Rehabilitation is essential for disabled people to achieve the highest level of functional independence, reducing or preventing impairments. Nonetheless, this process can be long and expensive. This fact together with the ageing phenomenon has become a critical issue for both clinicians and patients. In this sense, technological solutions may be beneficial since they reduce the costs and increase the number of patients per caregiver, which makes them more accessible. In addition, they provide access to rehabilitation services for those facing physical, financial, and/or attitudinal barriers. This paper presents the state of the art of the assistive rehabilitation technologies for different recovery methods starting from in-person sessions to complementary at-home activities. According to the World Health Organization (WHO), about 15% of the world's population suffers some form of disability. Due to the ageing phenomenon and the prevalence of chronic diseases such as epilepsy, cancer, or mental health disorders, this percentage has incessantly increased. This fact leads to a growing demand for rehabilitation services since they play an important role in enhancing functioning, reinforcing the person's autonomy, and improving the patient's quality of life . This dePhysical, to regain strength, mobility, and fitnessOccupational, to relearn the person's daily activitiesSpeech-language, to recover communication skills In this context, the key to technology success depends on its functionality and adaptability to the user's needs and environment. However, rehabilitation is a broad concept covering a wide range of responses to disability. Generally speaking, rehabilitation can be defined as the step-by-step process designed to reduce disability and to optimise functioning in individuals with health conditions, enabling them to better interact with their environment. For that, rehabilitation commonly includes three aspects:The duration of the rehabilitation can vary depending on several factors such as the patient's impairment level, the therapy intensity, or the individual activity and participation. For that reason, new ways without compromising patient wellbeing have been proposed. So, three different modalities can be found in the literature: (1) the in-person rehabilitation, where patients performs their program in presence of a therapist in an inpatient facility; (2) the combined in-person and at-home rehabilitation, where in-person rehabilitation takes place in an outpatient facility and is aided with at-home programs such that patients perform some therapeutic exercises prescribed by the clinician at home; and (3) the at-home rehabilitation, suitable for those requiring minor assistance or support, where a tailored therapy takes place entirely at home.Focusing on the individual's functioning, the technological solutions developed up to date have mainly aimed to physical recovery since mobility plays a main role in the independence and confidence of disabled people. More recently, research in occupational rehabilitation has emerged in response to Alzheimer's disease and neurocognitive impairments.This paper addresses the state-of-the-art assistive technologies for rehabilitation from the hospital to in-home programs. Despite its great importance in disabled people recovery, devices designed to replace the impaired limb  and video game can fill the gap. That is, computer-based programs designed to simulate real-life objects and events in an attractive environment may engage patients to stage on track. In fact, the use of this kind of systems has been shown to be an effective mean for rehabilitation treatments since they offer clinicians the ability to control and grade tasks to challenge the user while providing them with an enriched environment to achieve high user's engagement , 22.From this starting point, a treadmill can be combined with VR technology. This is the case of C-Mill , a treadAnother example is Lokomat , an exosGoing a step further, GestureTek developed IREX (Immersive Rehabilitation EXercise) , a virtuSimilarly, SeeMe  was deveA proper recovery is composed of two essential components: an in-clinic supervised learning and a recommended home exercise routine. In this way, the home exercise program may increase therapeutic gains. Note that this exercise routine must be designed to be practical, accessible, feasible, and easy to complete to maximise the patient's efforts without instruction. With that purpose, some technological solutions are designed to provide patients with assistance in both in-person and at-home rehabilitation. So, an in-clinic supervised learning facilitates the at-home therapy, which continuously reports the patient's progress to their care specialist.One of the early approximations was the incorporation of Nintendo Wii in the rehabilitation process , 33. DesStandardised assessments: this functionality offers an accurate measurement of user's movements based on its motion library with parameterised measurements and its implementation of body compensation