Bidirectional effect of CD200 on breast cancer development and metastasis, with ultimate outcome determined by tumor aggressiveness and a cancer-induced inflammatory response

Abstract

CD200 acts through its receptor (CD200R) to inhibit excessive inflammation. The role of CD200-CD200R1 interaction in tumor immunity is poorly understood. In this study, we examined the role of CD200-CD200R1 interaction in the progression and metastasis of highly aggressive 4THM murine-breast carcinoma using CD200 transgenic (CD200(tg)) and CD200R1 knock-out (CD200R1(-)(/-)) BALB/c mice. 4THM cells induce extensive visceral metastasis and neutrophil infiltration in affected tissues. CD200 overexpression in the host was associated with decreased primary tumor growth and metastasis, whereas lack of CD200R1 expression by host cells was associated with enhanced visceral metastasis. Absence of CD200R1 expression led to decreased tumor-infiltrating-cytotoxic T cells and increased the release of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-6. In contrast, CD200 overexpression led to increased tumor-induced interferon-γ and IL-10 response and decreased TNF-α and IL-6 release. Neutrophil infiltration of tissues was markedly decreased in CD200(tg) animals and increased in CD200R1(-/-) mice. These findings are contradictory to what has been reported in the EMT6 mouse breast-cancer model. Other distinguishing features of tumor elicited by EMT6 and 4THM cell injections were also examined. Visceral tissues from mice bearing EMT6 tumors showed a lack of neutrophil infiltration and decreased IL-6 release in CD200R1(-/-) mice. EMT6 and 4THM cells also differed in vimentin expression and in vitro migration rate, which was markedly lower in EMT6 tumors. These results support the hypothesis that CD200 expression can alter immune responses, and can inhibit metastatic growth of tumor cells that induce systemic and local inflammatory response. Increasing CD200 activity/signaling might be an important therapeutic strategy for treatment of aggressive breast carcinomas.Oncogene advance online publication, 29 September 2014; doi:10.1038/onc.2014.317.

Full text

ORIGINAL ARTICLE
Bidirectional effect of CD200 on breast cancer development
and metastasis, with ultimate outcome determined by tumor
aggressiveness and a cancer-induced inflammatory response
N Erin
1
, A Podnos
2
, G Tanriover
3
, Ö Duymuş
1
, E Cote
2
, I Khatri
2
and RM Gorczynski
2
CD200 acts through its receptor (CD200R) to inhibit excessive inflammation. The role of CD200-CD200R1 interaction in tumor
immunity is poorly understood. In this study, we examined the role of CD200-CD200R1 interaction in the progression and
metastasis of highly aggressive 4THM murine-breast carcinoma using CD200 transgenic (CD200
tg
) and CD200R1 knock-out
(CD200R1
−/−
) BALB/c mice. 4THM cells induce extensive visceral metastasis and neutrophil infiltration in affected tissues. CD200
overexpression in the host was associated with decreased primary tumor growth and metastasis, whereas lack of CD200R1
expression by host cells was associated with enhanced visceral metastasis. Absence of CD200R1 expression led to decreased tumor-
infiltrating-cytotoxic T cells and increased the release of inflammatory cytokines, such as tumor necrosis factor-α(TNF-α) and
interleukin (IL)-6. In contrast, CD200 overexpression led to increased tumor-induced interferon-γand IL-10 response and decreased
TNF-αand IL-6 release. Neutrophil infiltration of tissues was markedly decreased in CD200
tg
animals and increased in CD200R1
−/−
mice. These findings are contradictory to what has been reported in the EMT6 mouse breast-cancer model. Other distinguishing
features of tumor elicited by EMT6 and 4THM cell injections were also examined. Visceral tissues from mice bearing EMT6 tumors
showed a lack of neutrophil infiltration and decreased IL-6 release in CD200R1
−/−
mice. EMT6 and 4THM cells also differed in
vimentin expression and in vitro migration rate, which was markedly lower in EMT6 tumors. These results support the hypothesis
that CD200 expression can alter immune responses, and can inhibit metastatic growth of tumor cells that induce systemic and local
inflammatory response. Increasing CD200 activity/signaling might be an important therapeutic strategy for treatment of aggressive
breast carcinomas.
Oncogene advance online publication, 29 September 2014; doi:10.1038/onc.2014.317
INTRODUCTION
CD200 is a widely expressed cell surface glycoprotein that belongs
to the immunoglobulin family and regulates inflammation in
autoimmunity, transplantation and viral infections.
1
CD200 func-
tions through its receptor, CD200R, which is expressed mostly by
myeloid cells,
2,3
and some subsets of lymphoid-derived cells.
2
There are five described CD200R isoforms in mice, CD200R1–R5;
CD200R1 is reported to mediate the anti-inflammatory effects of
CD200.
4
The role of CD200-CD200R1 interaction in tumor
immunity is poorly understood. CD200 expression by leukemic
tumor cells inhibits activation of tumor-specificT cells in vitro.
5
Treatment with anti-CD200 antibody can enhance tumor rejection
by peripheral blood mononuclear cells (PBMC) in a hu-SCID
adoptive transfer model.
6
Although multiple studies have documented tumor-promoting
effects of CD200 in leukemia,
7–9
there are conflicting results
regarding its role in solid tumors. CD200 expression is associated
with increased metastatic survival of squamous cell carcinoma,
10
and CD200 expression by human basal carcinoma is associated
with tumor-initiating properties.
11
On the other hand, CD200
expression by melanocytes results in decreased lung metastasis,
and CD200R activation by an agonistic monoclonal antibody
(OX110) is associated with decreased CD200-negative melanoma
tumor formation in the lungs.
12
These conflicting results regarding
the role of CD200 in cancer progression and metastasis may be
due to the bidirectional role of immune activation in cancer.
Chronic activation of the immune system may induce carcino-
genesis and metastasis by creating an inflammatory micro-
environment, which increases DNA damage and malignant
transformation.
