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. Author manuscript; available in PMC: 2018 Jul 23.
Published in final edited form as: Methods Mol Biol. 2017;1612:199–212. doi: 10.1007/978-1-4939-7021-6_15

Figure 3.

Figure 3

Analysis of cancer cell invasion using carcinoma cells in an organotypic invasion assay. (a) Representative image of H&E staining of an organotypic invasion assay of 4T1 cells cultured in the presence of CAFs. The different parts of the organotypic gel are indicated. Right panel is a zoom up region that shows both collective and single cell invasion of 4T1 cells. Fibroblasts and ECM fibers are also observed. (b) Representative images of H&E staining of organotypic invasion assays of 4T1 murine breast cancer cells cultured in the absence and presence of either murine normal fibroblasts (NFs) or PyMT-CAFs. Invasion indexes of 4T1 cells for each experimental set-up are also indicated. Scale bar, 50 μm. (c) Representative images of H&E staining of ‘killing’ organotypic invasion assays of 4T1 murine breast cancer cells cultured in gels previously remodeled by murine normal fibroblasts (NFs), PyMT-CAFs or mock-remodeled (no fibroblasts). Invasion indexes of 4T1 cells for each experimental set-up are also indicated. Scale bar, 50 μm. (d) Representative images of H&E staining of organotypic invasion assays of MDA-MB-231 human breast cancer cells cultured in the absence and presence of human breast cancer CAFs. The invasion index is indicated. Scale bar, 50 μm. (e) Representative images of H&E staining of organotypic invasion assays of 410.4 murine breast cancer cells with murine NFs and CAFs. Note that 410.4 cells present a ‘collective’ mode of invasion. Scale bar, 50 μm. (f) Exemplars for image analysis of a representative image of H&E staining of an organotypic invasion assay of 4T1 cells cultured in the presence of CAFs (shown in panel A). First, the non-invaded area (red) and the total area (blue) are measured using Image J (http://imagej.nih.gov/ij/). The invading area is then calculated by subtracting the non-invading area from the total area. The invasion index is the ratio of invaded area vs. total area. Invasion index and areas are also indicated.