Fig 2: Immunohistochemical staining of 4T1 mammary tumors. pHLIPs distribution (Alexa546-pHLIPs, red), LDHA staining (yellow), hypoxia (Pimonidazole, green), and blood flow (Hoechst, blue) are compared on tumor sections (a,c,e). Intensity profiles of the fluorescent signals in the highlighted regions are shown in panels b, d, and f.

Fig 3: Glycolysis facilitates STING signaling in DCs from human NSCLC.(A–C) Correlation between LDHA transcripts and STING signature, including STING (A), IFNA (B), and IFNB (C), in TCGA data set of patients with lung cancer with low or high LDHA transcripts (n = 262). (D) ECAR of the freshly isolated DCs from the paracancerous tissue and NSCLC tissue under Bas or Max conditions. (E) Intracellular ATP of the freshly isolated DCs from the paracancerous tissue and NSCLC tissue. (F) Immunoblot analysis of indicated proteins in whole-cell lysates of the freshly isolated DCs from the paracancerous tissue and NSCLC tissue. (G) Immunoblot analysis of the freshly isolated NSCLC DCs treated with 2-DG (5 mM) for 8 hours. Ctrl, without 2-DG treatment. (H) qRT-PCR analysis of DCs isolated from the paracancerous tissue and NSCLC tissue and NSCLC DCs treated with 2-DG (5 mM) for 8 hours. (I) Immunoblot analysis of indicated proteins in whole-cell lysates of human NSCLC DCs that were pretreated with 2-DG (5 mM) for 6 hours and subsequently incubated in the presence or absence of ATP and SLO for 3 hours. The numbers indicate the relative densities of indicated protein bands normalized to ß-actin. Data are representative of 3 independent experiments (D–I). Data are shown as the mean ± SEM. Statistical analysis was performed using a paired 2-sided Wilcoxon signed-rank test (A–C), 2-tailed Student’s t test (D and E), and 1-way ANOVA (H); **P < 0.01.

Fig 4: Glycolysis promotes STING signaling via glycolytic ATP production.(A and B) Intracellular ATP of BMDCs stimulated with 2 µg/mL cGAMP (A) or 40 µg/mL Tu-DNA (B) for 4 hours. (C) Intracellular ATP of splenic DCs (Spl-DC) and tumor-infiltrating DCs (Tu-DC) isolated from WT mice inoculated s.c. with MC38 colon cancer cells at 14 days. (D and E) Intracellular ATP of BMDCs stimulated with 2 µg/mL cGAMP in the presence of streptolysin-O (SLO) and ATP for 2 hours. BMDCs were pretreated with 2-DG (1 mM; D) or DCA (10 mM; E) overnight and subsequently stimulated as indicated. (F) Intracellular ATP of WT and Ldha/b-DKO (DKO) BMDCs stimulated with 2 µg/mL cGAMP in the presence of SLO and ATP for 2 hours. (G and H) Immunoblot analysis of indicated proteins in whole-cell lysates of BMDCs stimulated with 2 µg/mL cGAMP in the presence of SLO and ATP for 2 hours. BMDCs were pretreated with 2-DG (1 mM; G) or DCA (10 mM; H) overnight and subsequently stimulated as indicated. (I) Immunoblot analysis of indicated proteins in whole-cell lysates of WT and Ldha/b-DKO BMDCs stimulated with 2 µg/mL cGAMP in the presence of SLO and ATP for 2 hours. The numbers indicate the relative densities of indicated protein bands normalized to ß-actin. Representative data are shown from 3 independent experiments. Data are shown as the mean ± SEM. Statistical analysis was performed using 2-tailed Student’s t test (A–C) and 1-way ANOVA (D–F); *P < 0.05; **P < 0.01.

Fig 5: The lactate dehydrogenase (LDH) inhibition by oxamate (Oxa) and GNE-140 (GNE) and a LDHA/B double knockout (LDH-/-) results in a reduced heat shock protein 70 (Hsp70) and globotriaosylceramide (Gb3) expression on the cell membrane. (a,b) Membrane-bound Hsp70 on untreated (Ctrl) and Oxa- or GNE-treated LS174T tumor cells (a) and membrane-bound Hsp70 on B16F10 and LS174T cells (b), as determined by flow cytometry using cmHsp70.1-FITC mAb. The proportion of positively stained cells is shown. Error bars show the standard deviations (SD) of at least three biological replicates (*: p = 0.05, **: p = 0.01 and ***: p = 0.001). (c) LS174T cells stained for the expression of membrane Hsp70 (cmHsp70.1-FITC mAb, green) and Gb3 (CD77-PE mAb, red) were analyzed by confocal microscopy. The colocalization of Hsp70 and Gb3 is visualized in yellow as a merge of the red and green staining. Scale bar: 5 µm. (d) Extracellular Hsp70 levels in the supernatant of the LS174T cells were measured by ELISA and the data normalized to 1 × 106 viable tumor cells. Error bars show the SD of five biological replicates (***: p = 0.001). (e) Proteome profiling of LS174T WT and LDH-/- cells. The most significant canonical pathways and top molecular functions altered by LDH depletion are illustrated. The analyses were generated through the use of Ingenuity Pathway Analysis (IPA) (QIAGEN Inc., https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis, accessed on 22 April 2021). Bars indicate the canonical pathways, and the y-axis displays the -(log p) enrichment significance. NRF2: nuclear factor erythroid 2–related factor 2, PPARa: peroxisome proliferator-activated receptor alpha.