13
Once malignant transformation occurs, cancer
cells may also induce a chronic inflammatory response in the host,
which enhances the metastatic process.
13
In this study, we examined the role of CD200 in a murine model
of breast cancer. The 4T1 breast carcinoma represents an
inflammatory, aggressive, triple-negative breast cancer, originally
obtained from a spontaneously formed breast cancer in a BALB/c
mouse.
14
We previously reported that heart metastases of
4T1 cells, named 4THM, grow more aggressively and induce a
systemic inflammatory response.
15–17
Recently, we have shown
that more than 50% of 4THM cells express CD44 and are CD24
negative, under in vitro conditions, which is a feature of ‘stemness’.
Primary tumors formed by 4THM cells are positive for vimentin,
another marker of cancer stem cells (CSCs).
18
The role of CD200 in
breast cancer progression and metastasis has so far only been
studied in an EMT6 breast carcinoma model, where CD200
overexpression resulted in increased lymph node metastasis.
19,20
1
Department of Medical Pharmacology, School of Medicine, Akdeniz University, Antalya City, Antalya, Turkey;
2
University Health Network, Toronto General Hospital, Toronto, ON,
Canada and
3
Department of Histology and Embryology, School of Medicine, Akdeniz University, Antalya, Turkey. Correspondence: N Erin, Department of Medical Pharmacology,
School of Medicine, Akdeniz University, B-blok kat 1 Immunoloji, Antalya 07070, Turkey.
Email: nerin@akdeniz.edu.tr
Received 1 March 2014; revised 2 August 2014; accepted 9 August 2014
Oncogene (2014), 1–11
© 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14
www.nature.com/onc

To our knowledge, there have been no reports examining the
effects of CD200 on visceral metastasis in an inflammatory, highly
aggressive breast cancer model. Here, we examined the effects of
CD200 on tumor growth, metastasis and anti-tumor immune
responses to orthotopically-injected 4THM breast carcinoma cells
using CD200
tg
and CD200R1
−/−
BALB/c female mice. We report
that overexpression of CD200 by the host is associated with
decreased metastatic growth of 4THM cells, the opposite of the
results seen using EMT6 breast tumors.
RESULTS
Growth of primary tumors and lung/liver metastasis after injection
of 4THM breast carcinoma cells is attenuated in hosts
overexpressing CD200
Wild type (WT) and CD200
tg
mice were injected with 4THM cells
(10
5
cells/mouse) orthotopically, with transgene induction using
doxycycline (DOX)-water beginning 2 days before tumor injection.
Primary tumor growth and visceral metastases were evaluated
24 days after the injection. As shown in Figure 1, 4THM primary
tumor growth was attenuated in CD200
tg
mice, although all
animals developed visible tumors at the site of injection by day 12.
Primary tumors regressed completely in 3/7 CD200
tg
mice with
~ 50% regression of the primary tumor observed in an additional
animal (days 24 vs 12). Lung metastases were also reduced in
CD200
tg
mice, consistent with the altered pattern of primary
tumor growth (Figure 1). Since macroscopic nodules were clearly
observed in lung tissues following fixation with Bouins solution
and correlated with the microscopic results,
18
only the macro-
scopic findings are shown in Figure 1. However, microscopic
analysis of lung tissues was performed in the three CD200
tg
mice
in which primary tumors had completely regressed and no visible
lung nodules were detected. No microscopic lesions were seen in
lung tissues of these animals (Supplementary Figure 1: micro-
scopic lung metastasis of WT and CD200R1
−/−
mice is shown).
Liver metastases were evaluated microscopically and analyzed
to determine the extent of metastases as well as the frequency of
metastatic lesions. As shown in Figure 1d, the degree of
metastases was significantly decreased in CD200
tg
mice compared
with WT mice. We have analyzed the data in the two groups of
CD200
tg
mice with regression/nonregression of their primary
tumors to determine whether liver metastases were detected in
each. We observed occasional metastatic lesions in the liver
tissues of mice with regression of primary tumors, but an overall
decrease in metastasis was observed (data not shown). The levels
of liver metastases in the CD200
tg
mice whose primary tumor
persisted throughout the study were similar to those seen in WT
mice, suggesting that CD200 over expression in the host resulted
in the attenuation of growth of lung metastasis more effectively
than liver metastasis.
Visceral metastasis of 4THM breast carcinoma was increased in
CD200R1
−/−
mice
Lack of CD200R1 expression increased liver and lung metastasis in
CD200RKO compared with both WT and CD200
tg
mice. The extent
of metastasis and the frequency of lesions in the liver tissue were
also increased in CD200R1
−/−
mice (Figure 1d). The rate and extent
of primary tumor growth, on the other hand, was not significantly
different among WT and CD200R1
−/−
animals (Figure 1b).
Flow cytometric analysis of CD45, CD200, CD3, CD8, CD4, Gr-1,
CD11b and F4/80 expression by tumor infiltrating immune cells
and draining lymph node cells
Lack of CD200 expression in CD200
−/−
mice has been reported
to result in increased CD200R expression by myeloid cells.
21
Interestingly, 12 days after 4THM cell injection, the percentage of
CD200-expressing tumor infiltrating CD45
+
cells was decreased in
CD200R1
−/−
animals compared with WT, along with a fourfold
decrease in the number of CD3
+
CD8
+
tumor infiltrating T cells in
the same mice (Figure 2a). However, the percentage of CD4
+
cells
did not differ among the groups, and the percentage of GR1
+
,
F4/80
+
and CD11b
+
cells was also unchanged (data not shown).
Levels of CD3
+
CD25
+
cells in draining lymph nodes (DLNs) of
CD200R1
−/−
animals injected with 4THM breast carcinoma cells
(1.54%) were decreased ~ twofold in comparison with levels in WT
animals (3.95%; see Figure 2b). This was similar to data from
primary tumors at 12 days following 4THM injections (0.61% vs
1.82%, respectively; see Figure 2b).
