Metabolic activity of the cell lines under different treatment conditions

3.2.1.5.1 Preliminary experiments First preliminary experiment

In order to determine the optimal incubation times and effective radiation doses for all planned experiments the preliminary CTB assay procedure was performed as described in Chapter 2.3.6.1. First preliminary experiments were performed with incubation times of 26, 48, 72 and 96 hours for ZMK-1 and HaCat cell lines (Figure 3.15). The viable cells can convert resazurin into resorufin, a highly fluorescent product. This intensity of the fluorescence is proportional to the number of viable cells that are able to perform this redox reaction.

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Figure 3.15 shows that a reduced viability of 4 Gy-irradiated ZMK-1 cells can been seen after a 96-hour incubation time, while HaCat cells would probably require a longer incubation time or a higher radiation dose to show alterations in metabolic activity.

Second preliminary experiment

A second preliminary experiment was performed to determine the optimal radiation dose. The incubation time for this second preliminary test was set at 72 hours after irradiation (see Chapter 2.3.6.1.).

Figure 3.15: First preliminary CTB assay experiments with 26, 48, 72 and 96 hours incubation time for ZMK-1 and HaCat cell lines. The incubation time, after which difference between the irradiated and non-irradiated cells was observable, was chosen as an optimal incubation time. A) In the ZMK-1 cell line a difference of metabolic activity of irradiated and non -irradiated cells was observed after 96 hours. B) In the HaCat cell line no impact of irradiation on cell viability was detected even after 96 hours incubation time. Each experimental condition was performed in triplicate. A total dose of 4 Gy was delivered with a dose rate of 2 Gy/min .

A B

C D

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Figure 3.16 illustrates that for the diverse radiation doses, no clear difference in cell viability of control cell lines, HaCat and DF-19, can be observed. The tumour cell line FaDu showed reduced metabolic activity following 6, 8, 10 and 12 Gy radiation, while the tumour cell line GR-145 showed slightly decreased cell viability only with 8 Gy irradiation (Figure 3.16).

Third preliminary experiment

In the final experiment, the number of cells in each well was reduced from 5000-6000 cells to 2000 cells. The incubation time was prolonged to one week (168 hours) to analyse the cell viability in an expanded period of time and a radiation dose of 8 Gy was used. The results are summarised in Figure 3.17. There was only a non-significant reduction in the fluorescence

Figure 3.16: Second preliminary CTB assay experiment with different radiation doses. Cells were incubated for 72 hours. A) The tumour cell line FaDu ( at 6 to 12 Gy) showed reduced cell viability compared to non-irradiated control cells after irradiation . B) The control DF-19 cell line showed reduced metabolic activity after 12Gy radiation . C) The tumour GR-145 cell line showed reduced cell viability compared to non-irradiated control cells after irradiation (only with 8Gy). D) In the HaCat cell line even after 12Gy radiation, the metabolic activity of the cells was still efficient. Each experimental condition was performed in triplicate. A total dose of 2, 4, 6, 8, 10 and 12 Gy was delivered with a dose rate of 2 Gy/min.

Figure 3.17: Optimising incubation time, radiation dose and cell densities in all used cell lines.

The cells were irradiated with 8 Gy and after one week incubation time, the cell viability was measured. Each cell line excep t DF-19 demonstrates reduced metabolic activity following irradiation.

Each experimental condition was performed in triplicate. A total dose of 8 Gy was delivered with a dose rate of 2 Gy/min.

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intensity in the DF-19 cells, while a notable reduction of fluorescence was detected in the other cell lines (Figure 3.17). In addition, between non-irradiated cells, the FaDu cells after one week incubation time showed more cell viability in compare to DF-19 cells.

After optimising the parameters for analysing cell viability the results suggested that the 8 Gy irradiation dose with a one-week incubation time should be used to detect the impact of irradiation on cell viability.

3.2.1.5.2 Main experiment: Analysis the cell viability of cells under different treatment conditions

This aforementioned irradiation dose and incubation time (Figure 3.15-17) was used in the following CTB assay to determine the impact of CXCL12 and AMD3100 on the metabolic activity of each cell line.

To this end, cell suspensions were prepared and the cells incubated with 100 ng/ml CXCL12, 5 µg/ml AMD3100 or both for 30 minutes at 37°C and 5% CO2. The cells were pipetted in two 96-well plates (2000 cells/well). One of two plates was irradiated at 8 Gy. The cell viability of each cell line under the different treatment conditions was analysed after one week. The non-irradiated or untreated cells were compared using the t-test, and p<0.05 was regarded significant. In each group the cell viability in the presence of CXCL12, AMD3100 or both compounds was compared with untreated cells. In addition, cells treated with CXCL12, AMD3100 or both and irradiated with 8 Gy were compared with non-irradiated cells to study the impact of irradiation on cell viability. Data are presented as mean differences ± standard deviations. Results are taken from two independent experiments, always performed in triplicate (Figure 3.18).

Of all cell lines, tumour cell lines ZMK-1, FaDu and GR-145 were rather radiosensitive, and irradiation reduced viability as shown by the results of the colony-forming assays (Figure 3.18 A-C). However, the metabolic activity of the control cell lines, DF-19 and HaCat, was not significantly reduced by irradiation (Figure 3.18 D-E).

As seen in Figure 3.18, the tumour cell lines, ZMK-1, FaDu and GR-145 as well as the control cell line DF-19 showed unchanged cell viability following treatment with CXCL12, AMD3100 or both compounds. However, when HaCat cells were treated with CXCL12,

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AMD3100 or even both together, there was a significant increase of metabolic cell activity compared to the untreated control cells.

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3.2.2 The role of CXCL12 and CXCR4 in the migration of HNSCC and control