Nature - USA (2020-01-02)

118 | Nature | Vol 577 | 2 January 2020

Article

subcutaneous tumour growth (Extended Data Fig. 7e–g). MCT1 is thus
able to increase metastasis in at least some melanomas.
We also inhibited MCT1 in mouse melanomas^30 in immunocompetent
C57BL mice (AZD3965 also has activity against mouse MCT1^31 ). MCT1
inhibition by treatment with AZD3965 (Fig. 2j, k) or CRISPR-mediated
deletion (Extended Data Fig. 7a–c) reduced metastatic disease burden
without significantly affecting the growth of subcutaneous tumours.
Human and mouse melanomas thus became more dependent on MCT1
function during metastasis in both immunocompromised and immu-
nocompetent environments.

MCT1 promotes cell survival during metastasis

Inhibition of MCT1 with AZD3965 did not impair the migration or

invasion of melanoma cells in culture (Extended Data Fig. 8a). Acute

treatment with AZD3965 for 7 days in mice with established subcutane-

ous and metastatic tumours did not significantly affect the growth of

subcutaneous or metastatic tumours, but did reduce the frequency of

melanoma cells in the blood (Extended Data Fig. 8b, c). This suggests

that MCT1 inhibition reduced the survival of melanoma cells during

metastasis.

b

0

2

4

6

8

M405 M481 UT10

CellRox DeepRed

(fold change)

DMSO

AZD

DMSO

AZD

DMSO

AZD

c

Days

0.0

0.5

1.0

1.5

2.0

2.5

30 40 50 60

Days

0.0

1.0

2.0

30 40 50 60

d

0

2

4

8

Metastatic burden(relative total flux)

0

1

2

3

4

M405 M481 UT10

CellRox Green(fold change)

5

ef

Metastatic burden(relative total flux)^0

5

20

10

15

0.0

0.5

1.0

Melanoma

cells in blood (%)

1.5

6

0.00

0.05

Melanoma

cells in blood (%)

0.10

DMSO, n = 5

AZD3965, n = 10

NAC, n = 5

AZD3965

+NAC, n = 5

DMSO, n = 10

AZD3965, n = 10

NAC, n = 5

AZD3965

+NAC, n = 5

70

3.0 0.15

diameter (cm)

M481

diameter (cm)

UT10

DMSO

AZDNACAZD

+NAC

CellRox DeepRed-APC

CellRox Green-FITC

a

DMSO AZD

Unst.

DMSO AZD

Unst.

10

P < 0.0001P < 0.0001

P < 0.0001

DMSO

AZD

DMSO

AZD

DMSO

AZD

DMSO

AZDNACAZD

+NAC

DMSO

AZ

D

NACAZ

D

DMSO +NAC

AZ

D

NACAZ

D

+NAC

P = 0.013

P = 0.0038

P < 0.0001

P = 0.0006

P = 0.0008

P < 0.0001

P = 0.0063

P = 0.010

P = 0.0006

P < 0.0001

P = 0.0001

P < 0.0001

17

15

11

15

18

16

17

15 1115

18

16

19 17

11

15

15

11

(^1917)
17
20
15
15
15 15
20
17
Fig. 3 | MCT1 inhibition causes oxidative stress in melanoma cells. a–c,
Representative f low cytometry histograms of ROS levels (a) and fold change in
mean f luorescence intensity (b, c) in melanoma cells from mice treated with
AZD3965 (blue) or DMSO control (black) (two experiments per melanoma).
The number of tumours or mice analysed per treatment is indicated. Unst.,
unstained. d–f, Growth of subcutaneous tumours (d) in xenografted mice
treated with DMSO, AZD3965, N-acetyl cysteine (NAC), or AZD3965 plus NAC,
as well as the frequency of circulating melanoma cells in the blood (e) and
metastatic disease burden based on bioluminescence imaging at end point (f).
Data in e and f reflect three experiments per melanoma, but only one
representative experiment per melanoma is shown in d. Data are mean ± s.d.
Statistical significance was assessed using log 2 -transformed t-tests (b),
Mann–Whitney tests (c), nparLD followed by Benjamini–Hochberg’s multiple
comparisons adjustment (d) and log 2 -transformed one-way ANOVA with
Holm–Sidak’s multiple comparisons adjustment (e, f).
c
Intracellular pH
DMSO
AZD
M405
7.0
7.2
7.6
7.4
8.0
7.8
Intracellular pH
DMSO
AZD
M481
6.0
6.5
7.5
7.0
8.5
8.0
Intracellular pH
DMSO
AZD
UT10
7.0
7.2
7.6
7.4
8.0
7.8
b
m+1 lactate
/m+2 lactate
(ratio)
d
0.0
0.1
0.2
0.3
0.4
M405 M481 UT10
(^4445450)
20
40
NAD
+/NADH
M405
a
0.0
0.2
0.4
0.6
Fractional enrichment
glucose m+2
0.8
DMSO
AZD
M481
Fractional enrichment
0.4
0.2
0.0
03090 180
DMSO, n = 5
e Glucose
Time (min)
Fractional enrichment
0.2
0.1
0.0
03090 180
Fructose-6-P
Time (min)
f g
Fractional enrichment
0.4
0.2
0.0
2-/3-Phosphoglycerate
Fractional enrichment
0.2
0.1
0.0
h Pyruvate
03090 180
Time (min)
03090 180
Time (min)
DMSOAZD DMSOAZD
i
Fractional enrichment
0.2
0.1
0.0
6-Phosphogluconolactone
Fractional enrichment
0.2
0.1
0.0
j 6-Phosphogluconate
03090 180
Time (min)
03090 180
Time (min)
P = 0.085
P = 0.012
P = 0.002
P = 0.008
P = 0.046 P = 0.007 P =
0.0003
P = 0.008 P = 0.001
P =
0.01
P =
0.008
12 14 55 55 55 55
5
5
AZD, n = 5
DMSO
AZD
DMSO
AZD
DMSO
AZD
Fig. 4 | MCT1 inhibition reduces f lux through the oxidative branch of the
PPP relative to glycolysis. a, Glucose m + 2 as a fraction of total glucose in
xenografted tumours after infusion of [1,2-^13 C]glucose (six experiments).
The number of tumours or mice per treatment is indicated. b, The lactate
m + 1/lactate m+2 ratio in subcutaneous tumours from the same mice (two
experiments per melanoma). c, d, Intracellular pH (c) and NAD+/NADH ratio (d)
in dissociated melanoma cells from subcutaneous tumours (one experiment
per melanoma). e–j, Fractional enrichment in glycolytic (e–h) and PPP (i, j)
metabolites, 30, 60 or 180 min after [U-^13 C]glucose infusion (two experiments).
Fructose-6-P, fructose-6-phosphate. Data are mean ± s.d. Statistical
significance was assessed using t-tests (a, b and d), nparLD (c) or repeated
measures two-way ANOVA (e–j).