Purpose: WST11 vascular targeted photodynamic therapy (VTP) is a local ablation approach relying upon rapid, free radical-mediated destruction of tumor vasculature. A phase III trial showed that VTP significantly reduced disease progression when compared with active surveillance in patients with low-risk prostate cancer. The aim of this study was to identify a druggable pathway that could be combined with VTP to improve its efficacy and applicability to higher risk prostate cancer tumors.

Experimental Design: Transcriptome analysis of VTP-treated tumors (LNCaP-AR xenografts) was used to identify a candidate pathway for combination therapy. The efficacy of the combination therapy was assessed in mice bearing LNCaP-AR or VCaP tumors.

Results: Gene set enrichment analysis identifies the enrichment of androgen-responsive gene sets within hours after VTP treatment, suggesting that the androgen receptor (AR) may be a viable target in combination with VTP. We tested this hypothesis in mice bearing LNCaP-AR xenograft tumors by using androgen deprivation therapy (ADT), degarelix, in combination with VTP. Compared with either ADT or VTP alone, a single dose of degarelix in concert with VTP significantly inhibited tumor growth. A sharp decline in serum prostate-specific antigen (PSA) confirmed AR inhibition in this group. Tumors treated by VTP and degarelix displayed intense terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining 7 days after treatment, supporting an increased apoptotic frequency underlying the effect on tumor inhibition.

Conclusions: Improvement of local tumor control following androgen deprivation combined with VTP provides the rationale and preliminary protocol parameters for clinical trials in patients presented with locally advanced prostate cancer. Clin Cancer Res; 24(10); 2408–16. ©2018 AACR.

Translational Relevance

Targeting of the androgen receptor pathway is a key therapeutic strategy for prostate cancer. Androgen deprivation therapy (ADT) is the most effective treatment of metastases and is used as adjunctive therapy in combination with radiotherapy for whole gland ablation of intermediate-/high-risk cancer. A recent multicenter phase III clinical trial in low-risk prostate cancer patients showed tumor ablation, minimal toxicity, and decreased localized progression by WST11 vascular targeted photodynamic therapy (VTP) compared with active surveillance. Transcriptome profiling provided herein shows an upregulated androgen response pathway following VTP. We hypothesized that targeting this pathway with short-course ADT in combination with VTP should significantly delay tumor growth compared with VTP or ADT monotherapies. Furthermore, because ADT and VTP have already been approved for treatment of prostate cancer, the proposed combination might translate rapidly into the clinic and expand the utility of VTP to populations with higher-risk, localized prostate cancer.

Current treatment choices for localized prostate cancer range from active surveillance to radical therapies (prostatectomy, external beam radiation; ref. 1). However, active surveillance can present a risk of progression for patients with higher-risk disease, whereas for some cancers, radical therapies may be an unnecessarily aggressive overtreatment and are associated with notable side effects (2–4). Therefore, there has been interest in developing partial gland ablation such as focal therapies that are less aggressive than radical therapies as an alternative treatment option for these patients (5). Vascular targeted photodynamic therapy (VTP) destroys targeted tissues using padeliporfin (TOOKAD Soluble, WST11) as a photosensitizer in association with a low-power near-infrared laser light in the presence of oxygen. Padeliporfin is intravenously infused and circulates systemically with no extravagation out of the circulation until clearance. Illumination confined to the cancerous lobe of the prostate using transperineal optic fibers induces ultrafast electron transfer to oxygen molecules in the circulation. The resulting short-lived super oxide and hydroxyl radicals (6, 7) initiate rapid destruction of the targeted vasculature followed by a cascade of biological events that end with coagulative necrosis of the tumor (6–8).

Positive outcomes from patients with low-risk, localized prostate cancer [Grade Group 1 (Gleason Score ≤ 6), no prior treatment] treated with VTP have recently been reported in U.S. and European multicenter phase II and III studies. In follow-up biopsies at 6 months after prostate hemiablation, up to 80.6% of patients were negative for cancer (9), and there was a decreased disease progression at 24 months when compared with active surveillance (28% vs. 58%, respectively; HR, 0.34; 95% confidence interval, 0.24–0.46; P < 0.0001; ref. 10). After a median follow-up of 68 months, 82% of patients treated with VTP were free of clinically significant cancer in the treated lobes and 76% of the treated patients had avoided a need for subsequent radical therapy (11). The efficacy of VTP could potentially be improved with a complementary, targeted combination therapy. Furthermore, combination therapy may allow for the extension of VTP treatment to additional cohorts of patients with high-risk localized prostate cancer.

