In the last decade, treatment for castration-resistant prostate cancer has changed markedly, impacting symptom control and longevity for patients. However, a large proportion of cases progress despite androgen deprivation therapy and chemotherapy, while still being fit enough for several more lines of treatment. Overstimulation of the androgen receptor (AR) activity is the main driver of this cancer. Targeting biological functions of the AR or its co-regulators has proven very effective in this disease and led to the development of several highly effective drugs targeting the AR signalling axis. Drugs such as enzalutamide demonstrated that the improvement in anti-tumour efficacy is closely correlated with an affinity for the AR and its activity and have established the paradigm that AR remains activity in aggressive disease. However, as importantly, key insights into mechanisms of resistance are guiding the development of the next generation of AR-targeted drugs. This review outlines the historical development of these highly specific agents, their mechanism of action in the context of defective AR activity, and explores the potential for the upcoming next-generation AR inhibitors (ARI) for prostate cancer by targeting the alternative domains of AR, rather than by the conventional ligand-binding domain approach. There is huge potential in these approaches to develop new drugs with high clinical activity and further improve the outlook for patients. Prostate cancer (PCa) is the most common cancer in men in the United Kingdom. It continues to be a highly significant issue and health burden affecting one in every six men during their lifetime, with around 50,000 new cases of the disease per year in total The main driver of the aggressive disease is the AR and its overstimulated, often constitutively active oncogenic signalling . Normal Three-dimensional crystal structures of the LBD and DBD of the AR protein have been solved, however the structure of the NTD is unknown, it is recognised to be intrinsically disordered, with its size and sequence varying greatly between nuclear hormone receptors. The NTD is primarily responsible for transcriptional activation functions and harbours activation function-1 (AF-1), which can be activated independently of the LBD. Within the AF-1 are two transactivation units (TAU), TAU-1 and TAU-5 that drive AR-dependent transcriptional activation. The DBD allows the AR to interact with DNA. It contains P-box and D-box structures that have two zinc finger motifs required for a stable interaction with specific DNA sites known as androgen response elements (AREs) within the chromatin. The DBD is highly conserved across nuclear hormone receptors, where alignment studies suggest a high overlap (at least 76%) between the AR-DBD and those of other nuclear hormone receptors Patients will follow a certain treatment pathway as the disease progresses. If the tumour is localised at diagnosis, surgical intervention is commonly used to remove the tumour mass or patients may be treated with radiation therapy to destroy the tumour, which may be more suitable for frailer patients. High intensity ultrasound (HIFU) and cryotherapy are also options. Patients with advanced disease extending outside of the prostatic capsule are often treated with androgen deprivation therapy (ADT) employing castration by either surgery While this hormone blockade is initially effective, many patients eventually progress to ARIs non-responsive disease termed castration-resistant prostate cancer (CRPC) characterised by individual or a combination of rising serum PSA levels and symptomatic/radiographic progression such as the formation of bone metastases. Despite the failure of ARI, most CRPC still relies on AR signalling as evident from the success of the next-generation of ARIs th Helical structure occurs within the LBD, and thus the AF-2 site is distorted. Consequently, interferences with transcriptional co-factor binding can occur, leading to transcriptional inhibition ARIs that target the LBD are the predominant form of anti-androgen therapy given to CRPC patients. They are competitive inhibitors of androgens that occupy the ligand-binding pocket and therefore block androgens from binding and activating the AR. X-ray crystal structures of AR-antagonist complexes suggest displacement of the 12Steroidal ARIs such as cyproterone acetate (CPA) , Nilutamide and Flutamide (Hydroxyflutamide is its active form) are first-generation, non-steroidal ARIs and competitive inhibitors of the AR , Apalutamide (ARN-509) and Darolutamide (ODM-201)  are more potent than first-generation inhibitors as they display greater binding affinity for the AR, less off-target effects and have additional novel mechanisms of blocking AR action. As well as blocking the binding of androgens to the LBD of the AR, these drugs inhibit nuclear translocation of the AR whilst also impairing its chromatin binding Enzalutamide showed such palpable results in the phase III AFFIRM trial, that the trial was stopped early since interim analysis determined that it was able to prolong survival of CRPC patients for five months compared to placebo. This trial determined the efficacy of Enzalutamide and safety in patients who had castrate levels of testosterone, failed with chemotherapy and had a progressive disease A new ARI Darolutamide was developed more recently to combat resistance mechanisms and other clinical manifestations that patients encountered with Enzalutamide and Apalutamide A receptors in the brain causing seizures and other central nervous system issues in some patients. Darolutamide has a low brain:plasma ratio in vivo and therefore is much less likely to cross the blood-brain-barrier A common problem with several previous ARIs is that the drugs can cross the blood-brain-barrier and inhibit the GABA5, 3β-hydroxyl moiety, which is also present in some testosterone derivatives , and therefore it is metabolised by the enzyme 3βHSD to Δ5A (D4A) 50 values vs Abiraterone. This therefore reduces ligand-activated AR signalling whilst also competitively antagonising the AR with comparable potency to Enzalutamide. D4A contains a 3-keto structure, similar to testosterone and DHT that allows for a modest affinity to the AR. These properties suggest that D4A is more potent than Abiraterone in vitro and in vivo and has potential as a promising clinical agent LBD-targeting ARIs dominate currently in the clinic intending to reduce AR signalling and thus curbing disease progression, but other approaches are also being exploited to curb the mechanisms of resistance to ARIs. Although not a direct ARI, Abiraterone acetate is commonly used in the clinic as a second-generation drug to reduce androgen levels in the blood, with Abiraterone being its active metabolite. Clinical trials involving Abiraterone administration in combination with second-generation LBD-antagonists or with ADT has also been assessed AR allow for the continued and overstimulated firing of the oncogenic AR signalling pathway.Following prolonged treatment with ARIs, oncogenic mutations in AR gene emerge allowing the AR to become activated despite ARIs based therapies. These “cis” resistance mechanisms such as AR LBD point mutations, AR gene/enhancer amplification and the formation of AR variants are summarised in AR gene sequence and therefore codons corresponding to amino acids can change Point mutations within the LBD of the AR often emerge due to treatment-induced selection pressure and are therefore uncommon in treatment-naive patients . NucleotAn example of this would be the mutation W742L, where the amino acid, tryptophan is changed to leucine at position 742  since its side chain is more flexible than the side chain of phenylalanine and thus the LBD can form an agonist conformation with Enzalutamide The treatment-emergent point mutations can also lead to AR promiscuity whereby the AR is activated by other non-androgenic ligands . The T87AR gene. The increased copy number of the AR gene leads to AR overexpression and increased AR protein levels  have emerged that can circumvent AR inhibition by conventional LBD-targeting ARIs. These AR-Vs lack some or all the LBD, with carboxy-terminal extensions replacing the LBD that are encoded by unique transcripts . The NTDOver 20 AR variants (AR-Vs) have been identified to co-express with wild-type AR in human prostate cancer cell lines, xenografts or clinical specimens AR. Alternative splicing occurs such that a different pre-mRNA sequence is included in the mature mRNA compared to the intended sequences  associates with the splicing factor SF2 but MALAT1 is regulated by suppressive androgen response element in the promoter and therefore AR antagonism  will increase MALAT1 expression and thus the SF2 splicing factor These truncated AR-Vs form due to alternative splicing and/or structural gene rearrangements of the equences  due to dequences  .AR that is commonly seen is a tandem duplication that causes the formation of variants (present in CWR22Rv1 cell line) AR exons and consequently synthesis of these cryptic exons is increased  have not only been linked to increased androgen synthesis but opposes Abiraterone suppression by Abiraterone, suggesting an important role of this enzyme as a key driver of CRPC progression In addition to the several “cis” mechanisms that cause gain-of-function mutations in the AR gene, it is now well established that the AR pathway can be stimulated within the tumours by the upregulation of androgen synthesis. 3β hydroxysteroid dehydrogenase (3βHSD1) an AR regulated enzyme, which catalyses the formation of extragonadal androgens within the tumours from adrenal precursors thus fuelling cancer progression Degradation of proteins is a natural manifestation, often directed at misfolded or unfolded proteins, but is also vital for the maintenance of cellular function. This is usually carried out via the process of ubiquitination of the protein in question and consequent degradation by the proteasome Niclosamide  is an FDA-approved anthelmintic drug, used to treat tapeworm infections that have recently shown to mediate solely AR-V7 protein degradation via the proteasome-dependent pathway (the exact mechanism has not been reported) Two-Phase I clinical trials are currently ongoing to assess dosing and tolerability of Niclosamide treatment when in combination with Enzalutamide for CRPC patients in vitro and in vivoASC-J9 or Dimethylcurcumin in vitro and in vivo, reducing the proliferation of cells and growth of tumours UT-34  was namein vitro has demonstrated efficient degradation of the AR protein by 95% and can inhibit cell viability with 100 times more potency than AR LBD antagonists. Furthermore, single doses of the compound were able to reduce AR protein levels in vivo .Proteolysis targeting chimaeras (PROTACs) are a promising, new agent class that can be employed to degrade the AR. PROTACs are hetero-bifunctional small molecules and structurally are made up of an AR binder/antagonist joined to an E3 ubiquitin ligase ligand via a linker With the NTD being critical for the transcriptional activity of the AR, inhibiting this part of the AR protein would be predicted to block AR activity regardless of the presence or absence of the LBD, and thus such inhibitors will have the potential to treat all AR-Vs since all functional AR isoforms rely on a functional NTD. It remains a challenge to create specific inhibitors for the NTD because of its intrinsically disordered nature and thus undetermined 3D structure and therefore current NTD inhibitors are often discovered using compound screening libraries.In vitro studies have shown EPI reduces the chromatin binding of AR thus decreasing transactivation, leading to growth inhibition. Furthermore, it has also shown to block the N/C interdomain interaction of the full-length AR and disrupt protein-protein interactions of the AR with factors that aid the stabilisation of the N/C interaction. However, it does not competitively prevent ligands binding to the LBD as it solely interacts with the NTD. Moreover, it blocks interactions between the AR and transcriptional cofactors such as CREB-binding protein and RAP74 Epoxide based EPI-001  and its Data first suggested that EPI interacts with the AF-1 domain of the NTD by direct binding and creating conformational change in the AR polypeptide. Further studies then showed that EPI reversibly interacted with TAU-5, in which EPI forms a reversible complex with a precise conformation of AF-1 which allows for nucleophilic attack by a protein side-chain on the C-Cl bond of EPI, thus forming an adduct incapable of activating transcription 50 to Bicalutamide and Darolutamide so can be administered at similar doses. EPI-7386 has shown to be effective in prostate tumour models driven by wild-type, mutant and AR-Vs and continues to inhibit transactivation EPI-001 showed to be non-specific to the AR, however, where it inhibited the growth of AR-negative prostate cancer cell lines as well as in a breast carcinoma cell line where it modulated both oestrogen and progesterone receptors Dysidea sp. family that antagonise and uniquely bind the AF-1 domain of the NTD Sintokamides  are polyin vitro and in vivo models that are driven by full-length AR and AR-Vs significantly better than EPI-001 A recent study showed QW-07 to be another compound that blocks the transcriptional activity of the NTD . It can in vitroin vivo remains be determined.Another approach in targeting the NTD is the use of monoclonal antibodies. These antibodies are often used to bind cell surface antigens or extracellular molecules since they cannot penetrate the cell membrane and flag them for degradation by the immune system. mAb 3E10, derived from an anti-DNA autoantibody from lupus nephritis-infected mice, however, can penetrate mammalian cells rapidly and its uptake is dependent on ENT2, a nucleotide salvage receptor. Bispecific antibodies to target the NTD were developed using 3E10 as a scaffold and combining single-chain variable fragments (scFv) additions joined by linkers. AR441  , a cyaniFurther results suggest that PP only interacts with the AR DBD once the AR is bound to DNA. While PP does not stop the AR nuclear localisation or binding to DNA, it can prevent RNA polymerase II recruitment causing transcriptional repression in vitro models of CRPC leading to suppression of growth in vivo CRPC model, VPC-14449 treatment reduced tumour size and repressed PSA synthesis FASN, FKBP5 and TSC2), whilst it was only able to block recruitment for FASN and FKBP5, but not the TSC2 gene in an AR-V model in vivo however, demonstrated that a higher dose of VPC was required to elicit an effect and therefore metabolic stability needs to be further explored A group of compounds were discovered to be solely selective to the AR DBD (as opposed to other NRs) such that AR signalling is inhibited. VPC compounds , and a Prostate Cancer Foundation (USA) young investigator Award (M.A).Not applicableNot applicableAll authors declared that there are no conflicts of interest.


