Biochemical and Biophysical Research Communications
Truncated CD200 stimulates tumor immunity leading to fewer lung metastases in a novel Wistar rat metastasis model
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.
References (22)
- et al.
Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function
Immunity
(2000) - et al.
CD200+ and CD200- macrophages accumulated in ischemic lesions of rat brain: the two populations cannot be classified as either M1 or M2 macrophages
J. Neuroimmunol.
(2015) - et al.
A truncated form of CD200 (CD200S) expressed on glioma cells prolonged survival in a rat glioma model by induction of a dendritic cell-like phenotype in tumor-associated macrophages
Neoplasia
(2016) - et al.
Expansion and activation of CD103(+) dendritic cell progenitors at the tumor site enhances tumor responses to therapeutic PD-l1 and BRAF inhibition
Immunity
(2016) - et al.
CD8alpha(+) dendritic cells are the critical source of interleukin-12 that controls acute infection by Toxoplasma gondii tachyzoites
Immunity
(2011) - et al.
Down-regulation of the macrophage lineage through interaction with OX2 (CD200)
Science
(2000) - et al.
The unusual distribution of the neuronal/lymphoid cell surface CD200 (OX2) glycoprotein is conserved in humans
Immunology
(2001) - et al.
Characterization of the CD200 receptor family in mice and humans and their interactions with CD200
J. Immunol.
(2003) - et al.
CD200 expression on tumor cells suppresses antitumor immunity: new approaches to cancer immunotherapy
J. Immunol.
(2007) - et al.
Importance of CD200 expression by tumor or host cells to regulation of immunotherapy in a mouse breast cancer model
PLoS One
(2017)