The initial aim of this study was therefore to identify potential druggable pathways active in prostate cancer tumors exposed to VTP using transcriptome analysis. We identified a compensatory, acute upregulation of AR pathway activation following VTP treatment. As ADT to inhibit prosurvival signaling via androgen receptor (AR) is the mainstay treatment for aggressive prostate cancer, we then went on to confirm that inhibition of AR activity enhanced the efficacy of VTP treatment in prostate cancer xenograft models.

General

Lyophilized WST11 was obtained from Steba Biotech. Human prostate cancer cell lines VCaP was purchased from the ATCC, and LNCaP-AR was kindly provided by Dr. Charles Sawyers (MSKCC). Both cell lines were tested negative for mycoplasma using the MycoAlert PLUS Assay from Lonza and authenticated using Short Tandem Repeat analysis by ATCC. LNCaP-AR cells were cultured in RPMI supplemented with 10% FBS, 2 mmol/L l-glutamine, whereas VCaP cells were cultured in DMEM with high glucose,10% FBS, and 2 mmol/L l-glutamine. All the components for cell culture were from Life Technologies. Degarelix was purchased from Ferring Pharmaceuticals Inc.

Animal models

All animal work was performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. Subcutaneous tumors were established in intact male mice through injection of LNCaP-AR or VCaP human prostate cancer cell lines. We subcutaneously injected 2 × 106 LNCaP-AR cells in 100 μL of 1:1 media/Matrigel (BD Biosciences) into the hindlimb area of 6- to 8-week-old, male, athymic nude mice (NCI, Fredrick, MD) or severe combined immunodeficiency (SCID) mice (C.B-Igh-1b/IcrTac-Prkdcscid, Taconic). We also injected 2 × 106 VCaP cells into SCID mice (Taconic). Tumor growth was monitored by caliper measurement weekly. When the volume of tumors reached approximately 100 mm3, the animals were randomly assigned to different cohorts for further experiments.

Treatments

VTP.

An anesthetic cocktail of 150 mg/kg ketamine and 10 mg/kg xylazine was administered intraperitoneally prior to treatment and was supplemented with inhaled isoflurane. A single dose of carprofen (5 mg/kg) and 1 mL of subcutaneous warm saline were administered. WST11 was reconstituted in sterile 5% dextran in water at 2 mg/mL under light-protected condition, and the aliquots were stored at −20°C. On the day of VTP treatment, an aliquot was thawed and filtered through 0.2 μm disc syringe filter (Sartorius Stedin Biotech North America). The mice were intravenously infused with WST11 via tail vein (9 mg/kg) followed immediately by 10-minute laser (Ceramoptec) illumination (755 nm, 100 mW/cm for transcriptome analyses, and 150 mW/cm for in vivo studies) through a 1-mm frontal fiber (MedLight S.A.). The light field was arranged to cover the entire tumor area plus 1 mm rim using red-light aiming beam.

ADT.

Single dose of degarelix was administered at 0.5 mg per mouse at 3 days before VTP treatment via subcutaneous or intraperitoneal injection. Drug administration was initiated when tumor size reached approximately 100 mm3.

PSA detection in serum

Free PSA and total PSA were measured with a dual-label immunofluorometric assay (DELFIA Prostatus PSA Free/Total PSA; Perkin-Elmer Life Sciences) according to the manufacturer's recommendations. This assay measures free PSA and complexed PSA in an equimolar fashion (12, 13), and the cross-reactivity of PSA-ACT for free PSA is less than 0.2% (14). The lower limits of detection are 0.1 ng/mL for both total PSA and free PSA. For detection, the 1235 automatic immunoassay system from Perkin-Elmer Life Sciences was used.

