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Characterization of 22RV1-Luc xenograft response to
androgen-deprivation therapy.
Katie M. O’Donovan1, Heather Nesbitt1, S. Nicole Williams2, Jenny Worthington2, Stephanie
R. McKeown1, Declan J. McKenna1
1Transcriptional
Regulation and Epigenetics Group, Biomedical Sciences Research Institute University of Ulster,
Coleraine, Northern Ireland
2Axis Bioservices, Coleraine, Northern Ireland
Figure 1. Successful generation of a 22RV1-Luc cell line which is not significantly
different to parental cells
A
10
9
B
3.50E+07
8
y = 317.68x - 404854
R² = 0.9878
3.00E+07
2.50E+07
Fold Difference
• Growth and survival of the prostate tumours is initially dependent
on androgens such as testosterone activating Androgen Receptor
(AR) signalling1,2.
Figure 5. Gene expression in bicalutamide treated 22RV1-Luc tumours is altered
Total Flux (Photons/second)
BACKGROUND
RESULTS
2.00E+07
1.50E+07
1.00E+07
7
6
5
3
20000
40000
60000
80000
100000
120000
0
Bcl2
Parental
0.4
Luc
Fold Difference
Absorbance (A450nm-A650nm)
0.5
0.3
0.2
2
• 22RV1-Luc cell line was created with similar growth, protein and
gene expression profile to Parental 22RV1 cells (Figure 1).
0
24
48
Hours
96
BCL2
168
VEGF NANOG POU5F1 SOX2
(A) Bioluminescence was measured using the IVIS imaging system and Living SYStems
Software. (B) Bioluminescence was measured as 287.84 ± 18.5 photons/sec/cell.
Results are mean of 2 independent experiments. (C) Student’s t test was performed
at each time point and proliferation between cell types was not found to be
significantly different. Results are mean ± SEM of 3 independent experiments.
(D) Expression of hypoxia and cancer stem cell related genes in Parental and Luc
expressing cells measured by qPCR were not significantly different, results are mean ±
SEM of 2 technical replicates (E) AR & PTEN expression as measured by western blot
was similar for Parental and Luc expressing cells .
Figure 2. Bicalutamide treatment has no significant effect on 22RV1-Luc tumour
growth or androgen receptor activity
B 0.14 PSA
Absorbance (A450nm)
2x106 cell implant: Tumour growth
A
0.1
0.08
0.06
0.04
Bicalutamide
0.02
0
5
10
15
20
0
D
4x106 cell implant: Tumour growth
Vehicle
0
5
AIM
10
Day
Veh
BCA
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0
15
14
PSA
Day
The tumour growth results shown (n=3) are mean ± SE. Student’s t test was
performed comparing treatments at each time point and differences were not
found to be significant. ELISA (n=2) was performed for prostate specific antigen
(PSA) on samples with no significant change in circulating PSA in bicalutamide
(BCA) treated mice compared to vehicle.
Figure 3. Bicalutamide treatment significantly reduces tumour oxygenation in a
time-dependent manner
Veh
BCA
pO2 (mmHg)
12
1. Bioluminescence of 22RV1-Luc cells. Cells were plated out as
indicated in 100uL of media in an opaque 96 well plate and
incubated for 24 hours. D-Luciferin was added and the plate
was imaged. Bioluminescence was measured using the IVIS
imaging system and Living SYStems Software.
10
3. qPCR. Performed using Sybr Green on the Lightcycler 480
system following Trizol RNA extraction.
CONCLUSIONS
6
4
*
*
*
*
2
*
0
7
Day
14
21
28
The results shown (n=3) are mean ± SE. Student’s t test was performed comparing
each time point back to day 0 [*p<0.05]. There was significant difference between
vehicle at day 0 and day 28, there was significant difference between bicalutamide
(BCA) at day 0 and days 3,7,14,21.
Figure 4. Bicalutamide treatment results in microvascular collapse in 22RV1 tumour
window chamber experiments
6. ELISA. Using Abnova PSA (Human) ELISA Kit.
7. Window chamber experiment. A dorsal skin fold chamber
was fitted onto the backs of male SCID mice and a 22Rv1
tumour fragment was implanted. The fragment was allowed 7
days to vascularise before commencement of treatment.
Animals were treated with BCA (2mg/kg/day) or vehicle
(0.2% DMSO in corn oil) daily via oral gavage.
• 22RV1-Luc cells were successfully generated which were not
significantly different to parental cells.
• Bicalutamide had no effect on 22RV1 tumour growth showing the
androgen receptor to be insensitive to external ligands in these
cells.
• However, bicalutamide had a marked effect on the tumour
microenvironment - reducing both the vasculature and causing a
profound hypoxia (2mmHg; ~0.2% oxygen).
• There were marked changes in gene expression following
bicalutamide treatment, suggesting that the treatment-induced
hypoxia may be selecting for more apoptosis-resistant cells and
cells with more stem-like properties.
4. Western blot. Primary antibodies used for blotting were antiPTEN, anti-AR and anti-GAPDH. Signal was detected on a
ChemiDoc™ XRS+ imaging system
5. In vivo experiment. 2x106- 4x106 cells were implanted onto
the backs of male SCID mice and allowed to form tumours.
