EGF has long been shown to affect the development of GBM ^[1]. Indeed, microscopic analyses revealed noticeable phenotypic shifts towards a spindle-like (mesenchymal) morphology in the T98G cell populations exposed to the EGF (1 and 10 ng/mL). Interestingly, EGFR inhibition by Erlotinib (Erl) entirely promoted the epithelial phenotype in both the EGF-treated and non-treated cell populations. These effects were accompanied by a pronounced dose-dependent pro-proliferative activity of the EGF (Figure 1A,B). Furthermore, microscopic and spectrophotometric experiments revealed intensified MTT substrate processing in the cells exposed to 48 h EGF treatment. This was manifested by a considerable accumulation of formazan crystals within the intracellular space (Figure 1C,D). Concomitantly, the effects of EGF on the cell morphology, proliferation, and metabolism were inhibited by erlotinib administration (Erl, a chemical inhibitor of the EGF receptor (EGFR); Figure 1A–D). Notably, no cytotoxic/pro-apoptotic effects of EGF/Erl were observed, as demonstrated by ca. 100% cell viability in all experimental variants (Figure 1E). This notion was confirmed by the DNA content analyses, which showed a strong pro-mitotic activity of the EGF, illustrated by a prominent increase of S and G2/M fractions after 24 h EGF treatment (Figure 1F). Again, the inhibition of EGFR signaling by Erl resulted in G1 arrest of GBM cells. These data show that EGF induced proliferation and an invasive morphology of GBM cells in an EGFR-dependent manner.

GBM cells efficiently invade the adjacent brain regions. To estimate the effect of EGF on the invasiveness of T98G cells, we performed time-lapse video microscopy analyses of their motility in the presence of EGF. Our data indicated a prominent pro-migratory activity of EGF at both applied concentrations (Figure 2A). This was illustrated by the increased (>200%) cell motility (cell speed and displacement) in the populations of EGF-stimulated cells in comparison to controls (Figure 2B). Again, Erl completely abolished this effect, decreasing the motility of T98G cells cultivated in the absence of EGF.

Furthermore, we examined the effect of the EGFR-dependent signaling on cellular metabolic homeostasis, and in particular on the mitochondrial ROS, ATP, and lactate production in the EGF-treated T98G cells (Figure 3). EGF-exposed cells showed a pronounced ROS upregulation (Figure 3A,B). This phenomenon was accompanied by a prominent modulation of lactate/ATP production. Specifically, the increase of lactate secretion (Figure 3C) was accompanied by a slight reduction of ATP levels within the motile EGF-treated cells (Figure 3D). This effect (as well as ROS production) was abolished by the application of Erl. EGFR inhibition also considerably perturbed the production of ATP/lactate in T98G cells, regardless of culture conditions. Thus, links exist between the EGF-dependent augmentation of invasiveness, metabolic reprogramming, and ROS production in T98G cells.

To further examine the mechanisms underlying the pro-invasive effects of the EGF, we performed detailed microscopic analyses of the actin cytoskeleton architecture in the EGF-treated cells. TIRF (Total Internal Reflection Fluorescence) microscopy revealed a substantial remodeling of F-actin filament architecture and focal adhesions within the T98G cells in response to the EGF (10 ng/mL) treatment. In particular, spindle-like (mesenchymal) cells with less prominent stress fibers, but pronounced F-actin polymerization regions in the leading edges were observed (Figure 4A). Moreover, dispersed aggregates of vinculin instead of vinculin + plaque-like structures were present in the leading edges of the EGF-stimulated GBM cells (Figure 4A). This phenomenon was correlated with the significant decrease of cellular circularity at the population level after 72 h EGF (10 ng/mL) exposure (Figure 4C), and with the prominent increase of the T98G transmigration potential (TMI; Figure 4D). Interestingly, leading-edge formation (Figure 4A) and the induction of T98G invasiveness (Figure 4D) were abolished by Erl in the EGF-treated T98G cells. Erl treatment also led to the disorganization of the F-actin cytoskeleton and focal adhesions’ sides, accompanied by their morphological switch into an epithelioid phenotype. In turn, the Western blot analysis did not reveal any changes in the intracellular vinculin levels under EGF/Erl stress (Figure 4B). Collectively, these observations confirmed the involvement of EGFR-dependent signaling in determination of T98G cell invasiveness.

Finally, we focused on the significance of ROS upregulation for EGF-induced T98G invasiveness. Interestingly, the administration of N-Acetyl-L-cysteine (NAC; 1 mM) did not abolish the EGF-induced morphological changes of the T98G cells and did not inhibit the pro-invasive remodeling of the F-actin cytoskeleton (Figure 5A). On the other hand, NAC remarkably attenuated the EGF-induced upregulation of ROS (Figure 5B,C). This effect was accompanied by a significant inhibition of the EGF-induced T98G motility. At the population level, this was illustrated by a decrease of the average speed of motility and displacement of the EGF/NAC-treated cells (Figure 5D). Accordingly, single cell trajectories of EGF/NAC-treated cells clearly demonstrated a retardation of their motility in comparison to the EGF-stimulated cells (Figure 5E). Notably, NAC had a negligible impact on T98G motility when administered alone (Figure 5D,E). These data show the cooperation of the ROS-dependent and ROS-independent EGFR-dependent pathways that regulate T98G motility and cytoskeleton architecture, respectively.