Nature 2020 01 30 Part.02

c

0102030

0

20

40

60

80

100

Days

Percent surviva

l

IC GFP-mRNA

IC PD1 CTLA4 TIM3

IC VEGF-C-mRNA + PD1 CTLA4 TIM3

P = 0.01

P = 0.001

* **

B16

0102030

0

20

40

60

80

100

Days

Percent surviva

l

FT IC GFP-mRNA

FT IC VEGF-C-mRNA

FT IC GFP-mRNA + PD1 CTLA4 TIM3

P = 0.01 FT IC VEGF-C-mRNA + PD1 CTLA4 TIM3

P = 0.45

P = 0.65

*

B16

0102030

0

20

40

60

80

100

Days

Percent surviva

l

IC VEGF-C-mRNA + PD1 CTLA4 TIM3

FT IC VEGF-C-mRNA + PD1 CTLA4 TIM3

P = 0.24

B16

Tumor kinetics

(^00510152025303540)
100
200
300
400
500
Days
Tumor
Vo
lume (m
(^3) m
)
VEGF-C-mRNA
(^00510152025303540)
100
200
300
400
500
Days
Tumor
Vo
lume (m
(^3) m
)
GFP-mRNA
b
0510152025303540
0
100
200
300
400
500
Days
Tumor
Vo
lume (m
(^3) m
)
GFP-mRNA + PD1 CTLA4
0510152025303540
0
100
200
300
400
500
Days
Tumor
Vo
lume (m
(^3) m
)
VEGF-C-mRNA + PD1 CTLA4
0510152025303540
0
100
200
300
400
500
Days
Tumor
Vo
lume (m
(^3) m
)
Ligation + VEGF-C-mRNA + PD1 CTLA4
YUMMER1.7 YUMMER1.7 YUMMER1.7
YUMMER1.7 YUMMER1.7
de
f
CD45.1-APC
CD45.2-BU
V737
Resident
Tr ansfer
Recipient
FSC-A
SSC
Lymphocytes
FSC-H
FSC-W
Lymphocytes
Single cells
SSC-H
SSC-W
Single cells
Single cells-2
CD45-PE
SSC-W
ResidentCirculating
Single cells-2
CD8-BUV395
CD3-APC-Cy7
CD3+CD8-
CD3+
CD8+
CD3-
Tr ansfer/Recipient
g
VEGF-C-mRNA/GFP-mRNA
Intracisternal injection
YUMMER1.7 YUMMER1. 7
a
Extended Data Fig. 10 | Therapeutic application of VEGF-C mediates
protection against intracranial melanoma cell lines when combined with
various checkpoint inhibitors. a, Schematic of experimental design for
Fig. 4a–c and b–e. b, Growth kinetics of f lank tumours from Fig. 4a–c were
measured using a caliper (n = 12 for all groups except the ligation group, n = 7).
c–e, Mice were given either only B16 intracranial tumours (IC) (c) or both a B16
intracranial tumour and a B16 f lank tumour (FT) (d) and treated with GFP mRNA
or VEGFC mRNA on day 7, and anti-PD-1 (RMP1-14), anti-CTLA4 (9H10) and anti-
TIM3 (RMT3-23) on days 7, 9 and 11. f, Flow cytometry gating strategy for Fig. 4e,
f. g, Schematic of VEGF-C-induced tumour rejection. P < 0.05; P < 0.01;
P < 0.001; ****P < 0.0001 (two-sided log-rank Mantel–Cox test).