Fig. 4

Radiosensitization in PMI ablated HNSCC models driven by combined PMI ablation and mannose mediated metabolic alterations. A) Schematic mannose treatment schedule of colony forming assay. Clonogenic assays in WT and PMI KO/KD B i & ii) FaDu and C i & ii) CAL27 cells, +/- mannose (20 mM) pre-treatment (48 h), followed by radiation (2–8 Gy). D) Relative ROS levels detected using 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) in WT and PMI KO/KD (i) FaDu and (ii) CAL27 cells +/- mannose (20 mM) pre-treatment (24 h) prior to radiation (4 Gy). ROS levels were measured 10 min post-radiation treatment. E) 53BP1 immunofluorescence in WT and PMI KO (i) FaDu cells with (ii) quantified differences calculated by scoring a minimum of 50 cells per independent replicate. Scale bars: 40 μm. F) 53BP1 immunofluorescence in WT and PMI KD (i) CAL27 cells with (ii) quantified differences calculated by scoring a minimum of 50 cells per independent replicate. Unlabelled LC-MS analysis of the metabolic profiles of WT and PMI KO FaDu cells treated with mannose, +/- radiation (4 Gy). Scale bars: 40 μm. G) Principal Component Analysis (PCA) depicting distinctive clustering of the metabolome in mannose exposed cells. H) A heatmap generated from thirty-nine dominant metabolites for each treatment group (n = 5 for each group). Data presented (panels B-F) are mean ± SD of three independent biological replicates. *p ≤ 0.05 - One-way ANOVA - Tukey’s multiple comparisons test B-D) and two-way ANOVA - Tukey’s multiple comparisons test A)