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286 | Nature | Vol 579 | 12 March 2020

Article

P = 0.017

P = 0.039

MockLD-AET

500

1,000

1,500

Donor-derived cells (%) 0

Mock LD-AET

0.2

0.4

0.6

(^0) Donor-derived cell number
(per 10
6 total cells)
P = 0.018
Mock LD-AET
0.1
0.2
0.3
0
0.4
Donor-derived cells (%)
P = 0.009
MockLD-AET
500
1,000
1,500
Donor-derived cell number^0
(per 10
6 total cells)
CD45.1
CD45
Mock
LD-AET
Ly6G
Ly6C
Mock
LD-AET
Microarray
(^00510)
5
10
15
20
25
0.0
0.5
1.0
1.5
(^0) Mock LD-AET
0.5
1.0
1.5
Relative
Ccr2
mRNA expression
Count
CCR2
Mock
LD-AET
0510
CCL2 concentrations (ng ml–1)
0
10
15
20
25
5
P = 0.01
NS
P < 0.001
P < 0.001
Ly6C
Ly6G
Ly6G
LLC (lung)
HNM007 (lung)
WT Ccr2 KO
(^005101520)
50
100
Lung-metastasis-free
survival (%)
0
50
100
0
50
100
0
50
100
010320 040
0510 15 20 010320 0
Lung-metastasis-free
survival (%)
Days after resection Days after resection
Days after resection Days after resection
WT (n = 10)
Ccr2 KO(n = 10)
WT (n = 10)
Ccr2KO(n = 10)
WT (n = 10)
Ccr2 KO (n = 10)
WT (n = 10)
Ccr2 KO (n = 10)
P < 0.001 P < 0.001
P < 0.001 P < 0.001
Survival (%)
Survival (%)
ab
c
d
e
f
g
0
2
4
6
–log
10
(P
value)
0
2
4
6
Ccr2
401 genes
downregulated
2,188 genes
upregulated
493 genes
downregulated
1,416 genes
upregulated
–3 –2 –1 0123
–3 –2 –1 0123
Fold change (log 2 transformed)
Fold change (log 2 transformed)
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−2
−1
0
1
2
Cx3cr1Ccr2Ccl20Ccr3Cxcl17Ccr4Cxcr5Ccl4Ccr7Cxcr1Ccl24Ccr9Cxcr2Cxcr4Cxcr3Cxcl10Ccr1l1Ccl5Ccr5
Fold change (log
transformed) 2
Chemokines/chemokine receptors of M-MDSCs from bone marrow
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−1
0
1
2
3
Ccr2Ccr6Ccl19Ccl5Cxcr5Cxcr4Ccr7Cxcl9Ccl20Ccl22Cxcr6Cxcl17Ccl27aCxcr1Cxcl11Cxcr7Ccr1Cx3cr1Cx3cl1Ccl9Ccl3Ccl4Cxcl5Ccl6Cxcl14Ccr1l1Ccl8Ccl17Cxcl15Ccr5Cxcl16Cxcl13Ccl2Ccl24Ccl12Cxcl3Ccl7
Fold change (log
transformed) 2
Chemokines/chemokine receptors of M-MDSCs from lung
Lung M-MDSCs
–log
( 10
P value)
BM M-MDSCs
Fold change in migration
Cx3cr1
CD45.1
CD45
Mock
LD-AET
105
104
103
100
100 103104105
105
104
103
100
100 103104105
105
104
103
100
100 103104105
105
104
103
100
100 103104105
105
104
103
0
0103 104 105
105
104
103
0
0103 104 105
0
100
0101102103104
105
104
103
0
–10^3
101102103104105
105
104
103
0
–10^3
101102103104105
105
104
103
0
–10^3
–10^30103104105
105
104
103
0
–10^3
–10^30103104105
Ccr6
Ccr2
Fig. 2 | Low-dose AET inhibits migration of monocytic MDSCs from the bone
marrow to the lung premetastatic microenvironment by downregulating
expression of CCR2. a, b, The effect of low-dose AET on monocytic MDSCs
transferred from CD45.1 to CD45.2 mice (a) and the trafficking ability of
adoptively transferred monocytic MDSCs from low-dose-AET- or vehicle-
treated CD45.1 mice in CD45.2 recipient mice (b). FACS and graphs showing
percentages and absolute numbers of the donor-derived-cell subset (CD45.1+
cells) in both lungs of the recipient LLC mice (n = 3 mice per group). c, Cell-
sorting schema for collecting monocytic MDSCs on day 3 after resection (left).
Volcano plots showing differences in RNA expression of monocytic MDSCs
from the bone marrow (BM) or lung between low-dose-AET- and vehicle-treated
LLC mice (right) (n = 3 biological replicates). d, Agilent cDNA array expression
of chemokines and chemokine receptors in monocytic MDSCs from bone
marrow or lungs of low-dose-AET- and vehicle-treated LLC mice (n = 3 biological
replicates). False-discovery-rate (FDR)-adjusted P values of CCR2 are 0.005
(bone marrow) and 0.016 (lung). C xcr7 is also known as Ackr3. e, Effect of
low-dose AET on Ccr2 expression in monocytic MDSCs from bone marrow
(BM-M-MDSCs) from LLC mice on day 3, by quantitative PCR (left)
(n = 3 biological replicates) and FACS (right). In the FACS plot, red represents
low-dose AET and blue represents mock treatment. f, Transwell migration
assay of sorted monocytic MDSCs from bone marrow of low-dose-AET- or
vehicle-treated LLC mice (day 3) induced by CCL2 for 60 min. Fold changes are
normalized to the migration of the cells in the unstimulated mock group (set
at 1) (n = 3 biological replicates). g, Left, FACS showing representative results of
lung MDSCs in Ccr2-knockout (KO) and wild-type (WT) LLC and HNM007 mice
at day 3. Right, Kaplan–Meier curves showing disease-free survival and overall
survival of Ccr2-knockout and wild-type LLC (top) and HNM007 (bottom) mice.
Two-sided log-rank test. In a–f, two-sample, two-sided t-tests were used. Bars
show mean ± s.e.m.