In almost all patients, the proliferation and growth of prostate cancer is driven by the androgen receptor. Given this critical role, inhibiting the androgen receptor is the standard of care following surgery or radiotherapy. However, the effectiveness of current anti-androgen therapeutics is almost always temporary, with the cancer eventually developing drug resistance. There are no curative treatment options for resistant prostate cancer and these patients have an average life-expectancy of only 20 months. Currently, all clinical inhibitors of the androgen receptor act by direct binding to the hormone binding site. These drugs are therefore vulnerable to mutations which frequently arise at this region or to the emergence of truncated forms of the androgen receptor that are constitutively active without any need of hormone binding. In this study, we aim to develop small molecules inhibitors that attack the molecular functions of the androgen receptor at the N-terminal domain, distinct from steroid recruitment to evade resistance to conventional treatments.Prostate cancer patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer. Resistance can occur when mutations in the androgen receptor (AR) render anti-androgen drugs ineffective or through the expression of constitutively active splice variants lacking the androgen binding domain entirely . In this study, we are reporting the discovery of a novel AR-NTD covalent inhibitor 1-chloro-3-[(5-([(2S)-3-chloro-2-hydroxypropyl]amino)naphthalen-1-yl)amino]propan-2-ol (VPC-220010) targeting the AR-N-terminal Domain (AR-NTD). VPC-220010 inhibits AR-mediated transcription of full length and truncated variant ARV7, downregulates AR response genes, and selectively reduces the growth of both full-length AR- and truncated AR-dependent prostate cancer cell lines. We show that VPC-220010 disrupts interactions between AR and known coactivators and coregulatory proteins, such as CHD4, FOXA1, ZMIZ1, and several SWI/SNF complex proteins. Taken together, our data suggest that VPC-220010 is a promising small molecule that can be further optimized into effective AR-NTD inhibitor for the treatment of CRPC. Prostate cancer (PCa) is one of the most common forms of cancer in men, with over 1.3 million new cases per year worldwide . HoweverMost, if not all, of the AR transcriptional activity is mediated via the N-terminal domain (NTD) ,8. GivenHere, we have developed novel AR NTD inhibitors that were extensively characterized in biological assays. We show that these compounds effectively inhibit both full length AR and its constitutive variant ARV7 by disrupting critical coactivator interactions. Of these, VPC-220010 demonstrated superior inhibitor activity in AR transcription and AR-dependent growth of cancer cells compared to EPI-001. While still needing further hit-to-lead optimization, the prototype AR NTD inhibitor VPC-220010 could provide avenue to overcome common CRPC resistance mechanisms by decreasing prostate cancer cell growth through an AR-directed mechanism of action.Medicinal Chemistry and synthesis of the VPC-220XXX series. Detailed protocols for the synthesis of the VPC-220010 enantiomers can be found in Cell culture: LNCaP, PC3 and 22Rv1 human PCa cells were obtained from the American Type Culture Collection and authenticated by IDEXX Laboratories . Cells were maintained in RPMI-1640 media  and 5% FBS . All cells were cultured at 37 °C and 5% CO2 and monitored weekly for mycoplasma contamination.AR fluorescent reporter assay: Stably transfected eGFP-expressing LNCaP human prostate cancer cells (LN-ARR2PB-eGFP) containing an androgen-responsive probasin-derived promoter (ARR2PB) were assayed, as previously described [escribed .Luciferase assay: For both AR and nuclear receptor assays, PC3 human PCa cells grown in RPMI 1640 media (Invitrogen) supplemented with 5% charcoal-stripped serum (CSS) were seeded in 96-well plates (5000 cells/well) and transfected with 50 ng of AR, estrogen receptor alpha , glucocorticoid receptor (GR), or progesterone receptor (PR) plasmids, 50 ng of ARR3tk-luciferase  or 3ERE-luc (ER) using 0.3 μL/well TransIT20/20 transfection reagent . Following 48 h incubation, cells were then treated with compounds at various concentrations and 0.1 nm R1881 (in 100% ethanol) for 24 h for AR assays. GR, ER, and PR activation was stimulated with 1 nm dexamethasone, estradiol, or progesterone, respectively. For NTD-transactivation assays, LNCaP cells were co-transfected with 50 ng of both Gal4UAS-luciferase and AR (1-558)-Gal4DBD 24 h prior to treatment with forskolin (50 μM). Cell lysis was carried out with 60 μL of 1× passive lysis buffer/well . 20 μL of cell lysate from each well were mixed with 50 μL of luciferase assay reagent , and luminescence was recorded on a TECAN M200pro plate reader.Proliferation assay: LNCaP, 22Rv1 and PC3 cells were plated at 3000 cells per well in RPMI1640 containing 5% CSS in a 96-well plate, treated with 0.1 nm R1881 (a synthetic androgen) and test compounds (0–25 μM) for 72 h. Cell proliferation was determined using the PrestoBlueTM cell proliferation assay following incubation with the compound (0–100 μM) over 72 h. In brief, 30 μL of the PrestoBlueTM reagent was added to cells in each well of the 96-well plate containing 200 μL of media and incubated for 60 min at 37 °C in 5% CO2. The production of formazan was measured at 490 nm. Fluorescence measurement was read on a TECAN F200.qPCR and RNA sequencing analysis: LNCaP cells (6 × 106) were seeded in a 10 cm plates and grown with RPMI 1640 media containing 5% CSS and antibiotics for 48 h. On the day of treatment, CSS media was refreshed and the LNCaP cells were treated with either EtOH + DMSO (EtOH Control), 10 nM DHT + DMSO (DHT Control), EtOH + VPC220010/ENZA, or DHT + VPC220010/ENZA for 6 h. The compounds were dissolved in 100% DMSO, using the IC90 concentration of VPC-220010 (6 µM) and Enzalutamide (1.5 µM). Total RNA was extracted from cells with Trizol and the Qiagen RNeasy RNA Extraction Kit . Approximately 8 × 106 cells were collected per sample, with three biological replicates for each treatment. The concentrations and the qualities of the RNA samples were checked by Nanodrop. cDNA was prepared busing MMLV-Reverse Transcriptase . qRT-PCR was performed with LightCycler® 480 SYBR Green I Master . AR full length regulated genes TMPRSS2, FKBP5 and KLK3 in LNCaP cells, AR variant 2 regulated genes UBE2C, AKT1, CDC20 in PC3 cells : LNCaP cells (4 × 107/treatment) were cultured in RPMI-1640 medium supplemented with 5% CSS and 1% penicillin/streptomycin for 48 h. After refreshing the CSS culture medium, cells were treated with the same conditions as RNAseq samples for 6 h. After treatment cells were cross-linked by incubating in freshly prepared serum-free RPMI-1640 medium (no serum) containing 1% (vol/vol) formaldehyde for 8 min at room temperature on shaker. The reactions were then quenched with 0.1M glycine for 5 min. After two ice-cold PBS washes, cells were harvested by gentle scrapping in ice-cold PBS containing 100X protease inhibitor cocktail. The crosslinked AR protein complex was enriched with an AR antibody (Millipore # 06-680). From the resulting mass spectrometry data, the AR interactome was defined as those proteins that had >1 log2 fold-change in label-free quantification (LFQ) intensity in both DHT replicas compared to the ethanol control. Those proteins with a positive LFQ intensity in any IgG control samples were eliminated. A total of 111 proteins were identified to interact with AR in the DHT treated samples. Known AR interacting proteins were identified from previous published studies [ studies . An inteAR Phase separation: The mEGFP-tagged AR plasmid was transfected into Charcoal Stripped Serum (CSS) starved LNCaP cells for 2 days using Lipofectamin 3000 , following the user guide. Cells were stimulated with 1 nM dihydrotestosterone (DHT) for 2 h and then treated with 10 µM VPC-220010 or EPI-001 AR for an additional 2 h. Fluorescence staining was visualized with an Olympus FV3000RS confocal microscope  using a 60× UPLAPO oil objective. The images were taken with the FV31S-SW software with the confocal pinhole set to “automatic” under the Z-stack model. The condensates were quantified by two independent investigators and presented as the percentage of cells containing the condensates to the cells expressing GFP-tagged proteins. The results shown are the average of three independent experiments.In previous work we demonstrated that in a panel of novel AR inhibitors many were active via an AR degradation mechanism . However50 of 2.7 μM, which is approximately three and five times more potent than VPC-2055 (IC50 = 8.8 μM) and compound EPI-001 (IC50 = 17.1 μM), respectively  was comparable to VPC-2055 (IC50 = 1 uM) but 20 times better than EPI-001 (IC50 > 25 uM). To further validate VPC-220010 as an AR inhibitor, we evaluated its ability to reduce the production of endogenous prostate specific antigen (PSA) in PCa cell lines. PSA is an AR-regulated serine protease and is widely used as a biomarker for PCa. As expected, VPC-220010 reduced the secreted PSA levels in LNCaP cells with an IC50 of 0.91 μM. In contrast, EPI-001 had an IC50 value > 25 μM  with a reporter gene cloned downstream of the Gal4-binding site. When incubated with forskolin, this system induces transcription activity through phosphorylation of the AR-NTD [To confirm the proposed mechanism of VPC-220010 we tested the effect of this compound on NTD-mediated transactivation. Using a mammalian two hybrid system, we co-transfected AR-NTD fused