Extended Data Fig. 6: In vitro and in vivo oncogenic capacities of Fgfr2 variants.

a, Representative images of 12-well plate wells and quantification of 3D soft agar colony formation assay using NMuMG cells expressing GFP or indicated Fgfr2 variants and treated with vehicle, 100 nM AZD4547, or 100 nM pemigatinib for 15 days. Data are represented as mean ± standard deviation (s.d.) of GFP, Fgfr2^FL, Fgfr2^ΔE18, vehicle, n = 33; AZD4547, pemigatinib, n = 18 independent replica from 4 individual experiments. Fgfr2^FL-Bicc1, vehicle, n = 18; AZD4547, pemigatinib, n = 9 independent replica from 3 individual experiments. Fgfr2^ΔE18-Bicc1, Fgfr2^FL-Ate1, Fgfr2^ΔE18-Ate1, Fgfr2^FL-Tacc2, Fgfr2^ΔE18-Tacc2, Fgfr2^ΔE18-IGR1, Fgfr2^ΔE18-IGR2, Fgfr2^E18-C2, Fgfr2^E18-C3, Fgfr2^E18-C4, Fgfr2^Y674*, Fgfr2^T678*, Fgfr2^L681fs*6, Fgfr2^P686*, Fgfr2^S694*, Fgfr2^S156W, Fgfr2^C287R, Fgfr2^N454K, vehicle, n = 12; AZD4547, pemigatinib, n = 6 independent replica from 2 individual experiments. Fgfr2^ΔE18-Bicc1^ΔSAM, Fgfr2^K422R, Fgfr2^K422R-ΔE18, Fgfr2^K422R-Bicc1, Fgfr2^K422R-ΔE18-Bicc1, Fgfr2^S687fs*3, Fgfr2^S697fs*4, Fgfr2^V702*, Fgfr2^S704fs*22, Fgfr2^Y717*, Fgfr2^K564E, n = 6; AZD4547, pemigatinib, n = 3 independent replica from 1 experiment. b, c, FACS to quantify traced EGFP⁺ EpCAM⁺ epithelial cells (b) and their FGFR2 MFI (c). Rosa26-mT/mG female reporter mice were intraductally injected with lentiviruses encoding Cre or indicated Fgfr2-P2A-Cre variants resulting in Cre-mediated mT/mG allele switching, thus cell membrane-localized tdTomato (mT) expression was replaced by membrane-localized EGFP (mG) expression. Mammary glands (MGs) were subjected to FACS analysis at indicated timepoints post injection. Data are represented as mean ± s.d. and each data point represents a MG pool of an individual mouse. Analyses were done in batches of 1–2 mice of each Fgfr2 variant and one timepoint. In (b), 1 week, uninjected MGs, n = 5; Cre, n = 7; other Fgfr2 variants, n = 4; 3 weeks, all groups, n = 4; 6 weeks, uninjected MGs, Cre, Fgfr2^K422R-P2A-Cre, Fgfr2^K422R-ΔE18-P2A-Cre, Fgfr2^K422R-Bicc1-P2A-Cre, Fgfr2^K422R-ΔE18-Bicc1-P2A-Cre, n = 5; other Fgfr2 variants, n = 6 mice. In (c), 1 week and 3 weeks, all groups, n = 3; 6 weeks, Cre, Fgfr2^FL-P2A-Cre, Fgfr2^ΔE18-P2A-Cre, Fgfr2^FL-Bicc1-P2A-Cre, Fgfr2^ΔE18-Bicc1-P2A-Cre, Fgfr2^ΔE18-Bicc1^ΔSAM-P2A-Cre, n = 4; other Fgfr2 variants, n = 3 mice. For FACS gating strategy, see Supplementary Fig. 2b. d, e, Kaplan-Meier curves showing mammary tumour-specific survival of female wild-type (WT) mice intraductally injected with lentiviruses encoding indicated Fgfr2 variants. Fgfr2^FL, n = 20; Fgfr2^ΔE18, n = 22; Fgfr2^FL-Bicc1, Fgfr2^ΔE18-Bicc1^ΔSAM, Fgfr2^FL-Ate1, Fgfr2^ΔE18-Ate1, Fgfr2^FL-Tacc2, Fgfr2^ΔE18-Tacc2, Fgfr2^ΔE18-IGR1, Fgfr2^ΔE18-IGR2, Fgfr2^E18-C2, Fgfr2^E18-C3, Fgfr2^E18-C4, n = 10; Fgfr2^ΔE18-Bicc1, n = 11 mice. Fgfr2^FL and Fgfr2^ΔE18 curves in a are duplicated in d, h. f, g, Kaplan-Meier curves showing mammary tumour-free (c) and -specific (d) survival of female Wap-Cre;Cdh1^F/F mice intraductally injected with lentiviruses encoding indicated Fgfr2 variants. Fgfr2^FL, n = 34 of 15; Fgfr2^ΔE18, n = 39 of 15; Fgfr2^FL-Bicc1, n = 19 of 10; Fgfr2^ΔE18-Bicc1, n = 21 injected MGs of 11 mice. h, Kaplan-Meier curves showing mammary tumour-specific survival of female wild-type (WT) mice intraductally injected with lentiviruses encoding indicated Fgfr2 variants. Fgfr2^Y674*, Fgfr2^L681fs*6, Fgfr2^S697fs*4, Fgfr2^S704fs*22, n = 10; Fgfr2^T678*, n = 4; Fgfr2^P686*, Fgfr2^S694*, Fgfr2^V702*, Fgfr2^Y717*, n = 5 mice. P values were calculated with one-tailed two-way ANOVA and FDR multiple-testing corrections using the two-stage step-up method from Benjamini, Krieger, and Yekutieli (a, c), one-tailed Kruskal-Wallis tests and Dunn’s multiple-testing corrections (b), or log rank (Mantel-Cox) tests (b–h). ****P < 0.0001; NS, not significant (P ≥ 0.05).

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