to reduce assessment errorsExercise platform: this functionality shows a virtual gym where a monitor is performing the exercise indicated at the bottom of the screen together with a digital user's representation who is continuously getting visual feedback about their performanceActivities: this functionality includes a wide range of virtual reality environments representing real-life scenarios to challenge the user to work on their motor, balance and mental skills.With that aim, three different functionalities have been implemented :StandardThese functionalities become JRS into a complementary tool to in-person therapy as well as a virtual therapist for at-home rehabilitation. In both cases, a therapist subscribes a set of exercises and activities (graded according to the patient's level) to stimulate their recovery. The remote patient's monitoring is possible thanks to the JRS register that provides care specialists with a detailed patient's progress in terms of session durations, movement accuracy, and progress with respect to previous performances and reaction time. In this way, the therapist can view and evaluate the patient's progress and adapt their activity program to their needs.Thinking of movement learning at neural level led to IKKOS , a VR soWith the purpose of diagnosing disease at an early stage and encouraging people to a healthy activity level, the PAMAP  assistive system was developed . So, a bThe need of combined therapies led Gomez-Donoso et al.  to develGoing a step further, autonomous rehabilitation systems could encourage and monitor the rehabilitation at home always under the remote therapist's supervision. Thus, these kinds of systems are appropriate for keeping improvement when specialist visits end, complementing in-person sessions, and/or avoiding problematic patient's displacement to healthcare facilities.Under the assumption that repetition is the key to a successful stroke recovery, the home-therapy tool FitMi was proposed . This soOn their behalf, Saebo Inc. proposed SaeboVR , a nonimExercise and staying active are an important part of rehabilitation for chronic diseases , 41. In Costa et al. proposed PHAROS , an inteAlmost a sixth of the world's population suffers some type of disability. This fact has led to an unceasing demand for rehabilitation services, impossible to satisfy with the limited healthcare professionals. In this context, technology can be a solution.Starting from the labor-intensive rehabilitation processes, the early rehabilitation technology was focused on robotic devices as a tool for repetitive movement training. In addition, these devices allow clinicians to quantitatively measure the patient's performance and progress. Nonetheless, the monotony of rehabilitation sessions resulted in a loss of patient's interest and, consequently, in a poor improvement. As a consequence, new mechanisms like virtual reality or video games were integrated to make the rehabilitation exercises fun, challenging, and engaging.From the rehabilitation's point of view, it is important to take intensive and continuous therapeutic exercise for a successful patient's recovery. This requirement can be satisfied when therapy is not only taken place in hospitals but also at home. Along this line, research responded with hybrid systems. So, the in-person sessions allow the patients to be familiar with the tool and set configuration parameters when necessary, while the at-home sessions provide the patients with the tailored exercise program set by the specialist. In this last case, the patients usually get immediate feedback on the performed exercises and, at the same time, these data are remotely available to the clinicians at any time. Going a step further, autonomous rehabilitation systems are being developed to prevent impairments, specially in the case of elderly, and to in reach for those with travelling difficulties.It is noteworthy that despite the adaptation to the patient's needs, most of the existing literature is focused on physical therapy. However, the ageing phenomenon and some diseases like Alzheimer are demanding new assistive solutions for occupational and speech-language therapies. Although some efforts have been done in this sense, there is still a long way to go.


However, it is poorly understood the mechanism(s) to control differentiation of Mo-MDSCs and PMN-MDSCs. Here, we found that lnc-C/EBPβ may promote PMN-MDSC but impede differentiation of Mo-MDSCs in vitro and in vivo. We demonstrated that lnc-C/EBPβ mediated differentiation of MDSCs was through downregulating multiple transcripts such as IL4il. Lnc-C/EBPβ not only bound to C/EBPβ isoform LIP to inhibit the activation of C/EBPβ but also interacted with WDR5 to interrupt the enrichment of H3K4me3 mark on the promoter region of IL4i1. Data also imply that conserved homo lnc-C/EBPβ has a similar function with mouse lnc-C/EBPβ. Since MDSC subsets exert different suppressive function, lnc-C/EBPβ may be acted as a potential therapeutic target for inflammatory and tumor-associated