To verify that aberrant expression of leukocyte markers on
4THM tumor cells themselves did not interfere with the reported
results, we examined CD45, CD3, CD200R and CD200 staining on
4THM cells grown in vitro. No cell-surface expression of CD3, CD45,
CD200 or CD200R1 was detected on 4THM cells growing in vitro,
nor was CD200 and CD200R expression detected by immuno-
histochemistry on 4THM and EMT6 primary tumors themselves
grown in WT mice (Supplementary Figure 2). Data in
Supplementary Figure 2 (Panels a, c, d and e) show, by
comparison, similar staining using primary tissue from mice
injected with a less aggressive EMT6 tumor.
19
Using liver tissue
from WT mice bearing 4THM tumors as positive control, we
observed, as expected, membranous staining of hepatocytes with
CD200, with no expression on metastatic lesions within the liver
tissue (Supplementary Figure 2b).
In the terminal stages of tumor growth (day 24) when animals
started to die and primary tumors became necrotic, most of the
cells obtained from tumor digestion were granulocytes, as
determined by GR-1 staining, with 64.6 ± 6.6 and 60.5 ± 2.3
GR-1
+
cells in the tumor tissue of WT and CD200R1
−/−
animals,
respectively. At this stage of primary tumor growth no significant
differences for any other markers were observed between the
different groups.
Increased tumor-induced IFN-γresponses in mixed leukocyte
cultures using cells from tumor-bearing CD200
tg
mice
Mixed leukocyte cultures (MLCs) were prepared 12 and 24 days
after 4THM injection, as described in Materials and methods, with
control MLCs using cells from mice not injected with 4THM. Day
12 was chosen, because 4THM cells grow sufficiently to start
metastasizing by this time, and the immune response to tumor
cells would be evoked by day 12; day-24 MLCs were used to assess
changes in the immune response in the presence of extensive
disease. MLCs were stimulated either with lipopolysaccharide
(LPS) or irradiated 4THM cells. IFN-γresponses were measured
40 h. after challenge.
The 4THM-induced IFN-γresponse was significantly higher in
MLCs of 4THM-injected CD200
tg
animals compared with WT and
CD200R1
−/−
mice at day 12 (Figure 3). In contrast, LPS-induced
IFN-γsecretion was higher in CD200R1
−/−
and CD200
tg
animals
compared with WT. In control non-tumor-bearing mice, the levels
of IFN-γinduced by LPS in CD200
tg
, CD200R1
−/−
and WT animals
were similar; challenge with irradiated 4THM cells did not induce
IFN-γsecretion.
Irradiated 4THM cells induced IFN-γresponses only in CD200
tg
animals at day 24, although this response was attenuated
compared with day 12. There was no LPS-induced IFN-γresponse
in WT and CD200
tg
animals at day 24, and again the LPS-induced
IFN-γresponse in CD200R1
−/−
animals at this time was diminished
compared with day 12. All of the above are consistent with a
generalized attenuation of IFN-γproduction in all mice at day 24.
Attenuation of LPS-induced interleukin-10 secretion in MLCs of
CD200
tg
mice 24 days after 4THM injection
Unstimulated MLCs of tumor-bearing animals secreted significant
amounts of interleukin (IL)-10 (50 −200 pg/ml), which was highest
Effect of CD200 on breast cancer development
N Erin et al
2
Oncogene (2014), 1 –11 © 2014 Macmillan Publishers Limited

Mean # of Lung Metastases/Mouse
0
10
20
30
40
50
WT CD200R1KO CD200tg
*
$
Mean weight of primary tumors
(g/mouse)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
WT CD200R1KO CD200tg
*
WT
CD200tg with
tumors
CD200tg with
regressed
tumors
Microscopic Liver Metastases
0
2000
4000
6000
8000
10000
12000
CD200R1KO
*
$
CD200R1KO
CD200tg
WT
Metastatic lesions Neutrophil infiltration
*
*
*
Figure 1. Changes in tumor growth and metastasis of 4THM cells in CD200
tg
and CD200R1
−/−
mice. (a) changes in the number of lung
nodules per mouse. *
,$
Po0.05 significantly different from WT mice. (b) changes in primary tumor growth
*
Po0.05 significantly different from
WT and CD200R1
−/−
mice. (c) changes in levels of microscopic liver metastases per mouse. *
,$
Po0.05 significantly different from WT mice.
(d) microscopic appearance of liver metastasis and neutrophil infiltration.
Effect of CD200 on breast cancer development
N Erin et al
3
© 2014 Macmillan Publishers Limited Oncogene (2014), 1 –11

in CD200R
−/−
mice (Figure 3c). In MLCs prepared 12 days
after 4THM injection, IL-10 production following LPS challenge
was significantly higher from cells of both CD200R1
−/−
and
CD200
tg
animals compared with WT, whereas irradiated 4THM
cells did not induce significant IL-10 secretion from any group
(Figure 3c). However, by 24 days after tumor injection, only
CD200R1
−/−
mice, but not WT or CD200
tg
, produced IL-10 after
LPS challenge.
LPS and irradiated 4THM cells induced IL-6 secretion by host
immune cells, which was suppressed in MLCs of CD200
tg
mice
24 days after 4THM tumor cell injection
MLCs from all the tumor-bearing animals secreted significant
amounts of IL-6 without challenge compared with control animals
not bearing a tumor. Challenge with LPS or irradiated 4THM cells
further enhanced IL-6 secretion in tumor-bearing animals 12 days
after injection of tumor cells (Figure 4a). Challenge with irradiated
4THM cells induced significantly higher levels of IL-6 secretion in
MLCs of CD200
tg
mice compared with WT or CD200R1
−/−
hosts at
day 12. Interestingly, IL-6 responses induced by both 4THM or LPS
challenge, as well as the basal cytokine secretion, were preserved/
elevated in MLCs of WT and CD200R1
−/−
mice, respectively,
24 days post tumor injection, but were essentially abolished in
CD200
tg
animals at this time (Figure 4b).
