Figure 3. Highly-metastatic mammary tumors differentially activate periostin-expressing CAFs.

(A) qPCR analysis of periostin expression in mammary tumor cell lines and murine mammary CAFs. Data represent the mean ± SEM. Experiments were performed in triplicate and results compared using Kruskal-Wallis test. (B) Study design for EO771 vs EO771.LMB experiment in Postn^iZSGreen lineage tracing mice. (C) Tissue tilescans of primary mammary tumors from Postn^iZSGreen mice. Tumor cells labelled with mCherry and periostin-expressing cells genetically labelled with ZSGreen. Nuclei counterstained with DAPI. Scale bars: 500 μm. (D) Percentage of tissue area positive for ZSGreen in low-metastatic (EO771) tumors versus highly-metastatic (EO771.LMB and PyVMT) tumors (n = 3–7 mice per group). Each data point represents a histological section. Statistics shown for Kruskal-Wallis, Dunn’s multiple comparisons test. (E) Representative immunofluorescence images of mammary tumors from Postn^iZSGreen mice stained for periostin (in blue), with periostin-expressing cells genetically labelled with ZSGreen. Scale bars: 100 μm. (F) Representative images of individual ZSGreen-labelled periostin-expressing cells in EO771 and EO771.LMB tumors. Scale bars: 10 μm. (G) Area of individual ZSGreen-labelled cells in primary tumors represented as a histogram (left) and scatter plot (right). Each data point in the scatter plot represents an individual cell. 120–180 cells were measured from multiple tumor sections (n = 3–6 mice per group) and results compared using Kruskal-Wallis, Dunn’s multiple comparisons test. (Study design schematic created using BioRender.com).