diseases.Myeloid-derived suppressor cells (MDSCs), which play an important role in tumor and inflammatory diseases, are divided into two subsets CD11b The Gr1+ subsets may be more accurately identified based on Ly6C and Ly6G markers as CD11b+Ly6ChiLy6G− monocytic MDSC (Mo-MDSC) and CD11b+Ly6Clow/neg(negative)Ly6G+ polymorphonuclear MDSC (PMN-MDSC) (+) MDSCs and PMN (CD15+) MDSCs, are described as lineage negative cells that co-express CD11b and CD33 but lack HLA-DR (Myeloid-derived suppressor cells (MDSCs) have emerged as a major regulator of immune responses in cancer and other pathological conditions –4. In miMN-MDSC) , 6. Humak HLA-DR . These Mk HLA-DR . Furtherk HLA-DR . Thus, iin vitro induce development of MDSCs. Transcription factors such as CCAAT/enhancer binding protein β (C/EBPβ), phosphorylated signal transducer of activator of transcription 3 (phospho-STAT3), and C/EBP homologous protein (CHOP) centrally regulate MDSC expansion . However, their development is governed by a complex network of signals including promoting accumulation of immature myeloid cells and providing for the pathological activation of these cells , 8. Inflxpansion , 9, 10. xpansion . They maxpansion . Multiplic cells , lincRNArophages –16, lncRid cells . Howeverlnc-C/EBPβ, which was identified by us  and maintained in a specific pathogen-free (SPF) facility. B6.SJL-CD45a (Ly5a) (CD45.1) mice were purchased from the Model Animal Research Center of Nanjing University . All animal experiments were carried out in accordance with Nankai University Guide for the Care and Use of Laboratory Animals. The peripheral blood and tissues samples from the patients and healthy human were obtained after signing informed consent at People Union Hospital . The collection and use of all human samples were approved by the Institute's Human Ethics Committee of Nankai University and in accordance with the Declaration of Helsinki.Murine melanoma B16, breast cancer 4T1, colon cancer CT26, and human embryonic kidney cell line HEK 293T cells were obtained from the American Type Culture Collection . Murine ovarian tumor cell line 1D8 was from Dr. Richard Roden .Recombinant murine GM-CSF and IL-6 were purchased from PeproTech . Mouse anti-C/EBPβ, anti-IL4i1, and anti-H3K4me3 were from Abcam . Rabbit anti-WDR5 was from Cell Signaling Technology . Mouse anti-V5 was purchased from Thermo Fisher Scientific . Anti-Gr1-PE, anti-CD11b-PerCP/Cy5.5, anti-Ly6G-PE, anti-Ly6C-FITC, anti-F4/80-FITC, anti-CD3-PerCP/Cy5.5, anti–CD45-APC, anti–CD45.1-Cy7, anti-CD33-PE, anti-HLA-DR-APC, anti-CD14-FITC, anti-CD15-PE were from BD Biosciences .IL4i1and WDR5 were listed in lnc-C/EBPβ lentivectors together with pMD2G and psPAX2 packaging plasmids were co-transfected into 293T cells. The full-length sequences of C/EBPβ LAP/LIP, IL4i1, and WDR5 were amplified using PCR . siRNA sequences for lnc-C/EBPβ lentivectors (50 MOI) or control empty lentiviruses (50 MOI) in the presence of 8 μg/ml polybrene  by centrifugation and then cultured with complete medium for 24 h. For transfection, the cells were transfected with WDR5 siRNA (100 nM), IL4il siRNA (100 nM), negative control siRNA (100 nM) or pcDNA3.1/WDR5 (4 μg/ml), pcDNA3.1/IL4il (4 μg/ml), and empty pcDNA3.1 control (4 μg/ml) using Lipofectamine™ 3000  or HiPerFect transfection reagent (siRNA transfection)  according to the manufacturer's instructions. The cells were then washed and cultured under GM-CSF or GM-CSF plus IL-6 or tumor supernatants for 4 days.BMCs were collected from C57BL/6 mice and cultured in six-well plate. Human monocytes were collected and isolated from peripheral blood. For transduction, the cells were infected with shRNA lentivectors [50 multiplicity of infection (MOI)] or +Gr1+ MDSCs in vitro, 5 × 104 1D8, 4T1, CT26, or B16 tumor cells (upper chamber) were co-cultured with 2 × 106 BMCs (lower chamber) in a 24-transwell plate in the presence of GM-CSF (40 ng/ml) for 4 days. Human MDSC-like cells were generated according to previous reported methods (Inflammatory factors associated MDSCs were generated by culturing bone marrow cells of C57BL/6 mice in flask or 6-well plate for 4 days in the presence of 5% FBS medium containing GM-CSF (40 ng/ml) only or GM-CSF (40 ng/ml) plus IL-6 (40 ng/ml) . To prep methods .lnc-C/EBPβ knockdown MDSC was performed using Affymetrix GeneChip mouse Genome 430 2.0 array by Beijing Capitalbio Technology Co., Ltd. Briefly, total RNA was extracted using Trizol (Life Technologies). Contaminating DNAs were removed using RNeasy spin columns (Qiagen). The quality of isolated RNA samples was evaluated with an Agilent Bioanalyzer 2100 (Agilent technologies) and the purified RNA was quantified using a NanoDrop ND-2000 spectrophotometer (Infinigen Biotechnology Inc.). Microarray of coding mRNA in lnc-C/EBPβ knockdown MDSC was performed using Affymetrix GeneChip mouse Genome 430 2.0 array. The R software (v.2.13.0) platform was applied to analyze the microarray data, and the limma (linear regression model) package was used to statistically analyze differentially expressed