Attenuation of tumor necrosis factor-αsecretion in cells from
CD200
tg
compared with WT or CD200RKO mice
Basal tumor necrosis factor (TNF)-αsecretion was below the limits
of detection in all tumor-bearing animals and challenge with
irradiated 4THM cells did not induce TNF-αsecretion in MLCs of
tumor-bearing or non-tumor-bearing mice. Challenge with LPS
induced higher levels of TNF-αsecretion in WT and CD200R1
−/−
tumor-bearing animals compared with non-tumor-bearing mice,
or tumor-bearing CD200
tg
animals at day 12. By day 24 after 4THM
injection MLCs of CD200
tg
animals failed to secrete TNF-αin
response to LPS. MLCs of CD200R1
−/−
mice showed a similar
TNF-αproduction at day 24 as compared with that observed at
day 12, whereas MLCs of WT animals secreted less TNF-αat day 24
compared with day 12 (Figure 4c).
CD8-PE.CY7
CD3-PE
CD8-PE.CY7
CD3-PE
CD200-PE
CD200R1KO
WT
WT-tumor CD200R1KO-tumor
Tumor
2.32% 0.59%
CD3-PE
CD25-FITC
WT-tumor
CD25-FITC
CD3-PE
CD3-PE
CD25-FITC
CD3-PE
CD25-FITC
WT-LN
CD200R1KO-tumor
CD200R1KO-LN
1.82% 0.61%
3.95% 1.54%
Figure 2. Flow cytometric analysis of immune cells within the primary tumor tissue and DLNs. (a) expression of CD200 on CD45
+
cells within
tumor tissue; CD3
+
CD8
+
cells infiltrating primary tumors from WT and CD200R1
−/−
mice. (b) CD3
+
CD25
+
cells within 4THM primary tumors as
well as DLNs of WT and CD200R1
−/−
mice.
Effect of CD200 on breast cancer development
N Erin et al
4
Oncogene (2014), 1 –11 © 2014 Macmillan Publishers Limited

γ
γ
*
*
*
*
*
*
*
Figure 3. IFN-ɣ(a,b) and IL-10 (c,d) secretion from MLCs prepared from WT, CD200
tg
and CD200R1
−/−
mice bearing 4THM tumors for 12 (a,c)
and 24 (b,d) days. None indicates unstimulated MLCs; LPS-MLCs stimulated with LPS for 40 h; 4THM-MLCs stimulated with irradiated 4THM
cells for 40 h. *Po0.05 significantly different from corresponding WT MLCs.
α
*
*
*
**
Figure 4. IL-6 (a,b) and TNF-α(c) secretion from MLCs prepared from WT, CD200
tg
and CD200R1
−/−
mice bearing 4THM tumors for 12 and
24 days. a,bshow IL-6 secretion at days 12/24, respectively. None indicates unstimulated MLC; LPS- MLC stimulated with LPS for 40h;
4THM- MLC stimulated with irradiated 4THM cells for 40 h. (): TNF-αsecretion from MLCs stimulated with LPS for 20 h. * o0.05 significantly
different from corresponding WT MLCs.
Effect of CD200 on breast cancer development
N Erin et al
5
© 2014 Macmillan Publishers Limited Oncogene (2014), 1 –11

Table 1 shows a compilation of the various parameters
discussed in Figures 1 −5, indicating tumor growth, metastasis
and cytokine production from the various mice used.
Significant differences in growth of, and host responses to, EMT6
and 4THM tumors
Examination of mice injected with 4THM or the less aggressive
EMT6 breast cancer showed numerous differences in growth rate
(Figures 5a and c) and host responses such as systemic
leukocytosis (Figures 5d and f). Cells from tumor explants
(Figures 5g and h) of 4THM injected mice spontaneously released
10 −20 times more TNF-αand IL-6 into culture media than
explants from EMT6 injected mice (Figure 5i), although interest-
ingly WT, and not CD200R1
−/−
mice, released more IL-6 after
challenge by EMT6 cells in MLC (Figure 5j) unlike the reverse
situation with 4THM tumor bearers (Figures 4a and b). We also
observed a ⩾twofold increase in the numbers of CD200
+
cells in
DLN of WT mice bearing EMT6 tumors compared with mice
bearing 4THM tumors (Figure 5k). Microscopic examination of
lung and liver tissues from EMT6 tumor-bearing animals failed to
demonstrate tumor metastasis (Supplementary Figure 3), unlike
4THM tumor bearers, where lung tissue was also found to stain
strongly for S100A8 infiltrating cells (Supplementary Figure 4A).
Interestingly, further comparison of primary tumor tissue of both
4THM and EMT6 tumor-injected mice showed an absence of
S100A8 staining by either 4THM or EMT6 tumor cells themselves
(Supplementary Figure 4B), but marked infiltration of 4THM
tumors by cells staining for S100A8, whereas S100A8
+
immune
cells were observed in the vascular structures of liver tissue of
both 4THM and EMT6-injected animals (Supplementary Figure 4C),
with more pronounced tissue infiltration by S100A8
+
cells in the
4THM-injected animals.
EMT6 and 4THM cells were also observed to differ in expression
of vimentin, and in their in vitro migration rate. Higher vimentin
expression was observed in 4THM primary tumors (Supplementary
Figure 4D) correlating with their faster migration in vitro than
EMT6 cells (Supplementary Figure 5). EMT6 cells were observed
to migrate in clusters, which would make invasion through
vascular structures less feasible, whereas 4THM cells migrated as
single cells.
DISCUSSION
Cancer is a heterogeneous disease in which the outcome and
response to treatment is determined by the tumor microenviron-
ment, systemic and local host responses, and intrinsic features of
the cancer cells. Given this complexity, it has proven difficult to
define a single and a definitive role for any given molecule. CD200
seems to be one of a number of molecules with a bidirectional
role in cancer development and metastasis. CD200 is known to
exert both anti-inflammatory and immunosuppressive effects. We
hypothesize that in the 4THM model described, CD200 exerts a
potent anti-tumoral/antimetastatic effect on inflammation driven
carcinogenesis and metastasis. Given that the degree of
inflammation correlates with the degree of aggressiveness and
treatment resistance in most carcinomas, CD200 agonists may
thus exert potent anti-tumoral and anti-metastatic effects.
