Lactate metabolism in human lung tumors

[U-¹³C]glucose infusion reveals two major patterns of ¹³C enrichment in human lung tumors

We used a previously-described protocol to characterize human NSCLC through pre-surgical imaging, intra-operative infusion of ¹³C-labeled nutrients, and post-surgical analysis of metabolic, molecular and histological features of tumor and adjacent lung (Hensley et al., 2016). Table S1 contains clinical information from the 35 patients in this cohort, including 9 from our previous study (Hensley et al., 2016). The tumors included adenocarcinoma (n=28), squamous cell carcinoma (n=3), large cell neuroendocrine carcinoma (n=1) and carcinoid tumors (n=3). A sequencing panel to assess oncogenes and tumor suppressor genes commonly mutated in NSCLC, including KRAS, EGFR, PIK3CA, BRAF, STK11 and TP53 confirmed molecular heterogeneity in the cohort (Table S1).

Thirty of these patients were infused with [U-¹³C]glucose. Metabolites extracted from benign lung revealed a progressive reduction in ¹³C enrichment downstream of glucose (Figure 1A). This pattern suggests dilution of ¹³C enrichment by the utilization of unlabeled nutrients. A similar pattern was observed in a subset of tumors, which as a group were practically indistinguishable from metabolite labeling in adjacent lung (Figure 1A). However, many tumors displayed a different pattern of ¹³C enrichment in which lactate had excess labeling relative to upstream glycolytic intermediates like 3-phosphoglycerate and phosphoenolpyruvate (3PG and PEP) (Figure 1A). Hereafter, we refer to these two phenotypes as Low and High Lactate/3PG labeling ratios. Labeling patterns in adjacent lungs did not differ between tumors with High and Low Lactate/3PG labeling ratios (Figure S1A). Complete ¹³C isotopologue distributions of all metabolites analyzed in each fragment are in Table S2.

Our protocol includes ligation of the pulmonary artery prior to surgical resection. Although tumor and adjacent lung tissue continue to receive blood flow from the bronchial artery and collateral vessels, occlusion of the pulmonary artery could in principle temporarily impact metabolism and ¹³C labeling. One of the tumors in our cohort could not be resected because it was affixed to the chest wall. In this patient, [U-¹³C]glucose was infused intravenously during a needle biopsy of the mass without first occluding blood flow. Metabolites extracted from the resulting 3 mg biopsy had a High Lactate/3PG labeling ratio similar to the ratio in tumors resected surgically (Figure 1B). These findings demonstrate that a) as previously suggested (Hensley et al., 2016), the labeling observed in these tumors is unrelated to perturbations of blood flow accompanying pulmonary artery clamping; and b) needle biopsies contain sufficient tissue for labeling analysis, thereby expanding the clinical contexts in which this type of data may be obtained.

Because the Lactate/3PG labeling ratio was heterogeneous, we sought to correlate this feature with biological factors that might influence tumor metabolism (Figure 1C-I). EGFR-mutant tumors had homogeneous Lactate/3PG labeling ratios similar to adjacent lung, whereas the KRAS-mutant group was more heterogeneous and included tumors with ratios far in excess of lung (Figure 1C). Similarly, adenocarcinomas with predominantly acinar and non-mucinous histology tended to be homogeneous with low ratios, whereas some tumors with papillary, solid and large cell neuroendocrine features had much higher ratios (Figure 1D). Histopathology revealed that high-ratio tumors had lower microvessel densities (Figure 1E), but there were no differences in MIB1/Ki67 staining or stromal content (Figure 1F, S1B).

At the time of infusion, the tumors differed in their degree of local invasiveness, with some tumors displaying no invasion and others having already spread to regional lymph nodes or the pleural cavity. None of the patients had evidence of distant metastasis, which would have contraindicated resection. The Lactate/3PG labeling ratio did not correlate with invasion or spread at the time of infusion (Figure 1G). Remarkably, however, this ratio did correlate with tumor progression over several years of follow up (Figure 1H). Stage I and stage II tumors that recurred in the lung or gave rise to distant metastases had, on average, higher Lactate/3PG labeling ratios at the time of the original [U-¹³C]infusion and surgery.

FDG-PET signal (SUV_max, SUV_peak, SUV_mean) correlated positively with ¹³C enrichment in 3PG, lactate and the Lactate/3PG labeling ratio, whereas clinical factors like age and smoking were not correlated (Figure 1I). Many tumors displayed regional heterogeneity in FDG-PET signal (Figure 1J), and in some but not all cases, areas of higher signal correlated with higher Lactate/3PG labeling ratios (Figure 1J). Altogether, these data indicate heterogeneity of the Lactate/3PG labeling ratio among human NSCLC tumors and within individual tumors.