to a Gal4 DNA binding domain  and EPI-001 (>25 μM)   C. Furthe> 50 μM) D. We did> 50 μM) E. OveralMultiple sequence alignment of related nuclear receptors shows marked amino acid diversity in the NTDs of AR, estrogen (ER), glucocorticoid (GR) and progesterone (PR) receptors. To rule out cross-reactivity with other receptors, we performed a reporter transcription assay for each of these nuclear receptors. We used reporter constructs containing the appropriate response regions for each nuclear receptor: ARR3tk for AR/GR/PR and estrogen response element for ER. Each receptor was stimulated with their specific agonist; the synthetic androgen methyltrienolone (R1881) for AR, estradiol (E2) for ER, progesterone for PR, and dexamethasone for GR . As expeKLK3, TMPRSS2 and FKBP5 as efficiently as enzalutamide and markedly better than EPI-001 , an approach that combines immunoprecipitation with mass-spectrometry to identify all AR interacting proteins on chromatin . Using tThe NTD is an attractive drug target for treating both early stage PCa and CRPC, as both ligand-dependent and -independent transcriptional activity of the AR is mediated, almost exclusively, by the N-terminal Tau1 and Tau5 regions. Targeting the AR NTD can therefore effectively bypass many mechanisms of resistance in CRPC including ARVs and point mutations. However, the identification of small molecules targeting the AR-NTD has proven to be an elusive goal as the NTD is a disordered protein domain.Based on our early work, we found that the small molecule compound VPC-2055 was inhibiting the AR through interaction with the NTD. With the help of our in-house computer-aided drug discovery (CADD) program, we developed its derivative VPC-220010 based on this novel scaffold. In this work we demonstrate that VPC-220010 can effectively inhibit the transcriptional activity of both full-length AR, and LBD-truncated variant ARV7 A,B. VPC-cis-regulatory enhancer elements [The AR-NTD mediates transcriptional activity through interactions with co-regulatory proteins ,8,30. Thelements . When ceelements  . However, further medicinal chemistry optimization is needed to make this scaffold stable enough to proceed to potential preclinical studies. Importantly, VPC-220057 is a 100-fold more stable in human microsomes than VPC-220010, albeit 10-fold less potent . Study oThis study broadens the repertoire of compounds targeting the AR NTD and provides useful insight for further development of novel antiandrogens with unique and novel mechanisms of action. The AR-NTD small molecule inhibitor prototype identified in this work possesses promising clinical utility for treating resistant forms of late-stage prostate cancer. Such drugs would be of immense value for CRPC patients that do not respond to conventional AR inhibitors.


To evaluate the correlations between the inflammatory factors in the aqueous humor and hyperreflective foci (HRF) in patients with intractable macular edema treated with antivascular endothelial growth factor (anti-VEGF).  This study included 17 patients with intractable macular edema (ME) treated with anti-VEGF agents. Inflammatory factors in the aqueous humor were measured by the Cytometric Beads Array before injection, and the numbers of HRF pre- and post-anti-VEGF treatment were counted from four different directions  in the SD-OCT images, respectively, before treatment and one month after treatment. The correlations between inflammatory factors and the numbers of HRF were assessed.  The numbers of HRF were reduced significantly after anti-VEGF treatment. The change in the HRFs at the 90-degree location was significantly positively correlated with IL-8 and VCAM-1. The change of all HRFs was significantly positively correlated with IL-8. The HRFs before the treatment also had a positive correlation with IL-8 and VCAM-1.  After anti-VEGF treatment, the numbers of HRF in intractable ME declined greatly. The higher the levels of IL-8 and VCAM-1 before treatment, the more significant the reduction of HRF after anti-VEGF treatment, which indicated that HRF could be an effective noninvasive imaging indicator for evaluating the effect of anti-VEGF on intractable macular edema. The OCT images at the 90-degree location could better show the inflammatory reaction of patients and also had better clinical significance for the prognosis evaluation of ME associated with inflammation. The macular is the most sensitive location of vision, and macular edema (ME) is one of the main reasons for visual impairment in the presence of macular lesions. In clinical circumstances, ME is mainly caused by diabetic retinopathy  and retiHyperreflective foci (HRF) is defined as “discrete, well-circumscribed dots with equal or greater reflectivity than the RPE band” in the images of spectral domain optical coherence tomography (SD-OCT) , 6. HRF In this study, we compared the changes in numbers of HRF and measured its correlation with inflammatory factors in intractable ME patients treated with anti-VEGF drugs, in order to find the possible roles and mechanism of HRF in the clinical follow-up of intractable ME.μl aqueous humor from every intractable ME patient before an intravitreal injection of anti-VEGF.This study included 17 patients with intractable ME, who had been treated with anti-VEGF agents at least twice based on a previous study . The lasThe levels of inflammatory factors ) in the aqueous humor were analyzed and quantified using Cytometric Beads Array  and flow cytometry.μm [A volume scan covering a 6 × 6 mm area of the macular area centered on the fovea was acquired using SD-OCT . HRF in the SD-OCT images is a small, well-circumscribed, dot-shaped lesion with similar or higher reflectivity than RPE in all retinal layers. The range of diameters of HRF was limited to 20 to 50 t-test was used to compare the differences in HRF pre- and posttreatments with anti-VEGF. The correlations between the levels of inflammatory factors and the change in the numbers of HRF were assessed with Pearson's or Spearman's correlation analysis. A value of was considered statistically significant.The statistical analysis was performed with SPSS software version 19.0 . All values are reported as mean ± standard deviation (mean ± SD). A paired Student's 17 eyes of 17 subjects were recorded and enrolled in this study . All the subjects had been treated with anti-VEGF agents at least twice prior to their aqueous humor measurement. The anti-VEGF agent names, the previous injections of anti-VEGF, and the inflammatory factors in aqueous humor for this treatment are listed in P < 0.01), which indicated that after more than two times anti-VEGF treatments, macular edema had been effectively controlled. In the meantime, the numbers of HRF significantly decreased at four locations  after the treatments compared with before the treatment.     (P > 0.05).In this study, the numbers of total HRF before the treatments had a positive correlation with IL-8 (= 0.006)  and VCAM= 0.025) , which i= 0.031)  and VCAM= 0.008) . In contr = 0.510, P = 0.044)  (P > 0.05).More interestingly, we found that the changes of total HRFs after treatments were also significantly positively correlated with its levels of IL-8 (= 0.044)  and the = 0.033) , which iME is clinically defined as an accumulation of serous fluid within the neurosensory retina, with increased thickness of the central retina. Some researches have shown that the leakage from the choroidal new vessels, predominantly VEGF-induced, may produce a large accumulation of fluid under the neurosensory retina . Anti-VEHRF may appear in vivo inflammatory components of ME, and HRF can be presumed to activate microglia , 18. InfResearch shows that the origin of HRF might be from activated microglia related with the inflammatory environment in age-related macular degeneration. Anti-VEGF treatment is associated with a reduction of HRF in DME, and HRF has been considered a marker of active inflammatory status . The detIL-8 is a well-known proinflammatory cytokine that acts as a neutrophil chemoattractant and a T-cell activator. Researchers have reported that IL-8 directly stimulates VEGF expression and the autocrine activation of VEGF receptor-2 in vascular endothelial cells. Some research also found the aqueous humor levels of IL-8 increased significantly in patients with DME . Many stThe previous further studies showed that IL-8 was secreted by microglia , and VCAThis study has still several limitations. Since aqueous humor accumulation is an invasive operation, we could not obtain the samples of aqueous humor from patients, which results in that we could not compare the change in inflammatory factors in the aqueous humor before and after treatments. However, according to our results, HRF, as an indirect indicator, can be used to evaluate changes in inflammatory factors postoperation. Although we obtained some significant results from this not too large sample, a further study can be conducted to test and verify our results based on large enough samples in the future.In conclusion, we have observed that the numbers of HRF in intractable ME significantly decreased after anti-VEGF treatments, and there was a positive correlation between the levels of IL-8 and VCAM1, and the numbers of HRF shown by SD-OCT. The marked change in HRF and the relationship with the inflammatory factors suggest that HRF can act as a clinical marker for inflammatory reaction in patients with intractable ME. Moreover, they can provide as an effective noninvasive imaging indicator for evaluating the effect of anti-VEGF on inflammation-related diseases.


