Figure 4. ROS scavenging prevents induced mTOR addiction.

a, Reseeding capacity of AOM/DSS^ATKN tumor organoids treated as indicated (n=3, one of three biological replicates).

b, Reseeding capacity of AOM/DSS^ATKN tumor organoids treated as indicated (n=3, one of three biological replicates).

c, Reseeding capacity of AOM/DSS^ATKN tumor organoids treated as indicated (n=3, one of four biological replicates).

d, Reseeding capacity of hCRC^shP2X4 tumor organoids treated as indicated (n=3, one of four biological replicates).

e, Fluorescence microscopic and flow cytometric ROS analysis using DCFDA in 5-FU (20h) treated colon tumor organoids. One representative of four biological replicates (scale bar = 1mm).

f, Immunoblot analysis of AOM/DSS^ATKN tumor organoids treated as indicated. p-γH2AX and the corresponding α-tubulin loading were probed on a separate gel (see SI) (n=3).

g, AOM/DSS^ATKN tumor organoids were injected subcutaneously. Once tumor reached 200 mm, mice were treated as indicated for 12h and analysed for cleaved caspase 3. Nuclei were counterstained with DAPI. (n=3 mice, scale bar = 200 μm).

h, Treatment regimen of subcutaneous hCRC tumors.

i, Growth rate of subcutaneous hCRC tumors in response to treatment as indicated in (h) (n=6 mice for vehicle treatment, n=7 mice for all other treatments. One set of mice was analysed.

j, Representative images of subcutaneous hCRC tumors on day 8 treated as indicated in (i), (n=6 for vehicle, n=7 for all other treatments, scale bar = 5 mm).

k, Representative H&E staining of subcutaneous hCRC tumors described in (i) (n=6 for vehicle, n=7 for all other treatments, scale bar = 200 μm). Staining was performed on all tumors indicated in (i).

l, Schematic representation of how dying cells activate mTOR in adjacent cells to counteract apoptosis induction by increased ROS levels.

All data are mean ±SD and analysed by 1-way ANOVA (a-d) or 2-way ANOVA (i) with Bonferroni’s multiple comparison.