116 | Nature | Vol 577 | 2 January 2020
Article
diameter, then examined labelling in metabolites extracted from the
blood and tumours. Infusion of uniformly labelled^13 C-glutamine ([U-
(^13) C]glutamine) enriched the circulating glutamine pool and produced
no differences in labelling between efficient and inefficient metastasiz-
ers (Extended Data Fig. 1b, c). Infusion of [U-^13 C]glucose modestly but
significantly increased glucose enrichments in inefficient metastasizers
compared with efficient metastasizers (Fig. 1a), despite no differences
in circulating glucose (Extended Data Fig. 1d, e). For this reason, we
normalized glucose-derived metabolites in the tumour to glucose
m + 6. After this normalization, the labelling of 3-phosphoglycerate
(3PG) was similar between the tumour types, but the efficiently metas-
tasizing tumours had increased labelling of lactate compared with 3PG
(Fig. 1b). In efficient, but not inefficient, metastasizers, the absolute
enrichment in circulating lactate also exceeded the enrichment in
tumour 3PG (Fig. 1c). These labelling features in efficient metastasiz-
ers are similar to some human lung cancers, in which excess lactate
labelling relative to 3PG was explained by the uptake of lactate derived
from infused glucose^17.
Next, we infused [U-^13 C]lactate using conditions that produced
steady-state labelling and abundance in the blood (Extended Data
Fig. 1e, f ), and found no differences in the abundance of tumour lactate
between efficient and inefficient metastasizers (Fig. 1d). To account
for labelling resulting from the transfer of^13 C from lactate to glucose
by gluconeogenesis, followed by glucose uptake and glycolysis in the
tumour, we normalized metabolite labelling to 3PG, which presumably
arises from glycolysis. Lactate enrichment was higher in efficient com-
pared with inefficient metastasizers, and exceeded enrichment in 3PG
or pyruvate (Fig. 1e). These data suggest that efficient metastasizers are
better than inefficient metastasizers at taking up circulating lactate.
Efficient metastasizers also had higher enrichments in metabolites
related to the TCA cycle (citrate, glutamate and malate) (Fig. 1e), which
suggests that^13 C from lactate was transferred to the TCA cycle. Both
efficiently and inefficiently metastasizing melanomas expressed lactate
dehydrogenase (LDH) A and B, indicating their capacity to metabolize
lactate (Extended Data Fig. 1i).
To verify lactate uptake directly, we infused [2-^2 H]lactate. Exchanges
between lactate and pyruvate transfer^2 H to NAD+, resulting in unla-
belled pyruvate (Extended Data Fig. 1h); thus, the appearance of label in
the tumours indicates the uptake of lactate, not pyruvate^17. As expected,
we observed label in tumour lactate but not pyruvate or alanine (Fig. 1f).
Lactate labelling was higher in efficient than in inefficient metastasizers
(Fig. 1f), despite similar labelling in the blood (Extended Data Fig. 1g).
Efficient metastasizers also contained labelled malate (Fig. 1f), which
could arise from the transfer of^2 H from NAD^2 H to malate^17 ,^25 (Extended
Data Fig. 1h).
Higher MCT1 in efficient metastasizers
We observed consistently higher levels of MCT1 in efficient metastasiz-
ers as compared to inefficient metastasizers by western blot analysis
(Fig. 2a; see Extended Data Fig. 2a for quantification). We confirmed this
difference using two other anti-MCT1 antibodies by immunofluores-
cence analysis (Extended Data Fig. 2e–j) and flow cytometry (Fig. 2d, e,
Extended Data Fig. 2c; see Extended Data Fig. 2d for quantification). The
difference in surface MCT1 staining between efficient and inefficient
metastasizers by flow cytometry was particularly notable. Immunofluo-
rescence analysis suggested that MCT1 staining tended to be associated
with the cell surface in efficient metastasizers (Extended Data Fig. 2j),
0.00
0.01
0.02
0.03
0.04
Lactatem+1Pyruvatem+1 Malatem+1
Fractional enrichment
e
3PG
0.0
0.5
1.0
1.5
2.0
3.0
2.5
LactatePyruvateCitrate
Glutamate
Malate
Fractional enrichment
(relative to 3PG)
[U-^13 C]Lactate
b
f
Glucose m+6
0.0
0.5
1.0
1.5
2.0
2.5
3PG m+3
Pyruvate m+3Lactate m+3Citrate m+2Glutamate m+2
Fractional enrichment (relative to glucose m+6)
[U-^13 C]Glucose
Malate m+2
EfficientInefficient
0.0
0.2
0.4
0.6
Fractional enrichment
glucose m+6
a
[2-^2 H]Lactate Efficient, n = 7
Inefficient, n = 9
3.5
Alan
ine
m+1
Efficient, n = 30
Inefficient, n = 36
Efficient, n = 19
Inefficient, n = 38
EfficientInefficient
d
0.2
0.4
0.6
0.8
0.0
Tumour lactate
(μmol mg
–1 protein)
P = 0.011
P = 0.0007
P < 0.0001
P = 0.0047P = 0.0066
P = 0.057
Fractional enrichment
0.0
0.1
0.2
0.3
0.4
0.5
3PG m+3SQ, n
= 30
Lactate m+3
Serum,
n = 303PG m+3
SQ,
n = 36
Lactate m+3
Serum,
n = 36
c Efficient Inefficient
P = 0.010
P = 0.050
P < 0.0001P < 0.0001
P < 0.0001 P^ = 0.0005P = 0.007
P = 0.010
P = 0.0008 P = 0.0001
99
30 36
Fig. 1 | Eff iciently metastasizing melanomas exhibit enhanced lactate
uptake in vivo. Isotope tracing in primary subcutaneous tumours xenografted
in NSG mice with efficiently (M405, M481, M487 and UT10) and inefficiently
(M715, UM17, UM22, UM43, UM47, M498, M528, M597 and M610) metastasizing
melanomas. The number of mice or tumours per treatment is indicated.
a, b, Glucose m + 6 as a fraction of the glucose pool (a) and enrichment of other
metabolites normalized to m + 6 glucose (b) in subcutaneous tumours after
infusion of [U-^13 C]glucose. c, The 3PG m + 3 fraction in subcutaneous tumours
(SQ) and lactate m + 3 fraction in the plasma of mice infused with [U-^13 C]glucose
(20 experiments). d, Tumour lactate concentration (3 experiments).
e, Enrichment of metabolites normalized to 3PG m + 3 in subcutaneous
tumours after [U-^13 C]lactate infusion (23 experiments). f, Isotope labelling
after [2-^2 H]lactate infusion (3 experiments). Data are mean ± s.d. Statistical
significance was assessed using t-tests (a, f), paired t-tests (c), log 2 -transformed
t-tests to compare efficient versus inefficient melanomas or Wilcoxon tests to
compare metabolites (b, e). Multiple comparisons were adjusted using the
Holm–Sidak’s method (b, c, e, f).