000) Suppression of NK cell activity and of resistance to metastasis by stress: a role for adrenal catecholamines and beta-adrenoceptors. Neuroimmunomodulation 8:154–164PubMedCrossRefGoogle Scholar
  41. 41.
    Kanaoka E, Takahashi K, Yoshikawa T et al (2002) A significant enhancement of therapeutic effect against hepatic metastases of M5076 in mice by a liposomal interleukin-2 (mixture). J Control Release 82:183–187PubMedCrossRefGoogle Scholar
  42. 42.
    Kedar E, Braun E, Rutkowski Y et al (1994) Delivery of cytokines by liposomes. II. Interleukin-2 encapsulated in long-circulating sterically stabilized liposomes: immunomodulatory and anti-tumor activity in mice. J Immunother Emphasis Tumor Immunol 16:115–124PubMedGoogle Scholar
  43. 43.
    Liu L, Sakaguchi T, Kanda T et al (2003) Delivery of interleukin-12 in gelatin hydrogels effectively suppresses development of transplanted colonal carcinoma in mice. Cancer Chemother Pharmacol 51:53–57PubMedCrossRefGoogle Scholar
  44. 44.
    Gately MK, Desai BB, Wolitzky AG et al (1991) Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor). J Immunol 147:874–882PubMedGoogle Scholar
  45. 45.
    Robertson MJ, Soiffer RJ, Wolf SF et al (1992) Response of human natural killer (NK) cells to NK cell stimulatory factor (NKSF): cytolytic activity and proliferation of NK cells are differentially regulated by NKSF. J Exp Med 175:779–788PubMedCrossRefGoogle Scholar
  46. 46.
    Eng VM, Car BD, Schnyder B et al (1995) The stimulatory effects of interleukin (IL)-12 on hematopoiesis are antagonized by IL-12-induced interferon gamma in vivo. J Exp Med 181:1893–1898PubMedCrossRefGoogle Scholar
  47. 47.
    Gately MK, Warrier RR, Honasoge S et al (1994) Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-gamma in vivo. Int Immunol 6:157–167PubMedCrossRefGoogle Scholar
  48. 48.
    Matsumoto G, Omi Y, Lee U et al (2000) Adhesion mediated by LFA-1 is required for efficient IL-12-induced NK and NKT cell cytotoxicity. Eur J Immunol 30:3723–3731PubMedCrossRefGoogle Scholar
  49. 49.
    Kawamura T, Takeda K, Mendiratta SK et al (1998) Critical role of NK1+ T cells in IL-12-induced immune responses in vivo. J Immunol 160:16–19PubMedGoogle Scholar
  50. 50.
    Lasek W, Golab J, Maslinski W et al (1999) Subtherapeutic doses of interleukin-15 augment the antitumor effect of interleukin-12 in a B16F10 melanoma model in mice. Eur Cytokine Netw 10:345–356PubMedGoogle Scholar
  51. 51.
    Okuno K, Jinnai H, Lee YS et al (1996) Interleukin 12 augments the liver-associated immunity and reduces liver metastases. Hepatogastroenterology 43:1196–1202PubMedGoogle Scholar
  52. 52.
    Gan X, Zhang L, Solomon GF et al (2002) Mechanism of norepinephrine-mediated inhibition of human NK cytotoxic functions: inhibition of cytokine secretion, target binding, and programming for cytotoxicity. Brain Behav Immun 16:227–246PubMedCrossRefGoogle Scholar
  53. 53.
    Mitsuhashi M, Liu J, Cao S et al (2004) Regulation of interleukin-12 gene expression and its anti-tumor activities by prostaglandin E2 derived from mammary carcinomas. J Leukoc Biol 76:322–332PubMedCrossRefGoogle Scholar
  54. 54.
    Panina-Bordignon P, Mazzeo D, Lucia PD et al (1997) Beta2-agonists prevent Th1 development by selective inhibition of interleukin 12. J Clin Invest 100:1513–1519PubMedCrossRefGoogle Scholar
  55. 55.
