Table 1.
Patient clinical parameters and lactate levels in LABC biopsies.
Figure 1.
Human tumor samples measured for lactate with bioluminescence ex vivo show lactate concentrations vary considerably between samples.
Bioluminescence color map for lactate concentrations measured in a LABC patient biopsy (A). Tumor regions are outlined and marked (T). Waterfall Box & Whisker plot for lactate concentrations (μmol/g) measured in benign breast tissue (bb) and LABC patient biopsies (B). Stars indicate separate biopsies from the same patient. LABC1 and LABC10 samples show a significant difference in lactate concentrations (†p<0.05, Student’s T-test). First quartile values represented in red; third quartile values are represented in green. Median values represented at the interface of red and green boxes. Error bars represent the 5th and 95th percentile lactate concentrations. All biopsies n = 3–4.
Figure 2.
R3230Ac cells take up and metabolize lactate to alanine and glutamate in vitro and in vivo.
1H NMR of R3230Ac cell lysates exposed to various concentrations of lactate for 4 h (glucose absent) showed a concentration-dependent lactate uptake (arrows, ∼1.3 and ∼1.6 ppm, A). 13C NMR plot for R3230Ac cell lysate after 12 h treatment with 5 mM 3-13C-lactate (no glucose) showing generation of 13C-labeled glutamate and alanine species (B). Dioxane was included as an internal standard which allowed quantification of labeled metabolites (C). 13C NMR plot from R3230Ac tumor after infusion with equimolar concentrations of universally labeled U-13C-lactate and 1-13C-glucose showing uptake of U-13C-lactate (∼20.75 and ∼21.25 ppm) and generation of U-13C-alanine species (∼16.9 and 17.2 ppm) in the presence of labeled glucose and during production of glycolytically-derived lactate (∼21 ppm) and alanine (∼17.1 ppm) (D). Tissue staining of R3230Ac tumor shows positive expression of MCT1 (green); areas of perfusion are indicated by Hoechst 33342 (blue) (E). Glucose-deprived R3230Ac cells show significantly increased oxygen consumption (n = 3, Student’s T-test, p < 0.05) with increasing concentrations of exogenous lactate compared to the untreated control (F).
Figure 3.
Kinetic analyses of metabolites with radioactive probes show fast plasma clearance of lactate and lactate uptake in perfused regions of R3230Ac tumors.
Plots of individual metabolite and fitted pharmacokinetic standard uptake values (SUV) of 14C-labeled glucose (100 µCi, A) and lactate (50 µCi, B) infused at a rate of 0.1 mL/min data for plasma, subcutaneous tissue (SQ) and R3230Ac tumor tissue over 160 minutes. R3230Ac tumors, grown in the flanks of Fischer 344 rats, show clearance of 14C labeled glucose (n = 6) from plasma from 6 SUV to 2 SUV over 40 mins and maximum uptake of 14C labeled glucose in the tumor after 16 mins (A). 14C labeled lactate (n = 3) was cleared from the plasma (from 6 SUV to 2 SUV) in 14 mins and showed maximum uptake in the tumor after 14 mins (B). SUV = standard uptake rates. Autoradiography images (D-F) of 14C-lactate uptake in R3230 Ac tumors show high lactate uptake (C&E) in well-perfused areas, as indicated by positive Hoechst 33342 staining (blue, D&F), compared to hypoxic tumor regions, as indicated by positive pimonidazole staining (orange, D&F). R3230Ac tumors (G) show presence of 13C-lactate, 13C-alanine and 13C-glutamate at 15, 30 and 60 minutes after 13C-lactate infusion. All 13C metabolites are increased compared to baseline levels (prior to infusion). 13C-lactate uptake and 13C-metabolite generation in brain (H) and liver (I) after 13C-lactate infusion show a slight increase in metabolites compared to baseline but do not reach concentrations found in R3230Ac tumors.
Table 2.
Kinetic Transfer Rates for 14C-glucose and 14C-lactatein R3230Ac Tumors.
Figure 4.
Lactate uptake and metabolism in human breast cells and metabolite excretion.
13C NMR spectra of human breast lines indicated evidence of 13C-lactate (arrow, 19 ppm) uptake after 24 h exposure to 10 mM 13C-3-lactate (A). Lactate measurements of cell media after 5 day incubation with 20 mM unlabeled sodium lactate in glucose-free media showed a significant difference in lactate consumption between MCF7 and MDA-MB-231 cells (mean overall decrease in lactate concentrations were 0.4 mM for the no-cell media control plate, 5.5 mM for MDA-MB-231 media and 18.3 mM for MCF7 media, n = 5, *p < 0.001 compared to MDA-MB-231 and media control, Student’s T-test, B). The increase in cell number of MCF7 and MDA-MB-231 cells at the beginning (day 0) and end (day 5) of lactate treatment show no significant difference (C). Heteronuclear multiple quantum coherence (HMQC) NMR plots of cell lysates treated for 24 h (n = 2) with 10 mM 3-13C-lactate (no glucose) showed 13C-lactate (dark green “L”) uptake and 13C-glutamate (blue “G”), 13C-alanine (orange “A”), and 13C-pyruvate (red “P”) generation in HMEC (D), MCF7 (E), and MDA-MB-231 cells (F). 13C NMR spectra of R3230Ac, MCF7 and MDA-MB-231 cell lysates (bottom) and media (top) show evidence of 13C-metabolites in the media of each cell line and in the lysate of MCF7 and MDA-MB-231 cells after 24 h incubation with 40 mM lactate (G). Numeric labels: 1 = 13C-Glu γ, 2 = 13C-Glu β, 3 = methyl 13C- lactate, 4 = methyl 13C-Ala
Figure 5.
Inhibition of exogenous lactate uptake and endogenous lactate excretion with addition of CHC.
