The Evolving Role of Succinate in Tumor Metabolism: An 18F-FDG-Based Study

Abstract

In recent years, inherited and acquired mutations in the tricarboxylic acid (TCA) cycle enzymes have been reported in diverse cancers. Pheochromocytomas and paragangliomas often exhibit dysregulation of glucose metabolism, which is also driven by mutations in genes encoding the TCA cycle enzymes or by activation of hypoxia signaling. Pheochromocytomas and paragangliomas associated with succinate dehydrogenase (SDH) deficiency are characterized by high ¹⁸F-FDG avidity. This association is currently only partially explained. Therefore, we hypothesized that accumulation of succinate due to the TCA cycle defect could be the major connecting hub between SDH-mutated tumors and the ¹⁸F-FDG uptake profile. Methods: To test whether succinate modifies the ¹⁸F-FDG metabolic profile of tumors, we performed in vitro and in vivo (small-animal PET/CT imaging and autoradiography) experiments in the presence of succinate, fumarate, and phosphate-buffered saline (PBS) in different cell models. As a control, we also evaluated the impact of succinate on ¹⁸F-fluorocholine uptake and retention. Glucose transporter 1 (GLUT1) immunohistochemistry was performed to assess whether ¹⁸F-FDG uptake correlates with GLUT1 staining. Results: Intratumoral injection of succinate significantly increased ¹⁸F-FDG uptake at 24 h on small-animal PET/CT imaging and autoradiography. No effect of succinate was observed on cancer cells in vitro, but interestingly, we found that succinate caused increased ¹⁸F-FDG uptake by human umbilical vein endothelial cells in a concentration-dependent manner. No significant effect was observed after intratumoral injection of fumarate or PBS. Succinate, fumarate, and PBS have no effect on cell viability, regardless of cell lineage. Intramuscular injection of succinate also significantly increases ¹⁸F-FDG uptake by muscle when compared with either PBS or fumarate, highlighting the effect of succinate on connective tissues. No difference was observed between PBS and succinate on ¹⁸F-fluorocholine uptake in the tumor and muscle and on hind limb blood flow. GLUT1 expression quantification did not significantly differ between the study groups. Conclusion: The present study shows that succinate stimulates ¹⁸F-FDG uptake by endothelial cells, a finding that partially explains the ¹⁸F-FDG metabotype observed in tumors with SDH deficiency. Although this study is an ¹⁸F-FDG-based approach, it provides an impetus to better characterize the determinants of ¹⁸F-FDG uptake in various tumors and their surrounding microenvironment, with a special emphasis on the role of tumor-specific oncometabolites.

Keywords: 18F-FDG; paraganglioma; succinate; succinate dehydrogenase; tricarboxylic acid cycle.

FIGURE 1.

(A) Representative PET/CT images of HT-29 tumor–bearing mice (n = 3) 40 min after ¹⁸F-FDG injection (5–10 MBq/50 μL, intraperitoneally) and 27 h after the first 10-μL intratumoral injection of 1 mM succinate (right shoulder, red arrow), 1 mM fumarate (left shoulder, green arrow), or PBS (right hind limb, yellow arrow) every 6 h for 24 h, along with graph showing quantifications from ¹⁸F-FDG small-animal PET/CT in tumors. **P < 0.01, 1-way ANOVA with Bonferroni post hoc test, 3 mice per condition. (B) Representative autoradiographic images of HT-29 tumors extracted from mice immediately after small-animal PET/CT imaging, along with graph showing quantifications from tumor autoradiography. *P < 0.05, 1-way ANOVA with Bonferroni post hoc test, 3 mice per condition. (C) Representative small-animal PET/CT images of mouse hind limbs 40 min after ¹⁸F-FDG injection (5–10 MBq/50 μL, intravenously) and 27 h after first succinate or PBS injection every 6 h for 24 h, along with graph showing quantifications expressed as percentage injected dose per gram of tissue (%ID/g) over time from dynamic small-animal PET/CT reconstruction. **P < 0.01, Mann–Whitney test, 3 mice per condition. (D) Representative small-animal PET/CT images 40 min after ¹⁸F-fluorocholine injection (5–7 MBq/50 μL, intravenously) and 27 h after first succinate or PBS injection every 6 h for 24 h, along with graph showing quantifications expressed as %ID/g over time from dynamic small-animal PET/CT reconstruction. P = 0.609, Mann–Whitney test, 3 mice per condition. DLU = digital light units.

FIGURE 2.

Influence of succinate pretreatment on ¹⁸F-FDG uptake in HUVECs (A, top), in HT-29 cells (B, top), and in primary cardiac fibroblasts (C, top) after pretreatment for 24 h with 0, 0.01, 0.1, 1.0, or 10 nmol of succinate per μL of culture medium. *P < 0.05, **P < 0.01, and ***P < 0.001, 2-way ANOVA with Bonferroni post hoc test, 3 mice per condition. Viability of HUVECs (A, bottom), HT-29 cells (B, bottom), and fibroblasts (C, bottom) by counting on Kova slides with trypan blue after 24-h incubation with 0.01 or 10 nmol/μL fumarate or succinate, compared with PBS treatment. P = 0.609, 0.991, and 0.715 for HUVECs, HT29, and fibroblasts, respectively, 2-way ANOVA with Bonferroni post hoc test, 3 mice per condition.

FIGURE 3.

(A) Representative small-animal PET/CT images of mouse hind limbs 40 min after ¹⁸F-FDG injection (5–10 MBq/50 μL, intravenously) and 27 h after first succinate (right hind limb) or fumarate or PBS (left hind limb) injection every 6 h for 24 h, along with graph showing quantifications in each hind limb expressed as percentage injected dose per gram of tissue (%ID/g) over time from dynamic small-animal PET/CT reconstruction. *P < 0.05, Mann–Whitney test, 3 mice per condition. (B) Representative small-animal PET/CT images of mouse hind limbs 40 min after ¹⁸F-fluorocholine injection (5–7 MBq/50 μL, intravenously) and 27 h after first succinate (right hind limb) or fumarate or PBS (left hind limb) injection every 6 h for 24 h, along with graph showing quantifications in each hind limb expressed as %ID/g over time from dynamic small-animal PET/CT reconstruction. P = 0.617 vs. PBS and P = 0.923 vs. fumarate, respectively, not statistically significant, Mann–Whitney test, 3 mice per condition. (C) At top are representative laser-Doppler perfusion images of hind limbs from 6 mice immediately after ¹⁸F-FDG PET (28 h after first succinate [right hind limb] or fumarate or PBS [left hind limb] injection every 6 h for 24 h). At bottom are corresponding quantifications of perfusion signal in each hind limb.