Extended Data Fig. 7. Comparative analysis of tumour growth by serial transplantation of constant and transient ph-KD EICs.

a- Schematic overview of the experimental allograft workflow. Flies of the same genotype were subjected to no ph-KD (control, 18 °C), constant ph-KD (29 °C) or transient ph-KD (24h ph-KD at 29 °C during L1 stage). L3 EDs expressing constitutive GFP were dissected from donor larvae and repeatedly allografted into the abdomen of host flies for 10 consecutive rounds until T10 of transplantation (T10≈ 3 months). All allograft experiments were performed at 18 °C to avoid ph-RNAi expression after transplantation. b-c- Tumour growth measured as the percentage of flies showing tumour progression 20 days after transplantation (b) or surviving 20 days after each allograft (c) constant (purple) or transient (blue) ph-KD tumours for 10 rounds of transplantation (x-axis). d- Tree representation of the allograft assay. A primary ED tumour derived from constant or transient ph-KD is dissected from L3 donor larvae and repeatedly allografted into the abdomen of a female host maintained at 18 °C to prevent re-expression of-ph-RNAi. Each injected fly is monitored every two days. When the host fly abdomen is completely filled with GFP positive cells, the host is dissected and the tumour cells are injected again into multiple hosts. The procedure was repeated until the tenth generation (T10). e- Host lifespan (x-axis) after the first transplantation (T1) of control (no ph-KD, in black), constant (in purple) or transient (in blue) ph-KD tumours. Statistical significance was assessed using log-rank test. f- Host lifespan (x-axis) after the fifth (T5) and the tenth (T10) rounds of transplantations of constant (T5 in pink, T10 in purple) or transient (T5 in light blue, T10 in blue) ph-KD tumours. Since control (no ph-KD) tissues do not grow and cannot be serially transplanted, PBS injections were used as control (in black). Statistical significance was assessed using log-rank test. g- Flies injected with dissected grafts after no ph-KD (control), constant or transient ph-KD. Only primary tumours generated by constant or transient ph-KD can invade the abdomen and surrounding tissues but not EDs resulting from no ph-KD (control) conditions. h- In order to score the frequency of metastases, the injected flies were monitored twice a week and the appearance of metastases in the thorax, head, proboscis, eyes and legs were noted for each generation. Values in the table represent the number and percentage (in brackets) of flies with metastases after the 1^st, 5^th or 10^th round of transplantation (T1, T5 and T10, respectively) of constant or transient ph-KD tumours. i-j- Tumour growth measured as the percentage of flies showing tumour progression 20 days after transplantation (d) and host fly survival 20 days after allograft of late L3 ph-KD (e) for 5 consecutive rounds of transplantation.