Daily treatment with bicalutamide (2-6mg/kg) or vehicle
(1%DMSO) began when tumour volume reached 200250mm3. Oxylite electrode measurements were taken at day
0,1,3,7,14,21 & 28. Tail vein bleeds were taken at day 0 & 14.
Future work on this project will further investigate the
mechanisms by which bicalutamide acts upon tumour vasculature
resulting in microvessel collapse and treatment-induced hypoxia.
We also intend to explore the role of bicalutamide/bicalutamideinduced hypoxia in selecting for cells which have a more cancer
stem cell-like phenotype.
8
0
2. XTT for 22RV1-Luc Vs Parental proliferation. 22RV1-Parental
and 22RV1-Luc cells were seeded at 1000 cells per well in a
96 well and viability was measured at 0, 24, 48, 96 & 168
hours using a Roche Cell Proliferation Kit II.
• This suggests that whilst bicalutamide is not having an effect
through direct action on the AR of 22RV1 cells, there is some effect
on the tumour microenvironment as indicated by Figure 5 which
shows a loss of microvessel density in bicalutamide treated
tumours compared to vehicle.
FUTURE WORK
16
METHODS
• These results indicate that bicalutamide has no effect on 22RV1Luc cells AR signalling and therefore tumour growth is unlikely to
be controlled by inhibition of this pathway.
• The effect of bicalutamide treatment on the tumour
microvasculature and the hypoxia associated with this effect
results in significant changes in gene expression, in both hypoxia
related genes such as Bcl-2 and in cancer stem cell related markers
Nanog and Sox2.
14
Day
14
• This corresponds with a lack of bicalutamide effect on circulating
PSA levels following 14 days of treatment (Figure 2-B, D).
0
25
Day
C
• When treated with bicalutamide, 22RV1-Luc tumours showed no
significant tumour growth delay compared to vehicle treated
tumours (Figure 2-A,C).
• However, oxygen electrode measurements (Figure 3) show that,
compared to day 0, only bicalutamide treated tumours show a
significant drop in pO2 at days 3, 7, 14 & 21. (Vehicle treated
tumours show a significant reduction at day 28).
0.12
Bicalutamide
Previously we have characterised the effect of bicalutamide in LNCaPLuc tumours.
We now aim to examine the physiological and molecular changes
caused by bicalutamide in a second prostate cancer model: 22RV1-luc
tumour xenografts. The expression of selected genes will be
considered in relation to the physiological changes found.
SOX2
Luc
1
Absorbance (A450nm)
• Changes in gene expression, driven by the treatment-induced
hypoxia, allow hypoxic cells to survive and within 3 - 4 weeks
revascularisation occurs despite continued exposure to
bicalutamide.
POU5F1
DISCUSSION
Parental
1.5
Vehicle
• This occurs in conjunction with microvessel collapse, with
resultant loss of oxygenation and gene expression changes5.
NANOG
E
D
0.5
0.1
0
• Previously we have shown that bicalutamide treatment of LNCaP
xenografts causes exacerbation of the already hypoxic tumour
microenvironment5.
VEGF
2.5
0.6
0
• Whilst treatment with androgen deprivation (bicalutamide) is
initially successful, most patients fail ADT within 2 years, and
tumours often recur in a treatment-resistant form, which is
inevitably lethal4.
**
4
Cell Number
Androgen Receptor Activation3.
**
1
0
3
RNA was extracted from Day 14 tumours (n=2) and
pooled before cDNA synthesis. qPCR was performed for
a range of hypoxia associated and cancer stem cell
related genes. The results shown are mean ± SE for 2
technical replicates. Student’s t test was performed
comparing day 14 vehicle to bicalutamide at each time
point and differences were found to be significant in
Bcl2, Nanog & Sox2(**p<0.01).
Bicalutamide
2
5.00E+06
0.00E+00
C
**
Vehicle
• Overall this study shows bicalutamide causes hypoxia that drives
selection for hypoxia-tolerant cells; this may explain why tumours
recur with a more treatment-resistance/ malignant phenotype.
Imaging of the vasculature was carried out by injecting anaesthetised mice I.V with
FITC- labelled dextran. Images were taken using a multiphoton confocal microscope.
Tumour fragments were imaged before treatment began; (A) vehicle pre-treated group
(E) bicalutamide pre-treated group, and on treatment days 7, 14 and 21; (B-D) vehicle
only (F-H) bicalutamide treatment. Images were taken at 10x magnification. Each
image is representative of a minimum of 5 animals per treatment group.
REFERENCES
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2.
3.
4.
5.
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DEHM, S.M. and TINDALL, D.J., 2007. Androgen receptor structural and functional elements: role
and regulation in prostate cancer. Molecular endocrinology (Baltimore, Md.), 21(12), pp. 28552863.
FELDMAN, B.J. and FELDMAN, D., 2001. The development of androgen-independent prostate
cancer. Nature reviews.Cancer, 1(1), pp. 34-45.
ACAR, O., ESEN, T. and LACK, N.A., 2013. New therapeutics to treat castrate-resistant prostate
cancer. TheScientificWorldJournal, 2013, pp. 379641.
MING, L., BYRNE, N.M., CAMAC, S.N., MITCHELL, C.A., WARD, C., WAUGH, D.J., MCKEOWN, S.R. and
WORTHINGTON, J., 2013. Androgen deprivation results in time-dependent hypoxia in LNCaP
prostate tumours: informed scheduling of the bioreductive drug AQ4N improves treatment
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