Telmisartan enhances anti-PD-1 efficacy in MSS colorectal cancer via modulation of PD-L1 and STAT3 signaling

Figures

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  Figure 1. Classification of CRC cell lines by mismatch repair protein expression and cytotoxicity assessment of telmisartan. (A) Western blot analysis of MLH1, MSH2, MSH6, and PMS2 expression in CRC cell lines. SW480 and CT26 showed intact expression of all four MMR proteins, indicating pMMR/MSS status. HCT116 showed loss of MLH1 and PMS2 expression, and HT29 showed loss of MSH6 expression, indicating dMMR/MSI-H status. (B) MMR protein expression in murine CT26 cells, showing intact expression (MSS). (C-E) Cell viability following telmisartan treatment (0–50 μM) for 24 hours in SW480 (C), HCT116 (D), and CT26 (E) cells as determined by CCK-8 assays. Data represent mean ± SD (n=5, three independent experiments). β-actin served as loading control. CCK-8, Cell Counting Kit-8; CRC, colorectal cancer; dMMR, deficient mismatch repair; DMSO, dimethyl sulfoxide; MMR, mismatch repair; MSI-H, high microsatellite instability; MSS, microsatellite stable; pMMR, proficient mismatch repair; SD, standard deviation.

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  Figure 2. Telmisartan inhibits CRC cell migration and counteracts Ang II-induced effects. (A,B) Representative images (A) and quantification (B) of Transwell migration assays in SW480 (MSS) cells treated with vehicle control, telmisartan (10 or 20 μM), Ang II (100 nM), or combinations for 16 hours. (C,D) Representative images (C) and quantification (D) of migration assays in HCT116 (MSI-H) cells under identical conditions. Migrated cells were stained with crystal violet and counted in five random fields per membrane. Data represent mean ± SD from three independent experiments. Scale bar =100 μm. Ang, angiotensin; CRC, colorectal cancer; MSS, microsatellite stable; MSI-H, high microsatellite instability; SD, standard deviation.

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  Figure 3. Telmisartan is associated with reduced PD-L1 expression and increased STAT3 phosphorylation. (A-C) Western blot analysis of PD-L1 protein expression in SW480 (MSS) (A), CT26 (MSS) (B), and HCT116 (MSI-H) (C) cells following treatment with telmisartan (0, 10, 20 μM) for 24 hours. (D) Western blot analysis of total STAT3 and phosphorylated STAT3 (p-STAT3, Tyr705) in SW480 cells treated with increasing concentrations of telmisartan (0–30 μM) for 24 hours. Densitometric quantification of p-STAT3/total STAT3 ratio is shown. β-actin served as loading control. Representative of three independent experiments. MSI-H, high microsatellite instability; MSS, microsatellite stable; PD-L1, programmed death-ligand 1; STAT3, signal transducer and activator of transcription 3.

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  Figure 4. Histopathological evaluation demonstrates absence of systemic toxicity. Representative H&E-stained sections of heart, liver, spleen, lung, and kidney from CT26 tumor-bearing mice treated with PBS, telmisartan (10 mg/kg/day), pembrolizumab (200 μg/mouse every 3 days), or combination therapy for 12 days. No treatment-related histopathological abnormalities were observed. Scale bar =100 μm (n=5 per group). H&E, hematoxylin and eosin; PBS, phosphate buffered saline.

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  Figure 5. Telmisartan enhances pembrolizumab efficacy in MSS-CRC without systemic toxicity. (A) Tumor growth curves in BALB/c mice bearing CT26 tumors over 12 days. Tumor volume calculated as V = 0.5 × length × width². Data represent mean ± SD (n=5 per group). ****P<0.001 by two-way repeated-measures ANOVA. (B) Body weight monitoring throughout treatment, showing no significant differences among groups. Data represent mean ± SD (n=5 per group). (C) Gross morphology of excised tumors at day 12 from each treatment group: PBS, pembrolizumab, telmisartan, and combination therapy. ANOVA, analysis of variance; CRC, colorectal cancer; MSS, microsatellite stable; PBS, phosphate buffered saline; SD, standard deviation.

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  Figure 6. Telmisartan promotes intratumoral T-cell infiltration in MSS-CRC. Representative H&E staining (top row) and immunofluorescence staining of tumor sections showing CD3⁺ (second and third rows), CD4⁺ (fourth and fifth rows), and CD8⁺ (sixth and seventh rows) T-cell infiltration in CT26 tumors following treatment with PBS, telmisartan, pembrolizumab, or combination therapy. Lower-magnification overview images show regions of interest (yellow boxes labeled “Enlarge“) with corresponding high-magnification images below. T-cell markers visualized in red (Alexa Fluor 594); nuclei counterstained with DAPI (blue). Scale bars =100 μm (n=5 tumors per group, five fields per tumor). CRC, colorectal cancer; DAPI, 4',6-diamidino-2-phenylindole; H&E, hematoxylin and eosin; MSS, microsatellite stable; PBS, phosphate buffered saline.

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  Figure 7. Transcriptomic association of the AT1R-STAT3-PD-L1 axis and its relationship with immune cell infiltration in colorectal cancer. (A) Spearman correlation analysis of AGTR1, STAT3, and CD274 (PD-L1) expression levels in colorectal cancer samples from the TCGA cohort. (B) Comparison of representative immune cell fractions, including CD8⁺ T cells, CD4⁺ memory resting T cells, CD4⁺ memory activated T cells, and M2 macrophages, among AGTR1-high tumor tissues, AGTR1-low tumor tissues, and adjacent normal tissues estimated using the CIBERSORT algorithm. The X-axis represents immune cell subsets, and the y-axis represents the estimated CIBERSORT infiltration score. Statistical significance was evaluated using the Kruskal-Wallis test followed by pairwise Wilcoxon rank-sum tests with Benjamini-Hochberg correction for multiple comparisons. Significance levels were based on FDR-adjusted P values: ns, P≥0.05; *adjusted P<0.05, **adjusted P<0.01, ***adjusted P<0.001, and ****adjusted P<0.0001. AGTR1, angiotensin II receptor type 1; AT1R, angiotensin II type 1 receptor; CIBERSORT, Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts; FDR, false discovery rate; ns, not…