Labeled glycolytic products in the blood contribute to tumor metabolism

A high Lactate/3PG labeling ratio is consistent with uptake from the circulation of labeled 3-carbon intermediates distal to 3PG in glycolysis. As previously reported (Hensley et al., 2016), our infusions produce measurable levels of plasma [U-¹³C]lactate (Figure 2A). We also detected plasma [U-¹³C]pyruvate and [U-¹³C]alanine (Figure 2A). Because labeling of plasma lactate and pyruvate exceeded labeling of tumor 3PG, uptake of one or both could contribute to a high Lactate/3PG labeling ratio.

We next used metabolic flux analysis (MFA) to explore the contribution of circulating lactate and related metabolites to the tumors. Quantitative MFA in cultured cells usually relies on definitive import/export rates (e.g. glucose import/lactate export) to constrain intracellular fluxes (Buescher et al., 2015). In vivo, estimating relative fluxes can provide an overview of metabolic activity even if definitive rates cannot be quantified (Hensley et al., 2016). We constructed a metabolic network to fit ¹³C mass isotopologues from plasma glucose and lactate, and tissue glucose, lactate, 3PG, PEP and TCA cycle intermediates (Figure 2B, Table S3). The model incorporates enzymes that convert 4-carbon TCA cycle intermediates to PEP and pyruvate to explain labeling patterns in those 3-carbon intermediates. Such enzymes could include malic enzyme (malate→pyruvate) and phosphoenolpyruvate carboxykinase (Oxaloacetate→PEP). The model does not exclude contributions from the pentose phosphate pathway, which could also produce complex labeling in 3-carbon intermediates. We also examined lactate exchanges between the plasma and tissue. In lungs and tumors with Low Lactate/3PG labeling ratios, these exchanges fit within a narrow range consistent with net lactate export (Figure 2C). However, tumors with high Lactate/3PG labeling ratios were more difficult to fit, with a much wider range of acceptable fluxes, including values consistent with net lactate import (Figure 2C). Although this model cannot define quantitative lactate exchange rates or the net direction of flow in vivo, it indicates that tumors with Low and High Lactate/3PG labeling ratios differ with respect to lactate transport, with ¹³C features of the latter group consistent with lactate import from plasma.

To assess expression of transporters and enzymes involved in taking up and metabolizing lactate, we ranked all tumors from the lowest to highest Lactate/3PG labeling ratio (Figure 2D). Several tumors below and above the median were chosen for western blot analysis. MCT1, MCT4, and lactate dehydrogenase isoforms A and B (LDHA and LDHB) were broadly expressed in these fragments, but the levels of all four proteins tended to be elevated in fragments with a high ratio (Figure 2E).

Metabolism of circulating lactate/pyruvate by human NSCLC

We next infused five NSCLC patients with ¹³C-lactate (one with [2-¹³C]lactate and four with [U-¹³C]lactate). The sterile, pyrogen-free [¹³C]lactate used in some of the infusions contained a small amount of [¹³C]pyruvate, which we estimated at 0.003% of the [¹³C]lactate concentration (Figure S2A). The material was introduced using a bolus followed by a continuous infusion during anesthesia, similar to the [U-¹³C]glucose infusions. Although the amount of lactate introduced was small relative to the total circulating pool, we monitored for signs of metabolic acidosis. Three patients were modestly acidotic, but in all cases the acidosis was respiratory in origin, as indicated by elevated partial pressure of CO₂ in arterial blood (Figure 3A). The acidosis rapidly resolved with increased mechanical ventilation. Plasma lactate and pyruvate concentrations were not significantly perturbed (Figure S2B).

These infusions resulted in essentially steady state labeling of circulating lactate in the 20-25% range (Figure 3B). Pyruvate and alanine were also labeled to appreciable levels, as expected because lactate exchanges with pyruvate and pyruvate exchanges with alanine. Labeling in pyruvate modestly exceeded labeling in lactate. This likely arose from a combination of the infused ¹³C-pyruvate, which had a proportionally large effect on the small circulating pyruvate pool, and excess dilution of lactate by unlabeled lactate secreted from the muscle and other tissues. Labeled glucose was also detected, indicating that infused ¹³C-lactate was used for gluconeogenesis (Figure 3B, S2C).

In the tumors, comparing labeling in 3PG to labeling in lactate, pyruvate, alanine and TCA cycle intermediates allowed us to infer whether ¹³C entry occurred predominantly through uptake of glucose or of three-carbon intermediates (Figure 3C). Inspection of tumor metabolites revealed barely detectable labeling in 3PG and PEP, consistent with the scarce labeling in circulating glucose (Figure 3D). However, the tumors contained measurable label in lactate, pyruvate, alanine and TCA cycle intermediates, most of which were more enriched than 3PG (Figure 3D). These findings indicate the propensity of these tumors to take up one or more 3-carbon glycolytic products from the circulation and use them to supply the TCA cycle.