genes. The expression levels of mRNAs at each time point were compared with control. Genes having a fold change > 2 or < −2 and an adjusted p < 0.05 were considered as differentially expressed. Microarray GEO accession number GSE104571.Microarray of coding mRNA in Cells were collected and rinsed twice with ice cold PBS, incubated with FITC-, PE-, percy5.5-, or APC-labeled antibodies for 30 min in PBS with 1% FBS according to our previous method . After wTotal RNA was extracted from the cells, tissues, and organs using TRIzol reagent (Invitrogen). First-strand cDNA was generated from total RNA using oligo-dT/random primer mix and reverse transcriptase (Invitrogen Corp). Quantitative real-time PCR (qRT-PCR) was conducted using QuantiTect SYBR Green PCR Master Mix (Qiagen) and specific primers in an ABI Prism 7000 analyzer (Applied Biosystems). GAPDH mRNA expression was detected in each experimental sample as an endogenous control. The fold changes were calculated using the ΔΔCt method according to the manufacturer's instructions (Applied Biosystems). All the reactions were run in triplicate.Cells were harvested at the indicated times and rinsed twice with ice cold PBS. Cell extracts were prepared with lysis buffer and centrifuged at 13,000 g for 10 min at 4°C. Protein samples were electrophoresed using 12% polyacrylamide gels and transferred to PVDF membranes. After the membranes were blocked with 5% skim milk powder for 1 h at room temperature, they were incubated with first antibody in TBST overnight at 4°C. Secondary antibodies with horseradish peroxidase (HRP) (1:5000) were labeled according to our previous method . The sigin situ hybridization (RNA-FISH) were performed according to reported protocol (Immunostaining and RNA Fluorescence protocol . Cells wChromatin immunoprecipitation (CHIP)-PCR was performed using EZ-CHIP™ chromatin immunoprecipitation kit  according to the reported method . MDSCs wRNA immunoprecipitation (RIP) was performed according to previously reported protocol . BrieflyImmunoprecipitation (IP)-MASS was performed according to our previous method . BrieflyLnc-C/EBPβ was transcribed  and labeled using RNA 3′ Desthiobiotinylation Kit (Thermo Scientific Pierce) in vitro. 50 ul beads and 50 pmol of labeled RNA were added into RNA capture buffer, and incubated for 30 min at room temperature with agitation to binding of labeled lnc-C/EBPβ to streptavidin magnetic beads. After washing beads with an equal volume of 20 mM Tris (pH 7.5), 100 μL of 1× protein-RNA binding buffer was added into the beads and mixed well. 100 μL of master mix of RNA-protein binding reaction was added to the RNA-bound beads, mixed by pipetting and then incubated 60 min at 4°C with rotation to binding of RNA-binding proteins to RNA. After washing beads with 100 ul wash buffer for twice, 50 μL of elution buffer was added, and incubated 30 min at 37°C with agitation. The samples were analyzed on a gel.RNA-protein pull-down analyses were performed using Pierce™ magnetic RNA-protein pull-down kit. MDSCs were harvested and cell lysates were prepared using IP lysis buffers  . Lnc-C/Et-test and GraphPad Prism 5 software. A 95% confidence interval was considered significant and defined as p < 0.05. * indicates p < 0.05, **p < 0.01, ***p < 0.001.Statistical analyses were performed using two-tailed student's lnc-C/EBPβ negatively regulates suppressive function of MDSCs    chimera model to investigate the effects of lnc-C/EBPβ on MDSC differentiation in vivo. CD45.1+ BMCs from homogeneous mice were transduced with lnc-C/EBPβ shRNA/lentiviruses and then injected into B16 tumor-bearing WT mice via tail vein at the indicated time  . Consist1b+MDSCs , indicats (BMCs) , suggestrophages . We furted cells , differeed cells . All of ted time . Once mi.1+ BMCs . PMN-MDS.1+ BMCs . CD45.1+ced BMCs , indicat5.1+ BMC . The pro.1+ BMCs . PMN-MDS5.1+ BMC . CD45.1+ced BMCs . Taken tlnc-C/EBPβ associated gene (s), which potentially affect the differentiation of MDSCs, we analyzed gene expression pattern of lnc-C/EBPβ knockdown MDSCs using a microarray (lnc-C/EBPβ knockdown affected multiple gene expression such as interleukin 4-induced gene-1(IL4i1). As shown (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE104571), IL4i1 was remarkably upregulated in lnc-C/EBPβ knockdown MDSCs. The higher levels of expression could be further confirmed by qRT-PCR and immunoblot in these lnc-C/EBPβ knockdown cells  by which findings , lnc-C/Ef C/EBPβ . Genome f C/EBPβ . In the of IL4il . All of C/EBPβ as a bait to investigate whether lnc-C/EBPβ also bind with other molecules, which are related to epigenetic modification. IP-MASS analyses showed that there were remarkable differences not only in the nuclear proteins but also in their signal molecule networks between GM-CSF and GM-CSF plus IL-6 mediated MDSCs . H3K4me3 mark in the 5′ promoter region of these genes was remarkable enrichment  after co-culturing with GM-CSF plus tumor supernatants or IL-6 (lnc-C/EBPβ could modulate the expression of IL4i1 (lnc-C/EBPβ on IL4il in vivo MDSCs, we