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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 1. 2. 3. 4. 5. BRINKMANN, A.O. and TRAPMAN, J., 2000. Genetic analysis of androgen receptors in development and disease. Advances in Pharmacology (San Diego, Calif.), 47, pp. 317-341. 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 response. International journal of cancer., 132(6), pp. 1323-1332.