Extended Data Fig. 8. Radiation-exposure boosts Notch-signalling and stemness in metastatic cells.

a, Quantitative RT-PCR analysis of Notch1 and target gene Hes1 in lineage-traced lung alveolar type 2 cells from SPC-Cre-ER^T2 control mice and SPC-Cre-ER^T2/Rosa26^{NICD-IRES-GFP} Notch-activated mice. Mice were administered tamoxifen by oral gavage (40 mg/kg) over three consecutive days. Lungs were harvested 14 days after the last tamoxifen dose and GFP⁺ cells were sorted by flow cytometry (n=3 control mice, n=4 Notch mice). b, Primary tumour weight from PyMT/Control (n=6) and PyMT/Notch mice (n=8) harvested two weeks post-tamoxifen induction. c, Quantification and representative images of Hes1 immunostaining in lung metastases from PyMT/Control (n=5) and PyMT/Notch mice (n=7) mice (metastatic lungs harvested over n=5 independent tamoxifen administrations, immunostaining quantification in methods). The enlarged inset shows nuclear localisation. Scale bar, 100 μm (main image), 10 μm (enlarged inset). d, Combined Uniform Manifold Approximation and Projection (UMAP) plot of cells from the mCherry⁺ niche and mCherry^- distant lung from irradiated mice (n=10 mice, pooled). The expression level of EpCAM (distinguishing epithelial cells), Pdgfrα and Pdgfrβ (fibroblasts) and Pecam1(CD31) (endothelial cells) is indicated in blue. e, Representative immunostaining for RBPJ (top panel) and Hes1 (lower panel) in metastatic lungs from control, irradiated, and neutrophil-depleted irradiated mice harvested at day 14 (n=7 mice per group, 2 independent experiments). Quantification shown in Figure 6d,e. The enlarged inset shows nuclear localisation within tumour cells. Hes1⁺ cells are indicated by arrows. Scale bar, 100 μm (main image), 10 μm (enlarged inset). f, Representative immunostaining and quantification of RBPJ staining intensity in metastatic lungs from irradiated FVB mice pre-treated with rGCSF or PBS prior to injection of MMTV-PyMT cancer cells (Figure 3e, n=5 mice per group). Staining intensity within the metastatic area was measured using ImageJ (see methods). Scale bar, 100 μm. All data represented as mean ± s.e.m. Statistical analysis by non-parametric two-tailed Mann-Whitney test for (a), a two-tailed unpaired t-test for (b), (c) and (f). UT, untreated; IR, irradiated. The violin plot displays the median, 25^th and 75^th percentiles as well as the density of data points. Dots represent individual mice. Gating strategies for FACS sorting for (d) provided in Extended Data Fig. 2. Source data.