isolated the MDSCs from patients with colorectal cancer, and examined the relationship between the expression of lnc-C/EBPβ and IL4il. Lin− HLA-DR− CD33+ CD11b+ MDSCs were isolated from the blood of patients with glioblastoma, breast cancer, colon cancer, lung cancer and kidney cancer (+CD14+ Mo-MDSC and Lin−HLA-DR−CD11b+CD15+ PMN-MDSC populations were much higher in patients with tumor as compared to tumor free-individuals (lnc-C/EBPβ could be detected in Lin−HLA-DR− CD11b+ cells isolated from peripheral blood of patients with colorectal cancer as compared to Lin−HLA-DR−CD11b+ cells from tumor free healthy individuals (lnc-C/EBPβ and IL-4il in different MDSC subpopulations (lnc-C/EBPβ in the samples of patients with colorectal cancer. FISH and immuno-staining of colitis/carcinoma tissues demonstrated the expression of lnc-C/EBPβ in CD11b+ monocytes/macrophages (lnc-C/EBPβ in the mononuclear cells of colitis and carcinoma (lnc-C/EBPβ may regulate the expression of MDSCs-associated genes in vitro, a high level of lnc-C/EBPβ may play a role in the differentiation and function of MDSCs in patients with colorectal cancer. Thus, human lnc-C/EBPβ is also involved in the differentiation of human MDSCs.Since human c-C/EBPβ , we exam or IL-6 . Interesof IL4i1 . To examy cancer . Consisty cancer –37, CD11ividuals . Higher ividuals . QRT-PCRulations . Finallyrophages  and nuclarcinoma . Since llnc-C/EBPβ with C/EBPβ and WDR5 affects the activity of C/EBPβ and enrichment of H3K4me3 to promote PMN-MDSCs but inhibit the differentiation of Mo-MDSCs. As lnc-C/EBPβ is present in human monocyte-derived cells, it may be acted as a potential therapeutic target for inflammatory and tumor-associated diseases.Previous data have already shown that transcription factor C/EBPβ plays a central role in regulating the differentiation and immune suppressive function of MDSCs , 38, andlnc-C/EBPβ may bind with C/EBPβ and WDR5 to affect differentiation of MDSCs through regulating expression of IL4il. IL4i1, a regulator of M2 macrophage polarization, may contribute to the regulation of monocyte/macrophage programming (lnc-C/EBPβ with C/EBPβ LIP affects LAP activity, MDSCs may not differentiate into Mo-MDSCs. WDR5 is involved in multiple biological processes and cell differentiation. It may mediate self-renewal and reprogramming of embryonic stem cell (Our data show that gramming , 31, 39.gramming , 40. Itstem cell , and regtem cell , 43. Othtem cell .lnc-C/EBPβ impedes MDSCs to differentiate into Mo-MDSC may have an important significance in the inflammation and tumor. Multiple reports have described the presence of MDSCs in patients with tumors such as colon cancer, lung cancer, breast cancer, pancreatic adenocarcinomas, urothelial carcinoma, kidney Cancer, glioblastoma (lnc-C/EBPβ to control differentiation of MDSCs into Mo-MDSCs.That blastoma , 34. Stublastoma . In the blastoma . These cblastoma , which hblastoma , 38. Lesblastoma . Thus, oThe datasets generated for this study can be found in Gene Expression Omnibus, GSE104571 GSE104558.All animal studies were conducted according to the Institutional Animal Care and Use Committee of the Model Animal Research Center. Animal experiments were approved by the Institute's Animal Ethics Committee of Nankai University.in vivo and in vitro experiments and immunoassay, participated in the study design, and performed the statistical analysis. WS was involved in vitro and in vivo assay. DZ, XZ, and SZ treated patient's samples. YZ offered assistances for the animal experiments. All authors read and approved the final manuscript.RY designed the research and wrote the paper. YG conducted The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


JQ1 exhibited its anti-proliferative activity by inducing apoptosis and cell cycle arrest. JQ1 activity was not mediated by MYC downregulation. Instead, JQ1 blocked the interactions among the ATPase-family AAA-domain-containing 2 protein (ATAD2), BRD2, BRD4, and AR; effectively suppressing the expression of AR associated targets. In addition, JQ1 showed significant anti-tumor activity in vivo in TNBC xenograft mouse models as a monotherapy and in combination with anti-AR therapy. Taken together, our results showed that the BET inhibitor JQ1 is a promising therapeutic agent for the treatment of AR-positive TNBC.Triple-negative breast cancer (TNBC) is a heterogeneous disease comprising several subtypes. Androgen-receptor (AR) signaling has been targeted by several investigational agents in luminal AR subtype TNBCs. Bromodomain (BRD) and extra-terminal motif (BET) protein inhibitors have been shown to attenuate AR signaling in metastatic castration-resistant prostate cancer and to overcome enzalutamide resistance. We demonstrated potent anti-tumor effects of the BET inhibitor JQ1 against AR-positive TNBC cell lines using cell viability and cell cycle analysis. To reveal the mechanisms of JQ1 effects, multiplex gene expression analysis and immunoblotting assays were used. We examined  Cytotoxic chemotherapy is still known as the primary