Cancer Immunology, Immunotherapy

, Volume 66, Issue 9, pp 1131–1142 | Cite as

Downregulation of neuropilin-1 on macrophages modulates antibody-mediated tumoricidal activity

  • Kosuke Kawaguchi
  • Eiji SuzukiEmail author
  • Mariko Nishie
  • Isao Kii
  • Tatsuki R. Kataoka
  • Masahiro Hirata
  • Masashi Inoue
  • Fengling Pu
  • Keiko Iwaisako
  • Moe Tsuda
  • Ayane Yamaguchi
  • Hironori Haga
  • Masatoshi Hagiwara
  • Masakazu Toi
Original Article


Neuropilin-1 (NRP-1)-expressing macrophages are engaged in antitumor immune functions via various mechanisms. In this study, we investigated the role of NRP-1 on macrophages in antibody-mediated tumoricidal activity. Treatment of macrophages with NRP-1 knockdown or an anti-NRP-1-neutralizing antibody significantly suppressed antibody-dependent cellular cytotoxicity and modulated cytokine secretion from macrophages in vitro. Furthermore, in vivo studies using a humanized mouse model bearing human epidermal growth factor receptor-2 (HER2)-positive breast cancer xenografts showed that antibody-mediated antitumor activity and tumor infiltration of CD4+ T lymphocytes were significantly downregulated when peripheral blood mononuclear cells in which NRP-1 was knocked down were co-administered with an anti-HER2 antibody. These results revealed that NRP-1 expressed on macrophages plays an important role in antibody-mediated antitumor immunity. Taken together, the induction of NRP-1 on macrophages may be a therapeutic indicator for antibody treatments that exert antibody-dependent cellular cytotoxicity activity, although further studies are needed in order to support this hypothesis.


Neuropilin-1 Breast cancer HER2 Humanized mouse Antibody-dependent cellular cytotoxicity 



Antibody-dependent cellular cytotoxicity


Ethylenediaminetetraacetic acid


Fetal bovine serum


Granulocyte colony-stimulating factor


Gaussia luciferase


Human epidermal growth factor receptor-2






Macrophage inflammatory protein-1α


Macrophage inflammatory protein-1β


Nod/Shi-scid, IL-2Rγ null




Peripheral blood mononuclear cells


Phosphate-buffered saline


Quantitative real-time PCR


Relative luminescence units


Standard error of the mean


Small interfering RNA


Tumor-infiltrating lymphocytes


Tumor necrosis factor


Vascular endothelial growth factor



We thank the medical staff of the Department of Breast Surgery of Kyoto University Hospital for their help in the recruitment of participants and collection of samples. We also thank Dr. Hitoshi Niwa (RIKEN CDB, Kobe, Japan) for providing the pCAGIPuro vector.

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interests.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Financial support

This research was supported by a grant from the Japan Society for the Promotion of Science (KAKENHI Grant Number 12907996).

Supplementary material

262_2017_2002_MOESM1_ESM.pdf (1.8 mb)
Supplementary material 1 (PDF 1815 kb)


