Figure 3

ASC-Ls promote tumors through increased vasculogenesis. (A) Gene ontology biological process categories associated with adipose stromal cells from lactating mice compared to mammary adipose stromal cells from nulliparous or involuting mice. The DAVID Functional Annotation Tool was used to define categories with an enrichment score >1.5, and the number of genes represented in the list and the P value of genes differentially expressed in the microarray are shown. (B) Gene set enrichment analysis indicates that ASC-Ls exhibit increased expression of genes associated with cytokine production and tumor angiogenesis. (C-E) Endothelial cell assays using primary mouse heart endothelial cells treated with conditioned media collected from ASCs isolated from nulliparous, lactating or regressed glands (ASC-N, ASC-L or ASC-R, respectively), n = 3 per group. Data are presented as means ± SEM (C) Proliferation assay day 3. Data are presented as means ± SEM. n = 3 experiments. (D) Quantification of tube formation assay four hours after plating. Data are presented as means ± SEM. n = 3 experiments. (E) Representative image and quantification of wound healing assay six hours after addition of conditioned media. Data are presented as means ± SEM. n = 3 experiments. (F) Representative immunofluorescence and quantification of 4 T1-12B tumors co-mixed with adipocytes for CD31 (red) and counterstained for nuclei with DAPI (blue) (n = 4 per group). Data presented as means ± SEM. (G) Quantitative RT-PCR of VEGF-A, VEGF-D, MMP-9 and IL-6 in ASC-N, ASC-R or ASC-L. Data are presented as average 2^(-ΔCt) ± SEM; n = 3 experiments. (H) Representative images of immunohistochemistry of 4 T1-12B tumors co-mixed with adipocytes for LYVE-1. n = 3 experiments. Scale bar = 100 μm. ASC-L, lactation-derived adipose stromal cells; ASC-N, nulliparous-derived adipose stromal cells; ASC-R, regressed-derived adipose stromal cells; DAPI, 4′,6-diamidino-2-phenylindole; MMP-9, matrix metalloproteinase 9; SEM, standard error of the mean; VEGF, vascular endothelial growth factor.