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Frame et al. Investigating models for prostate cancer research

Selection of stem cells, transit amplifying cells Colony forming assay
and committed basal cells Selected cells (SC and TA) were plated at 100-500
Following trypsinisation of primary cultures, cells cells per well on a collagen I-coated 6-well plate and
were first selected using collagen adherence. stem treated with 30 μmol/L etoposide or an appropriate
o
cells (SC) and transit amplifying (TA) cells are dilution of DMSO for 45 min at 37 C, washed twice
α2β1integrin and committed basal (CB) cells are with phosphate buffered saline (PBS) and fresh SCM
hi
o
α2β1integrin . A stringent selection of TA cells can was added to each well. Cells were kept at 37 C and
lo
be achieved with 5 min adherence to collagen I. SCM was changed every second day. An appropriate
Any non-adherent cells can be passed on to another amount of irradiated feeder cells were added to keep
plate, then any cells not adhered after 20 min are the wells confluent. After 6-14 days SCM was removed
the committed basal cells. A slightly less stringent and cells were washed once with PBS then stained
selection of TA cells can be achieved with a 20 min with crystal violet (1% crystal violet, 10% ethanol
adherence where any non-adherent cells represent in PBS) for 20 min, and after a final PBS wash, the
the committed basal population. This latter selection number of colonies was determined. Colonies with
can be used when trying to achieve maximum stem < 32 cells and ≥ 32 cells (5 population doublings)
cell (α2β1integrin /CD133 ) yield. To select stem were counted. Colony forming assays were also
hi
+
cells, positive selection using a CD133 microbead kit carried out with radiation and Vorinostat treatment.
(Miltenyi Biotec) was used [14] . For combination treatments, cells were treated with
0.625, 2.5 or 10 μmol/L of Vorinostat for 30 min then
Ethics approval and patient consent treated with a range of radiation doses.
Patient samples were collected with ethical Treatments with radiation and drugs
permission from Castle Hill Hospital (Cottingham, An RS2000 X-Ray Biological Irradiator was used,
Hull) (ethics number: 07/H1304/121). Use of patient which contains a Comet MXR-165 X-Ray Source
tissue was approved by the Local Research Ethics (Rad-Source Technologies Inc. GA, USA). A range
Committees. Patients gave informed consent and all of radiation doses were administered with a dose
patient samples were anonymized.
rate of 0.02 or 0.08 Gy/s. Addition of Vorinostat
Alamar blue assay (Cambridge Bioscience) was carried out at three
concentrations: low, 0.625 μmol/L; medium,
The alamarBlue reagent (ThermoFisher scientific) 2.5 μmol/L; high, 10 μmol/L.
®
was used as an assessment of cell viability. Briefly,
cells were plated at 5,000 cells per well in a 96-well Comet assays
plate and treated with drug. Radiation of cells was The comet assay was carried out as previously
carried out prior to plating. The alamar blue assay described [15,16] . Briefly, drug-treated cells were
was carried out 24-72 h post-treatment. Cells were in resuspended in 25 μL of PBS and mixed with 225 μL
200 μL and a 1:10 dilution of alamar blue reagent was of low melting point agarose. Following mixing, the
added. Fluorescence was measured on a plate reader cells and agarose were spread onto a glass slide that
2 h after addition of reagent. had been pre-coated with 1% agarose in PBS. A clean
coverslip was placed on top until the cell-agarose
Table 1: Patient samples mixture had set. Slides were placed in lysis buffer
Patient age
Sample Operation Diagnosis overnight and then incubated in alkaline solution for
(years) o
209/12 LA RP 64 Normal 40 min at 4 C then electrophoresed at 23V/300 mA
329/13 R RP 53 Normal in the alkaline solution for 40 min on ice. This was
434/14 LM RP 68 Normal followed by two washes in neutralising buffer. SYBR
048/11 RP - Gl6 (3+3)
018/11 RP - Gl7 Gold was applied at a concentration of 1:10,000 in TE
035/11 RP - Gl7 (3+4) buffer to stain the DNA. Following collection of images
054/11 RP 58 Gl7 (3+4)
665 RP 53 Gl7 (3+4) on a fluorescent microscope (Nikon Eclipse TE300),
049/11 RP - Gl7 (3+4) comets were quantified using CometScore freeware
087/11 RP 68 Gl7 (3+4)
031/10 RP - Gl7 (3+4) (TriTek Corp, VA, USA).
034/11 RP - Gl7 (3+4)
517/15 RM RP 65 Gl7 (3+4)
329/13 L RP 53 Gl7 (3+4) Immunofluorescence
209/12 RA RP 64 Gl7 (4+3) Immunocytochemistry was carried out to stain selected
545/15 LB RP 69 Gl7 (4+3) populations for DNA damage [γH2AX - anti-phospho-
307/13 LB RP 65 Gl7 (4+3)
545/15 RM RP 69 Gl7 (4+3) Histone H2A.X (Ser139) clone JBW301, Millipore,
RP: radical prostatectomy UK], proliferation (Ki67 - ab15580, abcam) and a cell
304 Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ December 6, 2017