Extended Data Fig. 7: In vivo oncogenic capacities of Fgfr2 variants in somatic models and GEMMs.

a, b, Kaplan-Meier curves showing mammary tumour-free (a) and -specific (b) survival of female Wap-Cre;Cdh1^F/F mice intraductally injected with lentiviruses encoding indicated Fgfr2 variants. Fgfr2^T678*, n = 13 of 7; Fgfr2^P686*, n = 13 of 4; Fgfr2^S694*, n = 12 of 4; Fgfr2^V702*, n = 12 of 5; Fgfr2^Y717*, n = 11 injected MGs of 4 mice. Fgfr2^FL and Fgfr2^ΔE18 curves in a, b are duplicates from Extended Data Fig. 6f, g. c, d, Kaplan-Meier curves showing mammary tumour-free (c) and -specific (d) survival of female WT mice intraductally injected with lentiviruses encoding indicated Fgfr2 variants. Fgfr2^S156W, Fgfr2^C287R, Fgfr2^N454K, Fgfr2^K564E, n = 20 injected MGs of 10 mice. Fgfr2^FL and Fgfr2^ΔE18 curves in c, d are duplicates from Fig. 2c and Extended Data Fig. 6d. e, Schematic representation of the engineered Fgfr2^FL and Fgfr2^ΔE18 alleles. Frt-invCAG-Fgfr2^FL-IRES-Luc and Frt-invCAG-Fgfr2^ΔE18-IRES-Luc were inserted into the Col1a1 locus using the genetically engineered mouse model – embryonic stem cell (GEMM-ESC) methodology⁵⁴. Cre activity inverts the CAG promoter resulting in coherent FGFR2 and luciferase (Luc) expression. IRES, internal ribosome entry site. f, RT-qPCR quantification of Fgfr2 and Luc expression in mouse mammary epithelial cells (MMECs) isolated from pooled MGs of 10-week-old WT control, Fgfr2^FL-IRES-Luc, and Fgfr2^ΔE18-IRES-Luc female mice and mock-treated or treated with adenoviral Ad5CMVCre (AdCre) to switch Fgfr2 alleles in vitro. Data are represented as mean ± s.d. of WT, n = 1; Fgfr2^FL-IRES-Luc, Fgfr2^ΔE18-IRES-Luc, n = 4 MMEC cultures each from MG pools of individual mice. g, h, Western blot showing FGFR2 expression of mock- or AdCre-treated MMEC cultures (g) and quantification of relative FGFR2 intensities normalized to β-actin (h). β-Actin was run on a separate gel as sample processing control, and membranes were stained with Ponceau S to ensure equal loading of total protein. For gel source data, see Supplementary Fig. 1h, i. In h, data are represented as mean ± s.d. of WT, n = 1; Fgfr2^FL-IRES-Luc, Fgfr2^ΔE18-IRES-Luc, n = 3 MMEC cultures each from MG pools of individial mice. i, Luciferase activity measured using luciferin and bioluminescence imaging on mock- or AdCre-treated MMEC cultures. Data are represented as simple linear regressions across Fgfr2^FL-IRES-Luc, n = 4; Fgfr2^ΔE18-IRES-Luc, n = 3 MMEC cultures (each from MG pools of individual mice) at indicated cell densities. j, Representative in vivo bioluminescence images showing luciferase activity following luciferin administration measured as photon flux in 10-week-old Wap-Cre;Cdh1^F/F, Wap-Cre;Cdh1^F/F;Fgfr2^FL-IRES-Luc, and Wap-Cre;Cdh1^F/F;Fgfr2^ΔE18-IRES-Luc female mice. Scale bars, 1 cm. k, Quantification of luciferase activity using recurrent bioluminescence imaging in indicated GEMMs. Wap-Cre;Cdh1^F/F female mice show background luminescence. Wap-Cre;Cdh1^F/F, n = 3; Wap-Cre;Cdh1^F/F;Fgfr2^FL-IRES-Luc, n = 6; Wap-Cre;Cdh1^F/F;Fgfr2^ΔE18-IRES-Luc, n = 4 mice. l, m, Kaplan-Meier curves showing mammary tumour-free (l) and -specific (m) survival of indicated GEMMs. Wap-Cre;Cdh1^F/+, n = 12; Wap-Cre;Cdh1^F/+;Fgfr2^FL-IRES-Luc, n = 5; Wap-Cre;Cdh1^F/+;Fgfr2^ΔE18-IRES-Luc, n = 6; Wap-Cre;Cdh1^F/F, n = 16; Wap-Cre;Cdh1^F/F;Fgfr2^FL-IRES-Luc, Wap-Cre;Cdh1^F/F;Fgfr2^ΔE18-IRES-Luc, n = 19 mice. P values were calculated with log rank (Mantel-Cox) tests (a–d, l, m), one-tailed two-way ANOVA and FDR multiple-testing corrections using the two-stage step-up method from Benjamini, Krieger, and Yekutieli (f), a two-tailed unpaired Student’s t-test (h), or one-way analysis of covariance (ANCOVA) to compare linear regression slopes (i). ****P < 0.0001.

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