Fig 1: The ALDH1A1-ZBTB7B signaling axis transcriptionally regulated LDHA levels in tumor cells.A Volcano plot of differentially expressed genes in the HT29 cell line transfected with shALDH1A1 and the control group (CTRL) (P < 0.05). The |log2 (fold change)| of −1, and 1. Purple indicated low expression genes, yellow were highly expressed genes. B GSEA enrichment analysis of the differential genes in Fig. 4A on the glycolysis pathway. C In CT26 cells, qPCR of shAldh1a1 (#M1 and #M2) versus the control group (Ctrl) was used to verify the candidate genes with significant differences in RNA-seq. D In CT26 cells, candidate genes with significant differences in qPCR were verified by western blot of shAldh1a1 (#M1 and #M2) and control group (Ctrl). E In CT26 cells, the expression differences of Aldh1a1 and Ldha were detected by Western blot of shZbtb7b (#M1 and #M2) and the control group (Ctrl). F Histogram of Ldha mRNA expression changes after Zbtb7b overexpression (-OE) detected by qPCR. G Schematic diagram of the relative position of Ldha promoter full-length (FL) and Zbtb7b binding. WT is the wild-type sequence, and MUT is the mutant sequence. H Histogram of the Zbtb7b binding site wild-type and mutant luciferase reporter assays. I Analysis of the chromatin sequence of the Ldha promoter region pulled down by Zbtb7b antibody by ChIP–qPCR. J Western blot detection of the main SUMO modification types of Zbtb7b in CT26 cells. Data were mean ± S.D. of three independent experiments. (ns not significant; *P < 0.05, **P < 0.01, ***P < 0.001).
Fig 2: ALDH1A1 deficiency promoted tumor suppression by the immune system in vivo.Tumor growth curves of (A) confounding negative control (Ctrl) and two shAldh1a1 (#M1 and #M2) transfected CT26 xenografts in BALB/c mice and (B) Kaplan–Meier survival curves. C T cell killing assay in CT26 cells transfected with shAldh1a1 (#M1 and #M2) or control (Ctrl). D, E CD3+ and CD3+ CD8+, GZMB+ CD8+ TILs and quantitative statistics of BALB/c mouse tumor tissue in shAldh1a1 (#M1 and #M2) and control group (Ctrl) by flow cytometry. F, G CD3+ and CD8+ PD-1+, CD8+ Tim3+ TILs and quantitative statistics of BALB/c mouse tumor tissue in shAldh1a1 (#M1 and #M2) and control group (Ctrl) by flow cytometry. Results were presented as mean ± S.D., n = 3–5. ns not significant; *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: Genetic deficiency of ALDH1A1 reduced levels of tumor glycolysis and was associated with tumor growth suppression in an immunodeficient mouse model.A Kaplan–Meier survival curves of PFS in groups with high (32 cases) and low (32 cases) ALDH1A1 expression in NSCLC patients. B Histograms of ALDH1A1 expression in low-grade (highly differentiated, 20 cases) and high-grade (poorly differentiated, 20 cases) NSCLC tumors. C Histograms of glycolysis markers—lactate and ATP in NSCLC tumor tissues of the ALDH1A1 high (20 cases) and low (20 cases) expression group. D Kaplan–Meier survival curve of PFS in groups with high (32 cases) and low (32 cases) expression of ALDH1A1 in colon cancer patients. E Histogram of ALDH1A1 expression levels in low-grade (20 cases) and high-grade (20 cases) tumor tissues of patients with colon cancer. F Histograms of glycolysis markers—lactate and ATP in the colon cancer tissues of the ALDH1A1 high (20 cases) and low (20 cases) expression group. G Histograms of key products of glycolysis pyruvate and lactate in confounding negative control (CTRL) and two shALDH1A1 (#H1 and #H2) transfected HT29 xenografts in BALB/c nude mice. H Histograms of the key products of glycolysis pyruvate and lactate in confounding negative control (Ctrl) and two shAldh1a1 (#M1 and #M2) transfected CT26 xenografts in BALB/c nude mice. I Tumor growth curves of confounding negative control (CTRL) and two shALDH1A1 (#H1 and #H2) transfected HT29 xenografts in BALB/c nude mice and Kaplan–Meier survival curves of these mice (n = 10). J Tumor growth curves of confounding negative control (Ctrl) and two shAldh1a1 (#M1 and #M2) transfected CT26 xenografts in BALB/c nude mice and Kaplan–Meier survival curves of these mice (n = 10). K Cell lines from different sources (human intestinal cancer cell HT29, human lung cancer cell A549, and murine intestinal cancer cell CT26) were transfected with a mixed negative control (CTRL/Ctrl) and two shALDH1A1 (#H1 and #H2; #M1 and #M2) In vivo imaging photos of xenograft tumor models constructed in BALB/c nude mice. L Western blot analysis of LDHA/Akt/p-Akt/Mtor/p-S6K protein expression levels after shAldh1a1 #M1 knockdown and compensation experiments in CT26 cells (with β-Actin as internal reference). P.S. We specified that grade 1 was a low-grade tumor and grades 2–3 was a high-grade tumor. Results were presented as mean ± S.D.; ns not significant; *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4: ALDH1A1 mediated tumor immune escape by regulating glycolysis in vivo.Tumor growth curves (A) of confounding negative control (Ctrl) and Aldh1a1-OE-transfected CT26 xenografts in BALB/c mice and Kaplan–Meier survival curves (B) of these mice. Histograms of key markers of glycolysis glucose uptake (C), lactate production (D) and ATP level (E) after Aldh1a1-OE overexpression and 2-DG inhibition of glycolysis in CT26 cells. F T cell killing assay in CT26 cells after transfection with sAldh1a1-OE overexpression and 2-DG inhibition of glycolysis. G–I TILs and quantitative statistics of CD3+ and CD8+ GZMB+, CD8+ PD-1+ TILs of BALB/c mouse tumor tissue in Aldh1a1-OE and control group (Ctrl) by flow cytometry. Results were presented as mean ± S.D., n = 3–5. ns not significant; *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 5: ALDH1A1 and ZBTB7B were positively correlated with PD-L1 expression, respectively, in NSCLC samples.A, B The correlation of ALDH1A1, ZBTB7B and CD274 expression detected by qPCR in clinical NSCLC tissues. A Scatterplot of the correlation of ALDH1A1 and CD274 expression and the correlation of ZBTB7B and CD274 expression. B Quantitative correlation between the expression levels of ALDH1A1, ZBTB7B, and CD274. Correlation coefficients r and p values were calculated based on Spearman’s rank correlation method. C Representative images of immunohistochemical staining for ALDH1A1, ZBTB7B, or PD-L1 expression in NSCLC patient tissues. D IF labeling using ALDH1A1, ZBTB7B, and PD-L1 antibodies in tumor tissues from two patients. E The radiologist annotated the tumor diameter based on CT imaging with a red line. F The difference in tumor diameter before and after treatment with PD-1 monoclonal antibody (Nivolumab) in all patients, where the patients with increased tumor diameter are indicated in purple, and those with decreased tumor diameter are indicated in blue. G Quantitative correlation between tumor diameter changes and ALDH1A1 expression levels. Correlation coefficients r and p values were calculated based on Spearman’s rank correlation method. H, I Kaplan–Meier survival curves of PFS in groups with high and low expression of ALDH1A1 (H) and ZBTB7B in NSCLC patients. *P < 0.05, **P < 0.01, ***P < 0.001.
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