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,Takahiro Funatsu,Masamichi Takami,Kiyohito Sasa,Kentaro Yoshimura,Tomio Inoue,Ryutaro Kamijo,Dai Suzuki,Ayako Mochizuki,Emi Saito,Katsunori Inagaki

doi:10.18632/oncotarget.25711

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.

Highlights

Malignant melanoma, one of the most dangerous types of skin cancer, develops from melanocytes, though it rarely occurs in the mouth, intestines, or eyes
Accumulations of blackened B16F10 cells were observed in the epiphyses of the femora and tibiae in mice treated with the vehicle (Figure 1B), while these accumulations were markedly reduced in mice treated with R848 (Figure 1B, 1C)
To determine whether immune cells were the source of these cytokines, we evaluated the concentrations of IL-6, IL-12 p40, and IFN-γ in the supernatant of bone marrow macrophages (BMMs) cultured with R848 (100 nM)

Summary

Introduction

One of the most dangerous types of skin cancer, develops from melanocytes, though it rarely occurs in the mouth, intestines, or eyes. The median survival of patients affected by bone metastasis ranges from 4 to 6 months [3], metastasis of a malignant melanoma to bone is a late event in the evolution of this disease. In those cases, pain is the most common presenting symptom and palliation is often the treatment goal. Survival of patients with spreading metastatic malignant melanoma may be improved by treatments such as chemotherapy including with dimethyl triazeno imidazole carboxamide (DTIC) and dacarbazine; immunotherapy including with interleukin (IL)-2; targeted therapy including with BRAF inhibitors such as vemurafenib and dabrafenib; and radiation therapy. Postoperative adjunctive biological therapy with interferon (IFN)-α has been shown to have a sustained effect on recurrence-free survival

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Results

Conclusion