Abstract 1041: Binding of soluble DC-HIL to endothelial cell creates immunosuppression on premetastatic niches
Authors: Ramani Vijay , Teshima Takahiro , Tamura Kyoichi , Chung Jin-Sung , Cruz Ponciano , Ariizumi Kiyoshi 
Published: July 2017

Abstract
Highly metastatic attribute of melanoma can be recapitulated in a mouse model by high potential of B16 melanoma-derived soluble factors that form more premetastatic niches (PMNs) in distal organs than those of LL2 lung carcinoma. Having shown that the T cell-inhibitory receptor DC-HIL is a potent promoter of B16 melanoma growth and that B16 cells secrete it into blood circulation as a soluble receptor (sDC-HIL), we hypothesized that sDC-HIL accounts for the metastatic nature. We tracked sDC-HIL in various organs of melanoma-bearing mice by immunoblotting and showed that sDC-HIL was expressed in particular organs; at highest levels in bone marrow (BM) and lung; and no-to-little in lymphoid organs and liver. We next examined effects of sDC-HIL on LL2 cells (no DC-HIL expression). In a model of spontaneous metastasis quantified by colonogenic assay, syngeneic mice implanted subcutaneously with LL2- transfected-sDC-HIL cells produced 6-fold more lung mets than mice with LL2-transfected-GFP. We obtained similar results in a model of experimental metastasis (EM), in which LL2 cells injected i.v. in tumor-bearing mice. To establish specificity, similar mice were treated i.v. with anti-DC-HIL mAb 2 h before and 24 h after infusion of LL2 cells: The mAb led to 80% reduction in lung mets in mice with sDC-HIL-LL2 tumor. Next we infected tumor-free mice with lentiviruses for sDC-HIL/GFP or GFP gene and then performed the EM assay: lung mets in mice expressing sDC-HIL were 5-fold greater than control. To determine whether sDC-HIL is responsible for the ability of B16-conditioned media (CM) to induce metastasis, tumor-free mice were injected i.p. daily with CM from LL2, B16, or DC-HIL-knocked down (KD) B16 cells and then assayed for EM. B16-CM induced significantly more lung mets than LL2-CM, with mets almost corresponding to that produced by KD-B16-CM. We tracked the fate of sDC-HIL in lung by immunofluorescence and showed sDC-HIL accumulated on particular (not all) endothelial cells (EC), at sites where LL2 and BM-cells migrated. Kinetic studies in BM-chimera mice showed that sDC-HIL arrives at lung sites before VEGFR1+ BM-progenitors, which initiate formation of PMNs. Since the mets-enhancing effect of sDC-HIL was not seen in immunodeficient mice, we asked whether lungs expressing sDC-HIL were immunosuppressive. CFSE-labeled CD8+ T cells were infused into mice with sDC-HIL- or GFP-LL2 tumors, and their proliferation assayed by FACS: T-cell proliferation in lungs expressing sDC-HIL was reduced significantly (28%) vs. control (81%). Because sDC-HIL by itself is not suppressive, we asked whether binding of sDC-HIL by EC makes immunosuppressive. CD8+ T cells were co-cultured with sDC-HIL-bound EC and then co-stimulated: sDC-HIL-bound EC completely suppressed the IFN-γ response. Thus, sDC-HIL critically contributes to the highly metastatic potential by making PMNs immunosuppressive even before the arrival of tumor cells.
Citation Format: Vijay Ramani, Takahiro Teshima, Kyoichi Tamura, Jin-Sung Chung, Ponciano Cruz, Kiyoshi Ariizumi. Binding of soluble DC-HIL to endothelial cell creates immunosuppression on premetastatic niches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1041. doi:10.1158/1538-7445.AM2017-1041