Figure 3.

Long-term microevolution of invasive T98G cells under TMZ stress. (A) Comparison of innate morphology and motility of U87 and T98G cells estimated with immunofluorescence microscopy (tubulin-green/DNA-blue), Nomarski Interference Contrast (NIC) and time-lapse videomicroscopy. Dot-plots and bar graphs show trajectories, movement parameters at the single cell and population level, respectively (for circular diagrams, see Figure S3A). Scale bar 50 μm. (B,C) T98G cells were cultivated in the presence of 25 μM TMZ for one to 30 days. Their motility (B) and morphology (C) was estimated with time-lapse video and (NIC) microscopy, respectively. Architecture of microtubular cytoskeleton (tubulin-green/DNA-blue) was estimated with immunofluorescence (see also Figure S3B,C). Scale bar 50 μm. (D) T98G cells were cultivated in the presence of 25 μM TMZ for 1–30 days. The fractions of mesenchymal (rear-front polarized) cells (upper panel) and Snail-1 levels (lower panel) were then estimated with morphometric and immunofluorescence assays. (E) Invasive potential of T98G cells undergone the long-term (30 days) TMZ-treatment in vitro. Cells were seeded into the inserts, the number of cells that transmigrated through the membranes was estimated at the indicated time points and calculated in relation to naive T98G cells. (F) Naive T98G cells and their TMZ-treated (one, seven, and 30 days) counterparts were prepared as in Figure 1A and Cx43 levels were analyzed with immunofluorescence. Scale bar 25 μm. (G) Cells were treated with 25 µM TMZ for one and 30 days and their apoptotic response was estimated with AnnexinV/PI assay. At least 5000 single cells were analyzed, gated according to their area/aspect ratio. Statistical significance was calculated with ANOVA/non-parametric Mann–Whitney test, * p < 0.05 vs. control. Data are representative of three independent experiments. Note the relatively TMZ-resistance of MGMT^high T98G cells and the microevolution of invasive Cx43^high T98G lineages under persistent TMZ stress.