Fig 2: LDHA is highly expressed in COAD. (A) The expression level of LDHA in COAD in two GEO datasets (GSE9348: 10 normal samples and 70 COAD samples; GSE23878: 24 normal samples and 35 COAD samples). (B) Relative expression of LDHA in different cancers compared with normal tissues in the Oncomine database. *p < 0.05, **p < 0.01, ***p < 0.01 compared with control.

Fig 3: Correlation between LDHA expression and tumor markers in COAD. (A) Correlation between LDHA expression and dendritic cell markers HLA-DRA, HLA-DPA1, ITGAX, and NRP1. (B) Correlation between LDHA expression and CD8+ T cell markers CD8A and GZMA. (C) Correlation between LDHA expression and neutrophil cell markers SIGLEC5 and ITGAM. (D) Correlation between LDHA expression and TAM markers CCL2 and IL10.

Fig 4: Expression levels of glucose metabolism-related enzymes, products and PD-L1 under different intervention conditions.A The expression of PD-L1 in the 786-O and OS-RC-2 cell lines was detected by western blotting after 12 or 24 h of culture under low-glucose conditions. B The expression of PD-L1 in the 786-O and OS-RC-2 cell lines was detected by RT-qPCR after 12 or 24 h of culture under low-glucose conditions. C Western blotting was used to detect changes in PD-L1 expression in the 786-O and OS-RC-2 cell lines after 12 h of low-glucose culture followed by culture medium replacement with that of normal glucose concentration. D Lactic acid (LD) production in the two cell lines were different under low-glucose conditions. E Western blotting was used to detect the expression differences in HIF-1α, PFKFB3 and LDHA, key enzymes in glycolysis. Independent experiments were performed in triplicate. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig 5: A primary screen of LDHA using the SplitLuc CETSA assay (63.5 °C for 9 min). (A) 1,850 samples from the mechanism interrogation plate (MIPE) library were screened in qHTS format as an 11-point concentration series for thermal stabilization of LDHA. The waterfall plot shows dose-response curves for three compounds classified as active in the screen. The response curve for GSK2837808A, a known LDHA inhibitor, is colored light blue. (B) Only the activity of GSK2837808A was confirmed when the three hits from the primary screen were re-tested (mean ± SD, n = 9). CETSA was performed at 63 °C for 3.5 min in 384-well PCR plates (thermal cycler). (C) MIPE compounds were screened for activity using an LDHA enzymatic assay. Three actives, including GSK2837808A (light blue curve), were identified. The remaining two actives did not overlap with the compounds identified in the CETSA assay. (D) MIPE compounds were screened for activity with the lactate production assay. Three-hundred and seventy-six compounds (20.3% hit rate) were identified as active (black curves).