Abstract 1255: Upregulation of neuropilin-1 in human monocyte is induced by RANKL and may be associated with trastuzumab mediated antibody dependent cellular cytotoxicity in breast cancer cells.
Authors: Suzuki Eiji , Sato Fumiaki , Kawaguchi Kosuke , Sugie Tomoharu , Toi Masakazu 
Published: August 2014

Abstract
Purpose
We found that neuropilin (NRP)-1high monocyte exhibited higher and NRP-1low monocyte exhibited lower trastuzumab mediated antibody dependent cellular cyototoxicity (ADCC) in HER2 positive breast cancer cells. Recently, it was reported that NRP-1 expression in bone marrow-derived monocyte/macrophage precursor cells (BMMs) was rapidly and markedly suppressed after receptor activator of nuclear factor-κB ligand (RANKL) stimulation in mice model (Nature 2012). Aim of this study is to explore effect of RANKL on NRP-1 expression in human monocyte and effect of RANKL on trastuzumab mediated ADCC.
Methods
Peripheral blood mononuclear cells (PBMCs) were prepared from blood of healthy volunteers by using Lymphoprep (AXIS SHIELD, Dundee, UK). The cells were cultured with various concentration of RANKL (0, 5, 50, and 250 ng/ml) for 2, 4, and 24 hours. The cells were collected and stained with FITC conjugated anti-CD14 antibody, FITC conjugated anti-CD3 antibody, and APC conjugated anti-NRP-1 antibody and analyzed by flow cytometry. In ADCC assay, various concentration of RANKL treated PBMCs were co-cultured with HER2 positive human breast cancer cell line, SKBR3 and 10μg/ml of trastuzumab for 2 hours (E:T=10:1). The cell mixture was stained with FITC conjugated anti-CD14 antibody, FITC conjugated anti-CD56 antibody, PE conjugated anti-NRP-1 antibody, and PE-Cy 5 conjugated anti-CD107a antibody. CD56+ NK cell and CD14+ monocyte degranulation assay was used to evaluate effect of RANKL on target cancer cell killing activity of those effector cells.
Results
On the contrary to the recent reports, analysis of flow cytometry indicated that expression of NRP-1 in monocyte 24 hours after RANKL treatment was upregulated while there was no effect of RANKL on expression of NRP-1 in CD56+ NK cell. MFI of NRP-1 was 373 in RANKL 0ng/ml, 427 in RANKL 5ng/ml, 469 in RANKL 50ng/ml, and 477 in RANKL 250ng/ml. Furthermore, in ADCC assay, higher concentration of RANKL treated PBMCs showed higher CD107a expression level in monocyte population, 10.8% (0ng/ml of RANKL), 14.7% (5ng/ml of RANKL), 16.2% (50ng/ml of RANKL), and 18.9% (250ng/ml of RANKL), respectively.
Conclusions
These data suggested that RANKL upregulated NRP-1 expression in human monocyte and this might be associated with higher trastuzumab mediated ADCC activity in this experimental settings.
Citation Format: Eiji Suzuki, Fumiaki Sato, Kosuke Kawaguchi, Tomoharu Sugie, Masakazu Toi. Upregulation of neuropilin-1 in human monocyte is induced by RANKL and may be associated with trastuzumab mediated antibody dependent cellular cytotoxicity in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1255. doi:10.1158/1538-7445.AM2013-1255