Prostate cancer can develop under hormone treatment and chemotherapy from a castration-sensitive towards a castration-resistant into a drug resistant-tumor. The main hormonal drug target is the androgen receptor (AR). Androgen deprivation therapy reduces body-own androgen production and AR antagonists inhibit androgen-mediated activation of AR. Here, molecular mechanisms are described that review knowledge about tumor cells escape therapy by developing bypass mechanisms of AR-signaling. This includes genomic and non-genomic signaling. Deciphering the involved molecules that mediate castration and drug resistance will provide the basis of potential novel drug targets that may be used in addition to AR inhibition as combinatory treatment.Androgen receptor (AR) is a main driver of prostate cancer (PCa) growth and progression as well as the key drug target. Appropriate PCa treatments differ depending on the stage of cancer at diagnosis. Although androgen deprivation therapy (ADT) of PCa is initially effective, eventually tumors develop resistance to the drug within 2–3 years of treatment onset leading to castration resistant PCa (CRPC). Castration resistance is usually mediated by reactivation of AR signaling. Eventually, PCa develops additional resistance towards treatment with AR antagonists that occur regularly, also mostly due to bypass mechanisms that activate AR signaling. This tumor evolution with selection upon therapy is presumably based on a high degree of tumor heterogenicity and plasticity that allows PCa cells to proliferate and develop adaptive signaling to the treatment and evolve pathways in therapy resistance, including resistance to chemotherapy. The therapy-resistant PCa phenotype is associated with more aggressiveness and increased metastatic ability. By far, drug resistance remains a major cause of PCa treatment failure and lethality. In this review, various acquired and intrinsic mechanisms that are AR‑dependent and contribute to PCa drug resistance will be discussed. Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer and the second leading cause of cancer deaths in males in Western countries . PCa groCurrently, CRPC patients are additionally treated with AR-antagonists such as enzalutamide (Enz) and darolutamide (ODM-201) to fully block the AR-axis. The use of these AR-antagonists shows long terms benefits, including significantly lowering serum PSA level, decreases the risk of metastasis and prolonged overall survival in CRPC patients ,8. HowevMechanistically, AR antagonists can induce cellular senescence in PCa cells in vitro as well as ex vivo in patients PCa samples. This indicates that AR antagonists do not completely inhibit the AR rather promote the induction of an AR-dependent cellular senescence pathway ,12,13. AClassically, androgens, preferably dihydrotestosterone (DHT), bind to the AR, induce a conformational change that leads to dissociation from heat shock factors. This allows dimerization and nuclear translocation of AR to regulate the target genes. The activated AR acts as a transcription factor and suppresses or activates directly androgen-responsive target genes such as prostate-specific antigen (PSA) and many other genes involved in the differentiation and proliferation of prostatic cells . AR antaThe most common documented genomic aberration in CRPC, with around 60% of tumors, is AR gene amplification or mutation, leading to increased AR activity . AR overIt is widely accepted that mutations of AR in PCa under selection by anti-androgen treatment are one of the main reasons for DRPC ,23. MostAnother adaptive mechanism that bypasses ADT and leads to therapy resistance is the expression of AR splice variants. Approximately one-third of Enz or abiraterone treated patients express truncated receptors arising from AR splice variants (AR-Vs) . Enz treCRPC patients who express AR-V7 have both a shorter progression-free survival and overall survival. These patients fail mostly to respond to Enz or abiraterone treatment, and thereby the suggested treatment is those alternative drugs that target the N-terminal domain (NTD) , such asSo far, several EPI-derived compounds have been designed, analyzed on the molecular level and included in clinical trials. A new derivative of EPI is EPI-506 or ralaniten acetate that is used in a clinical trial for mCRPC patients as an oral soft gel capsule (NCT02606123). In this study, different concentrations (80–1800 mg) of EPI-506 were used, while only high doses >1280 mg) showed a meaningful decrease in PSA level for a short period ,34. This0 mg showIn CRPC a specific AR enhancer located 650 kb centromeric to the AR gene body was identified to be activated, led to gain of function probably due to the selective pressure exerted by anti-androgen treatment. This AR enhancer gene is either co-amplified with the AR gene in CRPC or even amplified at a higher rate. Further, it was detected that AR enhancer is enriched with H3K27 acetylation . AnotherThe AR transcriptional activity is regulated by a series of co-regulator complexes such as chromatin remodelers, co-activators and co-repressors that are co-recruited by the AR to chromatin by binding directly or in a protein complex to AR to regulate gene activity. A battery of different co-activators and co-repressor are known that interact with the AR . In geneCHD1 encodes an ATP-dependent chromatin remodeler. CHD1 regulates AR occupancy at a subset of AR binding sites of lineage-specific enhancers. CHD1 loss alters AR binding at lineage-specific enhancers and modulates distinct transcriptional programs to drive prostate tumorigenesis [CHD1 in vivo is the basis of a heterogeneous drug resistance mechanisms to Enz [The binding of AR to androgen response elements (ARE) itself can be regulated by diverse factors. The prostate-specific tumor suppressor gene igenesis . It has s to Enz .Another AR co-regulator and chromatin pioneering factor is homeobox B13, HOXB13, which interacts with AR to bind to AR target loci. HOXB13 is a homeodomain transcription factor that is involved in the development of normal prostate, as well as the formation and maintenance of prostate cancer . InteresAlongside with HOXB13 other pioneering factors such as FOXA1 and GATA2 act as chromatin factors at enhancer sites to regulate on one hand the expression of AR and on the other hand the activity of AR and AR-V7 at chromatin in mCRPC. FOXA1 induces open chromatin conformation to allow the binding of other transcription factors . This piAlso, GATA2 promotes AR activity and enhances progression of androgen dependent PCa to CRPC status. High GATA2 expression correlated with more aggressiveness of PCa . Higher Another epigenetics regulator of AR is enhancer of zeste homolog 2 (EZH2), which is a member of the polycomb group protein. . Of noteAnother AR-coactivator is CREB5, being amplified and overexpressed in CRPC, and associates with resistant PCa. In a recent clinical study, studying more than 1000 PCa patients under Enz treatment has shown that the expression of the oncoprotein CREB5 was abnormally high in ADT resistant tumors, which is due to an increased copy number of CREB5. High expression of CREB5 enhances the proliferation of PCa cells under ADT condition and mediates also resistance to Enz treatment. Therefore, drugs targeting CREB5 might be useful in DRPC therapy . Also, AR co-repressors may mediate castration resistance. The AR co repressor LCoR is recruited to AR in the presence of androgen. This recruitment of LCoR to androgen-bound AR inhibits AR action associated with inhibition of tumor progression in xenograft tumors of CRPC cells . The oveBesides the genomic action of AR to regulate gene expression by recruitment to chromatin, the non-genomic action of AR includes the regulation of several signaling pathways in the cytoplasm, including membrane-bound protein receptors .It is known that not all androgen-activated AR translocates into the nucleus. Androgen-activated AR interacts in the cytosol with other factors at non-genomic level. This interaction leads on one hand to modulation of signal transduction pathways and on the other hand, to phosphorylation of AR. Factors that interact with AR include the non-receptor tyrosine kinase Src, AKT and PKA. In addition, AR interacts with PTEN-PI3K, and the mTOR-autophagy pathways. These pathways cross-regulate each other. They may therefore contribute to drug resistance and promotion of PCa progression ,64. VariPreclinical studies showed that nerve growth factor (NGF) signaling enhances proliferation and metastasis in PCa. NGF binds to tropomyosin receptor kinase A (TrkA), which in turn activates the Ras/MAPK and AKT pathways . In LNCaA further important aspect which promotes PCa progression and contributes to drug resistance is the PCa microenvironment. The tumor microenvironment consists of stromal and inflammatory cells in addition to cancer-associated fibroblasts (CAFs). CAFs lead to the reorganization of structure and composition of the connective tissue, as well as enhanced tumor growth, invasiveness, and drug resistance of PCa. Hence, CAFs promote PCa progression. In the presence of androgen, the AR co-localizes with the scaffold protein filamin A (FilA) in fibroblasts. The AR/FilA complex results in CAFs moving towards PCa cells in 2D and 3D culture by increasing migration and invasiveness. An AR-derived staple peptide, Rh-2025u, was developed to disrupt the AR/FilA complex. The disruption inhibited migration and invasion. This peptide also impaired the AR induced metalloprotease cascade and thereby inhibiting remodeling of extracellular matrix, implying to be an interesting new therapeutic drug useful in combination with AR inhibition .In addition to the expression of AR splice variants that lack the AR LBD, other mechanisms of alternative activation of AR signaling have been described. In line with the interference of PI3K/AKT signaling with AR, PTEN gene mutations are common in PCa cancerogenesis, with loss of PTEN being identified in many metastatic PCa. PTEN is a tumor suppressor that inhibits the PI3K/AKT pathway. This augments the upregulation of the PI3K-AKT pathway in PCa and especially in mCRPC. First studies focused on blocking PI3K/AKT pathways as a possible therapeutic approach to treat PCa, since PI3K, through basal activation of AKT, has been strongly related to PCa growth and progression. Pharmacological inhibition of PI3K increased AR protein levels and thereby activating HER3-mediated AR-related gene expression . This reMultiple studies, therefore, investigated the possible benefits of combined treatment compared to single-agent therapy. Treating xenograft mouse models of androgen insensitive LNCaP with AZD5363, an AKT inhibitor leads to transient inhibition of tumor growth and PSA stabilization but followed eventually by resistance to AZD5363. However, combined treatment of a CRPC mouse model with the AKT inhibitor, AZD5363, and the AR antagonist Bic prolonged disease stabilization without signs of drug resistance . AnotherSrc kinase also emerges as a major player in the resistance of PCa. Src belongs to the family of non-tyrosine kinase family (SFK). Src expression is often correlated with malignant tumors and has been targeted in many therapies in PCa . On one Mechanistically, it is suggested that the Src kinase-mediated phosphorylation of AR at Tyr267 in the NTD, harboring a potent AR transactivation domain, as well as Tyr551 and Tyr552 in the hinge region, harboring the AR NLS, promotes both the nuclear translocation and transactivation by AR. Inhibition of Src kinase with PP2 was able to reduce AR translocation . Pre-cliNoteworthy, an interesting link between Src and mutation in the tumor suppressor genes BRCA1 and BRCA2 was shown. It was reported that in mCRPC with loss of BRCA1/2 heterozygosity Src signaling is enriched, therefore being more sensitive to Src inhibitors relative to mCRPC with wildtype BRCA2. Furthermore, Src knockdown in BRCA-null PCa cells enhanced PARPi sensitivity and indicated that Src activation might be a mechanism for PARPi resistance. This is supported by the findings using a combined treatment of a Src inhibitor with a PARPi inhibitor revealing a synergistic effect to inhibit proliferation of BRAC2-null mCRPC. Thus, inhibition of both Src and PARP is a promising new approach to treating mCRPC .ADT and AR-antagonist treatment may also fail due to intratumoral androgen production and therefore can lead to castration resistance. It was shown that