Histology and immunohistochemistry

All tumor specimens were fixed in 10% buffered formalin (Fisher Scientific), processed routinely, embedded in paraffin, sectioned at 5-μm thickness, and stained with hematoxylin–eosin (H&E). Immunohistochemistry (IHC) of tumors was performed on 5-μm formalin-fixed paraffin embedded (FFPE) section following heat-induced epitope retrieval (HIER) in a buffer at pH 9.0. AR staining with anti-AR antibody (at 0.66 μg/mL; Abcam) and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining for cell death with terminal deoxynucleotidyl transferase dUTP nick-end labeling (Roche Diagnostics) were performed using Discovery XT processor (Ventana Medical Systems, Inc.; ref. 15) at the Molecular Cytology core facility. IHC staining for CD31 and Ki67 markers was performed on FFPE sections at the Laboratory of Comparative Pathology on a Leica Bond RX–automated stainer (Leica Biosystems). Following HIER at pH 9.0, the primary antibody against CD31 (DIA-310, Dianova) or Ki67 (ab16667; Abcam) was applied at a concentration of 1:250 and 1:100, respectively, followed by application of a polymer detection system (DS9800, Novocastra Bond Polymer Refine Detection; Leica Biosystems). For all IHC stains and TUNEL, the chromogen was 3,3 diaminobenzidine tetrachloride (DAB), and sections were counterstained with hematoxylin. For quantification of CD31, Ki67, and TUNEL staining, whole slide digital images were generated on a scanner (Pannoramic 250 Flash III, 3DHistech, 20x/0.8NA objective) at a resolution of 0.2431 μm per pixel. Staining quantification was performed with QuPath 0.1.2 software (Centre for Cancer Research & Cell Biology, Queen's University Belfast, UK). For CD31 and Ki67, the region of interest (ROI) was defined as viable tumor tissue excluding necrosis. For TUNEL, the ROI was defined as total tumor tissue including necrosis. For CD31 and TUNEL, the positive area, defined as the ratio of DAB-stained pixels to total ROI area, was measured using the positive pixel count algorithm. For Ki67, the ratio (percentage) of cells with positive nuclear staining to total cell number was measured with the positive cell detection algorithm. ROI selection, algorithm optimization and validation, and qualitative examination of H&E slides were performed by a board-certified veterinary pathologist (S. Monette).

Gene set enrichment analysis for transcriptome analysis of LNCaP-AR xenografts following VTP treatment

LNCaP-AR xenografts were established in intact SCID mice by injecting 2 million cells as described previously (16) and, once established, were treated with VTP at 9 mg/kg WST11 followed by 100 mW laser fluence. Tumors were collected at 3, 6, or 24 hours, 1 week, and 8.5 weeks post VTP, and RNA was isolated following the standard protocol using TRIzol (Fisher Scientific). Expression profiling was performed using Illumina HT-12 Expression BeadChip array, and the data were analyzed using Partek Genomics Suite (Partek Inc.). The microarray data then underwent secondary analysis by gene set enrichment analysis (GSEA; ref. 17) using gene sets from the Hallmark and C2, Canonical Pathways collections (Molecular Signature Databases v6.0 (MSigDB); Broad Institute: http://software.broadinstitute.org/gsea/msigdb). GSEA| MSigDB. Accessed 19 Jun 2017. Microarray data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GSE109681).

Statistical analysis

Two-way ANOVA test using GraphPad Prism (GraphPad Software) was used for therapeutic efficacy in affecting tumor growth, one-way ANOVA for PSA, and a Mann–Whitney test for CD31, Ki67, or TUNEL staining quantification. Differences with P values < 0.05 were considered statistically significant.

Transcriptome analysis of VTP-treated tumors by GSEA revealed an enrichment of androgen response pathways

To identify potential druggable pathways active in prostate cancer that could be exploited for combination therapy with VTP, we analyzed the transcriptome of LNCaP-AR xenograft tumors following acute VTP exposure. Unbiased GSEA identified statistically significant enrichments with gene sets related to hypoxia, HIF1A, and VEGFR pathways at 3 to 6 hours after VTP treatment (Fig. 1, Supplementary Tables S1 and S2), effects that have previously been shown to be associated with photodynamic therapies (PDT; ref. 18). Interestingly, AR signaling gene sets were also upregulated in VTP-treated tumors compared with control mice, suggesting that the AR may be a viable target for combination therapy with VTP.

Combination therapy of ADT with VTP suppressed tumor growth to a greater extent than either treatment alone

To test our hypothesis that the preemptive blocking of androgen signaling upregulation induced by VTP might improve the outcome of tumor growth control, we tested the combination of VTP with an androgen signaling pathway inhibitor in widespread clinical use for the treatment of prostate cancer. Degarelix is a long-acting, gonadotropin-releasing hormone antagonist that results in a rapid onset of medical castration (19, 20). To establish preexisting AR inhibition, treatment with degarelix was initiated 3 days prior to administering VTP to prostate cancer xenograft tumors. Prior studies with VTP established that components of the immune response contributed to the anticancer activity of VTP (21). We therefore compared the efficacy of the combination therapy against LNCaP-AR tumors in both athymic nude (T-cell deficient; Fig. 2A) and SCID (both T- and B-cell deficient; Fig. 2B) mice. Tumor-bearing nude mice were randomly assigned to four cohorts: control, degarelix, VTP, and degarelix and VTP combination. The combination of degarelix and VTP resulted in statistically significant improved tumor growth control compared with either degarelix (P < 0.01) or VTP alone (P < 0.005; Fig. 2A). As in the nude mouse, the combination of degarelix and VTP led to superior control of LNCaP-AR tumor growth in SCID mice (P < 0.0001 for either monotherapy vs. combination; Fig. 2B). The combination of degarelix and VTP was also significantly more effective than VTP alone (P < 0.005) or degarelix alone (P < 0.0001; Fig. 3) in delaying the growth of VCaP, a human prostate cancer model with AR gene amplification that also expresses the constitutively active AR splice variant, AR-V7.

