The inhibition of malignant melanoma cell invasion of bone by the TLR7 agonist R848 is dependent upon pro-inflammatory cytokines produced by bone marrow macrophages

// Yoko Manome 1, 2 , Dai Suzuki 1 , Ayako Mochizuki 3 , Emi Saito 1, 4 , Kiyohito Sasa 1 , Kentaro Yoshimura 1 , Tomio Inoue 3 , Masamichi Takami 5 , Katsunori Inagaki 4 , Takahiro Funatsu 2 and Ryutaro Kamijo 1 1 Departments of Biochemistry School of Dentistry, Showa University, Shinagawa, Tokyo, Japan 2 Department of Special Needs Dentistry, Division of Dentistry for Persons with Disabilities, Showa University Dental Hospital, Shinagawa, Tokyo, Japan 3 Departments of Oral Physiology School of Dentistry, Showa University, Shinagawa, Tokyo, Japan 4 Department of Orthopedic Surgery School of Medicine, Showa University, Shinagawa, Tokyo, Japan 5 Departments of Pharmacology School of Dentistry, Showa University, Shinagawa, Tokyo, Japan Correspondence to: Dai Suzuki, email: dai.suzuki@dent.showa-u.ac.jp Keywords: bone invasion; bone marrow macrophage; cytokines; malignant melanoma; R848 Received: December 23, 2017     Accepted: June 12, 2018     Published: July 06, 2018 ABSTRACT Distant metastasis remarkably worsens the prognoses of malignant melanoma patients. Toll-like receptors (TLRs) recognize molecules derived from many types of pathogens and activate the innate intravital immune system. In this study, we examined the effects of R848, a TLR7 ligand, on bone invasion by malignant melanoma cells. Mice underwent transplantation with cells of a malignant melanoma cell line B16F10, and were also administered R848 every three days. Hindlimbs were obtained 13 days after transplantation and invasion of bone marrow by B16F10 cells was evaluated. ELISA was used to determine the concentrations of cytokines in mouse serum and in the culture medium from bone marrow macrophages (BMMs) in the presence or absence of R848. In addition, MTS assays were used to examine the effects of media from BMM cultures on the proliferation of B16F10 cells. The rate of infiltration by B16F10 cells and the area of invasion were significantly reduced with R848 administration. Furthermore, serum levels of IL-6, IL-12, and IFN-γ were significantly increased in mice administered R848, with the same trend observed in the culture medium of BMMs treated with R848. In addition, B16F10 cell proliferation was suppressed by the addition of medium from cultured BMMs treated with R848. Neutralization by antibodies against IL-6, IL-12, and IFN-γ abrogated the suppression of proliferation of B16F10 cells by culture medium from BMMs treated with R848. Our results suggest that R848 drives the production of IL-6, IL-12, and IFN-γ in BMMs, which reduces proliferation and bone invasion by B16F10 cells.