Subcutaneous and orthotopic NSCLC xenografts use plasma lactate as a carbon source

We next turned to mouse models to more directly assess the role of lactate and related metabolites in live tumors. Mice bearing orthotopic lung or subcutaneous xenografts from H460 NSCLC cells were infused with [U-¹³C]glucose using a protocol analogous to the human studies (Marin-Valencia et al., 2012). These infusions resulted in labeling of 40% of circulating glucose as [U-¹³C]glucose (Figure 4A; isotopologues from mouse experiments are in Table S4). Plasma glucose contained m+1, m+2, m+3 and m+5 labeling in addition to the expected m+6, indicating that gluconeogenesis from labeled 3-carbon intermediates occurred during the experiment (Figure S3A). Plasma lactate, pyruvate and alanine were also labeled (Figure 4A). Healthy lung tissue from these mice displayed a low Lactate/3-PG labeling ratio, similar to human lung (Figure 4B,C). In contrast, the tumors displayed enhanced labeling in lactate, pyruvate and alanine relative to 3PG, regardless of the site of the tumor (Figure 4B,C). Subcutaneous xenografts of HCC827 and HCC15 NSCLC cells also displayed high Lactate/3PG labeling ratios after infusion with [U-¹³C]glucose (Figure 4C).

We next infused mice with [¹³C]lactate, [¹³C]pyruvate or [¹³C]alanine to assess the contribution of each nutrient. In mice bearing H460 tumors, infusion with [3-¹³C]lactate resulted in high levels of ¹³C labeling in tumor lactate and TCA cycle intermediates relative to 3PG, particularly in tumors grown in the lung (Figure 4D,E). These infusions did not significantly increase plasma lactate concentrations (Figure S3B) and resulted in steady-state plasma lactate enrichments of 23.5 ± 0.01%. HCC827 and HCC15 subcutaneous xenografts also displayed high Lactate/3PG labeling ratios under this protocol (Figure 4E). Infusion of either [U-¹³C]pyruvate or [U-¹³C]alanine also labeled tumor metabolites, but in neither case did labeling differ as dramatically from labeling in the lung as for infusions with [¹³C]lactate (Figure S3C,D).

Lactate is much more abundant in plasma than either pyruvate or alanine, so we focused further effort on documenting its utilization by these tumors. When lactate exchanges with pyruvate via LDH, the proton attached to lactate carbon 2 is transferred to NAD+, producing NADH (Figure 4F). We obtained [2-²H]lactate as a tracer for infusion studies, because LDH converts this tracer to unlabeled pyruvate. If the tumor takes up [2-²H]lactate, lactate m+1 appears in the tumor. On the other hand, no label is transferred to the tumor if the route of entry is via pyruvate or alanine derived elsewhere from [2-²H]lactate. Infusion of [2-²H]lactate resulted in 8-10% enrichment in the plasma lactate pool (Figure 4G). This low enrichment compared to labeling by [¹³C]lactate likely reflects loss of ²H by peripheral LDH-catalyzed lactate/pyruvate exchanges; during [¹³C]lactate infusions, ¹³C is retained during these exchanges. We detected enrichment in tumor lactate, but as expected, no labeling in 3PG, pyruvate or alanine (Figure 4G). The TCA cycle intermediate malate was also labeled, presumably through transfer of ²H from labeled NADH arising from LDH activity (Figure 4G). Although this experiment does not rule out uptake of pyruvate and alanine, it confirms that the tumors take up lactate from the circulation.

MCT1-dependent lactate uptake by HCC15 tumors in vivo

We explored the mechanism of lactate transport by NSCLC xenografts. Both MCT1 and MCT4 are expressed in human NSCLC (Figure 2E, S4A). For functional analysis of MCT1 and MCT4, we used HCC15 cells, an NRAS and STK11-mutant NSCLC cell line expressing both transporters. We used CRISPR/Cas9 editing to obtain clones of HCC15 lacking either MCT1 or MCT4 (Figure 5A). In culture, proliferation persisted after loss of either transporter, with no major effect on the doubling time (Figure S4B). MCT1 loss resulted in increased oxygen consumption and decreased extracellular acidification, whereas loss of MCT4 had no significant effect on either (Figure 5B,C). Direct measurements of glucose utilization and lactate secretion revealed a large suppression of glycolysis after MCT1 loss and a modest effect after MCT4 loss (Figure 5D), indicating that MCT1 is required for the glycolytic phenotype of HCC15 cells in culture.