In support of the above hypotheses CD200 overexpression in
CD200
tg
mice was associated with decreased metastasis of the
highly aggressive breast carcinoma cells (4THM), whereas lack of
CD200R1 expression resulted in significantly increased lung and
liver metastasis, consistent with the prediction that the protective
effects of CD200 were, at least partly, mediated by CD200:
CD200R1 interactions.
4
Lack of CD200R1 expression by the host
was associated with decreased numbers of tumor infiltrating CD8
+
and CD3
+
CD25
+
T cells, and increased production of inflammatory
cytokines including TNF-αand IL-6. In contrast, CD200 over-
expression by CD200
tg
hosts resulted in increased tumor-induced
IFN-γand decreased TNF-αand IL-6 responses.
Cancer-related inflammation is a hallmark of cancer
22
and many
cases is increased in parallel with tumor growth.
23
4THM breast
carcinoma cells induce extensive visceral metastasis and neutro-
phil infiltration of affected tissues, although neutrophil infiltration
occurred independently of metastatic lesions. Neutrophil infiltra-
tion of tissues was markedly decreased in CD200
tg
animals and
increased in CD200R1
−/−
animals, in accordance with the
proposed anti-inflammatory role of CD200.
1,2,4
Table 1. Comparison of tumor growth/metastases and cytokine production in WT, CD200RKO and CD200
tg
mice
WT CD200RKO CD200TG
Lung metastasis (nodule/mouse) 14.33 ±2.64 39.87 ±6.7 3.5 ±1.2
Liver metastasis (microscopic
μm/area)
2241 ±370 8972.46 ±1599 2371.8 ±370 (P(+))
223 ±85 (P(-))
Primary tumor growth (g/mouse) 1.05 ±0.08 1.16 ±0.14 0.31 ±0.16
Neutrophil infiltration of metastatic
tissue
++++ ++++++ ++
IFN-ɣ(pg/ml) 12 days 294 ±77.5(LPS)
327 ±55.8(T)
1617.08 ±126.8(LPS)
427.01 ±48.97(T)
1203.11 ±349.39(LPS)
1325.6 ±114.89(T)
IFN-ɣ(pg/ml) 24 days 65.85 ±27.68(LPS)
29.05 ±11.66(T)
86.96 ±36(LPS)
54.63 ±11.22(T)
54.1 ±20.47(LPS)
176.07 ±77.66(T)
IL-10 (pg/ml) 12 days 256.06 ±35.26(LPS)
65.64 ±10.74(T)
599.91 ±54.03(LPS)
138.32 ±15.9(T)
733.63 ±84.47(LPS)
141.16 ±11.69(T)
IL-10 (pg/ml) 24 days 172.61 ±55.86(LPS)
22.97 ±5.36(T)
548.96 ±54.36(LPS)
28.66 ±3.4(T)
54.52 ±32.42(LPS)
84.02 ±54.52(T)
TNF-α(pg/ml) 12 days 752.59 ±72.91(LPS) 753.65 ±73.11(LPS) 357.68 ±13.32(LPS)
TNF-α(pg/ml) 24 days 288.61 ±123.44(LPS) 835.30 ±31.76(LPS) o7
IL-6 (pg/ml) 12 days 885.95 ±61.8(LPS)
653.67 ±115.52(T)
919.53 ±117.07(LPS)
669.65 ±97.56(T)
1095.73 ±166.16(LPS)
1122.31 ±228.55(T)
IL-6 (pg/ml) 24 days 711.29±251.46(LPS) 1637.38 ±110.29(LPS) 101.56 ±18.97(LPS)
600.20 ±233.70(T ) 1382.47 ±151.62(T ) 96.73 ±48.75(T )
P(+), mice with persistent primary tumors; P(-), mice with regression of primary tumors. Cytokines were measured in MLC 12 and 24 days after injectionsof
4THM cells; LPS, indicates levels in MLCs with LPS stimulation; T, indicates levels in MLCs after stimulation with irradiated 4THM. In the absence of challenge
with LPS or tumor cells, cytokine levels were either low or below the limits of detection in all groups.
Effect of CD200 on breast cancer development
N Erin et al
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Oncogene (2014), 1 –11 © 2014 Macmillan Publishers Limited

4THM cells are not only very aggressive and induce local and
systemic inflammatory response, but they also include many
properties of CSCs.
18
Recent studies demonstrated that CSCs
might be responsible for the induction of local and systemic
inflammation. CSCs are involved in tumor initiation and display
increased metastatic potential.
24
Breast CSCs have been reported
to express chemokines, including interleukin-8 (IL-8, as well as
inflammatory cytokines, including TNF-αand IL-6. In our study,
4THM cells have been observed to secrete high levels of MIP-2
(the murine equivalent of IL-8: EN et al., manuscript in prepara-
tion). Inflammatory mediators originating from CSCs can alter the
tumor microenvironment and induce local inflammatory response,
which can further potentiate the growth of CSCs.
25
IL-6 is one of the main mediators of inflammation-induced
stemness in breast cancer.
26,27
IL-6 has also been linked to
metastasis through induction of EMT, increased cell invasion and
migration, and recruitment of mesenchymal stem cells.
28,29
Interestingly, LPS and 4THM tumor-induced secretion of IL-6 was
progressively increased in WT and CD200R1
−/−
mice and reached
its greatest level in the late stages of disease (Figures 4b vs 4a).