intratumoral testosterone and DHT levels in mCRPC patients were significantly higher than in the control group . Drug reThe AKT-mTOR pathway regulates autophagy, which is suggested to be also a crucial pro-survival mechanism for CRPC ,94. UndeIt was shown that CRPC cells can survive PI3K inhibition. The suggested underlying mechanism is that AR increases the expression of Integrin α6β1 and indirectly Bnip3. Active AR-Integrin signaling suppresses apoptosis and induces Bnip3-mediated autophagy and mitophagy. Notably, inhibition of integrin α6β1 re-sensitized PTEN-negative CRPC to PI3K inhibitors. Hence, this might explain the lack of successful clinical trials of PI3K inhibitors for AR-dependent CRPC and unfold on the other hand a new approach to therapy .A mechanism of therapy resistance by AR antagonists may be explained by the findings that Enz and Bic induce autophagy leading to a pro-survival response in PCa cells ,97. ApalTherapy resistance of PCa is not only caused by AR-dependent mechanisms but also by AR-independent mechanisms, still activating a part of AR signaling. A short overview of possible pathways, which results in primary and acquired resistance, is described briefly in the following. The glucocorticoid receptor  is also a nuclear steroid receptor and closely related to the AR. GR becomes increasingly expressed in primary PCa tissue after long-term treatment with Enz . TherebyMechanistically, GR may bind an overlapping set of AR target genes ,103,104 GR expression and activity is also associated PCa cells with resistance to radiation. After radiotherapy, resistant cells have constitutive activated GR and decreased expression of AR. In radioresistant PCa, results suggest that radiation-induced GR through increased cAMP levels. Survival is promoted by GR in androgen-dependent PCa, but not in CRPC . InsteadMYCN, which is required for PCa tumor maintenance [MYCN amplification or N-Myc overexpression [MYCN amplification, is associated with shorter survival [Combined ADT with AR-antagonists may also cause resistance and lead to the development of neuroendocrine PCa (NEPC), presumably due to selective pressure from ADT and AR antagonist treatment . NEPC isntenance . The conpression . It has pression . RNA-seqsurvival .N-Myc overexpression in C4-2 cell line confers resistance to Enz by increasing ATM kinase activity, which promotes migration and invasion of PCa cells . MechaniONECUT2 is a transcription factor identified as a master regulator of AR networks in mCRPC. ONECUT2 suppresses the AR transcriptional program by direct regulation of AR target genes and the AR licensing factor FOXA . ONECUT2p53 interferes with AR signaling through binding to the AR promoter and suppressing AR expression. Enhanced p53 protein level negatively regulates the AR mRNA and protein levels in LNCaP cells. Knock down of p53 enhances AR protein expression in LNCaP cells . MutatioAnother AR-bypass pathway is the activation of the AKT-mTOR pathway by p66Shc, which is an oxidase previously shown to promote androgen-independent cell growth through generation of reactive oxygen species (ROS) and being elevated in PCa tumor samples. P66Shc is also known as a sensor for oxidative stress-induced apoptosis and as a longevity protein in mammals. Interestingly, in cancer p66Shc seems to activate AKT, mTOR, ERK, and Rac1 . Since pMicroRNAs (miRNAs) are endogenous small non-coding RNAs that act as a tumor suppressor or oncogene post-transcriptionally. Emerging evidence has discovered the importance of aberrant expression of miRNAs that are involved in the development of CRPC. Deregulated miRNA mediate drug resistance through different mechanisms such as reducing apoptosis by downregulating E2F1, enhancing CSC properties to facilitate tumorigenicity and chemoresistance, promoting the EMT process to increase PCa aggressiveness. For example, upregulation of miR 221/-222 via targeting p27/Kip1, HECTD2, and RAB1A are responsible for 90% of CRPC .Cisplatin is commonly used as a chemotherapy agent for the treatment of cancer. Chemotherapy of patients with cisplatin is initially effective, but patients eventually develop resistance . The lncIn CRPC, the overexpression of cytokines such as IL-6 is known to promote tumor progression and drug resistance ,134,135.The IL-6 signaling pathway is seemingly important in the regulation of growth and drug resistance of PCa cells and it helps cancer cell survival. A phase I study with patients scheduled for radical prostatectomy received either siltuximab or no drug. Tumor samples of siltuximab treated patients showed a decrease in  phosphorAnother pathway that involves STAT3 is an aberrant TGF-β pathway that plays a major function in the occurrence of DRPC and induction of epithelial-mesenchymal transition (EMT) because members of TGF-β family induce the expression of Snail . Snail iMoreover, reduced or loss of AR expression results in activation of IL-6 signaling, which activates STAT3. Activation of STAT3 is associated with enhanced Sox2, CD44, Nanog, Musashi-1, and integrin α2β1 that are stemness regulators. Thus, this may lead to a cancer stem cell (CSC) phenotype. Mounting the CSC repertoire results then in an unwanted sustained malignancy of human PCa tumors and is suggested to be responsible for the emergence of DRPC and tumor relapse . Epidermal growth factor receptor (EGFR) is involved in regulating cellular growth and function. The EGF ligand causes activation of EGFR by autophosphorylation, which leads in multiple downstream signaling . The mRNThe AR is a critical genomic and non-genomic factor in PCa pathogenesis. Adaptive AR signaling and occurrence of therapy resistant signaling are selected through therapy leading to CRPC and DRPC with unwanted further PCa progression. It emerges that many different adaptive and bypass pathways can lead to DRPC, which emphasizes the need for personalized medicine . AR-depe


Augmented Reality (AR) headsets have become the most ergonomic and efficient visualization devices to support complex manual tasks performed under direct vision. Their ability to provide hands-free interaction with the augmented scene makes them perfect for manual procedures such as surgery. This study demonstrates the reliability of an AR head-mounted display (HMD), conceived for surgical guidance, in navigating in-depth high-precision manual tasks guided by a 3D ultrasound imaging system. The integration between the AR visualization system and the ultrasound imaging system provides the surgeon with real-time intra-operative information on unexposed soft tissues that are spatially registered with the surrounding anatomic structures. The efficacy of the AR guiding system was quantitatively assessed with an in vitro study simulating a biopsy intervention aimed at determining the level of accuracy achievable. In the experiments, 10 subjects were asked to perform the biopsy on four spherical lesions of decreasing sizes . The experimental results showed that 80% of the subjects were able to successfully perform the biopsy on the 5 mm lesion, with a 2.5 mm system accuracy. The results confirmed that the proposed integrated system can be used for navigation during in-depth high-precision manual tasks. Augmented Reality (AR) gives the user the sense that virtual objects co-exist with real ones in the physical world. In fact, the user’s perception of the surrounding environment is enhanced by overlaying contextually relevant computer-generated information, thus providing an interactive experience ,2. AR isAR systems based on head-mounted displays (HMDs) provide the user with an egocentric perception of the augmented workspace and allow a hands-free interaction with it. They are thus considered to be the most ergonomic and efficient visualization devices for supporting complex manual tasks performed under direct vision, e.g., in surgery ,4. In faOST devices provide a full-scale resolution of the real scenario, which, in VST devices, depends on the resolution of the mediating camera.OST visors present an intrinsic virtual-to-real latency due to the time needed to render the virtual elements, which leads to them being spatially and temporally misaligned. On the other hand, VSTs enable the real scene to be delayed in order to match the virtual content, thus ensuring both accurate spatial and temporal virtual-to-real registration, at the expense of a higher system lag.In OST devices, the perceptual conflict due to the interaction between real and virtual elements ,9,10 proThe sub-optimal ergonomics  of both OSTs and VSTs prevents long-term use.OST visors need an accurate user-specific calibration mechanism between the virtual camera and the user’s eye in order to ensure the correct virtual-to-real alignment; whereas, for VST visors, the calibration procedure does not need to be user-specific, as it solely concerns the real and virtual camera .The lack of a standardized software framework for surgical application ,13.There are two types of AR: video see-through (VST) and optical see-through (OST) . In VST OST devices with a single focal plane fail to stimulate natural eye accommodation. This leads to visual fatigue and reduced user performance in completing a task that requires simultaneous focusing of real and virtual information ,15. SincIn this work, we assessed the efficacy of wearable VST AR devices for guiding in-depth high-precision manual tasks, such as needle localization of occult lesions before surgical biopsy, biopsy interventions, or cyst drainage. Learning and performing these interventions under ultrasound guidance is challenging. The ultrasound data are typically displayed on a conventional video monitor, which forces the clinician to shift his/her gaze away from patients and therefore requires considerable hand–eye coordination.Of the ultrasound probes currently available, 2D probes are mostly used for these types of intervention. These probes provide a real-time, single-slice ultrasound image, which is displayed on the remote 2D monitor. This means that the surgeons have to have a good sense of the three-dimensional relationship, as they have to mentally superimpose the ultrasound slice over the patient to determine both the position and orientation of the needle with respect to the slice. To obtain a good three-dimensional vision of the anatomical area of interest, one possibility is to scan the area continuously and over several planes. However, keeping both the needle and the lesion within the same ultrasound slice is problematic.Overcoming such issues entails using AR HMDs to simplify the learning and performing of ultrasound-guided interventions ,20,21,22In this study, we propose the integration of a commonly available 3D ultrasound imaging system with a wearable VST AR visor. In real-time, the system shows the 3D model of the target (extracted from the 3D ultrasound volume) superimposed on the patient’s anatomy. It also shows two crosshairs that guide the user in the puncture and insertion of the needle according to the planned entry point and insertion trajectory. The aim is to eliminate any hand–eye coordination problems and, at the same time, to improve the three-dimensional perception of the patient’s anatomy, thus improving targeting accuracy.To evaluate the feasibility of our idea, we studied and implemented custom-made calibration and tracking methods and verified that the global AR registration error is consistent with the clinical needs. To assess the efficacy of our solution, we designed an experimental study that simulates an ultrasound-guided biopsy task. We performed the biopsy task on a tailor-made phantom, using the AR surgical navigation platform provided by the VOSTARS project for the in situ visualization of medical imaging data derived from a Philips 3D ultrasound acquisition system.In this section, we detail the hardware and software components for the integration of the 3D ultrasound acquisition system and the AR platform. We also describe the in vitro testing performed to evaluate the accuracy and precision of our system.Similarly to magnetic resonance imaging (MRI), 3D ultrasound provides volumetric information on the patient’s anatomy, preventing exposure to the procedural radiation dose for both patients and clinical staff. However, in contrast to MRI, 3D ultrasound does not require a dedicated room; it provides immediate results; it is less expensive; and it is portable. This means that it can be used in both extra-operative  and intra-operative environments.We used a 3D ultrasound