Combination therapy of ADT and VTP was more effective than VTP alone in downregulation of total PSA levels and induction of apoptosis/necrosis

To verify that AR activity was inhibited by the treatments, we measured the levels of total PSA (tPSA) in serum of mice bearing LNCaP-AR tumors (Fig. 4A). tPSA values were determined in separate cohorts of mice at 1, 3, or 7 days post-VTP (4, 6, or 10 days post degarelix). tPSA values declined by either VTP or degarelix alone, but the sharpest drop in tPSA levels was seen with the combination of degarelix and VTP (P < 0.05 vs. control across all time points).

In parallel, we assessed the histology of degarelix and/or VTP-treated tumors on days 3 and 7 post-VTP by both H&E and TUNEL assay to detect cell death (apoptotic and/or necrotic cells; Fig. 4B). VTP-treated tumors displayed partial cell death characterized by large foci of TUNEL staining, but with significant TUNEL-negative areas. Tumors treated with combination therapy appeared to display more extensive areas of TUNEL staining. Although not statistically significant compared with VTP alone, there were fewer tumors that escaped cell death in the combination group, suggesting that increased cell death underlies the effect on tumor inhibition. In contrast, degarelix alone–treated tumors exhibited little TUNEL staining, but still showed reduced Ki67 signal compared with controls, as expected (P < 0.05, Supplementary Fig. S1). These findings are reminiscent of patient studies, which have reported overall low frequencies of apoptosis in prostate cancer following ADT (22, 23). Nuclear AR staining was inversely correlated with TUNEL, suggesting that viable AR-positive cells had escaped focal therapy effects of VTP alone. Notably, the tumors treated with combination therapy were absent of AR staining, suggesting that remaining viable tumor cells were few in number.

ADT reduces tumor vessel staining

CD31 is primarily a marker for endothelial cells which can help evaluate the degree of intratumoral vessel formation. The degarelix-treated tumors appear to have less vessels than tumors in the control group as shown in Fig. 5A. The quantification of staining area depicts a 38% decrease in CD31 in degarelix-treated tumors compared with tumors (P < 0.05) in the control group. This might be a contributing factor of reduction in tumor recurrence in ADT/VTP combination group, although we did not observe any significant difference in CD31 vessel counts between the combination and VTP groups with the caveat that there was minimal remaining viable tumor material in these two groups (Supplementary Fig. S2).

The effective adoption of prostate cancer screening has led to earlier detection of small, clinically significant prostate cancers amenable to the newly developed treatment strategies for partial gland ablation which are well tolerated and associated with fewer adverse side effects than aggressive radical therapies such as surgery and radiotherapy. Positive oncologic outcomes in clinical studies of VTP have led to the recent approval of TOOKAD Soluble for the treatment of low-risk prostate cancer and highlight the potential of VTP to serve as an alternative to active surveillance or radical therapies (9–11). To potentially extend VTP treatment to larger cohorts of patients, we have recently launched a VTP clinical trial (NCT03315754) for patients with localized prostate cancer of intermediate risk, Grade Group 2 [Gleason score 7 (3+4)]. However, adaptation of this treatment to slightly larger and more aggressive tumors has potential risk for undertreatment, prompting the need for augmenting the mechanism of VTP-mediated tissue necrosis.

A byproduct of the oxidative damage triggered by PDT is the induction of intrinsic cellular stress response pathways, and these are thought to contribute to cancer cell survival and therapy resistance (18). To identify stress response pathways amendable to pharmaceutical intervention that could be used in conjunction with VTP, we performed transcriptome and GSEA of VTP-treated human prostate xenograft tumors. Consistent with earlier studies of PDT in cancer (18), we observed the upregulation of gene sets related to hypoxia/HIF1α, VEGFR, AP-1, and NF-κB in the immediate hours following VTP exposure. In addition, other potential cell survival pathways such as EGF/EGFR were also enriched after VTP. Although EGFR inhibitors (erlotinib, gefitinib, and lapatinib) have been clinically evaluated for prostate cancer, these phase II trials were limited to those with advanced or metastatic disease and displayed clinical benefit in only a small subset of patients [lapatinib (24, 25), erlotinib (26, 27), and gefitinib (28)]. However, of particular interest to our study was the finding that VTP treatment resulted in the acute upregulation of pathways related to AR signaling. Targeted AR inhibition forms the cornerstone of therapy for metastatic prostate cancer (29) and is the standard of care in the neoadjuvant or adjuvant setting for high-risk localized disease treated with external-beam radiotherapy (EBRT; ref. 30). AR inhibition is also being increasingly explored in clinical trials as neoadjuvant or adjuvant treatment for surgical patients with intermediate- to high-risk localized prostate cancer. Thus, there is supporting rationale for targeting AR in the setting of VTP-mediated tissue ablation for prostate cancer.