  1. 1.
    Bellati F, Napoletano C, Ruscito I, Liberati M, Panici PB, Nuti M (2010) Cellular adaptive immune system plays a crucial role in trastuzumab clinical efficacy. J Clin Oncol 28:e369–e370. doi: 10.1200/JCO.2010.28.6922 (author reply e71) CrossRefPubMedGoogle Scholar
  2. 2.
    Ferris RL, Jaffee EM, Ferrone S (2010) Tumor antigen-targeted, monoclonal antibody-based immunotherapy: clinical response, cellular immunity, and immunoescape. J Clin Oncol 28:4390–4399. doi: 10.1200/JCO.2009.27.6360 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Park S, Jiang Z, Mortenson ED et al (2010) The therapeutic effect of anti-HER2/neu antibody depends on both innate and adaptive immunity. Cancer Cell 18:160–170. doi: 10.1016/j.ccr.2010.06.014 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Stagg J, Loi S, Divisekera U, Ngiow SF, Duret H, Yagita H, Teng MW, Smyth MJ (2011) Anti-ErbB-2 mAb therapy requires type I and II interferons and synergizes with anti-PD-1 or anti-CD137 mAb therapy. Proc Natl Acad Sci USA 108:7142–7147. doi: 10.1073/pnas.1016569108 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Piccart-Gebhart MJ, Procter M, Leyland-Jones B et al (2005) Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 353:1659–1672CrossRefPubMedGoogle Scholar
  6. 6.
    Pollard JW (2004) Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 4:71–78. doi: 10.1038/nrc1256 CrossRefPubMedGoogle Scholar
  7. 7.
    Leek RD, Lewis CE, Whitehouse R, Greenall M, Clarke J, Harris AL (1996) Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res 56:4625–4629PubMedGoogle Scholar
  8. 8.
    Gwak JM, Jang MH, Kim DI, Seo AN, Park SY (2015) Prognostic value of tumor-associated macrophages according to histologic locations and hormone receptor status in breast cancer. PLoS One 10:e0125728. doi: 10.1371/journal.pone.0125728 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Casazza A, Laoui D, Wenes M et al (2013) Impeding Macrophage Entry into Hypoxic Tumor Areas by Sema3A/Nrp1 Signaling Blockade Inhibits Angiogenesis and Restores Antitumor Immunity. Cancer Cell 24:695–709. doi: 10.1016/j.ccr.2013.11.007 CrossRefPubMedGoogle Scholar
  10. 10.
    Carrer A, Moimas S, Zacchigna S et al (2012) Neuropilin-1 identifies a subset of bone marrow Gr1- monocytes that can induce tumor vessel normalization and inhibit tumor growth. Cancer Res 72:6371–6381. doi: 10.1158/0008-5472.CAN-12-0762 CrossRefPubMedGoogle Scholar
  11. 11.
    Tordjman R, Lepelletier Y, Lemarchandel V, Cambot M, Gaulard P, Hermine O, Romeo PH (2002) A neuronal receptor, neuropilin-1, is essential for the initiation of the primary immune response. Nat Immunol 3:477–482. doi: 10.1038/ni789 PubMedGoogle Scholar
  12. 12.
    Ji JD, Park-Min KH, Ivashkiv LB (2009) Expression and function of semaphorin 3A and its receptors in human monocyte-derived macrophages. Hum Immunol 70:211–217. doi: 10.1016/j.humimm.2009.01.026 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Takamatsu H, Takegahara N, Nakagawa Y et al (2010) Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II. Nat Immunol 11:594–600. doi: 10.1038/ni.1885 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Broussas M, Broyer L, Goetsch L (2013) Evaluation of antibody-dependent cell cytotoxicity using lactate dehydrogenase (LDH) measurement. Methods Mol Biol 988:305–317. doi: 10.1007/978-1-62703-327-5_19 CrossRefPubMedGoogle Scholar
  15. 15.
    Wurdinger T, Badr C, Pike L, de Kleine R, Weissleder R, Breakefield XO, Tannous BA (2008) A secreted luciferase for ex vivo monitoring of in vivo processes. Nat Methods 5:171–173. doi: 10.1038/nmeth.1177 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chung E, Yamashita H, Au P, Tannous BA, Fukumura D, Jain RK (2009) Secreted Gaussia luciferase as a biomarker for monitoring tumor progression and treatment response of systemic metastases. PLoS One 4:e8316. doi: 10.1371/journal.pone.0008316 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Tannous BA (2009) Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nat Protoc 4:582–591. doi: 10.1038/nprot.2009.28 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Salgado R, Denkert C, Demaria S et al (2014) Harmonization of the evaluation of tumor infiltrating lymphocytes (TILs) in breast cancer: recommendations by an international TILs-working group 2014. Ann Oncol. doi: 10.1093/annonc/mdu450 Google Scholar
  19. 19.