treatment modality for TNBC due to the lack of specific targets, including the estrogen receptor, progesterone receptor, and human epidermal growth factor 2 and its outcomes are poor compared to those of other breast cancer subtypes2. TNBC is a heterogeneous disease, encompassing distinct molecular subtypes5. The luminal androgen-receptor (LAR) subtype, accounting for ~20% of TNBCs, demonstrates increased expression of mRNA encoding the androgen receptor (AR), enhancing AR-signaling sensitive to AR antagonists3. AR expression in TNBC attenuates chemotherapy sensitivity and reduces the rates of complete remission after neoadjuvant treatment7. Several preclinical and early clinical studies suggest that AR is a potential therapeutic target in AR-positive (AR+) TNBC10. In a recent phase II clinical trial, anti-androgen therapy with bicalutamide or enzalutamide demonstrated relatively modest clinical efficacy in a selected group of patients, with a clinical benefit rate of 19 to 29% at 24 weeks12.Triple-negative breast cancer (TNBC) accounts for ~20% of metastatic breast cancer13. Actually, the mechanisms of resistance to AR blockade have been extensively characterized in prostate cancer. Acquired resistance to AR blockade comes from in various ways including AR overexpression, copy number amplification, as well as the expression of splice variants of AR to sustain AR activity14.Considering the complexity of AR signaling and crosstalk in AR + breast cancer, AR antagonists have been investigated in combination with other targeted therapies. Such studies have focused on elucidating the mechanisms of primary and secondary resistance to AR inhibitors and on improving the efficacy of AR inhibitors in AR + breast cancers15. The BET family consists of four members, including BRD-containing protein 2 (BRD2), BRD3, BRD4, and BRD testis-specific protein BRDT16. BET inhibitors exhibit anti-tumor activity in various types of malignancies by inhibiting BRD4 and suppressing transcription of the MYC oncogene19. In addition, BRD4 interacts with the N-terminal domain of AR and induces AR-mediated gene transcription and BET inhibitor, JQ1 effectively disrupts the interaction between BRD4 and AR leading to cytotoxic efficacies in advanced prostate cancer model17. Recently, androgen regulated BRDs such as BRD2 and ATAD2 increased chromatin accessibility with enhancing AR recruitment to chromatin, which may drive cancer progression20. Given that BET family proteins interact with AR, BET inhibitors could be an alternative strategy for targeting AR-driven cancers. Consequently, we assessed the ability of the BET inhibitor JQ1 to enhance the efficacy of AR blockade in AR-expressing TNBCs.Bromodomain (BRD) and extra terminal domain (BET) family proteins control transcriptional activities by modulating protein assembly, and protein-protein interactions, contributing to a variety of cellular events, such as cell cycles, proliferation, and differentiation2 atmosphere. All other cell lines were cultured at 37 °C in free gas exchange with atmospheric air.Breast cancer cell lines, purchased from ATCC, were cultured according to supplier protocols. MDA-MB-231 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. MDA-MB-453 and MDA-MB-468 cells were cultured in Leibovitz’s L-15 medium. BT-20 cells were cultured in Eagle’s Minimum Essential Medium. All cultures were supplemented with 1% antibiotics  and 10% fetal bovine serum (GIBCO BRL). MDA-MB-231 and BT-20 cells were cultured at 37 °C in a 5% COFor cell viability measurement, the breast cancer cells were treated with 0.1–50 μM (+)-JQ1 and enzalutamide  diluted in dimethyl sulfoxide (DMSO). Cells were seeded in 96-well culture plates 24 hours prior to drug treatment, followed by exposure to indicated concentrations of compounds for 0–72 hours. 10 nM 5α-Dihydrotestosterone  was added to charcoal-stripped serum media  to evaluate drug effects on DHT-mediated proliferation of breast cancer cell lines. Cell viability was measured using the Cell Counting Kit-8 . For cell cycle analysis, drug-treated cells were stained with propidium iodide for quantitative analysis of DNA content. Analysis of the stained cells was performed using a BD LSRFortessa™ flow cytometer . Data was analyzed using FlowJo 10 Software .2 for 24–72 hours. After 6 hours, the complex-containing medium was removed and replaced with fresh medium. Transfection efficiency was determined using relative RNA and protein levels of each siRNA target in the transfected cells.AR signaling in MDA-MB-231 and MDA-MB-453 cells was inhibited using siRNA silencing of genes encoding AR , BRD2 , BRD4 , ATPase family, AAA domain containing 2 , and MYC . Cells were transfected with 10 pmol of indicated siRNA or of negative control siRNA using Lipofectamine® RNAiMAX (Invitrogen) according to manufacturer’s protocols. Briefly, cells were treated with the siRNA complex and incubated at 37 °C and 5% CO6 cells were seeded in 10-cm dishes in medium supplemented with 5% charcoal-stripped serum  and incubated for 72 hours. Then, cells were