Development of the novel radioresistant Rectal Cancer cell lines

Radioresistant novel derivatives were developed from the rectal cancer cell lines SW-837 and HRA-19 in collaboration with Mr Sajid Mehmood. Treatment resistance was produced by using clinically relevant doses of radiotherapy. Irradiation was performed at Castle Hill Hospital, Hull, UK in conjunction with a radiation physicist (Gary Liney or Matthew Bush) using X-rays produced by a clinical Linear Accelerator treatment machine (6 Mv) as described previously (Smith et al., 2009) (see Figure 16). A cradle was manufactured therefore allowing the vial containing the cell suspension to be suspended inside a water filled vessel (phantom) (see Figure 17). The purpose of this operation was to enable irradiation of cells to mimic the in vivo environment, with the surrounding water representing normal body tissue. The cradle was designed such that the vial containing the cell suspension was mounted precisely in the centre of the water filled phantom. From this assembly, it was calculated that the dose given to any cells at the centre of the vial represented that given to the cells throughout its total volume. Using this experimental set- up it was possible to deliver consistent doses to the cell samples throughout the course of the study.

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Figure 16: The experimental set-up required for the irradiation of cell populations.

Irradiation was performed at Castle Hill Hospital, Hull, UK using X-rays produced by a clinical Linear Accelerator treatment machine. The vial containing the cell suspension was suspended in a water-filled vessel (phantom), which was arranged so that the X-rays were delivered to the centre of the phantom.

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Figure 17: A schematic diagram of the phantom used during radiotherapy treatment.

The cradle was designed such that the vial containing the cell suspension was mounted precisely in the centre of the water filled phantom. From this careful assembly, it was assumed that the dose given to any cells at the centre of the vial represented that given to the cells throughout its total volume.

4.3.1 Cell counting

Cells were harvested using enzymatic dissociation (see section 4.1.3) and resuspended in 6 ml of RPMI or DMEM medium. Twenty five µl of the cell suspension was then mixed thoroughly with 25 µl 0.4% (w/v) trypan blue, giving a 1:1 concentration. Twenty five µl of this resultant cell suspension was applied to a haemocytometer under a glass coverslip. Cells were counted under a light microscope using a hand-held counter. Cells were counted in 5 squares, 4 corner squares and the central square of the grid (see Figure 18). The cell concentration per ml was calculated using the following formula:

[(Σ(1+2+3+4+5)/5) x 2] x 104

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Figure 18: Grid on haemocytometer used for cell counting.

Cells were counted in 4 corner squares and the central square of the grid. The cell concentration per ml was then calculated.

4.3.2 Modified colony counting assay for assessment of radiotherapy response

Prior to establishing a radioresistant cell line, the inherent sensitivity of SW837 and HRA- 19 was first established. This was performed by constructing dose response curves (DRCs) for each cell line using doses ranging from 0-10 Gy. For each DRC cells were harvested by enzymatic dissociation as described in section 4.1.3, and 1x106 cells were seeded in screwed-cap 7ml polypropylene containers. A total of 6 containers were used, each filled with 5ml of the cell suspension. The containers were then labelled with the dose of radiation each was going to receive i.e. 0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy and 10 Gy. The samples were then irradiated as described in section 4.3.A proportion of the cell suspension from each container, which corresponded to 1000 cells, was then removed and plated in triplicate into six well tissue culture plates. This was done in triplicate. The plates were then incubated at 37 ˚C for 12-14 days until control cells (0 Gy) reached a critical mass and individual colonies became distinguishable. At that point, the medium was removed and the cells were fixed in 3ml of ice cold Carnoy’s fixative (3:1 methanol: acetic acid) for 5 min. The cells were left to air-dry overnight. The following day, the cells were stained with 3ml 0.005% crystal violet for 5 min. The residual stain was then removed in slowly running tap water and the plates left to air-dry. In order to calculate the number of surviving cells after each dose of radiotherapy the stained colonies were photographed using a 14 mega-pixel

72 camera in order to produce a high-resolution image. Colonies of cells of >50 in number were deemed to represent surviving cells from the original cell line. The plates were examined under a light microscope and a colony of 50 cells was identified. This was then correlated with the photographed image and a measurement taken. Any group of cells of this size or greater was then counted independently, in triplicate, by 2 people and an average taken. A DRC of number of colonies against dose of radiotherapy was then produced (section 4.3.3).

4.3.3 Dose response curve for radiotherapy resistance

Plating efficiency (PE) and survival fraction (SF) were calculated for both parental cell lines using the following formulas:

PE = (Number of colonies counted/ number of cells plated) x 100 SF = (PE of treated sample/ PE of control) x 100

A survival curve was then generated by plotting the SF (Y axis) against radiation dose (X axis). Each experiment was done in triplicate for each dose and a mean value of SF for each dose was calculated. The whole experiment was repeated and the mean SF of two independent experiments was plotted on the DRC.

4.3.4 Incremental irradiation dose

Results generated from the DRC enabled the selection of an appropriately high sub-lethal dose, which was used during a fortnightly fractionation regimen. For these experiments, 8 Gy was selected for the SW-837 cell line, and 4Gy was selected for the HRA-19 cell line, a decision made based on the guidance from the DRC and also existing clinical treatment regimens. In these experiments, the parental cell line refers to the cell line which had received no radiotherapy, from which a radioresistant cell line was created. For each of the two cell lines, a sample of 6x106 from the parental cell line was placed into a 7ml polypropylene container and made up to a volume of 5 ml with RPMI/DMEM culture medium. This was then taken to the Radiotherapy Department and dosed at 8 Gy (SW837) and 4 Gy (HRA-19). The cells were then returned to the incubator and allowed to grow before the next dose. The cells were checked under the light microscope, and when approximately 80% confluence was reached the cells were counted and a further 6x106

73 were dosed. This process was repeated until a final total dose of 48 Gy was reached for both cell lines.

4.3.5 Confirmation of radioresistance

In order to determine whether the 48 Gy treated cell lines were more resistant to radiotherapy than their parental counterparts, a DRC was constructed, as per section 4.3.3 and compared to the DRC for SW837 and HRA-19 parental cells using the Student’s t-test for statistical analysis.