Morphological changes in cell lines with primary drug sensitivity

During the cultivation of the different A431 cells, it became obvious that the populations were subject to significant morphological changes. Phenotypical alterations were first observed after two to three months of cetuximab treatment. While the concentration of cetuximab was kept constant (33µg/ml), doses were steadily increased in the gefitinib-treated population. Here, the starting concentration

was 0,5µM (corresponding to the EC50 in a monolayer growth assay), which

significantly reduced cell numbers in culture and caused a longer period of growth stasis in the viable cells. After two to three months cells resumed growth and from this point, gefitinib concentrations were increased in steps of dose doublings (0,5µM; 1µM; 2µM; 5µM; 10 µM). The morphology of A431 was documented fifteen months after the start of treatments (figure 26).

Cetuximab Medium control

(low passage)

Gefitinib Medium control

(high passage) Cetuximab

Medium control (low passage)

Gefitinib Medium control

(high passage)

Figure 26 A431 cells showed distinct morphological alterations upon long-term treatment with cetuximab, gefitinib or long-term cultivation in full medium. A431 cells treated with cetuximab or gefitinib for 15 months, as well as medium-treated control cells of similar passage number (high passage) and medium- treated cells from the starting population (low passage) were seeded and photographed under 40 fold (up) and 400 fold (down) magnifications with a light microscope.

The A431 cell line is widely used as a model for cancer cells that display typical epithelial features. Epithelial cells are closely attached to each other through tight cell-cell contacts and are mainly assembled in compact colonies in cell culture. The A431 starting population (medium control, low passage) was a classic example for that (figure 26). In contrast, A431 cells showed a completely different morphology after a long-term cultivation in regular growth medium (medium control - high passage). In this population most cells had a longitudinal cell shape and a fibroblastic morphology with many cell extensions. Cell-cell contacts were loose and colonies were mainly broken up with single cells or small aggregates spreaded over the surface of the culture dish. This population displayed typical features of mesenchymal cell lines. Obviously, cultivation of A431 cells under standard cell

culture conditions for a period of fifteen months provoked morphological changes, which have been described as epithelial-mesenchymal transition (EMT).

The population that had been long-term exposed to cetuximab also showed morphological alterations in comparison to the starting population (low passage). In proportion, cells from the cetuximab-treated population were slightly bigger, appeared flattened and contained a more prominent nucleus than cells from the starting population. Also these features pointed towards a more mesenchymal phenotype. However, the cetuximab-treated population still displayed fundamental epithelial characteristics, including tight cell-cell contacts and development of organized, defined colonies.

In contrast, the morphology of A431 cells that were treated with gefitinib appeared very similar to that from long-term untreated A431 cells with similar passage number (medium control – high passage). Cell extensions protruding from gefitinib-resistant A431 cells were even a little more pronounced than in the high passage reference cells, possibly indicating an increased migratory activity within this population (figure 26).

In parallel to A431 cells, Difi cells were as well long-term exposed to cetuximab and gefitinib. Since these cells are hypersensitive to either drug under monolayer growth conditions, treatment doses were increased for cetuximab (from a starting concentration of 0,2µg/ml to a maximal dose of 5µg/ml) and gefitinib (from a starting concentration of 75nM to a maximal dose of 1,5µM). The starting doses were chosen to be close to the calculated EC50 value for each drug. Both therapeutics significantly

reduced cell numbers in culture and caused a long period of growth stasis in the living cells. Even after 15 months of drug exposure, cetuximab and gefitinib-treated Difi cells still growed slowly as compared to medium-treated reference cells.

Medium control low passage Medium control high passage Cetuximab Gefitinib Medium control low passage Medium control high passage Cetuximab Gefitinib

Figure 27 Difi cells showed distinct morphological alterations upon long-term treatment with cetuximab and gefitinib. Difi cells treated with cetuximab or gefitinib for 15 months, as well as medium-treated control cells of similar passage number (high passage) and medium-treated cells from the starting population (low passage) were seeded and photographed under 40 fold (up) and 400 fold (down) magnifications with a light microscope.

Plain cultivation of Difi cells under standard cell culture conditions did not confer any significant change with regard to the cell population’s morphology, as it had been observed for A431 cells (figures 26 and 27). On the other hand, higher magnifications of Difi populations that were exposed to gefitinib or cetuximab revealed considerable alterations. Especially cetuximab-treated cells showing a diffuse morphology and unorganized colony formation and some cells show an enlarged hyperchromatic nucleus (figure 27). Notably, gefitinib-treated Difi cells displayed an unusual colony formation pattern and single cell morphology. Figure 27 exemplarily highlights one gefitinib-treated cell with five nuclei. Along with multinucleated cells in the gefitinib- treated population, a high ratio of Difi cells with two nuclei was found after exposure to either drug. This may point towards impaired cellular cytokinesis.

Similarly, H460 lung cancer and SW707 colon cancer cell lines were long-term treated with cetuximab and gefitinib for 12 months and 8 months, respectively. Drug concentrations were kept constant for cetuximab (33µg/ml) and gefitinib (10µM), since no growth inhibition was seen for cetuximab at variable doses and the EC50 for

In contrast to the cell lines with primary sensitivity for gefitinib and cetuximab, A431 and Difi, no morphological changes were observed between medium-treated control populations and cells that were exposed for several months to either drug (figure 28). This implied that the morphological changes seen for A431 and Difi cells upon drug exposure were primarily due to effects of both drugs on EGFR and not on cellular off- targets. Cetuximab Gefitinib Medium control high passage Cetuximab Gefitinib Medium control high passage

H460

SW707

H460

SW707

Figure 28 H460 and SW707 cells did not show morphological alterations upon long-term treatment with cetuximab or gefitinib. H460 and SW707 cells treated with cetuximab or gefitinib for 12 months or 8 months, respectively, as well as medium-treated control cells of similar passage number (high passage) were seeded and photographed under 40 fold magnification with a light microscope.

3.2.4 Characterization of biological and biochemical changes in long-term-