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Vitamin D potentiates STING agonist-induced antitumor immunity in NSCLC

Ke Li, Guichao Liu, Wenhui Yuan, Xinhai Zhu and Peng Li
Cancer Biology & Medicine July 2026, 20260211; DOI: https://doi.org/10.20892/j.issn.2095-3941.2026.0211

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Figures

  • Vitamin D, in synergy with STING activation, potentiates CD8+ T cell-mediated antitumor immunity. (A) Overview of experimental design. (B) CT scans showing changes in tumor sizes in patients with NSCLC. Responder (R) vs. non-responder (NR). (C) Waterfall plot depicting changes in tumor size in patients with NSCLC who received the specified treatment (n = 11 per group). (D) VDR (n = 10) and STING (n = 11) expression levels in PBMCs from patients with NSCLC. (E) Relative levels of VDR and STING in R vs. NR groups, determined by western blotting. (F) Serum levels of 25(OH)D3 in R vs. NR groups (n = 11 per group). (G) Immunohistochemistry detection of CD8 expression in tumor tissues from patients with NSCLC. Scale bar = 100 μm. (H, I) Expression of IFN-γ+ and TNF-α+ in circulating CD8+ T cells isolated from patients with NSCLC classified as responders or non-responders (n = 11 per group). (J) Experimental design for LLC tumor models. (K) Tumor growth in mice in each group at the end of the experiment (n = 6 per group). Comb: 1α,25(OH)2D3 + diABZI. (L–N) tumor weight (L, M) and tumor growth (N) (n = 4 per group). (O) Tissue pathology of the specified organs, assessed through hematoxylin and eosin staining on day 18 post-tumor implantation. Scale bar = 100 μm. (P–R) Levels of IFN-γ and TNF-α in TILs-CD4+/CD8+ T cells after 1α,25(OH)2D3 (1.25D3) or diABZI treatment, alone or in combination (n = 4 per group). All mouse experiments were conducted as independent biological replicates. Two-tailed unpaired Student’s t-test (E, F, and I). Two-way ANOVA with Tukey’s multiple comparisons test (K and N). One-way ANOVA with Tukey’s multiple comparisons test (M, Q, and R). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant.
    Vitamin D, in synergy with STING activation, potentiates CD8+ T cell-mediated antitumor immunity. (A) Overview of experimental design. (B) CT scans showing changes in tumor sizes in patients with NSCLC. Responder (R) vs. non-responder (NR). (C) Waterfall plot depicting changes in tumor size in patients with NSCLC who received the specified treatment (n = 11 per group). (D) VDR (n = 10) and STING (n = 11) expression levels in PBMCs from patients with NSCLC. (E) Relative levels of VDR and STING in R vs. NR groups, determined by western blotting. (F) Serum levels of 25(OH)D3 in R vs. NR groups (n = 11 per group). (G) Immunohistochemistry detection of CD8 expression in tumor tissues from patients with NSCLC. Scale bar = 100 μm. (H, I) Expression of IFN-γ+ and TNF-α+ in circulating CD8+ T cells isolated from patients with NSCLC classified as responders or non-responders (n = 11 per group). (J) Experimental design for LLC tumor models. (K) Tumor growth in mice in each group at the end of the experiment (n = 6 per group). Comb: 1α,25(OH)2D3 + diABZI. (L–N) tumor weight (L, M) and tumor growth (N) (n = 4 per group). (O) Tissue pathology of the specified organs, assessed through hematoxylin and eosin staining on day 18 post-tumor implantation. Scale bar = 100 μm. (P–R) Levels of IFN-γ and TNF-α in TILs-CD4+/CD8+ T cells after 1α,25(OH)2D3 (1.25D3) or diABZI treatment, alone or in combination (n = 4 per group). All mouse experiments were conducted as independent biological replicates. Two-tailed unpaired Student’s t-test (E, F, and I). Two-way ANOVA with Tukey’s multiple comparisons test (K and N). One-way ANOVA with Tukey’s multiple comparisons test (M, Q, and R). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant.
    Figure 1

