Fig 1.
Histology and immunohistochemistry of myofibroblasts and endothelial cells in human and mouse breast cancer tumors.
Slides stained with hematoxylin, eosin and safranin (HES) and Masson’s trichrome, and stained for α-SMA and CD31: (a) human PDXs and (b) murine PyMT and ErbB2 tumors (original magnification: 400x).
Fig 2.
Heterogeneity of mouse-derived stroma in breast cancer tumors.
(a) Flow cytometry analysis of tumor-infiltrating hematopoietic stromal cells after dissociation of the HBCx-9 tumor graft and staining with anti-EpCAM, CD45, F4/80, Ly-6G, Ly-6C and CD19 antibodies. Viable murine macrophage-like cells (DAPI-EpCAM-CD45+CD11b+F4/80+), monocytes (DAPI-EpCAM-CD45+CD11b+F4/80+Ly-6Chi), granulocytes (EpCAM-CD45+CD11b+Ly-6G+) and B lymphocytes (EpCAM-CD45+CD11b-CD19+) were identified as separate populations. (b) Flow cytometry analysis of individual leukocyte populations as a percent of total CD45+ cells in MMTV-PyMT/BC-PyMT and MMTV-Erbb2/BC-Erbb2 mammary tumors and in 20 PDX. The data shown are the mean percentages of viable cells ± SEM (standard error of the mean) for three mice per cohort. BC models were compared in Kruskal-Wallis tests. (c) Comparison of dissociated cell profiles showing the similarity of spontaneous and allografted MMTV-PyMT/BC-PyMT and MMTV-Erbb2/BC-Erbb2 tumors on the basis of anti-EpCAM, CD45, Ly-6C and F4/80 staining. (d) Specific stromal profiles of BC tumors. Clustering of stromal population percentage data from flow cytometry shown in (2b) above.
Fig 3.
Transcriptome profiles of macrophage-like cells in BC tumors.
(a) Results of principal component analysis (PCA) for the subset of 1238 genes up- or downregulated in at least one comparison between MMTV-PyMT and BC-PyMT, HBCx-5, -24 or -34. The 15 samples, triplicates of MMTV-PyMT (green), BC-PyMT (orange), HBCx-5 (red), HBCx-24 (yellow) and HBCx-34 (blue), were projected oton the first three principal components, which accounted for ~58% of the total variability. Hierarchical clustering of the genes from the (b) “Immune system process” or (c) “Metabolic process” pathways from Gene Ontology analysis identified as significantly up- or downregulated in at least one comparison between BC-PyMT and HBCx-5, -24 and -34.
Fig 4.
M1/M2 macrophage-like cell phenotype in BC tumors.
(a) The expression of genes associated with the M1 (MHC-II, CD86, Cd11c, Il1b, Cxcl10 and Cxcl11) or M2 (Mrc1, Scara4, Scarb3, Arg1, Igf1 and Ccr2) phenotype was assessed by the dissociation of five tumors (MMTV-PyMT, BC-PyMT, HBCx-5, -24 and -34), the sorting of macrophage-like cells, and microarray analysis. Levels of gene expression in BC models were compared in unpaired Student’s t-tests (b) Protein levels for M1 (MHC-II and Cd11c) and M2 (Mrc1) markers on macrophage-like cells from the five tumors MMTV-PyMT, BC-PyMT, HBCx -5, -24 and x-34), as measured by flow cytometry. Three tumors were analyzed per model. For each model, Mann-Whitney tests were performed to compare the results obtained with those for the MMTV-PyMT tumor (* p< 0.05, **p <0.01, ***p <0.001). (c) Examples of flow cytometry findings for the levels of M1 and M2 marker proteins (the corresponding isotype is shown in gray).