ADT can be accomplished by orchiectomy, but is more commonly achieved medically through the use of gonadotropin-releasing hormone (GnRH) agonists (leuprolide, goserelin, triptorelin) or more recently, antagonists (degarelix). GnRH agonists are known to cause an initial surge in circulating testosterone levels before achieving castration levels. In contrast, degarelix results in rapid castration in both men and mice without the testosterone flare (31, 32). In nude mice, a GnRH antagonist modestly outperformed an agonist in growth inhibition of a prostate cancer xenograft (33).

We chose degarelix to test the hypothesis that neoadjuvant ADT may improve antitumor efficacy of VTP in mice bearing prostate cancer xenograft tumors. In multiple model systems, the combination of degarelix and VTP offered superior local tumor growth inhibition compared with either degarelix or VTP alone. The combination-treated group displayed fewer tumors that escaped cell death compared with VTP alone. We did not detect a substantial induction of apoptosis in the tumors treated with degarelix alone, which is consistent with clinical reports demonstrating that prostate cancer apoptosis is not commonly seen in patients after ADT (22, 23). Treatment success was also demonstrated by AR inhibition that was reflected in the significant reduction in serum PSA levels, which was most extensive in the combination group.

Induction of tumor hypoxia following PDT with subsequent activation of cellular survival pathways and angiogenesis are potential factors that could adversely affect VTP treatment response (18). In prostate cancer cells, androgens promote the expression of HIF1α and VEGF (34, 35), and HIF1α enhances the activity of AR signaling (36, 37). Thus, the hypoxic tumor microenvironment induced by VTP with corresponding HIF1α upregulation could result in promotion of protumorigenic AR signaling, which is consistent with the results of our gene set analysis. Studies in prostate cancer patients have demonstrated that tumor hypoxia and HIF1α are decreased following ADT (38, 39). In prostate cancer model systems, ADT decreases VEGF production and reduces tumor vascularization (40–42) corresponding to the decrease in CD31-positive staining of endothelial cells following degarelix treatment in this study. Thus, we propose that ADT potentiates the efficacy of VTP treatment at least in part by counterbalancing the protumorigenic effects of hypoxia and angiogenesis.

Our findings draw parallels with the use of neoadjuvant and adjuvant ADT in combination with EBRT, which was first demonstrated 20 years ago to extend survival in patients with locally advanced prostate cancer (43, 44). If the combination of ADT and VTP is found effective in the clinical setting, this strategy may provide means for effective treatment of locally advanced prostate cancer with significantly less side effects than the current approaches.

K. Kim, P.A. Watson, A. Scherz, and J.A. Coleman are listed as co-inventors on a provisional patent application on VTP and combination therapy that is owned by Memorial Sloan Kettering Cancer Center and the Weizmann Institute of Science. H. Lilja holds ownership interest (including patents) in free PSA, hK2, and intact PSA assays, and is named on a patent for a statistical method to detect prostate cancer licensed by OPKO Health. No potential conflicts of interest were disclosed by the other authors.

Conception and design: K. Kim, A. Scherz, J.A. Coleman

Development of methodology: K. Kim, S. Monette, A. Scherz, J.A. Coleman

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Kim, S. Jebiwott, A.J. Somma, S. La Rosa, K.S. Murray, H. Lilja, S. Monette, J.A. Coleman

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Kim, P.A. Watson, S. Lebdai, D. Mehta, K.S. Murray, D. Ulmert, S. Monette, A. Scherz, J.A. Coleman

Writing, review, and/or revision of the manuscript: K. Kim, P.A. Watson, S. Lebdai, K.S. Murray, H. Lilja, D. Ulmert, S. Monette, A. Scherz, J.A. Coleman

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Kim, S. La Rosa, K.S. Murray, H. Lilja, J.A. Coleman