pretreated with or without 30 uM enzalutamide for 3 hours followed by co-treatement with DHT for 3 hours. After that, cells were washed with PBS and cellular fractionation was performed using the NE-PER Nuclear and Cytoplasmic Extraction Kit  as per manufacturer’s instructions. Protein of the isolated nucleus and cytoplasm was separated on SDS-PAGE and transferred to PVDF membrane. Immunoblot was performed using rabbit-anti AR, TOPO-I, a-tubulin and HRP-conjugated secondary antibodies.A total of 1 × 10Total RNA was extracted from the breast cancer cells using the RNeasy Mini Kit . Genomic DNA was removed using DNase I . Synthesis of cDNA was performed using 500 ng of total RNA and the SuperScript ® III First-Strand Synthesis System  at 50 °C for 50 min. Quantitative (qPCR) was performed using a LightCycler® 96 Real-Time PCR System . Relative mRNA expression was quantified using the LightCycler® 96 software (Roche Diagnostics) and normalized to ß-actin transcript levels. The following qPCR conditions were used: 45 cycles of 95 °C for 15 sec and 60 °C for 60 sec.To identify cell signaling pathways affected by JQ1, multiplex gene expression analysis was performed using the nCounter® PanCancer Pathways Panel. Following JQ1 treatment, RNA was extracted from MDA-MB-231 and MDA-MB-453 cells as described above. The extracted RNA was subjected to an nCounter® PanCancer Pathways Panel  according to the manufacturer’s protocol. Briefly, capture probes, reporter probes, and total RNA were mixed with hybridization buffer and hybridized at 65 °C overnight. Then, the samples were processed on the nCounter Prep Station with wash reagents and an imaging cartridge. Hybridization occurred over 20 hours and was monitored in selected experiments.Mouse IgG against AR, BRD2, BRD4, ATAD2, and MYC  were used for immunoprecipitation assays, and rabbit IgG against AR, BRD2, BRD4, ATAD2, MYC, poly (ADP-ribose) polymerase (PARP), cappase-9, caspase-3, and β-actin  were used for immunoblotting. All antibodies were used at dilutions suggested by the manufacturers. For Western blot analysis, 50 μg of total protein extract was separated by SDS-PAGE and transferred to aNovex™ polyvinylidene fluoride (PVDF) membrane  using an iBlot2™ Gel Transfer Device . The blots were incubated overnight at 4 °C with primary antibodies diluted 1:500 or 1:1,000, and binding was detected using horseradish peroxidase (HRP)-conjugated secondary antibodies . Binding was detected using X-ray film via enhanced chemiluminescence with an Amersham ECL Western Blotting Detection Reagent .For co-immunoprecipitation experiments, protein extracts (0.1 mg) were incubated overnight at 4 °C with antibodies against AR, BRD2, BRD4, ATAD2, or MYC or with mouse IgG antibody. The antibodies and bound proteins were purified using Protein A/G Sepharose beads . The protein complexes were washed 3 times at 4 °C with lysis buffer and subsequently subjected to SDS-PAGE under reducing conditions and immunoblotting as described above.7 cells. Tumor cells resuspended in a Matrigel® matrix  were implanted subcutaneously on the dorsal hind flank of 6–7 week-old BALB/c nude mice. Tumor size was measured every 3 days using an in vivo imaging system or a caliper, and drug treatment was initiated when tumors reached 100 mm3 in volume. The mice were divided into the following randomized groups: JQ1 treatment group , enzalutamide treatment group , combination group , and vehicle group . Mice were administered JQ1 (5% DMSO in 5% dextrose) and/or enzalutamide  daily via intraperitoneal injection and oral gavage for 3 weeks. This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the National Cancer Center Research Institute, an AAALAC International-accredited facility that abides by the Institute of Laboratory Animal Resources guidelines.Female BALB/c nude mice, aged 5–6 weeks, were purchased from ORIENT BIO Inc. MDA-MB-231 cells were collected, washed with phosphate-buffered saline (PBS), and re-suspended in serum-free medium at a concentration of 1 × 10TM), according to the manufacturer’s protocol. The staining was visualized using a Zeiss LSM780 confocal Microscope System .Indicated TNBC cells were stained using an anti-AR antibody  and 4′,6-diamidino-2-phenylindole . The cells were washed with PBS, fixed for 5 min with 4% paraformaldehyde at room temperature, washed again with PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and then blocked with 3% normal goat serum in PBS for 10 min. Following overnight incubation at 4 °C with the primary antibody diluted 1:200, the cells were incubated for 60 min with rabbit-Alexa Fluor 488 conjugated secondary antibody  of the National Cancer Center Research Institute, an AAALAC International-accredited facility that abides by the Institute of Laboratory Animal Resources guidelines.To explore the correlation between enzalutamide sensitivity and AR expression, we evaluated AR transcript and protein abundance in selected TNBC cell lines . Despite confirmed AR suppression, the si-AR produced a cytotoxic effect comparable to that