    Valabrega G, Montemurro F, Aglietta M (2007) Trastuzumab: mechanism of action, resistance and future perspectives in HER2-overexpressing breast cancer. Ann Oncol 18:977–984. doi: 10.1093/annonc/mdl475 CrossRefPubMedGoogle Scholar
  20. 20.
    Flieger D, Spengler U, Beier I, Kleinschmidt R, Hoff A, Varvenne M, Sauerbruch T, Schmidt-Wolf I (1999) Enhancement of antibody dependent cellular cytotoxicity (ADCC) by combination of cytokines. Hybridoma 18:63–68. doi: 10.1089/hyb.1999.18.63 CrossRefPubMedGoogle Scholar
  21. 21.
    Hiramatsu H, Nishikomori R, Heike T, Ito M, Kobayashi K, Katamura K, Nakahata T (2003) Complete reconstitution of human lymphocytes from cord blood CD34+ cells using the NOD/SCID/γcnull mice model. Blood 102:873–880. doi: 10.1182/blood-2002-09-2755 CrossRefPubMedGoogle Scholar
  22. 22.
    Ito A, Ishida T, Yano H et al (2009) Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rgamma(null) mouse model. Cancer Immunol Immunother 58:1195–1206. doi: 10.1007/s00262-008-0632-0 CrossRefPubMedGoogle Scholar
  23. 23.
    Denkert C, von Minckwitz G, Brase JC et al (2015) Tumor-infiltrating lymphocytes and response to neoadjuvant chemotherapy with or without carboplatin in human epidermal growth factor receptor 2-positive and triple-negative primary breast cancers. J Clin Oncol 33:983–991. doi: 10.1200/JCO.2014.58.1967 CrossRefPubMedGoogle Scholar
  24. 24.
    Shaw GM, Levy PC, LoBuglio AF (1978) Human monocyte antibody-dependent cell-mediated cytotoxicity to tumor cells. J Clin Investig 62:1172–1180. doi: 10.1172/JCI109236 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Wallerius M, Wallmann T, Bartish M et al (2016) Guidance molecule SEMA3A restricts tumor growth by differentially regulating the proliferation of tumor-associated macrophages. Cancer Res 76:3166–3178. doi: 10.1158/0008-5472.CAN-15-2596 CrossRefPubMedGoogle Scholar
  26. 26.
    Denkert C, Loibl S, Noske A et al (2010) Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer. J Clin Oncol 28:105–113. doi: 10.1200/JCO.2009.23.7370 CrossRefPubMedGoogle Scholar
  27. 27.
    Loi S, Michiels S, Salgado R et al (2014) Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial. Ann Oncol 25:1544–1550. doi: 10.1093/annonc/mdu112 CrossRefPubMedGoogle Scholar
  28. 28.
    Dufour JH, Dziejman M, Liu MT, Leung JH, Lane TE, Luster AD (2002) IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J Immunol 168:3195–3204CrossRefPubMedGoogle Scholar
  29. 29.
    Wu L, Adams M, Carter T, Chen R, Muller G, Stirling D, Schafer P, Bartlett JB (2008) Lenalidomide enhances natural killer cell and monocyte-mediated antibody-dependent cellular cytotoxicity of rituximab-treated CD20+ tumor cells. Clin Cancer Res 14:4650–4657. doi: 10.1158/1078-0432.CCR-07-4405 CrossRefPubMedGoogle Scholar
  30. 30.
    Donia M, Hansen M, Sendrup SL, Iversen TZ, Ellebaek E, Andersen MH, Straten P, Svane IM (2013) Methods to improve adoptive T-cell therapy for melanoma: IFN-gamma enhances anticancer responses of cell products for infusion. J Invest Dermatol 133:545–552. doi: 10.1038/jid.2012.336 CrossRefPubMedGoogle Scholar
  31. 31.
    Friedman KM, Prieto PA, Devillier LE, Gross CA, Yang JC, Wunderlich JR, Rosenberg SA, Dudley ME (2012) Tumor-specific CD4+ melanoma tumor-infiltrating lymphocytes. J Immunother 35:400–408. doi: 10.1097/CJI.0b013e31825898c5 CrossRefPubMedGoogle Scholar
  32. 32.
    Quezada SA, Simpson TR, Peggs KS et al (2010) Tumor-reactive CD4(+) T cells develop cytotoxic activity and eradicate large established melanoma after transfer into lymphopenic hosts. J Exp Med 207:637–650. doi: 10.1084/jem.20091918 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Kosuke Kawaguchi
    • 1
  • Eiji Suzuki
    • 1
    Email author
  • Mariko Nishie
    • 1
  • Isao Kii
    • 2
  • Tatsuki R. Kataoka
    • 3
  • Masahiro Hirata
    • 3
  • Masashi Inoue
    • 1
    • 5
  • Fengling Pu
    • 4
  • Keiko Iwaisako
    • 4
  • Moe Tsuda
    • 1
  • Ayane Yamaguchi
    • 1
  • Hironori Haga
    • 3
  • Masatoshi Hagiwara
    • 2
  • Masakazu Toi
    • 1
  1. 1.Department of Breast Surgery, Graduate School of MedicineKyoto UniversityKyotoJapan
  2. 2.Department of Anatomy and Developmental Biology, Graduate School of MedicineKyoto UniversityKyotoJapan
  3. 3.Department of Diagnostic PathologyKyoto University HospitalKyotoJapan
  4. 4.Department of Target Therapy Oncology, Graduate School of MedicineKyoto UniversityKyotoJapan
  5. 5.Faculty of MedicineGunma UniversityGunmaJapan

Personalised recommendations