Extended Data Figure 1. Oncogene ablation leads to tumor regression in vitro and in vivo.

a, In vivo experimental scheme. Tumor cells isolated from primary tumors or tumor spheres were injected in nude mice fed with doxycycline in drinking water (+Dox). When mice developed tumors, doxycycline was withdrawn (-Dox) and tumors underwent a complete macroscopic regression after 2-3 weeks (arrows indicate regressed tumors). In residual lesions few tumor cells remain quiescent for months and they can quickly reform tumors upon KRas reactivation (+Dox). b-c, Tumor expressing KRas (+KRas) and tumor remnants after regression (-KRas) are positive for ductal epithelial marker CK19 (b) (40×). Tumor expressing KRas (+KRas) and epithelial remnants after tumor regression (-KRas) were stained for phosphorylated-p42/44 (pErk). No signal is detected in surviving cells (c) (20×). d, In vitro experimental scheme. After digestion to a single cells suspension, tumor cells isolated from primary tumors were plated in stem cell medium in presence of doxycycline (+Dox, +KRas). Spherogenic cells form tumor spheres (+KRas) that can be maintained by serial replating in presence of doxycycline. Upon doxycycline withdrawal (-Dox) tumor spheres undergo involution and only a minority of cells survives the ablation of KRas (surviving cells, -KRas). Surviving cells readily reform tumor spheres upon re-activation of KRas (+Dox). e, The amount of active Ras in KRas expressing cells (+KRas) and surviving cells (-KRas) has been evaluated in three independent tumor spheres by detecting the fraction of Ras protein that co-precipitates with Raf kinase. Total lysates were probed with anti-phospho-p42/44 (pErk), total p42/44 (Erk) antibodies. f, AnnexinV staining in tumor spheres after 3 days +/-KRas (n=3). g, Sphere formation is a regulated process and tumor cells enter and exit cell cycle. BrdU incorporation (pulse of 3 hours) has been evaluated at different time points during sphere formation and regression. KRas expressing fully formed spheres (Day 0 and 8) are quiescent. Upon sphere dissociation and replating (D0), spherogenic cells enter cell cycle (D1) and tumor cells continue to grow till day 3-4, when spheres reach their maximal S-phase. Then tumor cells gradually exit the cell cycle and become quiescent (D8). After doxycycline withdrawing (-KRas) tumor spheres undergo involution and surviving cells remain quiescent till KRas is re-expressed (-KRas 24hs +KRas) and spheres are reformed. Ruling out the effect of the cell cycle, transcriptomic and metabolomic characterizations have been done matching quiescent surviving tumor cells to quiescent fully formed KRas expressing spheres at D8 (n=3). h, HE and IHC of regressed tumors after three 8-hour pulses of BrdU show that epithelial remnants in regressed tumors after KRas ablation (-KRas) are completely quiescent (left panels). 48 hours after KRas reactivation (doxycycline IP injection) tumor cells re-enter massively the cell cycle (right panels). Red arrows indicate mitotic cells (20×). i, Representative AnnexinV staining with respect to CD133 and CD44 after three days of KRas ablation, two independent tumors are represented. Data are mean ± s.d.