transducer (VL 13-5) and a Philips iU22 ultrasonic system   with a ″ OmniVision CMOS 4 M pixel sensor  and M12 lens support  with a focal length of 6 mm, which was chosen to restore the 1:1 scale factor at arm’s distance.The switching between the two see-through mechanisms is provided through a pair of liquid-crystal (LC) optical shutters placed in front of the displays, which can be electronically dimmed to change their transparency. The VST camera-mediated view is provided by a pair of world-facing RGB cameras rigidly incorporated within the HMD. The cameras have an anthropometric interaxial distance (∼6.3 cm) and a fixed convergence angle of 3.4The computing unit runs on a laptop with an Intel Core i7-8750H CPU@2.20 GHz with 12 cores and 16 GB RAM  and an Nvidia GeForce RTX 2060 (6 GB) with 1920 CUDA Cores  graphic card processing unit. The software framework is designed to support in situ visualization of medical imaging data using the VTK library, an open-source library for 3D computer graphics, modeling, and volume rendering. The software features an inside-out optical tracking algorithm based on OpenCV API 3.4.1, which performs the stereo localization of a triple of spherical markers, using our ad hoc algorithm . The traTo summarize, the software provides the locationally registered in situ visualization of task-oriented digital content in terms of both optical and video see-through-based augmentations. The framework is highly configurable in terms of tracking capabilities and renderable content and ensures significant computational efficiency, thanks to the CUDA-based multi-threaded architecture. This architecture enables an average frame rate of ∼30 fps to be obtained for each eye.The definition of a local reference system associated with the ultrasound probe FRS in a.T in The tracking of the ultrasound probe pose in the world reference system associated with the visor WRS in a, i.e., X between FRS and the reference system of the ultrasound acquisition volume  dedicated calibration routine. For the probe to be used in combination with the AR platform, and thus, for the ultrasound virtual content to be correctly spatially registered over the real scene, the relative pose between the AR visor and the acquisition volume of the ultrasound probe needs to be known at all times. This entails:With regard to the third requirement , we had to identify the coordinates of the salient points acquired within the ultrasound volume with respect to the reference system of the probe itself. This is usually performed by imaging a phantom, namely an object with known physical properties and dimensions. Following , calibraFinally, we defined the transformation matrix that links the reference system of the probe (PRS) to the reference system of the optical frame (FRS). For this purpose, an additional MATLAB subroutine was created, which incorporates the hand–eye calibration method by Park .X is the unknown transformation between the optical frame and the ultrasound probe.A is the homogeneous transformation that describes the relative pose between two consecutive poses, henceforth referred as T, of the optical frame anchored to the ultrasound probe, in the world reference system (WRS) associated with the HMD.B is the homogeneous transformation that describes the relative pose, henceforth referred as U, between two consecutive poses of the comb (CRS) in the PRS.A and B were stored with the 14 relative poses, derived from two consecutive time instants, pose at time i+1 and pose at time i. In The ultrasound probe was moved in 15 different positions; therefore, transformation matrices X is derived, the calibration procedure is finished.Once The evaluation of the system included a quantitative analysis of the accuracy of the AR overlay and a user study to estimate the accuracy achievable during an in-depth high-precision task. The following subsections describe the experimental setups and the protocols implemented to test the system.System testing entailed selecting an echogenic material with acoustic properties resembling those of human tissue. Particular attention was paid to the speed of sound in the material, which is crucial to preventing distortions of shape and size during reconstruction, and to the stability of the acoustic properties over time.There are various material for creating ultrasound phantoms, from the most common organic materials such as agar, agarose, gelatine, gellan gum, and polyvinyl alcohol (PVA), to non-organic materials, such as polyvinyl chloride plastisol (PVC-P), room-temperature-vulcanizing (RTV) silicones, polydimethylsiloxane (PDMS), and polyurethane (PU). Biopolymers contain a high percentage of water (>80%), which makes them similar to soft biological tissues . HoweverPolyvinyl chloride plastisol is the best candidate for ultrasound phantom manufacturing. Compared with biopolymers, PVC-P is resistant to bacterial attack, while compared with chemically synthesized polymers, it has clear acoustic advantages over silicones and PDMS for ultrasound imaging ,39. In aThe AR overlay accuracy can be assessed by calculating the 2D target visualization error (TVE2D), namely the offset, expressed in pixels, between the real and the virtual objects in the image plane, evaluated as the reprojection error in 2D onto the displayed AR image.To quantitatively evaluate the AR overlay accuracy, tests were performed on an ad hoc developed ultrasound phantom in the shape of a spherical lollipop (20 mm in diameter). The phantom was manufactured in PVC-P, with a supporting structure made of ABS. The choice of shape is linked to the ease of identifying the centroids of spherical objects, both real and virtual, which made it easier to assess the accuracy of the AR overlay. To create the virtual sphere to be registered over the head of the lollipop, the phantom was dipped in the usual 37° water bath, and the ultrasound probe was positioned to have the head of the lollipop centered in the acquisition volume, thus mimicking the positioning of a target lesion to be biopsied in standard protocols.imfindcircles function, and thus to evaluate the TVE2D, namely the offset of the two centroids in the image plane. To facilitate the work of the algorithm, the real image was cropped around a region of interest containing solely the real sphere. Once calibrated, the AR HMD was anchored over a flexible mounting arm and moved in 10 different positions to simulate plausible user points of view. For each pose of the visor, four different images were acquired: three AR images and a real image with the AR switched off, with a total of 40 images. This set of images was processed in Matlab to determine the centroids of both virtual and real spheres through the The target visualization error in 3D (TVE3D), which is the estimation at a fixed distance of the visualization error in space (in terms of Euclidean distance), was derived from the TVE2D by proportioning the sphere’s size in pixels for each image with the actual size of the lollipop’s head.The level of precision and accuracy that the integrated system can ensure in performing an in-depth high-precision task was also checked. Ten participants were recruited and were asked to perform a simulated 3D ultrasound-guided biopsy procedure in a non-anthropomorphic parallelepiped-shaped phantom.The phantom is made of PVC-P doped with 2% by weight with graphite to increase the speed of sound in the material to make it comparable to human tissues. Four different-sized spherical lesions were randomly included to evaluate the system’s accuracy in guiding an in-depth high-precision manual task. More specifically, sizes of 3, 5, 7, and 10 mm in diameter were used to test an accuracy level of 1.5 mm, 2.5 mm, 3.5 mm, and 5 mm, respectively. Ten participants with normal or corrected-to-normal visual acuity were recruited among university students, staff, and faculty members. None of the participants had previous experience in performing an ultrasound-guided biopsy.3 was acquired, and the lesion was extracted via segmentation. The segmented mesh was then transformed into a virtual reality modeling language (VRML) file ready to be uploaded to the AR platform and displayed onto the HMD, coherently registered to the real phantom. Due to the phantom material’s excellent healing capabilities, it was possible to use a single phantom containing the four lesions for all of the users. For each lesion embodied in the phantom, a volume of 38 × 91 × 27 mmEach participant was provided with a 21 G syringe   and was asked to wear the HMD and to perform the biopsy task reaching the target lesion with the tip of the needle. The participants were also asked to mention if they perceived any visual discomfort or any spatial displacement of the virtual content while performing the task.To guide the needle insertion, the lesion concerned and two crosshairs were virtually added to the augmented scene and were thus visible through the visor. This visualization technique has already been tested in other studies and is successful in guiding high-precision tasks in different anatomical districts, such as neuro- and spine surgery ,42. One The participants conducted four different sessions, each session involving one of the four target lesions at a time. The objective was considered achieved if the real-time ultrasound image showed the needle within the target lesion. The participants were given only one chance to perform the biopsy on each lesion. The results of the AR overlay accuracy test are reported in All 10 participants completed the four biopsy tasks without perceiving any visual discomfort or any spatial displacement of the virtual elements. We proposed the use of an AR surgical navigation platform and a customized AR headset for the guidance of in-depth high-precision manual tasks. We addressed the navigation of 3D ultrasound image-guided interventions, such as biopsies, with the in situ visualization of the target. To this end, we developed a dedicated calibration routine, to integrate the data from the 3D ultrasound imaging system within the AR platform, and thus the AR visor.We designed an experimental study to quantitatively evaluate the accuracy level that the integrated system could provide in guiding a simulated biopsy task. First, we analyzed the TVE to determine the intrinsic accuracy achievable in the AR visualization, obtaining a TVE3D ranging from 0.87 mm to 2.79 mm, with a mean value of 2.02 mm. To also consider any user inaccuracy, we designed a user study and engineered an ad hoc ultrasound phantom with different-sized lesions randomly enclosed within it. The results of this second test, given in terms of success rates, suggest that the integrated system could be successfully used to guide in-depth high-precision manual tasks. Despite their inexperience in performing the biopsies, all of the participants were able to reach the target lesion of 10 mm, which is the average lesion size targeted during histopathological diagnosis biopsies , highligOne limitation of this work is due to a technological issue. To date, ultrasound imaging does not enable the real-time video streaming of volumetric acquisitions. These can only be stored on a disk and exported for external processing. For this reason, in the experimental setup, we were forced to constrain the probe in a predetermined pose; thus, the participants were not given the opportunity to orient it as they wished. Another limitation is the time needed to prepare the setup. Currently, the time required for preparation varies from 20 to 25 min. The goal of future studies is to reduce the waiting time (probably to the order of a few minutes) by automating the generation of the AR content, starting from segmentation. Ideally, exporting the acquired volume in real-time would positively boost the waiting time reduction.In conclusion, the results of this study show the potential of integration of a 3D ultrasound imaging scanner and AR visualization system (software framework combined with a dedicated headset) for the guidance of in-depth high-precision manual tasks. We also provided guidelines on how to perform the calibration between these two systems and on the materials that can be used to fabricate an ultrasound phantom. We are planning to investigate whether the proposed integrated system could decrease both the inter- and intra-operator variability in performance. In addition, we will also challenge our system with the simulation of more complex in-depth tasks, such as a tumorectomy.


