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. 2014 Jun 25;3(5):1099–1111. doi: 10.1002/cam4.291

Figure 2.

Figure 2

Isolation and characterization of cells with Cancer Stem Cells (CSC) characteristics from H460 and A549 cells. (A) H460 (A–C) and A549 (D–F) cells were cultured in defined media, under nonadhesive conditions for 1–10 days. Pictures were taken using a Nikon TS100 microscope. The bar represents a distance of 100 μm. (B) H460 and A549 cells were cultured in 96 well plates at an average density of 48 cell/plate and the number of clones or spheres present after 10 days of culture was counted. The upper row represents the number of clones obtained culturing the cells in standard serum-containing media under adherent conditions. The middle row represents the total number of spheres containing more than four cells obtained culturing the cells in defined media and under nonadherent conditions. The percentage of spheres (%CSC), as compared to the number of clones, is represented in the lower row of the table. (C) Cisplatin sensitivity. H460 and A549 CSC (H460C, A549C), cisplatin resistant (H460R, A549R), CSCs isolated from H460-resistant cells (H460R-C) or untreated cells (H460, A549) were incubated with 0–2.5 μg/mL of cisplatin for three days. The number of viable cells was estimated using the MTS reagent at the end of the treatment. Average values and standard deviations of three independent experiments are represented. (D) Migration of H460 untreated (H460), cisplatin-resistant (H460R) and CSC (H460C) cells through a matrigel cushion. Fetal Calf Serum was used as chemoatractant. HT1080 and MCF7 cells were used as controls. The lower panel shows microscopic images of the cells that migrated through the matrigel cushion. The quantification of the number of cells that migrated (ten fields for each cell population) is indicated on the upper panel. Significant differences between H460/H460R and H460R/H460C cells are indicated by asterisks (*P < 0.05, **P > 0.01). (E and F) Angiogenic capacity of H460 cisplatin-resistant and CSC cells. Conditioned media obtained for untreated (H460), cisplatin-resistant (H460R) of CSC (H460C) cell lines was embedded in matrigel and subcutaneously implanted in Nu/Nu mice. Matrigel plugs were extracted 10 days after implantation and the presence of endothelial cells analyzed by immunohistochemistry using anti-CD31 antibodies. Panel E shows microscopic pictures of the sections where CD31 expression is indicated in red and DAPI nuclear staining in blue. The lower pictures show the superposition of DAPI staining and CD31 expression. Panel F shows the quantification of CD31 staining. FGF was used as a positive control and the buffer PBS as a negative control. Significant differences between H460/H460C and H460R/H460C cells are indicated by asterisks (**P > 0.01).