Fig. 6: Metformin improves the outcome of fractionated radiotherapy only in the subgroup of tumors with attenuated cytokine response.

Cal 33 cells were heterotopically transplanted onto NMRI nude mice and subjected to fractionated irradiation with 5 × 2 Gy ± 250 µg/g metformin treatment (n = 10 animals for all groups). A Tumor volume was followed up, and means with upper and lower 95% confidence intervals are shown. p-values were calculated by two-way ANOVA with post-hoc Bonferroni-Holm correction. B Time to tumor volume > 200 mm³ was determined from growth curves shown in A, and group comparisons were performed by log-rank tests with post-hoc Bonferroni-Holm correction. C Schematic of tumor sampling for cytokine analyses shown in D, E (n = 6 animals for vehicle, 5 × 2 Gy, and metformin groups, n = 7 for 5×2 Gy + metformin group). D Cytokine levels as measured in lysates of tumor explants by multiplex ELISAs. Results from individual mice are shown with superimposed boxes (1st, 2nd, and 3rd quartiles). Asterisks indicate means, and whiskers depict extreme values. p-values were determined by Student’s t-tests with Benjamini-Hochberg correction. E Subgroup analysis in the 5 × 2 Gy + metformin group separated into tumors with attenuated and without attenuated cytokine response. Tumor volumes and tumor weights were determined on the day of explantation, and group comparisons were performed by Student’s t-tests. F Gene set enrichment analysis (GSEA) of differentially expressed human transcripts in xenografts with attenuated cytokine response vs. tumors without attenuated cytokine response (MSigDB hallmark gene sets). Left panel: global enrichment plot, right panel: Hallmark gene sets with FDR q-values < 0.1.