Metabolic imaging: a link between lactate dehydrogenase A, lactate, and tumor phenotype

Abstract

Purpose: We compared the metabolic profiles and the association between LDH-A expression and lactate production in two isogenic murine breast cancer cell lines and tumors (67NR and 4T1). These cell lines were derived from a single mammary tumor and have different growth and metabolic phenotypes.

Experimental design: LDH-A expression, lactate concentration, glucose utilization, and oxygen consumption were measured in cells, and the potential relationship between tumor lactate levels [measured by magnetic resonance spectroscopic imaging (MRSI)] and tumor glucose utilization [measured by [(18)F]2-deoxy-2-fluoro-D-glucose positron emission tomography ([(18)F]FDG-PET)] was assessed in orthotopic breast tumors derived from these cell lines.

Results: We show a substantial difference in LDH-A expression between 67NR and 4T1 cells under normoxia and hypoxia. We also show that small orthotopic 4T1 tumors generate 10-fold more lactate than corresponding 67NR tumors. The high lactate levels in small primary 4T1 tumors are associated with intense pimonidazole staining (a hypoxia indicator). Less-intense hypoxia staining was observed in the larger 67NR tumors and is consistent with the gradual increase and plateau of lactate concentration in enlarging 67NR tumors.

Conclusions: Lactate-MRSI has a greater dynamic range than [(18)F]FDG-PET and may be a more sensitive measure with which to evaluate the aggressive and metastatic potential of primary breast tumors.

Figure 1. Metabolic features of isogenic 67NR and 4T1 breast cancer cells

LDH-A mRNA expression in 67NR and 4T1 cells by the semi-quantitative RT-PCR (A). LDH-A, HXKII, PKM2 proteins expression assessed by Western blotting (B). Total LDH enzyme activity in 67NR cells and 4T1 cells (C). Cell culture medium acidification; supernatant pH (D). Oxygen consumption of 67NR and 4T1 cells in normal growth media (DMEM HG+10%FCS+2mM L-Glutamine, 25 mM HEPES) and upon treatment with 1 μM of rotenone (E) using the Oxylite system. Glucose utilization; clearance of glucose from the culture medium (F). Lactate production; appearance of lactate in the culture medium (G).

Figure 2. Lactate CSI Spectra

Different size 67NR non-metastatic tumors (A) and 4T1 metastasis-prone tumors (B) are shown. The lactate signal was detected using chemical shift imaging (CSI) and a Selective Multiple Quantum Coherence (SelMQC) editing sequence. LDHA protein expression assessed by Western blotting in different sizes of 67NR and 4T1 tumors (TV- tumor volume)(C).

Figure 3. MRSI measurements of lactate concentration in orthotopic 67NR and 4T1 breast tumors during tumor growth

Lactate concentration in 67NR tumors (solid circles, dashed line) (A), and in 4T1 tumors (solid squares, solid line) (B) plotted vs tumor volume. Average lactate concentration at three different tumor volumes (small<150 mm³, medium 150-400 mm³, large>400 mm³) (C). Values are the mean, ± SD, where * = P<0.05 and ** = P<0.01. Tumor lactate concentration plotted vs days post-orthotopic implantation (D).

Figure 4. PET/FDG measurements of [¹⁸F]FDG accumulation in orthotopic 67NR and 4T1 breast tumors during tumor growth

[¹⁸F]FDG accumulation in 67NR tumors (solid circles, dashed line) (A), and in 4T1 tumors (solid squares, solid line) (B) plotted vs tumor volume. Average [¹⁸F]FDG accumulation at three different tumor volumes (small<150 mm³, medium 150-400 mm³, large>400 mm³) (C). Values are the mean, ± SD. Representative [¹⁸F]FDG microPET images of tumor-bearing mice (D); small and large 4T1 orthotopic tumors are visualized (white arrows). High radioactivity is seen in the cervical brown fat, heart and bladder. Values are color-coded to a range of values (%ID/cc).

Figure 5. The effect of hypoxia on the cells growth and LDH-A expression

Growth profiles of 67NR cells and 4T1 cells under normoxic (N, 21%) and hypoxic (H, 1%) conditions (A). Immunoblot of LDH-A and LDH-B subunits from 67NR and 4T1 cells exposed to normoxia and hypoxia for 24 hours (B). The β-actin normalized intensity of LDH-A and LDH-B levels in cells under normoxic and hypoxic conditions (C). The ratio of β-actin normalized LDH-A and LDH-B expression under normoxic and hypoxic conditions (D).

Figure 6. Immunohistochemical staining of 67NR and 4T1 tumors

Histology (H&E), endothelial cell density (CD31, red), blood flow (Hoechst 33342, blue) and hypoxia (Pimonidazole, green) are compared. Small 67NR tumors showed no necrosis, a uniform endothelial cell density and blood flow and no hypoxia. Medium size 67NR tumors showed some Pimonidazole staining. This small 4T1 tumor shows some central necrosis, a decrease in endothelial cell density and blood flow, with a corresponding zone of hypoxia surrounding the necrotic zone. These observations were amplified in medium size 4T1 tumors.