    Pockaj BA, Basu GD, Pathangey LB et al (2004) Reduced T-cell and dendritic cell function is related to cyclooxygenase-2 overexpression and prostaglandin E2 secretion in patients with breast cancer. Ann Surg Oncol 11:328–339PubMedCrossRefGoogle Scholar
  56. 56.
    Ben-Eliyahu S (2003) The promotion of tumor metastasis by surgery and stress: Immunological basis and implications for psychoneuroimmunology. Brain Behav Immun 17:27–36CrossRefGoogle Scholar
  57. 57.
    Dithmar S, Rusciano D, Lynn MJ et al (2000) Neoadjuvant interferon alfa-2b treatment in a murine model for metastatic ocular melanoma: a preliminary study. Arch Ophthalmol 118:1085–1089PubMedGoogle Scholar
  58. 58.
    Gallagher WJ, Dubinett SM, Hoover HC Jr et al (1989) Efficacy of adjuvant interleukin-2 after excision of BALB/c fibrosarcomas. Surgery 106:120–125PubMedGoogle Scholar
  59. 59.
    Mu J, Zou JP, Yamamoto N et al (1995) Administration of recombinant interleukin 12 prevents outgrowth of tumor cells metastasizing spontaneously to lung and lymph nodes. Cancer Res 55:4404–4408PubMedGoogle Scholar
  60. 60.
    Strohlein MA, Grutzner KU, Schildberg FW et al (2002) Induction of cytotoxicity against autologous tumour cells by interleukin-12: evidence for intrinsic anti-tumor immune capacity in curatively resected gastrointestinal tumour patients. Cancer Immunol Immunother 51:505–512PubMedCrossRefGoogle Scholar
  61. 61.
    Brivio F, Lissoni P, Alderi G et al (1996) Preoperative interleukin-2 subcutaneous immunotherapy may prolong the survival time in advanced colorectal cancer patients. Oncology 53:263–268PubMedCrossRefGoogle Scholar
  62. 62.
    Brivio F, Lissoni P, Fumagalli L et al (1999) Pre-operative IL-2 immunoprophylaxis of cancer recurrence: long-term clinical results of a phase II study in radically operable colorectal cancer. Oncol Rep 6:1205–1207PubMedGoogle Scholar
  63. 63.
    Deehan DJ, Heys SD, Ashby J et al (1995) Interleukin-2 (IL-2) augments host cellular immune reactivity in the perioperative period in patients with malignant disease. Eur J Surg Oncol 21:16–22PubMedCrossRefGoogle Scholar
  64. 64.
    Luksch R, Perotti D, Cefalo G et al (2003) Immunomodulation in a treatment program including pre- and post-operative interleukin-2 and chemotherapy for childhood osteosarcoma. Tumori 89:263–268PubMedGoogle Scholar
  65. 65.
    Coughlin CM, Salhany KE, Gee MS et al (1998) Tumor cell responses to IFNgamma affect tumorigenicity and response to IL-12 therapy and antiangiogenesis. Immunity 9:25–34PubMedCrossRefGoogle Scholar
  66. 66.
    Dias S, Boyd R, Balkwill F (1998) IL-12 regulates VEGF and MMPs in a murine breast cancer model. Int J Cancer 78:361–365PubMedCrossRefGoogle Scholar
  67. 67.
    Voest EE, Kenyon BM, O’Reilly MS et al (1995) Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst 87:581–586PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Yossi Schwartz
    • 1
  • Roi Avraham
    • 1
  • Marganit Benish
    • 1
  • Ella Rosenne
    • 1
  • Shamgar Ben-Eliyahu
    • 1
  1. 1.Department of Psychology, Neuroimmunology Research UnitTel Aviv UniversityTel AvivIsrael

Personalised recommendations