Identification of proteins in BC and MCF-10A cell secretomes.

<p>A. A workflow was established to analyze the secretomes of BC and benign mammary epithelial cells. B. Benign (MCF-10A), ERα-positive BC (MCF-7), and TNBC (MDA-MB-231, DT22, and DT28) cells were established in 3D cultures using Matrigel extracellular matrix. Live (top row) and fixed (bottom row) cultures were imaged by phase contrast or immunofluorescence, respectively. Markers of proliferation (Ki67-red), basement membrane (laminin-green), and nuclei (DAPI-blue) were utilized. C. 3D cultures of MCF-10A cells were exposed to CM from MCF-10A, MCF-7, MDA-MB-231, DT22, or DT28 cells. Proliferation (red) and nuclei (blue) are indicated by Ki67 and DAPI staining, respectively. Scale bars indicate 25 μm. D. 3D cultures described in panel C were analyzed to determine the percent of proliferating MCF-10A cells for each treatment. An asterisk (*) indicates a p-value <0.05. E. CM from each cell line was subjected to MS and compiled results are shown. F. CM from 3D cultures of BC and MCF-10A cells were subjected to Western blotting analysis using antibodies to cathepsin D (CTSD), extracellular matrix protein 1 (ECM1), peroxiredoxin 1 (PRDX1), or 14-3-3 sigma (SFN). The number of spectral counts (SCs) is indicated above each lane. To visualize SFN, the CM was concentrated before blotting. MDA-MB-231 and MCF-10A cells are labeled as 231 and 10A, respectively. (Abbreviations: BC = breast cancer, TNBC = triple negative breast cancer, 3D = three dimensional, CM = conditioned medium, MS = mass spectroscopy)