IL-6 secretion was observed from lymphocytes of tumor-bearing
WT and CD200R1
−/−
animals even in the absence of additional
challenge in MLCs. In contrast, IL-6 secretion was essentially
abolished by 24 days after inoculation of tumor cells in CD200
tg
animals, although IL-6 secretion 12 days after inoculation of
CD200
tg
mice with tumor cells were similar to levels observed in
Spleen
4THM injected
EMT6-injected
Cells / Area (40x magnification)
0
10
20
30
40
4THM
EMT6
Neutrophils Lymphocytes Monocytes
**
*
Primary Tumor
4THM EMT6
Spleen Primary tumor
Weight (gram/mouse)
0.0
0.5
1.0
1.5
2.0
2.5
4THM
EMT6
*
*
4THM EMT6
20
40
60
600
900
*
*
IL-6
TNF
α
pg/ml cytokine in
culture supernatant
4THM EMT6
IL-6 (pg/ml)
0
500
1000
1500
2000
2500
3000
3500
WT
CD200R1KO
Un-stimulated EMT6-stimulated
Draining Lymph Nodes
CD200
4THMEMT6
CD200
*
*
Figure 5. Growth and cytokine production from 4THM and EMT6 injected mice. (a−c) increased growth rate of EMT6 (aand c), and spleen
weight in 4THM-injected animals (band c). d−fgreater numbers of neutrophils (black arrow), monocytes (black arrowhead) and lymphocyes
(white arrow) in 4THM-injected animals (dand e; peripheral blood smears). (f) shows the mean number of immune cells per area (n=4 mice).
(gand h) appearance of 4THM and EMT6 tumor explants (harvested at 20d) 72 h after plating and immediately before harvesting CM for
cytokine analysis. (i) cytokine production (24h cultures) of explants (see g,h) of primary 4THM or EMT6 tumors in WT mice. (j) IL-6 secretion
from MLCs from mice with EMT6 tumors. MCLs were either unstimulated or stimulated with irradiated EMT6 cells for 40 h. *Po0,05
significantly different from corresponding WT MLCs. (k) CD200
+
cells (Flow cytometry (FACS) staining) in DLNs of 4THM and EMT6-tumor
bearing WT mice.
Effect of CD200 on breast cancer development
N Erin et al
7
© 2014 Macmillan Publishers Limited Oncogene (2014), 1 –11

WT mice. This might imply that the ‘early’inflammatory IL-6
response observed in WT, CD200
tg
and CD200R1
−/−
mice is
responsible for fostering initial tumor growth, whereas further
propagation and metastasis of tumor depends upon the absence
of CD200-CD200R1 interactions which will otherwise contribute
to an anti-inflammatory response providing effective immune
surveillance.
30
Taken together, these findings are also in
accordance with clinical studies that demonstrated increased
serum concentrations of IL-6 in breast cancer patients are strongly
associated with tumor stage and poor prognosis.
31
TNF-α, a major pro-inflammatory cytokine, has been shown to
induce stem cell-like features in breast cancer cells.
32
TNF-α
mediates interactions between tumor and stromal cells, leading to
an upregulation of genes implicated in tumor cell growth, survival,
invasion, metastasis, inflammatory cell trafficking to the tumor site
and neoangiogenesis.
33
Tumor-pomoting effects of TNF-αwere
demonstrated in triple negative breast carcinoma,
34
and clinical
studies have also documented an important role for TNFα
in advanced breast carcinoma resistant to conventional
treatments.
35
As reported above, we found that WT animals
bearing 4THM tumors produced TNF-αin response to LPS
challenge, an effect which was even more pronounced in
CD200R1
−/−
mice in the late stages of disease. In contrast, in
CD200
tg
mice, negligible LPS-induced TNF-αproduction occurred
by 24 days post-tumor injection. In all mice no detectable TNF-α
was observed following 4THM challenge using cells harvested at
either day 12 or 24 post tumor injection, although IL-6 production
was observed under these circumstances (see above). This may
reflect a different threshold for induction of these inflammatory
cytokines by tumors growing in situ. In all cases however, the
persistence of LPS-induced cytokine production in WT and
CD200R1
−/−
mice at day 24 suggests the absence of a generalized
‘tolerance’to an inflammatory stimulus within the tumor
environment at this time period in the same animals.
IFN-γis produced mainly by natural killer cells and specific T-cell
subsets that play a critical role in an antitumor immune
response.
36
IFN-γinduces activation of effector macrophages,
which can lyse tumor cells, increase antigen presentation,
and inhibit angiogenesis,
37
all of which are consistent with an
anti-IFN-γprotective effect in the model described. Thus we
observed that 4THM-induced IFN-γproduction was greatest in
MLCs of 4THM-injected CD200
tg
animals compared with WT and
CD200R1
−/−
mice at day 12, and by day 24 irradiated 4THM cells
were capable of inducing IFN-γresponses only in cells from
CD200
tg
animals, consistent with increased growth in WT and
CD200R1
−/−
.
IL-10 can be secreted by both Th1 and Th2 cells and has a dual
role on tumor development and carcinogenesis.
38
IL-10 has been
reported to function as an anti-anti-inflammatory molecule
preventing tumor development in animal models of chronic
inflammation-induced carcinogenesis,
39
and inducing regression
of established breast cancer metastases.
40
Conversely, IL-10 has
been associated with tumor progression, and tumor IL-10 levels
correlate with disease severity.
38
Interestingly, our data showed a
biphasic effect of CD200 expression on IL-10 secretion during
tumor growth. Thus in CD200
tg
mice IL-10 production induced by
LPS/4THM was increased relative to unstimulated controls at
12 days post 4THM injection, but was essentially abolished by
24 days after injection, whereas persistence of LPS-induced IL-10
production was seen at 24 days in WT and CD200R1
−/−
mice. We
speculate (see also above) that CD200 expression may be most
important in later stages of tumor growth in this model (as IL-10
production declines) when it can later function as an alternative
(to IL-10) modulator of anti-tumoral immune responses in
carcinomas characterized by excessive inflammation.
Intra-tumoral CD8 T-cell infiltration is associated with delayed
recurrence and extended survival in patients,
41
and infiltration of
breast cancer with CD8
+
cytotoxic T lymphocytes is associated
with a good response to chemotherapy.
42
Somewhat surprisingly,
infiltration of ER
(−)
breast cancer with Treg cells has been reported
to be associated with increased anti-tumor immunity and a more
favorable prognosis.
