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Frame et al. Investigating models for prostate cancer research

marker (CD49b - anti-human CD49b:RPE, Serotec shows a varied response and reduces colony forming
MCA743PET). Primary cells were plated at 10,000 ability of primary prostate cells more effectively than
cells per well in collagen I coated 8-well chamber slides either treatment alone.
or in the case of rare stem cells, all stem cells collected
were plated on the slide. Staining of γH2AX was carried A previous investigation had shown that prostate
out as described previously . Fixation for CD49b and cancer stem-like cells were more radio-resistant than
[15]
Ki67 staining was with 4% paraformaldehyde, with no progenitor (TA) cells and more differentiated (CB)
permeabilisation step when staining CD49b and with cells from primary prostate epithelial cells cultured
[15]
permeabilisation using 0.3% Triton X-100 for Ki67 from patient tissue . Pre-treatment with a low dose
staining. Alexa Fluor secondary antibodies (goat anti- of a histone modifier, Trichostatin A, resulted in radio
mouse and goat anti-rabbit) with fluorescent tags were sensitisation of the stem-like cells, which was observed
used at a concentration of 1:1000. as an increase in DNA damage and a decrease in
colony forming ability. To follow on from this, a clinically
Flow cytometry approved histone modifier, Vorinostat, was tested in
Flow cytometry was used to measure expression combination with radiation treatment. First, a panel of
levels of CD49b on primary cells. All cell populations cell lines including normal prostate (PNT1a), benign
(WP, TA and CB) were harvested and resuspended (BPH-1), localized cancer (P4E6) and metastatic
in 300 μL MACs buffer. Control (REA control (I)- cancer (PC3), were tested using alamar blue assays to
APC) and target (CD49b-APC human clone REA188) measure viability following treatment with a combination
antibodies were used (Miltenyi Biotec). Ten μL of of seven drug concentrations (0.156/0.3125/0.625
antibody was added and incubated with rotation for /1.25/2.5/5/10 μmol/L) and six radiation doses (2, 5,
10 min at 4 C. Cells were washed, resuspended in 10, 25, 50, 75 Gy) with measurements taken at 24,
o
MACs buffer and analyzed by flow cytometry including 48 and 72 h. The percentage viability of each of the
a cell only control to set gates. highest doses alone and in combination in the cell line
panel is shown in Figure 2A-C. There is a significant
Image capture using ptychography and image decrease in viability in all cell lines with the combination
analysis using cell analysis toolbox treatment compared to drug only, however the effect
Quantitative phase imaging (QPI) was carried out on PC3 cells is minimal, whilst the effect on the cell
using a VL21 Live Cell Imaging System (Phase line derived from the localized cancer, P4E6, is most
Focus Limited, Sheffield, UK), which utilises a significant. Viability of primary prostate epithelial cell
method known as ptychography in image formation. cultures (n = 6) was then measured following single
The high contrast images generated by the system and combination treatments [Figure 2D and E].
are label-free and exempt from focal drift, allowing There was a significant reduction in viability with the
extended time-lapse imaging [17-19] . The high-contrast combination treatment in normal and cancer cells,
nature of the images facilitates automated individual however the cancer cells showed less of a reduction in
cell segmentation and tracking with the Cell Analysis viability. We have previously shown that radiation can
Toolbox software, which outputs extensive and cause senescence of primary prostate epithelial cells
®
specific feature measurements for each cell. As a rather than cell death, and so the small reduction in
result, data analysis can include information on cell viability as measured by alamar blue could be because
populations in addition to individual cell information the cells are senescing rather than dying . Therefore,
[20]
such as cell morphology and cell kinetics. we tested the effect of three drug doses with and
without 2 Gy of radiation on colony formation [Figure 2F].
Statistical analysis As previously seen, 2 Gy of radiation results in a 50%
Alamar blue assays were performed in triplicate and surviving fraction. We used 0.625, 2.5 and 10 μmol/L
data presented as % cell viability with percentage of Vorinostat, with and without 2 Gy radiation. Drug
standard error. Significance calculations were carried alone only reduced the surviving fraction by 10%-50%
out using the unpaired, nonparametric Mann-Whitney with patient variability observed. The combination
U-test. The P values indicating statistical significance treatments reduced surviving fraction by 65%-95%.
are displayed (*P < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.001). Cancer stem-like cells from patient tumor tissue are
more resistant to etoposide than the progenitor cells
RESULTS due to a quiescent phenotype.

A combination of Radiation and Vorinostat treatment Previous studies had identified the cancer stem-
on a panel of cell lines and on primary prostate cells like cells of primary prostate epithelial cell cultures
Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ December 6, 2017 305