Extended Data Fig. 3. (related to Figure 3): Dying tumor cells activate mTORC1 via ATP/P2X₄ in human CRC and PDAC.

a, Relative mRNA expression levels of the indicated genes in the colon tumors from Lgr5^EGFP-DTR(+) mice 24h after injection of DT injection or vehicle determined by qRT-PCR. (n=10 tumors for controls and n=9 tumors for DT).

b, Relative mRNA expression levels of the indicated genes in Lgr5^EGFP-DTR(+) tumor organoids at 24h after DT treatment determined by qRT-PCR. Expression of vehicle treated organoids was set to 100 arbitrary units (n=4 biological replicates).

c, Immunoblot analysis of Lgr5^EGFP-DTR(+) tumor organoids treated as indicated for 30 min (n=3).

d, Immunoblot analysis of Lgr5^EGFP-DTR(+) tumor organoids treated as indicated for 16h (n=3).

e, Immunoblot analysis of Sting WT and Sting KO Lgr5^EGFP-DTR(+) tumor organoids treated as indicated (n=4).

f, Immunoblot analysis of murine (CMT93 and CT26, left panel) and human CRC cells (HCT116 and RKO, right panel) treated as indicated (n=2).

g, Immunoblot analysis of Lgr5^EGFP-DTR(+) tumor organoids treated as indicated for 12h (n=3).

h, Left: Immunoblot analysis of ATP (100 μM) treated CT26 cells transfected with control or Rheb siRNA (n=3). Right: Relative mRNA expression levels of Rheb in siRNA transfected cells determined by qRT-PCR (n=3 biological replicates).

i, Left: Immunoblot analysis of ATP (100 μM) treated CT26 cells transfected with control or a combination of RagA and RagB siRNA (n=3). Right: Relative mRNA expression levels RagA and RagB in siRNA transfected cells determined by qRT-PCR (n=3 biological replicates).

j, Relative mRNA expression levels of P2x and P2y receptors in mouse colon tumor organoids tissues determined by qRT-PCR (n=3 biological replicates).

k, Immunohistochemistry analysis of P2x4 expression in healthy murine colon or colon tumors of AOM/DSS treated mice. Images from one representative staining (n=3, scale bar = 300 μm).

l, Reseeding capacity of hCRC tumor organoid line 2 treated as indicated (n=3, one of three biological replicates).

m, Reseeding capacity of doxycycline-inducible hCRC^shP2x4#² tumor organoids treated as indicated (n=4, one of three biological replicates).

n, P2x4 qRT-PCR analysis of doxycycline treated hCRC^shP2x4#² tumor organoids (n=3 biological replicates).

o, Reseeding capacity of human pancreatic tumor organoids treated as indicated (n=3, one of three biological replicates).

p, Reseeding capacity of human pancreatic tumor organoids treated as indicated (n=3, one of three biological replicates)

All data are mean ±SD and analysed by two-tailed Student’s t-test (a, b, h, i, n, o, p) or by 1way ANOVA with Bonferroni’s multiple comparison (l, m).