43
In a similar vein, we observed increased
metastasis in the absence of CD200-CD200R signaling, a putative
immunosuppressive signaling interaction, in association with a
decreased number of CD8
+
and CD4
+
CD25
+
T cells within the
tumor tissue. It is important to note, however, that we cannot
exclude the possibility that the altered phenotype of T-cells within
the tumor reflects a direct role for CD200:CD200R1 interactions in
regulation of cell migration per se, rather than an indirect one on
the environmental milieu (chemokines/cytokines and so on). Such
an interpretation is amenable to experimental investigation using,
for instance, adoptive transfer of WT (CD200R1
+
T cells) into
CD200R1
−/−
tumor-bearing mice to investigate the effect of
possible replenishment of CD8
+
tumor-infiltrating cells in such
circumstances-such studies are currently underway.
The effects of CD200-CD200R interactions on development and
metastasis of mouse 4THM breast carcinoma seemed to be the
opposite of what was observed previously with EMT6 breast
tumor cells.
19,44
Importantly, EMT6 tumors induce a systemic
inflammatory response to a far lesser degree than 4THM tumors
(see Figures 5d–f and I, comparing inflammatory cytokine
production and systemic leukocytosis) and do not express
vimentin (Supplementary Figure 4C) which is a marker for EMT
and increased metastatic potential.
45
In agreement with these
observations we observed (Supplementary Figure 5) that the
migration ability of EMT6 cells was less than that of 4THM cells,
and EMT6 cells migrated in clusters, which makes invasion
through vascular structures less feasible, whereas 4THM cells
migrated as single cells. Consistent with these differences,
metastasis is more pronounced for 4THM than EMT6 tumors.
These different features in turn might in part contribute to the
differences observed in the host’s response to the tumor. Thus the
IL-6 response in MLCs for cells from EMT6 tumor bearers was
greater in WT mice compared with CD200R1
−/−
animals,
suggesting that despite a lack of CD200R1 signaling, an
accelerated inflammatory response did not occur in EMT6 tumor
bearers. This reduced IL-6 response might in turn explain the
decreased EMT6 tumor growth and metastasis observed in
CD200R1
−/−
animals.
19,44,46
Immunoediting of cancer cells has
also been reported to lead to EMT and a more aggressive
phenotype,
47
and thus we predict that EMT6 tumors are likely to
be more immunogenic/less immunoedited. We suggest that
CD200 expression increases the malignant potential of tumors
that are highly immunogenic and poorly metastatic, whereas
acting to inhibit the growth of highly aggressive carcinomas
which present with extensive local and systemic inflammation.
Interestingly, S100A8, a calcium binding protein expressed by
cells of myeloid lineage, has been reported to induce proin-
flammatory chemokines both intracellularly and extracellularly.
Murine S100A8, also called CP-10, is a potent chemotactic factor
for neutrophils, and induces sustained leukocyte recruitment
in vivo.
48
More recently, evidence for S100A8 induction of
chemokines in lung tissue was correlated with increased invasive
and migratory abilities of tumor cells.
49
Carcinoma patients
express higher serum S100A8 protein concentrations than
adenoma patients,
50
and increased S100A8 expression was
associated with lung metastasis.
51
In a recent study, Liu et al.
52
reported that bone marrow-derived CD11b
+
Gr1
+
myeloid cells
expressing S100A8 were increased in number in premetastatic
brain lesions of mice bearing 4T1 tumors, and treatment of 4T1
tumor-bearing mice with the cyclooxygenase-2 inhibitor attenu-
ated inflammatory chemokine expression, brain infiltration by
CD11b
+
Gr1
+
cells and formation of brain metastasis.
The data shown above and in supplementary figures are
consistent with these observations, and show that premetastatic
liver and lung tissue of mice injected with highly inflammatory
Effect of CD200 on breast cancer development
N Erin et al
8
Oncogene (2014), 1 –11 © 2014 Macmillan Publishers Limited

4THM tumors are heavily infiltrated with S100A8
+
cells
(Supplementary Figures 3 and 4).
In conclusion, comparison of the data above with those
previously observed in the EMT6 breast cancer model, suggests
that the effects of CD200-CD200R1 interaction on tumor progres-
sion in these models may reflect differences in immunogenicity
and metastatic potential, and the capacity of the tumor cells
themselves to induce chronic inflammation. The prognosis of
triple negative breast cancers with a cancer stem cell phenotype is
poor and effective treatments are lacking. 4THM cells originally
derived from triple negative murine breast carcinoma cells
(4T1).
14,17
The results reported in this manuscript indicate that
systemic exposure to CD200 or CD200R1 agonists may prevent
metastasis as well as local growth of this aggressive breast cancer.
Inflammatory breast carcinoma is a highly aggressive, poorly
differentiated malignancy that is associated with resistance to
treatment and poor survival rates. The 4THM model mimics many
aspects of inflammatory breast carcinoma, including the presence
of breast CSCs, epithelial mesenchymal transition, extensive
visceral metastases, lack of estrogen receptor expression and an
undifferentiated state.
18
Our data are the first to suggest that
CD200-CD200R1 interactions can decrease progression of meta-
static inflammatory breast carcinoma, and taken together with our
previous observations in an EMT6 tumor model, imply that
modulating CD200 activity/signaling might be an important
therapeutic strategy for the treatment of breast tumors, even in
those characterized by increased systemic and local inflammatory
response.
MATERIALS AND METHODS
Mice
Wild-type (WT) female BALB/c mice were purchased from the Jackson
Laboratories (Bar Harbor, ME, USA). Mice were housed five per cage under
specific pathogen-free conditions and were allowed food and water ad
libitum. All mice were used at 8–12 week of age. All animal experimenta-
tion was performed following the guidelines of an accredited animal care
committee. The derivation of CD200R1
−/−
and CD200
tg
mice, both on a
BALB/c background was described previously.
19
Homozygous CD200
tg
females received DOX (1 μg/ml) in their drinking water (DOX-water) to
induce CD200 expression.