    Vitamin D, in synergy with STING activation, potentiates CD8+ T cell-mediated antitumor immunity. (A) Overview of experimental design. (B) CT scans showing changes in tumor sizes in patients with NSCLC. Responder (R) vs. non-responder (NR). (C) Waterfall plot depicting changes in tumor size in patients with NSCLC who received the specified treatment (n = 11 per group). (D) VDR (n = 10) and STING (n = 11) expression levels in PBMCs from patients with NSCLC. (E) Relative levels of VDR and STING in R vs. NR groups, determined by western blotting. (F) Serum levels of 25(OH)D3 in R vs. NR groups (n = 11 per group). (G) Immunohistochemistry detection of CD8 expression in tumor tissues from patients with NSCLC. Scale bar = 100 μm. (H, I) Expression of IFN-γ+ and TNF-α+ in circulating CD8+ T cells isolated from patients with NSCLC classified as responders or non-responders (n = 11 per group). (J) Experimental design for LLC tumor models. (K) Tumor growth in mice in each group at the end of the experiment (n = 6 per group). Comb: 1α,25(OH)2D3 + diABZI. (L–N) tumor weight (L, M) and tumor growth (N) (n = 4 per group). (O) Tissue pathology of the specified organs, assessed through hematoxylin and eosin staining on day 18 post-tumor implantation. Scale bar = 100 μm. (P–R) Levels of IFN-γ and TNF-α in TILs-CD4+/CD8+ T cells after 1α,25(OH)2D3 (1.25D3) or diABZI treatment, alone or in combination (n = 4 per group). All mouse experiments were conducted as independent biological replicates. Two-tailed unpaired Student’s t-test (E, F, and I). Two-way ANOVA with Tukey’s multiple comparisons test (K and N). One-way ANOVA with Tukey’s multiple comparisons test (M, Q, and R). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant.