All experiments represented were carried out in glucose-deprived conditions. 13C spectra of R3230Ac cell lysates incubated for 4 h with 5 mM 13C-lactate without CHC treatment shows peaks corresponding to lactate (“3”), alanine (“4”) and glutamate (“1”) (A, top). 13C spectra of R3230Ac cell lysates incubated for 4 h with 5 mM 13C-lactate with 5 mM CHC treatment show no peaks corresponding to lactate or metabolites (A, bottom). 13C spectra of R3230Ac cell lysates incubated for 4 h and 24 h with 40 mM 13C-lactate + 5 mM CHC shows a peak corresponding to lactate (arrow) but no other metabolites (B). 13C spectra of R3230Ac cell media incubated with 5 mM 13C-lactate for 4 h shows peaks corresponding to alanine and glutamate without CHC treatment (C, top); metabolite peaks are absent or smaller with 5 mM CHC (C, bottom). 1H spectra of R3230Ac cell lysate with the following treatments: 20 mM 13C-lactate + 0.1 mM CHC for 4 h (D) 20 mM 13C-lactate + 5 mM CHC for 4 h (E), 20 mM 13C-lactate + 0.1 mM CHC for 24 h (F), 20 mM 13C-lactate + 5 mM CHC for 24 h (G). 13C spectra of R3230Ac cell media with the following treatments: 20 mM 13C-lactate + 0.1 mM CHC for 4 h (H) 20 mM 13C-lactate + 5 mM CHC for 4 h (I), 20 mM 13C-lactate + 0.1 mM CHC for 24 h (J), 20 mM 13C-lactate + 5 mM CHC for 24 h (K).Endogenous lactate = green “Len”, endogenous alanine = orange “Aen”, 13C-lactate = dark green “13C-L”, 13C-glutamate = blue “13G”, DMSO = brown “D”
Figure 6.
Cell viability as measured by Annexin V –/7-AAD – labeling (n = 3) in MCF and R3230Ac cells treated with high (5 mM) or low (0.1 mM) CHC with and without glucose (no lactate) and with 40 mM lactate (with and without glucose). Viable MCF7 cells show significant decreases with 5 mM CHC (A, #p≤0.004 compared to control (no CHC, no lactate + glucose), ‡‡p < 0.01 compared to 40 mM lactate-treated MCF7 cells, *p = 0.003 compared to all no CHC and low CHC groups). Percentage of apoptotic (Annexin V+/7-AAD-) MCF7 cells show significant increases with the –glucose-lactate+high CHC treatment and the +glucose+lactate+high CHC treatment (#p < 0.05) and between the –glucose–lactate+high CHC group and 40 mM lactate treatments without CHC (+ or – glucose) (†p < 0.05, B). Percentage of MCF7 cells with loss of membrane integrity (Annexin V–/7-AAD+) show no significant differences with CHC treatment (C). Percentage of MCF7 cells marked for both cell death pathways (Annexin V+/7-AAD+) show significant increases with 5 mM CHC compared to the no CHC and low CHC groups (**p < 0.05, D). Percentage of viable R3230Ac cells show significant decreases with 5 mM CHC compared all no CHC and low CHC groups (*p < 0.008, E). Percentage of apoptotic (Annexin V+/7-AAD-) R3230Ac cells show significant increases with 5 mM CHC compared to compared to no CHC and low CHC groups (*p < 0.02, F). Percentage of R3230Ac cells marked for loss of membrane integrity (Annexin V–/7-AAD+) show no significant differences except–glucose-lactate+high CHC treatment (‡p < 0.05, G). Percentage of R3230Ac cells marked for both cell death pathways (Annexin V+/7-AAD+) show no significant changes with any treatment (H). Results analyzed with One-Way ANOVA and Bonferroni/Dunn post-hoc tests.
Figure 7.
Hypoxic R3230Ac cells take up 13C-lactate.
Treatment with 4013C-lactate in glucose-deprived, normoxic R3230Ac cancer cells result in lactate uptake and metabolism after 12 h (A). Under hypoxic conditions, evidence of lactate uptake (arrow) but no additional labeled metabolites can be seen in the cell lysates after 12 h (B). Arrow: 13C-methyl lactate.
Figure 8.
Summary diagram of lactate metabolism in high lactate-consumers vs. low lactate-consumers.
The blue gradient represents oxygen diffusion. The cell on the left is hypoxic; the cells on the right are aerobic. Arrow colors correspond to substrates, and arrow size corresponds to relative amount. Hypoxic tumor cells take up glucose (gray “Glc”) and produce lactate (dark green “L”), leading to higher concentrations of lactate. Lactate may be taken up by the hypoxic tumor cells, but it is not catabolized. Aerobic tumor cells that are high lactate-consumers and likely express high MCT1/low MCT4 can take up lactate and catabolize it to alanine (orange “A”) and glutamate (blue “G”), which will be exported from the cell. With the aerobic high lactate-consumer cell consuming lactate, more glucose can potentially be spared for hypoxic tumor cell use, potentially conferring a survival advantage. Aerobic tumor cells that are low lactate-consumers and likely express low MCT1/high MCT4 take up less lactate than the high lactate-consumers, consequently producing and exporting less alanine and glutamate. Low lactate-consumers utilize more glucose, which will not allow glucose to reach the hypoxic tumor cells (A). One proposed strategy for starving hypoxic tumor cells of glucose in a high lactate-consuming tumor is to treat with a MCT1- inhibitor, like CHC. CHC prevents lactate uptake and catabolism in cells, forcing the aerobic high lactate-consumer to use glucose, which starves the hypoxic tumor cell of glucose. Lactate transport out of hypoxic cells is also inhibited, which would also lead to hypoxic cell death (B).