Targeting androgen signaling with the second-generation anti-androgen drugs, such as enzalutamide (Enza), abiraterone (Abi), apalutamide , and darolutamide (Daro), is the mainstay for the treatment of castration-resistant prostate cancer (CRPC). While these treatments are effective initially, resistance occurs frequently. Continued expression of androgen receptor (AR) and its variants such as AR-V7 despite AR-targeted therapy contributes to treatment resistance and cancer progression in advanced CRPC patients. This highlights the need for new strategies blocking continued AR signaling. Here, we identify a novel AR/AR-V7 degrader (ARVib) and found that ARVib effectively degrades AR/AR-V7 protein and attenuates AR/AR-V7 downstream target gene expression in prostate cancer cells. Mechanistically, ARVib degrades AR/AR-V7 protein through the ubiquitin-proteasome pathway mediated by HSP70/STUB1 machinery modulation. ARVib suppresses HSP70 expression and promotes STUB1 nuclear translocation, where STUB1 binds to AR/AR-V7 and promotes its ubiquitination and degradation. ARVib significantly inhibits resistant prostate tumor growth and improves enzalutamide treatment in vitro and in vivo. These data suggest that ARVib has potential for development as an AR/AR-V7 degrader to treat resistant CRPC. Targeting androgen signaling via androgen deprivation therapy (ADT) is the first line treatment for prostate cancer (PCa). While initially effective, the majority of men eventually develop castration-resistant prostate cancer (CRPC). Enzalutamide (Enza), abiraterone (Abi), apalutamide , and darolutamide (Daro) are the second-generation anti-androgen drugs used for the treatment of CRPC and function by inhibiting androgen receptor (AR) signaling –4. Even Disruption of protein homeostasis (proteostasis) in cells causes aberrant folding and aggregation of pro-oncogenic proteins which is integrated by molecular chaperones, the ubiquitin-proteasome system, and their regulators , 13. CanMany studies have investigated ways to target persistent AR signaling with the goal to overcome resistance to AR-targeted therapies. These include finding methods to promote AR degradation, targeting the N-terminal domain and DNA binding domain of the AR, inhibition of AR synthesis and targeting AR co-regulators –19. Our In the present study, we synthesized a library of niclosamide analogs according to predicted bioavailability in order to improve its bioavailability and potency. We have identified several analogs (named ARVib) that can target AR/AR-V7 for degradation. In particular, analog #7 (ARVib-7) has better bioavailability than niclosamide and can inhibit prostate cancer cell proliferation and AR transcriptional activity. We demonstrate that ARVib degrades AR/AR-V7 protein expression through the ubiquitin-proteasome pathway mediated by HSP70/STUB1 machinery modulation. ARVib suppresses HSP70 expression and promotes STUB1 nuclear translocation, where STUB1 binds to AR/AR-V7 and promotes its ubiquitination and degradation. ARVib significantly inhibited resistant prostate tumor growth and improved Enza treatment in vitro and in vivo.Our previous studies discovered that niclosamide is a potent inhibitor of AR/AR variants and functions to enhance Enza and Abi treatment , 21. HowSince AR-V7 is critically involved in driving resistance to anti-androgens such as enzalutamide and abiraterone, we hypothesized that compounds with the ability to inhibit AR-V7 would synergize with anti-androgens and improve their therapeutic activity in resistant prostate cancer. To test this hypothesis, we determined the effects of these compounds at sub low dose (0.25 μM) either alone or in combination with Enza or Abi on cell growth. The effects of ARVib-7 and ARVib-31 (which inhibit AR-V7 expression) on cell growth of resistant prostate cancer cells were compared to the effects of compounds #1, and #34 (which do not inhibit AR-V7 expression). As shown in Fig .To explore the gene regulating mechanisms underlying ARVib treatment in drug resistant prostate cancer cells, we performed RNA-sequencing analyses using C4-2B MDVR cells treated with ARVib-7 and ARVib-31 to identify gene programs affected by treatment. There are 10,224 genes and 10,753 genes that were differentially expressed in ARVib-7 and ARVib-31 treated C4-2B MDVR cells, respectively, and 8850 genes that were commonly regulated by both ARVib-7 and ARVib-31 , ARVib-7, or ARVib-31 for 3 days, total cell numbers were determined. As shown in Fig. max and AUClast of ARVib-7 were 6-fold higher than niclosamide, indicating that ARVib has improved bioavailability over parental niclosamide. To determine ARVib-7 effects in vivo, we first used CWR22Rv1 tumor xenografts. Mice bearing tumors were treated with niclosamide (25 mg/Kg) and ARVib-7 (25 mg/Kg) through intraperitoneal (i.p) injection. As shown in Fig. Niclosamide has poor bioavailability in vivo. To determine if ARVib has improved bioavailability, we measured PK parameters in male Sprague-Dawley rat plasma. As shown in Table We next tested ARVib-7 against niclosamide in vivo through oral administration using the LuCaP 35CR PDX model, which has AR-V7 overexpression , 29. Ourp = 0.28), ARVib-7 treatment significantly suppressed PSA (p = 0.029) and the combination treatment further reduced PSA levels (p = 0.0053)  Fig. . Surviva53) Fig. . Treatme53) Fig. . Immunoh53) Fig. . CollectIn the present study, we identified a new AR/AR-V7 degrader called ARVib from a library of synthetic analogs of niclosamide, which was previously identified as a potent inhibitor of AR and AR-V7. We demonstrate that ARVib has better bioavailability and PK parameters than niclosamide, and can effectively degrade AR/AR-V7 and attenuate AR/AR-V7 downstream target gene expression in prostate cancer cells. Mechanistically, ARVib degrades AR/AR-V7 protein expression through the ubiquitin-proteasome pathway mediated by modulation of HSP70/STUB1 machinery. In addition, ARVib significantly inhibited resistant prostate tumor growth and improved Enza treatment in vitro and in vivo.Large efforts have been placed in order to identify potential inhibitors of AR-V7. ASC-J9® was developed as an AR degradation enhancer, and has shown efficacy in preclinical studies in slowing progression of Enza-resistant prostate cancer . PROTAC-Niclosamide initially was discovered as a treatment for tapeworm infection and functions by inducing mitochondrial uncoupling which causes tapeworm energy deficiency. However, its mechanism of action in human cancer cells remains unclear. Niclosamide has been reported to interfere with multiple signaling pathways, including mTORC1, IL-6/JAK/STAT3, ERK, Src, and Wnt signaling in different lineages of cancer cells . The ARVCumulative evidence shows that deficiency of proteostasis promotes tumorigenesis, which frequently causes abnormal aggregation of oncogenic proteins, such as c-myc, p53, BCL-2, etc , 38–40. Targeting the ubiquitin-proteolysis pathway to degrade oncogenic proteins has been emerging as an attractive strategy to treat cancer , 43. VarIn conclusions, we have identified a new AR/AR-V7 degrader named ARVib and showed that ARVib effectively degrades AR/AR-V7 protein through the ubiquitin-proteasome pathway mediated by the HSP70/STUB1 complex and attenuates AR/AR-V7 downstream target gene expression in prostate cancer cells. ARVib significantly inhibits resistant prostate tumor growth and improves Enza treatment in vitro and in vivo, suggesting a clinical potential for development as an AR/AR-V7 degrader to treat resistant CRPC.C4-2B and CWR22Rv1 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 units per ml penicillin and 0.1 mg per ml streptomycin. 293 and VCaP cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units per ml penicillin and 0.1 mg per ml streptomycin. All experiments with cell lines were performed within 6 months of receipt from the American Type Culture Collection (ATCC) or resuscitation from cryopreservation. C4-2B cells were kindly provided and authenticated by Dr. Leland Chung, Cedars-Sinai Medical Center . The resistant cells are referred to as C4-2B MDVR  . C4-2B M5 cells per well in 12-well plates or 2 × 105 cells per well in six-well plates and transfected with 20 nM of siRNA (Invitrogen) targeting the HSP70 sequence , STUB1 sequence  or control siRNA  using Lipofectamine-iMAX (Invitrogen). The effect of siRNA-mediated gene silencing was examined using qRT-PCR and western blot 2–3 days after transfection. Cells were transiently transfected by expressing plasmids for vectors, AR-FL, AR-V1, AR-V3, AR-V7, AR-V9, AR-V12 (AR-V567es), wild-type AR, AR mutants , Flag-STUB1, HA-Ubiquitin or HSP70  using Lipofectamine 2000 (Invitrogen).For small interfering RNA (siRNA) transfection, cells were seeded at a density of 0.5 × 10oC with the indicated primary antibodies AR (441), AR (N20), AR (C-19), HSP70 (F-3 and H-300), STUB1(H231 and G-2), HA (F-3), Ubiquitin (P4D1 and FL76), 1:1000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA; STUB1 ; AR-V7 ; FLAG® M2 monoclonal antibody ; Tubulin . Following secondary antibody incubation, immunoreactive proteins were visualized with an enhanced chemiluminescence detection system . The bands were quantified by ImageJ.Whole-cell protein extracts were incubated for 16 h at 45) and grown to 80% confluence and transiently transfected using Lipofectamine 2000 (Invitrogen). pGL3-PSA6.0 luciferase construct was co-transfected with pRL-TK . Briefly, pGL3-PSA6.0 luciferase construct and pRL-TK along with WT-AR, AR variants or HSP70 were mixed and transfected. Cells were then treated with anti-androgens or ARVib-7 or ARVib-31 with or without DHT. The luciferase activity was determined 24 h after treatment using a dual-luciferase reporter assay system (Promega). Cell lysates (35 μL per well) were used for measurement of luciferase activity in a luminometer by first mixing the cell lysates (25 μL) with 20 μL luciferase assay reagent for measuring firefly luciferase activity and subsequently adding 20 μL Stop-Glo reagent for measuring Renilla luciferase activity. Data were normalized to Renilla luciferase activity.C4-2B cells were plated in 12-well plates  was calculated. Cell survival rate (%) = (Treatment group cell number/Control group cell number) ×100%. CWR22Rv1 cells, C4-2B MDVR cells were seeded on 12-well plates at a