Fig. 2

Generation of 3-D human breast cancer microenvironment in vitro. a Schematic representation for generation of 3-D human breast stroma aggregates for in vitro model. Human breast cancer associated breast stromal cells were generated from patient specific primary or brain met tumor tissues ex vivo. Aggregates were then cultured on nucleopore membranes floating in D10 medium supplemented with human epidermal growth factor for 2 weeks for in vitro analyses. b Immuno-histochemical staining comparison between human breast cancer tissues and cancer-associated stromal aggregates. Paraffin-embedded stromal aggregates were sectioned and stained for vimentin, and activated fibroblast markers including alpha smooth muscle and fibroblast activating protein (FAP). Expression of ECM components was analyzed in both tissue section and aggregates. Antibody staining directed against fibronectin and collagen IV showed the presence of ECM in all aggregates. c Immuno-fluorescent antibody staining against Ki67 (red), cytokeratin (yellow) and vimentin (green) in patient-derived aggregates composed of cancer cells mixed with either normal, primary or metastatic stroma. Bar graph illustrates relative expression of Ki67 in cancer cells combined with either normal, primary or metastatic stroma patient samples