Study supervision: K. Kim, K.S. Murray, A. Scherz, J.A. Coleman

This work was supported by Thompson Family Foundation (K. Kim, S. Lebdai, S. Jebiwott, A.J. Somma, S. La Rosa, K.S. Murray, A. Scherz, and J.A. Coleman). Contributions from core facilities were supported in part by NIH/NCI Cancer Center Support Grant P30 CA008748. H. Lilja is supported in part by a Cancer Center Support Grant from the NIH/NCI made to Memorial Sloan Kettering Cancer Center (P30 CA008748), the MSKCC SPORE in Prostate Cancer (P50CA092629), the Sidney Kimmel Center for Prostate and Urologic Cancers, David H. Koch through the Prostate Cancer Foundation, and Oxford Biomedical Research Centre Program in the UK.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Heidenreich
A
,
Bastian
PJ
,
Bellmunt
J
,
Bolla
M
,
Joniau
S
,
van der Kwast
T
, et al
EAU guidelines on prostate cancer. part 1: screening, diagnosis, and local treatment with curative intent-update 2013
.
Eur Urol
2014
;
65
:
124
37
.
2.
Garisto
JD
,
Klotz
L
. 
Active surveillance for prostate cancer: how to do it right
.
Oncology (Williston Park)
2017
;
31
:
333
40, 345
.
3.
Wilt
TJ
,
Brawer
MK
,
Jones
KM
,
Barry
MJ
,
Aronson
WJ
,
Fox
S
, et al
Radical prostatectomy versus observation for localized prostate cancer
.
N Engl J Med
2012
;
367
:
203
13
.
4.
Hamdy
FC
,
Donovan
JL
,
Lane
JA
,
Mason
M
,
Metcalfe
C
,
Holding
P
, et al
10-Year outcomes after monitoring, surgery, or radiotherapy for localized prostate cancer
.
N Engl J Med
2016
;
375
:
1415
24
.
5.
Cathelineau
X
,
Sanchez-Salas
R
. 
Focal therapy for prostate cancer: pending questions
.
Curr Urol Rep
2016
;
17
:
86
.
6.
Ashur
I
,
Goldschmidt
R
,
Pinkas
I
,
Salomon
Y
,
Szewczyk
G
,
Sarna
T
, et al
Photocatalytic generation of oxygen radicals by the water-soluble bacteriochlorophyll derivative WST11, noncovalently bound to serum albumin
.
J Phys Chem A
2009
;
113
:
8027
37
.
7.
Brandis
A
,
Mazor
O
,
Neumark
E
,
Rosenbach-Belkin
V
,
Salomon
Y
,
Scherz
A
. 
Novel water-soluble bacteriochlorophyll derivatives for vascular-targeted photodynamic therapy: synthesis, solubility, phototoxicity and the effect of serum proteins
.
Photochem Photobiol
2005
;
81
:
983
93
.
8.
Borle
F
,
Radu
A
,
Fontolliet
C
,
van den Bergh
H
,
Monnier
P
,
Wagnières
G
. 
Selectivity of the photosensitiser Tookad for photodynamic therapy evaluated in the Syrian golden hamster cheek pouch tumour model
.
Br J Cancer
2003
;
89
:
2320
26
.
9.
Azzouzi
AR
,
Barret
E
,
Bennet
J
,
Moore
C
,
Taneja
S
,
Muir
G
, et al
TOOKAD® Soluble focal therapy: pooled analysis of three phase II studies assessing the minimally invasive ablation of localized prostate cancer
.
World J Urol
2015
;
33
:
945
53
.
10.
Azzouzi
A-R
,
Vincendeau
S
,
Barret
E
,
Cicco
A
,
Kleinclauss
F
,
van der Poel
HG
, et al
Padeliporfin vascular-targeted photodynamic therapy versus active surveillance in men with low-risk prostate cancer (CLIN1001 PCM301): an open-label, phase 3, randomised controlled trial
.
Lancet Oncol
2017
;
13
:
181
91
.
11.
Lebdai
S
,
Bigot
P
,
Leroux
P-A
,
Berthelot
L-P
,
Maulaz
P
,
Azzouzi
A-R
. 
Vascular targeted photodynamic therapy with padeliporfin for low risk prostate cancer treatment: midterm oncologic outcomes
.
J Urol
2017
;198:335–44.
12.
Ulmert
D
,
Evans
MJ
,
Holland
JP
,
Rice
SL
,
Wongvipat
J
,
Pettersson
K
, et al
Imaging androgen receptor signaling with a radiotracer targeting free prostate-specific antigen
.
Cancer Discov
2012
;
2
:
320
27
.
13.
Mitrunen
K
,
Pettersson
K
,
Piironen
T
,
Björk
T
,
Lilja
H
,
Lövgren
T
. 
Dual-label one-step immunoassay for simultaneous measurement of free and total prostate-specific antigen concentrations and ratios in serum