density of 0.5 × 105 cells/well in RPMI 1640 media containing 10% FBS and treated with 0.25 or 0.5 μM ARVibs with or without 20 μM Enza in media containing FBS. Total cell numbers were determined after 3 days. The coefficient of drug interaction (CDI) [C4-2B MDVR and CWR22Rv1 cells were seeded on 12-well plates at a density of 0.5 × 10on (CDI)  is calcuC4-2B MDVR or CWR22Rv1 cells were treated with different doses of ARVib-7 or ARVib-31 in media containing 10% complete FBS with or without 20 μM Enza. Cells were plated at equal density (1000 cells/dish) in 100 mm dishes for 14 days; the medium was changed every 7 days. The colonies were rinsed with PBS before staining with 0.5% crystal violet/4% formaldehyde for 30 min and the number of colonies was counted.5 cells/well) in RPMI 1640 media containing 10% FBS and treated with DMSO or different doses of ARVib or Nic#31 for 3 days. Mono- and oligonucleosomes in the cytoplasmic fraction were measured by Cell Death Detection ELISA kit  according to the manufacturer’s instructions. Briefly, floating and attached cells were collected and homogenized in 400 µL of incubation buffer. The wells were coated with antihistone antibodies and incubated with the lysates, horseradish peroxidase–conjugated anti-DNA antibodies, and the substrate. Absorbance was measured at 405 nm.C4-2B MDVR cells were seeded on 12-well plates . cDNAs were prepared after digestion with RNase-free RQ1 DNase (Promega). The cDNAs were subjected to real-time reverse transcription-PCR (RT-PCR) using Sso Fast Eva Green Supermix (Bio-Rad) according to the manufacturer’s instructions and as described previously . Each reEqual amounts of cell lysates (1500 µg) were immunoprecipitated using 1 µg of Flag antibody, HSP70 antibody, AR-V7 antibody, AR (N20) antibody or STUB1 antibody with 50 µL of protein A/G agarose with constant rotation 16 h. The immunoprecipitants were washed with 10 mM HEPES (pH 7.9), 1 mM EDTA, 150 mM NaCl, and 1% Nonidet P-40 twice with 1 mL each. The precipitated proteins were eluted with 30 µL of SDS-PAGE sample buffer by boiling for 10 min. The eluted proteins were electrophoresed on 8% SDS-PAGE, transferred to nitrocellulose membranes, and probed with indicated antibodies.4 293 cells were plated in four-well Nunc™ Lab-Tek™ II Chamber Slides and transfected with AR-V7, HSP70 and Flag-STUB1 for 3 days and then treated with ARVib-7 or ARVib-31 with 5 µM MG132 for 16 h. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and incubated with 1% BSA to block nonspecific binding. Cells were incubated with anti-AR  and anti-Flag antibodies (Sigma) overnight. Intracellular AR-V7 was visualized with FITC-conjugated secondary antibodies, Flag-STUB1 was visualized with Texas red conjugated secondary antibodies and nuclei were visualized with DAPI by All-in-One Fluorescence Microscope (BZ-X700).1 × 106 reads per sample). Data analysis was performed with a HISAT-StringTie-Cuffnorm pipeline for mapping/alignment of raw sequence reads (FASTQ format) to the reference human genome assembly (GRCh38/hg38), transcript assembly, and quantitation of gene and transcript expression as FPKM (Fragments Per Kilobase of transcript per Million mapped reads). Data were annotated for 60,658 unique genes/transcripts with GENCODE, Release 26 (GRCh38.p10). Principal component analysis (PCA) was conducted on the FPKM gene-level data for all genes/transcripts passing filter (Filtered on Expression > 0.1) in the raw data (GEO accession number: GSE158556). The relatedness of the differentially expressed genes from treatment with different ARVibs was depicted with a Venn diagram. Expression patterns of genes commonly regulated by ARVib treatment were examined by hierarchical clustering analysis with StrandNGS software (Strand Life Sciences).C4-2B MDVR cells were treated with vehicle, or 1.5 μM ARVib-7, or ARVib-31 for 24 h before RNA extraction. Indexed RNA-seq libraries were prepared from total RNA (1 μg) using the KAPA Stranded mRNA-Seq Library Kit (Kapa Biosystems), according to the manufacturer’s instructions. Subsequently, libraries were combined for multiplex sequencing on an Illumina HiSeq 4000 System  [GSEA was performed using the Java desktop software (dex.jsp) . Genes w2O with 0.1% (v/v) of formic acid  was used as mobile phase A (A) and ACN with 0.1% of formic acid was used as mobile phase B (B). The following UPLC gradient program was used for the separation: 0–1.5 min, 10% B; 1.51–3.5 min, 95% B; 3.51–5 min, 10% B. The output of the UPLC was fed to a Waters Xevo TQ-S triple quadrupole MS/MS system, which was used to ionize target molecules with the ESI- probe and monitor the ion m/z fragmentation transitions from 325 → 171 for niclosamide quantification, 348 → 194 for ARVib-7 quantification, and 331 → 177 for 13C6-niclosamide quantification at multiple reaction monitoring (MRM) mode. The calibration curve was fitted with weighted (1/x2) least-squares linear regression algorithm. The extraction yield was 58.29 ± 9.45% for niclosamide and 84.45 ± 15.24% for ARVib-7. The matrix effect enhanced the niclosamide MS signal by 25.16% and inhibited ARVib-7 MS signal by 3.8%. Both inter- and intra-batch accuracy of all levels of QC samples were within 0 to 11% (%deviation) and both intra- and inter-batch precision of all levels of QC samples were also within 0 to 11% (%CV).Blood samples (around 50 µl) from SD rats were collected from the tail veins punctured by a lancet at 0, 15 mins, 30 mins, 60 mins, 2 h, 4 h, 8 h, and 24 h. To quantify niclosamide, ARVib-7 and ARVib-31 concentrations in the plasma samples, rat plasma standards at 0, 0.5, 1, 5, 10, 50, 100, 500, and 1000 ng/ml for each drug were made for establishing a rat plasma calibration curve. All calibrators were prepared in blank rat plasma with lithium heparin as the anticoagulant . ARVib-31 plasma calibration curve could not be established as this was converted to niclosamide in the blank rat plasma immediately after spiking. Hundred microliters of the resulting rat plasma standard calibrators and rat plasma samples were loaded into each well of a 96-well Isolute PLD+ protein and phospholipid removal plate . One hundred eighty microliters of the internal standard (IS), 13C6-niclosamide , 100 ng/ml in acetonitrile , was then added to each well. Five microliters of the filtrate was injected into a Waters  Acquity UPLC with a BEH C18 1.7 µm, 2.1 mm × 50 mm column. To run the UPLC, HAll animals used in this study received humane care in compliance with applicable regulations, policies, and guidelines relating to animals. All experimental procedures using animals were approved by the Institutional Animal Care and Use Committee of UC Davis. CWR22Rv1 cells (3 million) were mixed with matrigel (1:1) and injected subcutaneously into the flanks of 4-5 week old male C.B17/lcrHsd-Prkdc-SCID mice (ENVIGO). Tumor-bearing mice (tumor volume around 50–100 mm3) were randomized into six groups (7 mice per group) and treated as follows: (1) vehicle control , intraperitoneal (i.p.)), (2) niclosamide (25 mg per Kg i.p.), (3) ARVib . Tumors were measured using calipers twice a week and tumor volumes were calculated using length × width × width × 0.52. Tumor tissues, liver, and kidney were harvested and weighed after 3 weeks of treatment. Tumor tissues, liver and Kidney were paraffin embedded and H/E stained.3 pieces of LuCaP 35CR tumor were implanted into the precastrated SCID mice. When tumors reached 50–100 mm3, mice were randomized into four groups (six mice per group) and treated as follows: (1) vehicle control (0.5% weight/volume (w/v) Methocel A4M p.o.), (2) niclosamide , (3) ARVib . Tumors were measured using calipers twice a week and tumor volumes were calculated using length × width × width × 0.52. Tumor tissues were harvested and weighed after 5 weeks of treatment. Serum was collected for PSA determination.To assess the effect of oral ARVib administration on the growth of PDX tumors, the LuCaP35 CR model was obtained from the University of Washington and established in the UC Davis Cancer Center. Briefly, 3-4 weeks C.B17/lcrHsd-Prkdc-SCID mice (ENVIGO) were surgically castrated. Two weeks later, ∼20- to 30-mm6) were mixed with matrigel (1:1) and injected subcutaneously into the flanks of 3–4 week male SCID mice. After PSA levels are detectable (5–10 ng/mL), mice were castrated and the tumor volume was monitored, after the tumor grew back to 50–100 mm3, tumor-bearing mice were randomized into three groups (five mice in each group) and treated as follows: (1) vehicle control (0.5% weight/volume (w/v) Methocel A4M p.o.), (2) Enzalutamide (20 mg/Kg p.o.), (3) ARVib , (4) Combination. Tumors were measured using calipers twice a week and tumor volumes were calculated using length × width × width × 0.52. Tumor tissues were harvested after 3 weeks of treatment.To assess the combination effects of ARVib and Enza in vivo, VCaP cells  according to the manufacturer’s instructions.Tumors were fixed by formalin and paraffin embedded tissue blocks were dewaxed, rehydrated, and blocked for endogenous peroxidase activity. Slides were incubated with anti-HSP70 , anti-Ki67 , or anti-AR-V7  at 4 °C overnight. Slides were then washed and incubated with biotin-conjugated secondary antibodies for 30 min, followed by incubation with avidin DH-biotinylated horseradish peroxidase complex for 30 min . The sections were developed with the diaminobenzidine substrate kit (Vector Laboratories) and counterstained with hematoxylin. Nuclear staining of cells was scored and counted in 5 different vision fields. Images were taken with an Olympus BX51 microscope equipped with DP72 camera.t-tests for single comparisons or one-way analysis of variance (ANOVA) followed by the Scheffé procedure for multiple group comparisons. In the tumor growth experiments, size of the tumor at sacrifice serves as the primary response measure. Tumor growth and PSA across groups was analyzed by ANOVA. p < 0.05 was considered statistically significant.Statistical analyses were performed with SPSS16.0. Raw data are summarized by means, standard deviations (SD), and graphical summaries and transformed if necessary to achieve normality. Data from the in vitro experiments are presented as means ± SD from three independent experiments. Differences between individual groups were analyzed by two-tailed Student’s supplementary figures and tables