Fig. 2

Metastasis expansion is not dependent on malignant MDCs. A H&E staining (left panel) or GFP immunohistochemistry (middle panel) of lung metastases forming in MMTV–PyMT-Green (MPG) mice at 12 (a) or 16 weeks (b). Blue, DAPI staining. To quantify the proportion of GFP-positive cells (right panel), the layers of images were decomposed by Image J software and restained using chromophore fluorescence for GFP expression (red) and DAPI (blue). Scale bar, 50 μm. B The absolute numbers (a) and proportion (b, number of GFP-positive cells divided by the total number of cells) of malignant mammary gland-derived cells (MDCs) in lung metastases of MPG mice (7 metastases/group, n = 5) at different ages (from 12 to 18 weeks) was quantified. n = number of groups. Data are expressed as mean ± standard deviation. C Single-cell RNA-Seq was performed on single-cell suspensions generated from the infiltrating cells (excluding T lymphocytes) or peritumoral cells of the lung metastatic tissues of 16-week-old MPG mice. a Cells were clustered using a graph-based shared nearest neighbor clustering approach and visualized using a t-distributed Stochastic Neighbor Embedding (tSNE) plot. b Cells on the tSNE plot were colored as originating either from the infiltrating cells (Infil) or peritumoral cells (Peri). D Schema of the genesis of MMTV–PyMT-Green/Sftpc-Tomato mice (MPG-ST) mice. E Reconstructed confocal images of the lung sections collected from MPG-ST mice at 12 (left panel) and 16 (right panel) weeks. The white frames showed metastases, and the arrows showed interspersed red fluorescent cells. Blue, DAPI; Green, malignant MDCs; Red, AT2 cells; Scale bar, 20 μm. F The proportion of malignant MDCs and AT2 cells in lung metastases of MPG-ST mice at different ages was quantified. Data are expressed as mean ± standard deviation