Fig. 3. Cytotoxic effect of anti-LDH-A compounds alone and in combination with pemetrexed or gemcitabine on MPM and DMPM monolayers and spheroids.

a Evaluation of the inhibition of the aggregation of the spheroids treated with pemetrexed (PMX), alone or in combination with 1 µM NHI-2, or NHI-Glc-2, compared with untreated spheroids originating from H2452 cells. The spheroids had similar volumes (below 500 µm³) at the start of drug exposure, and the untreated spheroids were still growing during the following 72 h. Columns, mean values obtained from three independent experiments; bars, SEM. *Significantly different (P < 0.05) from cells treated with PMX alone. b Cell viability bar graph of MesoII and STO cell lines treated with PMX. Columns, mean values obtained from three independent experiments; bars, SEM. c Combination index values, calculated at FA > 0.5, with Calcusyn software, as described in the “Methods” section. Columns, mean values obtained from three independent experiments; bars, SEM. d Evaluation of the inhibition of the aggregation of the spheroids treated with gemcitabine, alone or in combination with 10 µM NHI-Glc-2, compared with untreated spheroids originating from MesoII cells. Columns, mean values obtained from three independent experiments; bars, SEM. *Significantly different (P < 0.05) from cells treated with gemcitabine alone. Right panel: representative images of DMPM spheroids. The spheroids had similar volumes (below 500 µm³) and density at the start of drug exposure. However, after 7 days, the volume of untreated spheroids was reduced, while the density was significantly increased. In comparison with untreated spheroids (control), the treatment for 7 days with 25 µM of NHI-Glc-2 dramatically reduced the volume of STO spheroids, while the MesoII spheroids showed about a threefold decrease in their aggregation (considering both the volume and density, as explained in the “Methods” and Supplemental Methods and Supplementary Fig. S6). Scale bar, 100 µm. Columns, mean values obtained from three independent experiments; bars, SEM. *Significantly different (P < 0.05) from cells untreated.