To examine the role of MCT1 and MCT4 in vivo, subcutaneous xenografts were established from vector control, MCT1-deficient and MCT4-deficient HCC15 lines, then tumor-bearing mice were infused with [2-¹³C]lactate. MCT1 deficiency reduced labeling in tumor metabolites, nearly eliminating the elevated Lactate/3PG labeling ratio observed in control tumors (Figure 5E). Reduced labeling was not an artifact of a large accumulation of unlabeled lactate, because the total lactate pool was not significantly altered by MCT1 deficiency (Figure S4C). Because the MCT1-deficient tumors had nearly equivalent ¹³C enrichment in lactate and 3PG, it is conceivable that labeling in both metabolites arose largely from import of ¹³C-glucose labeled via gluconeogenesis. MCT4 deletion only modestly reduced labeling of tumor metabolites (Figure 5E). Infusing these tumors with [U-¹³C]glucose also revealed a prominent effect of MCT1 loss, which suppressed the Lactate/3PG labeling ratio and resulted in a pattern similar to normal lung, whereas MCT4 loss had no effect on labeling from [U-¹³C]glucose (Figure 5F and S4D). Thus, in HCC15 cells and tumors, MCT1 is the predominant isoform involved in lactate transport. In culture under high-glucose conditions, it promotes a glycolytic phenotype and in vivo, it permits entry of lactate carbon from the circulation. These data also demonstrate cancer cell-autonomous lactate uptake in HCC15 xenografts and indicate that MCT1 is required for the high Lactate/3PG labeling ratio observed during infusion of either ¹³C-lactate or ¹³C-glucose.

Lactate is preferred to glucose as a fuel for the TCA cycle in vivo

We next sought to compare the contributions of lactate and glucose to tumor TCA cycle metabolism. To do this, we established subcutaneous HCC15 tumors and infused mice concurrently with [U-¹³C]glucose and [3-¹³C]lactate. In this scheme, pyruvate and lactate derived from glucose are initially uniformly labeled (m+3) and TCA cycle intermediates arising downstream of PDH are labeled as m+2 (Figure 6A). Metabolites arising from lactate are labeled as m+1. Short (30 min) infusions were used to limit labeling complexity. Glucose and lactate concentrations in the plasma were not altered (Figure 6B). At the end of these infusions, the plasma lactate m+1 and m+3 fractions were equivalent (Figure 6C), indicating that some of the glucose-derived labeling in the tumors likely arises from peripheral conversion of glucose to lactate followed by entry of labeled lactate. Thus, the method underestimates lactate's contribution and may somewhat overestimate the contribution of glycolysis within the tumor. ¹³C-Lactate was also converted to ¹³C-glucose during the infusion, but the glucose m+6 far exceeded glucose m+1 in the circulation (Figure 6C). Tumor metabolite labeling was normalized to labeling of the precursor (glucose or lactate) in the plasma so that relative contributions of each nutrient could be estimated. Although glucose and lactate made similar contributions to tumor pyruvate, lactate made a much larger relative contribution to the TCA cycle (Figure 6D). MCT1-deficient tumors displayed reduced lactate-derived labeling in pyruvate and TCA cycle intermediates, consistent with MCT1's role in lactate import in HCC15-derived tumors (Figure 6E). Interestingly, MCT1 deficiency blunted glucose's relative contributions and reduced the absolute enrichment in tumor lactate from either precursor (Figure 6E,F). This suggests that MCT1 enables lactate uptake from the circulation and also supports glycolytic metabolism in vivo as it does in culture.

Finally, we compared glucose and lactate metabolism within tumors at two different sites in the same mice, where both tumors were exposed to the same supply of labeled glucose and lactate. H460-derived tumors were grown in the subcutaneous space and orthotopically in the lung, then the mice were co-infused with [U-¹³C]glucose and [3-¹³C]lactate. As with the HCC15 tumors, glucose and lactate made comparable contributions to pyruvate, but lactate made a more prominent contribution to the TCA cycle (Figure 6G). This difference was much more dramatic in lung tumors, where there was little dilution of label between circulating lactate and TCA cycle intermediates (Figure 6G). Thus, NSCLC-derived xenografts consume glucose and lactate concomitantly, with lactate predominating as a carbon source for the TCA cycle. However, the extent to which lactate is used as a respiratory fuel is at least partially related to the anatomic site of the tumor.

Contact for Reagent and Resource Sharing

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Ralph J. DeBerardinis ([email protected]).

Experimental Model and Subject Details

Method Details

Quantification and statistical analysis

Patient-matched tumor and non-cancerous lung samples, as well as intra-tumoral samples processed on the same day, were analyzed as described in the figure legends. All data were considered significant if p < 0.05. Statistics were calculated using PRISM software. The correlation analysis in Figure 1I was performed using the corrplot package in R Studio (Figure1I). Statistical details can be found in the figure legends of each experiment.