.
Clin Chem
1995
;
41
:
1115
20
.
14.
Pettersson
K
,
Piironen
T
,
Seppälä
M
,
Liukkonen
L
,
Christensson
A
,
Matikainen
MT
, et al
Free and complexed prostate-specific antigen (PSA): in vitro stability, epitope map, and development of immunofluorometric assays for specific and sensitive detection of free PSA and PSA-alpha 1-antichymotrypsin complex
.
Clin Chem
1995
;
41
:
1480
88
.
15.
Gavrieli
Y
,
Sherman
Y
,
Ben-Sasson
SA
. 
Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation
.
J Cell Biol
1992
;
119
:
493
501
.
16.
Chen
CD
,
Welsbie
DS
,
Tran
C
,
Baek
SH
,
Chen
R
,
Vessella
R
, et al
Molecular determinants of resistance to antiandrogen therapy
.
Nat Med
2004
;
10
:
33
39
.
17.
Subramanian
A
,
Tamayo
P
,
Mootha
VK
,
Mukherjee
S
,
Ebert
BL
,
Gillette
MA
, et al
Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles
.
Proc Natl Acad Sci U S A
2005
;
102
:
15545
50
.
18.
Broekgaarden
M
,
Weijer
R
,
van Gulik
TM
,
Hamblin
MR
,
Heger
M
. 
Tumor cell survival pathways activated by photodynamic therapy: a molecular basis for pharmacological inhibition strategies
.
Cancer Metastasis Rev
2015
;
34
:
643
90
.
19.
Broqua
P
,
Riviere
PJ-M
,
Conn
PM
,
Rivier
JE
,
Aubert
ML
,
Junien
J-L
. 
Pharmacological profile of a new, potent, and long-acting gonadotropin-releasing hormone antagonist: degarelix
.
J Pharmacol Exp Ther
2002
;
301
:
95
102
.
20.
Klotz
L
,
Boccon-Gibod
L
,
Shore
ND
,
Andreou
C
,
Persson
B-E
,
Cantor
P
, et al
The efficacy and safety of degarelix: a 12-month, comparative, randomized, open-label, parallel-group phase III study in patients with prostate cancer
.
BJU Int
2008
;
102
:
1531
8
.
21.
Preise
D
,
Oren
R
,
Glinert
I
,
Kalchenko
V
,
Jung
S
,
Scherz
A
, et al
Systemic antitumor protection by vascular-targeted photodynamic therapy involves cellular and humoral immunity
.
Cancer Immunol Immunother
2009
;
58
:
71
84
.
22.
Westin
P
,
Stattin
P
,
Damber
JE
,
Bergh
A
. 
Castration therapy rapidly induces apoptosis in a minority and decreases cell proliferation in a majority of human prostatic tumors
.
Am J Pathol
1995
;
146
:
1368
75
.
23.
Colecchia
M
,
Frigo
B
,
Del Boca
C
,
Guardamagna
A
,
Zucchi
A
,
Colloi
D
, et al
Detection of apoptosis by the TUNEL technique in clinically localised prostatic cancer before and after combined endocrine therapy
.
J Clin Pathol
1997
;
50
:
384
8
.
24.
Whang
YE
,
Armstrong
AJ
,
Rathmell
WK
,
Godley
PA
,
Kim
WY
,
Pruthi
RS
, et al
A phase II study of lapatinib, a dual EGFR and HER-2 tyrosine kinase inhibitor, in patients with castration-resistant prostate cancer
.
Urol Oncol
2013
;
31
:
82
6
.
25.
Liu
G
,
Chen
YH
,
Kolesar
J
,
Huang
W
,
Dipaola
R
,
Pins
M
, et al
Eastern Cooperative oncology group phase II trial of lapatinib in men with biochemically relapsed, androgen dependent prostate cancer
.
Urol Oncol
2013
;
31
:
211
8
.
26.
Nabhan
C
,
Lestingi
TM
,
Galvez
A
,
Tolzien
K
,
Kelby
SK
,
Tsarwhas
D
, et al
Erlotinib has moderate single-agent activity in chemotherapy-naïve castration-resistant prostate cancer: final results of a phase II trial
.
Urology
2009
;
74
:
665
71
.
27.
Gravis
G
,
Bladou
F
,
Salem
N
,
Gonçalves
A
,
Esterni
B
,
Walz
J
, et al
Results from a monocentric phase II trial of erlotinib in patients with metastatic prostate cancer
.
Ann Oncol
2008
;
19
:
1624
8
.
28.
Pezaro
C
,
Rosenthal
MA
,
Gurney
H
,
Davis
ID
,
Underhill
C
,
Boyer
MJ
, et al
An open-label, single-arm phase two trial of gefitinib in patients with advanced or metastatic castration-resistant prostate cancer
.
Am J Clin Oncol
2009
;
32
:
338
41
.
29.
Watson
PA
,
Arora
VK
,
Sawyers
CL
. 
Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer
