Fig. 3: FLASH RT induces lipid peroxidation in mouse xenograft tumors while sparing normal lung and intestine tissues.

A Lung tumor tissues (A549 and Calu-6) from subcutaneous mouse xenograft models were stained with 4-HNE, a lipid peroxidation marker. After conventional and FLASH RT (15 Gy for A549 and 20 Gy for Calu-6), there was a significant increase in 4-HNE staining intensity (n = 4 for A549 0 Gy and conventional RT, n = 3 for FLASH RT, n = 6 for Calu-6 0 Gy and FLASH RT, n = 8 for Calu-6 conventional RT). B BALB/c mouse lung tissues were stained with 4-HNE after 10 Gy of conventional or FLASH RT. Lipid peroxidation was significantly enhanced 24 h and 7 days after conventional RT. However, FLASH RT did not change lipid peroxidation (n = 6 per group at each time point). C, D BALB/c mice were treated daily with 2 mg/kg Ferr-1 or DMSO vehicle, starting one day prior to RT, to inhibit ferroptosis. Lipid peroxidation, detected by 4-HNE staining, was markedly increased after 10 Gy conventional RT compared to 0 Gy, but not after 10 Gy FLASH RT in the DMSO-treated group (C). This increase in lipid peroxidation was reversed by Ferr-1 treatment. Tissue damage in the upper intestines was assessed by H&E staining, based on the number of remaining intestinal crypts (D, arrowheads). Both 10 Gy conventional and FLASH RT significantly reduced crypt numbers, with conventional RT causing more severe damage. Ferr-1 treatment improved crypt preservation in the conventional RT group but had no significant effect in the 0 Gy or FLASH RT groups (n = 4/group). Error bars indicate standard deviation (SD). Statistical tests were performed by One-way ANOVA (A) or Two-way ANOVA (B–D) with Tukey’s multiple comparisons test. IntD: Integrated Density.