Visualizing the effects of lactate dehydrogenase (LDH) inhibition and LDH-A genetic ablation in breast and lung cancer with hyperpolarized pyruvate NMR

Abstract

In many tumors, cancer cells take up large quantities of glucose and metabolize it into lactate, even in the presence of sufficient oxygen to support oxidative metabolism. It has been hypothesized that this malignant metabolic phenotype supports cancer growth and metastasis, and that reversal of this so-called "Warburg effect" may selectively harm cancer cells. Conversion of glucose to lactate can be reduced by ablation or inhibition of lactate dehydrogenase (LDH), the enzyme responsible for conversion of pyruvate to lactate at the endpoint of glycolysis. Recently developed inhibitors of LDH provide new opportunities to investigate the role of this metabolic pathway in cancer. Here we show that magnetic resonance spectroscopic imaging of hyperpolarized pyruvate and its metabolites in models of breast and lung cancer reveal that inhibition of LDH was readily visualized through reduction in label exchange between pyruvate and lactate, while genetic ablation of the LDH-A isoform alone had smaller effects. During the acute phase of LDH inhibition in breast cancer, no discernible bicarbonate signal was observed and small signals from alanine were unchanged.

Keywords: GNE140; LDH; LDH-A; MRSI; Warburg effect; cancer metabolism; hyperpolarized 13C pyruvate.

Figure 1

Images acquired in a model of triple-negative BRCA1-related breast cancer, a) before, and b) after administration of GNE140. From the left: proton; ¹³C MRSI voxels overlaid onto proton; representative ¹³C spectra from the tumor; pyruvate; and lactate, images. Pyruvate and lactate images from the same ¹³C MRSI acquisition are displayed on the same scale. The expected location of the bicarbonate signal (aliased due to the spectral width of the MRSI acquisition) is indicated in the lower right spectrum of the bottom center panel. c) Column plot of the ratio of lactate and alanine peaks to pyruvate peaks integrated over the ¹³C MRSI data acquisition. Values and error bars represent the mean and standard deviation calculated over the breast cancer model mice in each group (i.e. n=5) respectively. Brackets highlight p-values from paired two-tailed Student’s t-tests of the results pre- and post- treatment with the drug GNE140 and DMSO or the vehicle DMSO only. d) Scatter plots showing the lactate-to-pyruvate ratios in the breast cancer model before and after administration of vehicle (left) and GNE140 (right).

Figure 2

Images acquired in a model of lung cancer, a) before, and b) after administration of GNE140. From the left: ¹³C MRSI voxels overlaid onto proton; representative ¹³C spectra from the tumor; pyruvate; and lactate, images. A vial of acetate used for reference (located approximately 50Hz from lactate in ¹³C spectra) appears in proton and lactate images to the left of the mouse. The signal intensity in the lactate image from ¹³C MRSI post administration of the LDH inhibitor is multiplied by 3 (relative to signal in the pyruvate image) to highlight the absence of lactate signal. c) Scatter plots show the change in lactate-to-pyruvate ratios from hyperpolarized ¹³C MRSI in the EFGR-driven lung cancer tumors before and after administration of vehicle (left) and GNE140 (right). N=3 for both control and treated groups.

Figure 3

Images acquired in a model of lung cancer, a) before, and b) after administration of tamoxifen to initiate LDH-A deletion. From the left: ¹³C MRSI voxels overlaid onto proton; representative ¹³C spectra from the tumor; pyruvate; and lactate, images. Pyruvate and lactate images from the same ¹³C MRSI acquisition are displayed on the same scale. c) Scatter plot shows the change in lactate-to-pyruvate ratios from hyperpolarized ¹³C MRSI in the lung cancer model before and after administration of tamoxifen to initiate LDH-A deletion.

Figure 4

Images from a) IHC staining for LDH in a sample of lung tissue, and b) the matching proton MRI, showing correspondence between the tissue sample and imaging. c) Scatter plot showing the lactate-to-pyruvate ratios from hyperpolarized ¹³C MRSI against the percentage of LDH-A positive pixels from corresponding samples of lung masses.

Figure 5

IHC staining for the transporters MCT1 and MCT4 in three representative animals, a) following tamoxifen administration to induce deletion of LDH-A, and b) untreated animals. Tumors from the three mice shown in the upper panel are the same that contribute to data plot in Figure 4c. Robust staining of the musculature (rightmost panels) as well as mono-nuclear cells (red arrow) served as an internal control for the staining. Consistent with prior reporting, expression of the transporters was low. Slides were processed according to standard protocol for IHC with antibodies against MCT1 and MCT4. Scale bar = 100 μm.