Fig. 4
Myoepithelial cells (MEPs) reduce proteolysis of extracellular matrix (ECM) and invasive phenotype of ductal carcinoma in situ (DCIS) structures formed in mammary architecture and microenvironment engineering (MAME) and modified MAME cultures, respectively. a Representative angled en face views of 3D reconstructions of structures formed by MCF10.DCIS-lenti-RFP (DCIS, red) cells seeded alone (top row) or in coculture with N1ME (MEPs, unlabeled; second row), WS-12T-lenti-YFP (cancer-associated fibroblasts [CAFs], pseudocolored fuchsia; third row), or both CAFs and MEPs (bottom row). The reconstituted basement membrane (rBM) overlay cultures contained dye-quenched collagen IV (DQ-collagen IV) and were imaged live at 21 days. One grid unit = 92 μm. b Volume of degraded dye-quenched collagen IV (dDQ-IV, green) per cell in the 4 MAME culture variants was quantified in 64–96 fields (16 contiguous fields per experiment from 4–6 independent experiments) with Volocity software. Unlabeled cells were used to eliminate possible interference with analysis of fluorescent dDQ-IV degradation products. Degradation products were quantified per cell number as determined by counting nuclei labeled with Hoechst 33342 in 3D reconstructions assembled from optical sections through the entire depth of the cultures. Intensities of dDQ-collagen IV fragments in 3D are the sum of intensities in individual optical sections. Using the nuclei counts, these data were then calculated as volume per cell [(μm3 × 103)/cell]. Data are presented as box-and-whisker plots where the box represents the interquartile range and whiskers represent minimum and maximum values. * p ≤ 0.05; ** p ≤ 0.01 as determined by one-way analysis of variance and Bonferroni’s multiple comparisons test (n = 4–6). Representative angled (c and d, top rows) and en face (c and d, bottom rows) views of 3D reconstructions of 8- and 21-day MAME cultures with the following culture modifications: WS-12T-lenti-YFP (CAFs, pseudocolored fuchsia) were embedded in a bottom layer of collagen I containing DQ-collagen I (dDQ-colI, green) and MCF10.DCIS-lenti-RFP (DCIS, red) cells with (c; 1 grid unit = 92 μm) and without (d; 1 grid unit = 90 μm) MEPs were seeded onto a second layer composed of rBM containing DQ-collagen IV (dDQ-IV, green). An overlay of 2% rBM was placed on top of the cocultured cells. Images are representative of at least three independent experiments. Additional results are shown in Additional file 6: Figure S4