  • Vitamin D alleviates immune exhaustion, enhances TCR signaling, and acts in synergy with STING agonists in potentiating antitumor immunity. (A) Heatmaps showing transcriptome analysis of human CD8+ T cells after pretreatment with vehicle or 1.25D3, and subsequent TCR stimulation under diABZI treatment for 4 h (n = 4 per group). (B) GSEA of the cytosolic DNA-sensing pathway and oxidative phosphorylation signaling pathway. (C, D) Flow cytometric analysis of antitumor-related cytokine levels in human CD8+ T cells (n = 5). (E–H) Diagram of the in vitro exhaustion assay. Surface phenotype of human CD8+ T cells on day 13 of the T cell exhaustion assay (n = 6). (I) Workflow for human CD8+ T cell pretreatment and phosphoproteomic analysis. (J) Principal component analysis (PCA) of vehicle and 1.25D3 treatments (n = 3). (K) Volcano plot and statistical chart showing differential phosphorylation modification sites. (L) KEGG pathway analysis. (M, N) Data divided into four sections (Q1–Q4) according to fold changes in differential expression (M), followed by KEGG pathway enrichment in each group (N). (O) Expression of the vitamin D receptor and STING is significantly elevated in patients responding to chemotherapy combined with anti-PD-1 therapy, and is associated with improved clinical outcomes in non-small cell lung cancer. Mechanistically, vitamin D mitigates CD8+ T cell exhaustion and enhances T cell receptor signaling, thereby acting in synergy with STING agonists in eliciting durable and robust antitumor immunity. One-way ANOVA with Tukey’s multiple comparisons test (D). Paired Student’s t-test (G and H). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant.
    Vitamin D alleviates immune exhaustion, enhances TCR signaling, and acts in synergy with STING agonists in potentiating antitumor immunity. (A) Heatmaps showing transcriptome analysis of human CD8+ T cells after pretreatment with vehicle or 1.25D3, and subsequent TCR stimulation under diABZI treatment for 4 h (n = 4 per group). (B) GSEA of the cytosolic DNA-sensing pathway and oxidative phosphorylation signaling pathway. (C, D) Flow cytometric analysis of antitumor-related cytokine levels in human CD8+ T cells (n = 5). (E–H) Diagram of the in vitro exhaustion assay. Surface phenotype of human CD8+ T cells on day 13 of the T cell exhaustion assay (n = 6). (I) Workflow for human CD8+ T cell pretreatment and phosphoproteomic analysis. (J) Principal component analysis (PCA) of vehicle and 1.25D3 treatments (n = 3). (K) Volcano plot and statistical chart showing differential phosphorylation modification sites. (L) KEGG pathway analysis. (M, N) Data divided into four sections (Q1–Q4) according to fold changes in differential expression (M), followed by KEGG pathway enrichment in each group (N). (O) Expression of the vitamin D receptor and STING is significantly elevated in patients responding to chemotherapy combined with anti-PD-1 therapy, and is associated with improved clinical outcomes in non-small cell lung cancer. Mechanistically, vitamin D mitigates CD8+ T cell exhaustion and enhances T cell receptor signaling, thereby acting in synergy with STING agonists in eliciting durable and robust antitumor immunity. One-way ANOVA with Tukey’s multiple comparisons test (D). Paired Student’s t-test (G and H). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant.
    Vitamin D alleviates immune exhaustion, enhances TCR signaling, and acts in synergy with STING agonists in potentiating antitumor immunity. (A) Heatmaps showing transcriptome analysis of human CD8+ T cells after pretreatment with vehicle or 1.25D3, and subsequent TCR stimulation under diABZI treatment for 4 h (n = 4 per group). (B) GSEA of the cytosolic DNA-sensing pathway and oxidative phosphorylation signaling pathway. (C, D) Flow cytometric analysis of antitumor-related cytokine levels in human CD8+ T cells (n = 5). (E–H) Diagram of the in vitro exhaustion assay. Surface phenotype of human CD8+ T cells on day 13 of the T cell exhaustion assay (n = 6). (I) Workflow for human CD8+ T cell pretreatment and phosphoproteomic analysis. (J) Principal component analysis (PCA) of vehicle and 1.25D3 treatments (n = 3). (K) Volcano plot and statistical chart showing differential phosphorylation modification sites. (L) KEGG pathway analysis. (M, N) Data divided into four sections (Q1–Q4) according to fold changes in differential expression (M), followed by KEGG pathway enrichment in each group (N). (O) Expression of the vitamin D receptor and STING is significantly elevated in patients responding to chemotherapy combined with anti-PD-1 therapy, and is associated with improved clinical outcomes in non-small cell lung cancer. Mechanistically, vitamin D mitigates CD8+ T cell exhaustion and enhances T cell receptor signaling, thereby acting in synergy with STING agonists in eliciting durable and robust antitumor immunity. One-way ANOVA with Tukey’s multiple comparisons test (D). Paired Student’s t-test (G and H). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant.
    Figure 2

    Vitamin D alleviates immune exhaustion, enhances TCR signaling, and acts in synergy with STING agonists in potentiating antitumor immunity. (A) Heatmaps showing transcriptome analysis of human CD8+ T cells after pretreatment with vehicle or 1.25D3, and subsequent TCR stimulation under diABZI treatment for 4 h (n = 4 per group). (B) GSEA of the cytosolic DNA-sensing pathway and oxidative phosphorylation signaling pathway. (C, D) Flow cytometric analysis of antitumor-related cytokine levels in human CD8+ T cells (n = 5). (E–H) Diagram of the in vitro exhaustion assay. Surface phenotype of human CD8+ T cells on day 13 of the T cell exhaustion assay (n = 6). (I) Workflow for human CD8+ T cell pretreatment and phosphoproteomic analysis. (J) Principal component analysis (PCA) of vehicle and 1.25D3 treatments (n = 3). (K) Volcano plot and statistical chart showing differential phosphorylation modification sites. (L) KEGG pathway analysis. (M, N) Data divided into four sections (Q1–Q4) according to fold changes in differential expression (M), followed by KEGG pathway enrichment in each group (N). (O) Expression of the vitamin D receptor and STING is significantly elevated in patients responding to chemotherapy combined with anti-PD-1 therapy, and is associated with improved clinical outcomes in non-small cell lung cancer. Mechanistically, vitamin D mitigates CD8+ T cell exhaustion and enhances T cell receptor signaling, thereby acting in synergy with STING agonists in eliciting durable and robust antitumor immunity. One-way ANOVA with Tukey’s multiple comparisons test (D). Paired Student’s t-test (G and H). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s., not significant.

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