Lactic Acid Accumulation in the Tumor Microenvironment Suppresses 18F-FDG Uptake

Abstract

The process by which tumor cells take up 2-[¹⁸F]fluoro-2-deoxy-D-glucose (FDG) is heterogeneous and influenced by a multitude of factors. In mouse tumor grafts, the core of the tumor often presents lower FDG uptake than the periphery. Whether this pattern is caused by the intrinsic avidity of individual cells for FDG, the density of viable cells in the tumor, or the perfusion of the radiotracer remains unknown. In this study, we used radioluminescence microscopy to measure FDG uptake in single cells isolated from the core and periphery of the tumor and found that differences in FDG uptake persist on the level of single cells. Single cells from the core of 4T1 and MDA-MB-231 tumors grafts took up 26% to 84% less FDG than those from the periphery. These differences were observed in mice with large tumors (>8 mm diameter) but not in those with smaller tumors. To explain the origin of these differences, we examined the influence of three microenvironmental factors on FDG uptake. Hypoxia was ruled out as a possible explanation because its presence in the core would increase and not decrease FDG uptake. Higher cell proliferation in the periphery was consistent with higher FDG uptake, but there was no evidence of a causal relationship. Finally, lactate was higher in the core of the tumor, and it suppressed FDG uptake in a dose-dependent fashion. We therefore conclude that lactic acidosis-the combination of lactate ion buildup and acidic pH-can increase the heterogeneity of FDG uptake in MDA-MB-231 and 4T1 tumor grafts. SIGNIFICANCE: Analysis of single cells from heterogeneous tumors reveals the role played by the tumor microenvironment, lactic acidosis in particular, on the uptake by tumor cells of ¹⁸F-FDG, a PET imaging agent.

Figure 1.

Heterogeneous FDG uptake in tumor grafts. (A) Photograph of a 4T1 tumor graft. (B) Autoradiography of the same tumor shows FDG shows lower FDG uptake at the center of the tumor compared to the periphery. (C) PET/CT of mice bearing 4T1 tumor displaying lower uptake of FDG at the core of the tumor. (D) Schematic representation of this study, which aims to measure the effect of the tumor microenvironment on the uptake of FDG by single cells.

Figure 2.

Single-cell FDG uptake of 4T1 and MDA-MB-231 cells grown as tumor grafts. (A) Bioluminescence imaging (BLI) and radioluminescence microscopy (RLM) of FDG uptake by cells from core and periphery of a representative 4T1 tumor. Cells selected for analysis are circled in black. (B) Number of FDG molecules per cell obtained by analyzing the RLM image, for single 4T1 cells from core and periphery (grey circles), with mean (red line), standard error of the mean (pink rectangle), and 75^th percentile (blue rectangle). (C, D) Same for MDA-MB-231 cell line. (E) Percent difference in mean FDG uptake per cell between tumor core and periphery, as a function of tumor diameter. 4T1 tumors (n=6) are displayed in blue and MDA-MB-231 (n=3) in red. Statistical difference between core and periphery (P < 0.05) is indicated using (+). Other samples are marked as (●). Evidence for a systematic difference between FDG uptake in the core and periphery is strongest for larger tumors. (F) Coefficient of variation (CV) computed from single-cell FDG uptake, for the seven tumors larger than 8 mm (i.e. those with significant differences in FDG uptake), in the core (+) and the edge (•) of the tumor in the same animal (dashed box).

Figure 3.

Microenvironment of tumor grafts. (A) qPCR analysis of a panel of 84 genes associated with hypoxia. Upregulation (red) of many hypoxia-related genes indicates a more hypoxic environment in the core of the tumor grafts. (B) Fraction of tumor cells from core and periphery staining positive for EdU incorporation into DNA. (C) Tumor histology and Ki-67 staining for core and periphery of 4T1 tumor. Scale bars, 50 µm and 20 µm, respectively. (D) Same for MDA-MB-231 tumor. (E) Quantitation of the fraction of tumor cells staining positive for Ki-67 for both cell lines shows higher proliferation in the periphery than in the core. (F) Lactate concentration in the tumor core and periphery for both cell lines. *P<0.05.

Figure 4.

In vitro FDG uptake for different environmental conditions, for 4T1 cells. (A) Single-cell FDG uptake after 7 day under normoxia or chronic hypoxia (2 % O₂). (B) Single-cell FDG uptake after 48h culture with and without serum. Serum-starved 4T1 cells took up 63% less FDG (P<0.005). Serum removal also caused a decrease in cell proliferation (Fig. S4). (C) Single-cell FDG uptake in 4T1 cells exposed to 15 mM lactic acid for 48 h. Lactic acidosis caused a decrease in mean FDG uptake of 85% (P<10⁻⁵). (D) Bulk FDG uptake (gamma counting) in cells normalized by protein weight (CPM/mg), as a function of the concentration of sodium lactate (⊗), hydrochloric acid (●), and lactic acid (○). Top: corresponding pH measured prior to incubation with FDG. Significance tests: NS not significant, *P<0.01, **P<0.001, ***P<0.0001.