Journal: bioRxiv
Article Title: Dual inhibition of lactate transporters MCT1 and MCT4 in pancreatic neuroendocrine tumors targets metabolic heterogeneity and functional redundancy
doi: 10.1101/2025.01.31.635625
Figure Lengend Snippet: Pharmacological inhibition of MCT1 and MCT4 reduces glycolysis and lactate secretion of PanNET cells. (A) Chemical structures of the MCT4 inhibitor AZD0095, MCT1/4 inhibitor syrosingopine, and MCT1/2 inhibitor AZD3965. Graphical representation of MCT1/2/4 function, their expected lactate transport directions and inhibitor specificities. (B) After treatment of the indicated five PanNET cell lines with increasing doses of syrosingopine (SYRO), AZD3965, and AZD0095, cells were cultured in normoxia or hypoxia (1.5% O 2 ) for 48h, and metabolic activity was measured by PrestoBlue assay. Data are presented as mean±SD from a representative experiment repeated three times (n=3 technical replicates). (C) Seahorse metabolic flux assay to measure extracellular acidification rate (ECAR) as a function of glycolysis and lactate secretion. The indicated PanNET cell lines were cultured for 48h in normoxia prior the start of the glycolytic stress test. Two hours before the start of the measurements, the culture media of the indicated PanNET cell lines was changed to serum- and glucose-free media and contained only glutamine to support oxidative phosphorylation (Suppl. Fig. 4B). The dashed vertical lines indicate the start of the different phases of the glycolytic stress test assay (inhibitor injection to block MCT1/2/4 activity, glucose injection to start glycolysis, ATP synthase inhibition with oligomycin for maximum glycolytic rate, glycolysis inhibition with 2-deoxyglucose). The bar charts represent the last measurement of the glucose injection phase (∼81min). Data are presented as mean±SD of 5-6 replicates. (D) Measurement of secreted lactate in conditioned media of the indicated PanNET cell lines treated for 48h with increasing doses of syrosingopine (SYRO), AZD3965, and AZD0095 in normoxia (saturated colors) and hypoxia (1.5% O 2 , pale colors). Data are presented as mean±SD of two experiments with n=4 technical replicates. Statistical significance for B-D: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Article Snippet: Syrosingopine (Sigma-Aldrich, SML1908-5MG), AZD0095 (MedChemExpress, HY-148517), AZD3965 (MedChemExpress, HY-12750), Sunitinib (Selleckchem, S1042), everolimus (Selleckchem, S1120) and SCH772984 (Selleckchem, S7101) were reconstituted in dimethylsulfoxide (DMSO) (Sigma-Aldrich, 472301).
Techniques: Inhibition, Cell Culture, Activity Assay, Prestoblue Assay, Flux Assay, Injection, Blocking Assay
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