.
Nat Rev Cancer
2015
;
15
:
701
11
.
30.
Bolla
M
,
Verry
C
,
Long
J-A
. 
High-risk prostate cancer: combination of high-dose, high-precision radiotherapy and androgen deprivation therapy
.
Curr Opin Urol
2013
;
23
:
349
54
.
31.
Van Poppel
H
,
Klotz
L
. 
Gonadotropin-releasing hormone: an update review of the antagonists versus agonists
.
Int J Urol
2012
;
19
:
594
601
.
32.
Hopmans
SN
,
Duivenvoorden
WCM
,
Werstuck
GH
,
Klotz
L
,
Pinthus
JH
. 
GnRH antagonist associates with less adiposity and reduced characteristics of metabolic syndrome and atherosclerosis compared with orchiectomy and GnRH agonist in a preclinical mouse model
.
Urol Oncol
2014
;
32
:
1126
34
.
33.
Redding
TW
,
Schally
AV
,
Radulovic
S
,
Milovanovic
S
,
Szepeshazi
K
,
Isaacs
JT
. 
Sustained release formulations of luteinizing hormone-releasing hormone antagonist SB-75 inhibit proliferation and enhance apoptotic cell death of human prostate carcinoma (PC-82) in male nude mice
.
Cancer Res
1992
;
52
:
2538
44
.
34.
Mabjeesh
NJ
,
Willard
MT
,
Frederickson
CE
,
Zhong
H
,
Simons
JW
. 
Androgens stimulate hypoxia-inducible factor 1 activation via autocrine loop of tyrosine kinase receptor/phosphatidylinositol 3′-kinase/protein kinase B in prostate cancer cells
.
Clin Cancer Res
2003
;
9
:
2416
25
.
35.
Joseph
IB
,
Nelson
JB
,
Denmeade
SR
,
Isaacs
JT
. 
Androgens regulate vascular endothelial growth factor content in normal and malignant prostatic tissue
.
Clin Cancer Res
1997
;
3
:
2507
11
.
36.
Park
S-Y
,
Kim
Y-J
,
Gao
AC
,
Mohler
JL
,
Onate
SA
,
Hidalgo
AA
, et al
Hypoxia increases androgen receptor activity in prostate cancer cells
.
Cancer Res
2006
;
66
:
5121
9
.
37.
Mitani
T
,
Harada
N
,
Nakano
Y
,
Inui
H
,
Yamaji
R
. 
Coordinated action of hypoxia-inducible factor-1α and β-catenin in androgen receptor signaling
.
J Biol Chem
2012
;
287
:
33594
606
.
38.
Milosevic
M
,
Chung
P
,
Parker
C
,
Bristow
R
,
Toi
A
,
Panzarella
T
, et al
Androgen withdrawal in patients reduces prostate cancer hypoxia: implications for disease progression and radiation response
.
Cancer Res
2007
;
67
:
6022
5
.
39.
Al-Ubaidi
FLT
,
Schultz
N
,
Egevad
L
,
Granfors
T
,
Helleday
T
. 
Castration therapy of prostate cancer results in downregulation of HIF-1α levels
.
Int J Radiat Oncol Biol Phys
2012
;
82
:
1243
8
.
40.
Joseph
IB
,
Nelson
JB
,
Denmeade
SR
,
Isaacs
JT
. 
Androgens regulate vascular endothelial growth factor content in normal and malignant prostatic tissue
.
Clin Cancer Res
1997
;
3
:
2507
11
.
41.
Stewart
RJ
,
Panigrahy
D
,
Flynn
E
,
Folkman
J
. 
Vascular endothelial growth factor expression and tumor angiogenesis are regulated by androgens in hormone responsive human prostate carcinoma: evidence for androgen dependent destabilization of vascular endothelial growth factor transcripts
.
J Urol
2001
;
165
:
688
93
.
42.
Byrne
NM
,
Nesbitt
H
,
Ming
L
,
McKeown
SR
,
Worthington
J
,
McKenna
DJ
. 
Androgen deprivation in LNCaP prostate tumour xenografts induces vascular changes and hypoxic stress, resulting in promotion of epithelial-to-mesenchymal transition
.
Br J Cancer
2016
;
114
:
659
68
.
43.
Bolla
M
,
Gonzalez
D
,
Warde
P
,
Dubois
JB
,
Mirimanoff
RO
,
Storme
G
, et al
Improved survival in patients with locally advanced prostate cancer treated with radiotherapy and goserelin
.
N Engl J Med
1997
;
337
:
295
300
.
44.
Horwitz
EM
,
Bae
K
,
Hanks
GE
,
Porter
A
,
Grignon
DJ
,
Brereton
HD
, et al
Ten-year follow-up of radiation therapy oncology group protocol 92-02: a phase III trial of the duration of elective androgen deprivation in locally advanced prostate cancer
.
J Clin Oncol
2008
;
26
:
2497
504
.