Truncated CD200 stimulates tumor immunity leading to fewer lung metastases in a novel Wistar rat metastasis model

https://doi.org/10.1016/j.bbrc.2018.01.065Get rights and content

Highlights

  • Subcutaneous transplantation of C6 glioma cells in rat back metastasized into the lung.

  • Two CD200 variants are full-length CD200 (CD200L) and truncated CD200 (CD200S).

  • CD200S-expressing C6 cells reduced lung metastasis and elongated survival period of rats.

  • Multiple subsets of dendritic cells were abundant in CD200S tumors in the back.

  • DCs activated cytotoxic T lymphocytes, causing elimination of metastasizing tumor cells.

Abstract

CD200 mediates immunosuppression in immune cells that express its receptor, CD200R. There are two CD200 variants; truncated CD200 that lacks the part of N-terminal sequence necessary for CD200R binding (CD200S) and full-length CD200 (CD200L). We established a novel lung metastasis model by subcutaneously transplanting C6 glioma cells into the backs of neonatal Wistar rats. All transplanted rats developed large back tumors, nearly 90% of which bore lung metastases. To compare the effects of CD200S and CD200L on tumor immunity, CD200L (C6-L)- or CD200S (C6-S)-expressing C6 cells were similarly transplanted. The results showed that 100% of rats with C6-L tumors developed lung metastases, while metastases were found in only 44% of rats with C6-S tumors (n = 25). Tumors disappeared in approximately 20% of the C6-S-bearing rats, and these animals evaded death 180 d after transplantation, while all C6-L tumor-bearing rats died after 45 d. Next generation sequencing revealed that C6-S tumors expressed chemokines and granzyme B at much higher levels than C6-L tumors. Flow cytometry revealed that C6-S tumors contained more dead cells and more CD45+ cells, including natural killer cells and CD8+ lymphocytes. In particular, multiple subsets of dendritic cells expressing CD11c, MHC class II, CD8, and/or CD103 were more abundant in C6-S than in C6-L tumors. These results suggested that CD200S induced the accumulation of multiple dendritic cell subsets that activated cytotoxic T lymphocytes, leading to the elimination of metastasizing tumor cells.

Introduction

CD200 (OX-2) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily that is an immunosuppressive ligand for cells expressing its cognate receptor, CD200R [1,2]. A variety of tissues and cells express CD200, including lymphocytes, neurons and endothelial cells [3]. CD200R is primarily expressed by myeloid cells, such as granulocytes, monocytes and macrophages, but also by many other kinds of cells and tissues [2,4]. CD200 may help cancer cells grow and metastasize by suppressing anti-tumor immunity [4,5] [6]. However, Wang et al. [7] reported that CD200 downregulation could promote tumor progression in glioma. Liu et al. [8] also showed that CD200R mediates signals that inhibit the growth and metastasis of CD200+ tumors. A truncated form of CD200 may also be responsible for the contradictory observations [9] [10,11]. The truncated form, a CD200 splice variant, lacks the N-terminal domain encoded by exons 1, 2, and part of exon 3. As a result, the truncated form has a shorter amino acid sequence than full-length CD200.

We have established rat C6 glioma cells that stably express either full-length CD200 (CD200L) or the truncated form (CD200S), and transplanted these cell lines into the brain parenchyma of neonatal Wistar rats as an experimental glioblastoma model [11]. Rats transplanted with C6-S cells survived for 33.6 d (mean value), while those with C6-L tumors survived only 22.1 d. The increased survival period of C6-S tumor-bearing rats was attributable to changes in the phenotypes of tumor-associated macrophages (TAMs) into dendritic cell (DC)-like antigen presenting cells that stimulated cytotoxic T cell (CTL)-mediated tumoricidal effects.

To further clarify the actions of CD200S on tumor-immunity, we have established a new distant metastasis model. Subcutaneous transplantation of the original C6 glioma cells into the backs of neonatal Wistar rats was found to cause distant metastasis to the lung by 28 d after birth in the majority of transplanted rats. When transplanted with C6-L cells, all rats developed metastatic lung tumors, while rats transplanted with C6-S cells showed decreased lung metastasis. To elucidate the mechanisms underlying the suppression of distant metastasis, we analyzed tumor cells in the back by flow cytometry, which showed increased expression of chemokines in the C6-S tumors as well as increased accumulation of T lymphocytes and DCs, suggesting CD200S or its related signaling molecules may represent a novel therapeutic intervention to prevent the distant metastasis of solid tumors.

Section snippets

Generating C6 cells stably expressing CD200L or CD200S

C6 cells stably expressing CD200L or CD200S were established as described previously [11]. Cells were cultured in Dulbecco's modified Eagle's medium (Wako, Osaka, Japan), containing 10% fetal bovine serum (FBS; Wako), 4.5 g/L glucose, and a penicillin/streptomycin/amphotericin B mixture (Wako). Original C6 glioma cells and cells transfected with empty vector were cultured in the same conditions.

Wound-healing assay

Confluent C6 cells on glass coverslips placed in 4-well plates were linearly scratched to remove

Distant metastasis to the lung of C6 cells

Four lines of C6 cells (1 × 106) were subcutaneously transplanted into the backs of neonatal Wistar rats. Four lines are C6-original cells (C6-ori), C6 cells transfected with empty vector (C6-empty), C6-L and C6-S cells. Tumor masses became visible 2 weeks after transplantation. A few tumor-bearing rats started to die after 3 weeks, probably due to lung metastases. Fig. 1A shows a rat transplanted with C6-L cells that developed a number of tumor masses in its lung 35 d after transplantation.

Discussion

In this study, we established a distant metastasis model using immunocompetent rats that could easily be prepared with good reproducibility. Although C6 glioma cells are rat cells, the cells were rejected when transplanted 7 d after birth. For better reproducibility, we performed subcutaneous transplantation on neonatal rats within 24 h of birth when their immune system is immature. Kaplan–Meier survival plots showed that there were no differences in death rates at the early time points (22 d

Conflicts of interest

Nothing to declare.

Funding

This study was supported in part by grants from Ehime University (#0503003000) and the Ministry of Education, Culture, Sports, Science, and Technology, Japan (16K20014 to SM; 15K10324 to HT).

Acknowledgements

We are grateful to the staff of the Animal Center of INCS, Ehime University, for their careful and gentle handling of the animals. We thank Dr. Kenji Kameda at the Division of Analytical Bio-Medicine of the Advanced Research Support Center (ADRES), Ehime University for his technical assistance when performing flow cytometry experiments. We also thank James P. Mahaffey, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

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