53
Cell lines
4T1 breast cancer cells were previously derived from a spontaneously
appearing breast tumor in a BALB/c female mouse. The 4THM cell line
was derived from cardiac metastases of 4T1 cells by EN et al.
16,54
4THM
and EMT6 cells were grown in α-minimum essential medium (α-MEM)
supplemented with penicillin and streptomycin and 10% fetal bovine
serum.
Metastasis assay
4THM cells (1 ×10
5
cells/mouse) or EMT6 cells (5 × 10
5
cells/mouse) were
injected orthotopically into the right upper mammary gland of 8–10 week
female recipients. Necropsies were performed either 12 or 24 days after
injection, with lung tissues stored in Bouin’sfixative to visualize
macroscopic nodules, as described before.
17
Liver tissue was originally
fixed in 10% buffered formalin and sectioned after embedding in paraffin
wax. Five sections from each tissue were stained with hematoxylin and
eosin to determine the extent of metastases microscopically. For each
animal, 20 photographs from 5 different sections were randomly taken at
20x magnification; areas of microscopic metastatic lesions were then
selected and measured as mm
2
with Spot advanced 4.6 software
(Diagnostic instruments, Inc 2006, Sterling Heights, MI, USA).
In some experiments (as noted in the text) equal numbers of EMT6 and
4THM cells (1x10
5
/mouse) were injected into mammary fat pad of mice
and animals were killed 20 days later. Blood samples were obtained before
kiling to prepare peripheral blood smears stained with hematoxylin and
eosin. Neutrophil, lymphocyte and monocyte numbers in the peripheral
blood were enumerated using 10 slides at 40x magnification from each
animal.
MLCs
Spleen and DLNs of animals injected with 4THM cells were removed
aseptically and single-cell suspensions prepared for MLCs. Cells
(4 × 10
6
/well in 48-well tissue culture plate) were cultured alone (control
for basal cytokine release) or stimulated with LPS 3 μg/ml or irradiated
(20 Gray) 5 × 10
4
4THM cells. MLCs from control animals (not injected with
4THM cell) were also prepared. TNF-αlevels were measured 20 h after
challenge, whereas measurements of IFN-γ, IL-6 and IL-10 were performed
in supernatants obtained 40 h after challenge. Standard ELISA kits were
used for cytokine measurements (for IL-6, TNF-αand IL-10, from BD
Biosciences (Mississauga, ON, Canada); for IFN-γfrom eBioscience (San
Diego, CA, USA)).
Analysis of cytokine expression in tumor tissue of tumor-bearing
mice
Tumor tissue was minced into small pieces in medium (1 −2 mm), passed
through an 18-gauge needle to dissociate the tumor cells and plated in
24-well plates. Dulbecco's modified Eagle medium: nutrient mixture F-12
(DMEM-F12) medium containing 0.1% FBS was added to cultures for 24 h
three days after plating to obtain CM. IL-6 and TNF-αlevels were
determined in CM using enxyme linked immunosorbant assay (ELISA).
Antibodies and cell staining
All anti-mouse monoclonal antibodies used for cell-surface phenotype
characterization (CD200-PE, CD200R-FITC, CD45-PeCy.7, CD3-FITC, CD3-
APC, CD8-PE, CD8-FITC, CD8-PeCy.7, CD4-FITC, CD4-PE, CD25-PeCy.7,
GR-1-FITC, CD11b-PE and F4/80-PeCy.7) were purchased from BioLegend
(San Diego, CA, USA). Multicolour flow cytometric analyses were conducted
to characterize DLN cells, splenocytes, and tumor infiltrating cells. Tumors,
DLNs and spleens were digested as described before.
44
The optimal
concentration of antibody for staining was titrated individually for each
antibody. Single colour controls were included in each experiment for
compensation purposes, as well as fluorescence-minus-one (FMO) controls;
all samples were analyzed in a CANTO-II flow cytometer, using FloJo
software (Ashland, OR, USA). Samples from 2 −3 mice were pooled per
staining group and staining was performed in triplicate.
In vitro migration assay
Migration assays were performed using an Oris Cell Migration Assembly Kit
(Platypus Technologies, Madison, WI, USA, Cat No: CMAUFL4). Cells were
seeded in 5% FBS in the presence of stopper, which was removed 16 h
later, and initial detection zones were photographed (time 0). 18 h later,
photographs of the detection zone were taken. Areas of cell movement
were marked as μm using Spot advanced 4.6 programme. Experiments
were repeated twice with four replicates in each experiment.
Immunohistochemistry
Primary tumor tissue was fixed in 10% formalin and embedded in paraffin.
5μm thick serial sections were collected. Staining with heterologous rabbit
antibodies for mouse CD200 and CD200R (prepared after immunization in
Freund’s adjuvant with protein purified from supernatants of CHO cells
transduced to express the respective cloned genes: Gorczynski et al.
unpublished), S100A8 (GTX62287, Genetex, Irvine, CA, USA) and Vimentin
antibody (1/100 dilution; Milipore Corporation, Billerica, MA, USA; Cat No.
AB5733) was performed essentially as described elsewhere.
18
Statistics
Student’st-test was used to compare metastatic indexes. When the
variance expressed as s.d. between the groups differed, the Welch t-test or
non-parametric (Mann–Whitney test) was used. Analysis of variance with
Dunnett’s post test was used for the analysis of cytokine results. P-values
o0.05 were considered biologically significant. Statistical analyses were
performed using GraphPad InStat 3 software (San Diego, CA, USA).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Effect of CD200 on breast cancer development
N Erin et al
9
© 2014 Macmillan Publishers Limited Oncogene (2014), 1 –11

ACKNOWLEDGEMENTS
This work was supported by funds from the TÜBİTAK (the scientific and technological
research council of Turkey; project no: 109S449); Akdeniz University Research Units,
Antalya, Turkey (project no: 2013.12.0103.001); and the Canadian Cancer Society
(grant to RMG). We thank Nilüfer Ekinci for her technical help.
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