Supplementary Figure 7: Lgr6⁺ cells contribute to tumour maintenance in the MMTV-PyMT model of breast carcinoma.

(a) Experimental setup to analyse chemotherapeutic resistance of Lgr6-expressing PyMT tumour cells in vitro. Tumour-bearing PLT mice were treated with tamoxifen and tumour organoids were cultured 24 h later. The organoids were treated with 2 μM doxorubicin for 48 h and allowed to recover until analysis. (b) Combined light microscopy and fluorescence images of tumour organoids comparing tdTomato tracing between untreated PLT organoids (upper panels) and doxorubicin-treated organoids (lower panels). tdTomato + cells survive chemotherapeutic treatment (lower middle panel) and are able to form completely traced, healthy organoids upon recovery (lower right panel). (c) Extent of tdTomato tracing within untreated and doxorubicin-treated organoids. (n = 8 pooled tumours from n = 4 mice, collected in 3 independent experiments). Mean ± s.e.m. ^∗∗P < 0.01, ^∗∗∗P < 0.001. (multiple unpaired t-tests). Scale bars, 200 Scale bars, 200 m, 100 μm (insets). (d) Scheme of PDL tumour cell isolation, allele recombination and DT treatment in vitro before colony formation analysis. Images show examples of tumour spheres from untreated (left) and DT-treated (right) cells. (e) mRNA expression for β-actin (Actb), Lgr6 and DTR (normalized over GAPDH) in tamoxifen- and DT-treated tumour cells from PDL mice. (n = 3 wells per group from n = 3 independent mice). Mean ± s.d. ^∗∗∗P < 0.001 (unpaired two-tailed t-test) (f) Analysis of colony formation of primary PDL tumour cells after treatment with tamoxifen alone or tamoxifen plus diphtheria toxin. (n = 12 wells per group from n = 3 independent mice). Mean ± s.d. ^∗P < 0.011 (Two-way ANOVA). (g) H&E staining of tumours formed by control and DT-treated PDL primary tumour cells injected subcutaneously into nude mice as shown in Fig. 7g. Scale bars, 500 μm (1x), 50 μm (40x). See Supplementary Table 2 for exact P values and source data for c,e,f.