elicited by the enzalutamide treatment alone and did not significantly alter MYC and ATAD2 levels relative to the control siRNA  for mouse experiments was selected by referring to the previous papers25. JQ1 treatment exhibited robust anti-tumor activity  had chromatin accessibility which may drive cancer progression and AR/androgen -regulated BRDs including ATAD2 mediated this effect20. They also suggested that ATAD2 and BRD2 overexpression have poor prognostic value. In line with their results in CRPC, our findings indicate that the BET inhibitor JQ1 is more efficacious than direct AR blocade in AR + TNBC cell lines and in xenograft models. These results suggest that clinical evaluation of BET inhibition, either as a monotherapy or in combination with anti-AR agents, is warranted in the exploration of novel approaches for targeting AR + TNBCs.In this study, expression of MYC target of JQ1 showed no clear association with JQ1 sensitivity, which was in line with previous studyThe BET inhibitor JQ1 enhances cytotoxic efficacy of therapeutic interventions against of AR + TNBCs. Anti-proliferative effects of JQ1 are likely driven by the disruption of ATAD2 interactions with BRD2, BRD4, and AR which leads to down-regulate AR associated genes. Clinical trials are needed to establish the efficacy and utility of using BET inhibition in combination with anti-AR therapy for the treatment of AR + TNBC.supplementary figures


Xenograft modeling demonstrates that AR+ CRPC is enzalutamide-sensitive but AR−/lo CRPC is resistant. Genome editing-derived AR+ and AR-knockout LNCaP cell clones exhibit distinct biological and tumorigenic properties and contrasting responses to enzalutamide. RNA-Seq and biochemical analyses, coupled with experimental combinatorial therapy, identify BCL-2 as a critical therapeutic target and provide proof-of-concept therapeutic regimens for both AR+/hi and AR−/lo CRPC. Our study links AR expression heterogeneity to distinct castration/enzalutamide responses and has important implications in understanding the cellular basis of prostate tumor responses to AR-targeting therapies and in facilitating development of novel therapeutics to target AR−/lo PCa cells/clones.Expression of androgen receptor (AR) in prostate cancer (PCa) is heterogeneous but the functional significance of AR heterogeneity remains unclear. Screening ~200 castration-resistant PCa (CRPC) cores and whole-mount sections (from 89 patients) reveals 3 AR expression patterns: nuclear (nuc-AR), mixed nuclear/cytoplasmic (nuc/cyto-AR), and low/no expression  expression in prostate cancer is unknown. Here the authors show AR expression heterogeneity is associated with distinct castration/enzalutamide responses and identify BCL-2 as a potential therapeutic target in castration-resistant prostate cancer. Most PCa patients are first treated by radical prostatectomy and/or radiation therapy. When post treatment serum PSA (prostate-specific antigen) levels rise, the patient is treated by first-line androgen deprivation therapy (ADT) using GnRH analogs, which suppress gonadal production of testosterone (T), and PCa cells at this stage are castration sensitive , a steroid hormone receptor normally activated by androgens, plays an essential role in prostate cancer (PCa) development, progression, and therapy response+), as well as AR low-expressing or non-expressing (AR−/lo) cells and this AR heterogeneity is accentuated in advanced metastatic and relapsed PCa14. Whether the heterogeneity in AR expression levels impacts PCa biology and therapy response remains unclear. This project is undertaken to address this important question and to fill a critical gap in our knowledge. Through extensive xenograft modeling, development of AR-tagged (AR+) and AR-knockout (KO) LNCaP cell clones, and performing in vitro biological and in vivo tumor regeneration assays, RNA-Seq, and multiple combinatorial therapeutic experiments, we link the AR expression status to distinct tumorigenic behavior and castration/Enza responses. Critically, our studies uncover signaling molecules and pathways underlying the development of, and also establish proof-of-principle therapeutic regimens targeting, the two distinct castration resistance modes mediated by AR+/hi and AR−/lo PCa cells, respectively.Both chemical castration (using ADT and abiraterone) and antiandrogens  target AR signaling. However, human PCa is heterogeneous containing both AR-expressing ; (2) both nuclear and cytoplasmic AR , and (3) lack of appreciable AR expression . The remaining 17 cores (9%) contain both AR+ and AR−/lo cells  that contain 195 CRPC cores derived from 81 patient CRPC samples Fig. , most oflls Fig. . Similarlls Fig.  whereas + PCa xenograft models, i.e., LAPC9, LAPC4, LNCaP, and VCaP17. Interestingly, when these 4 models transition from androgen-dependent  to a castration-resistant  state during serial propagation in castrated mice  CRPC  signaling whereas the LAPC9 1° CRPC becomes AR/lo−and shows a prominent upregulation of CSC molecules.Characterization of multiple proteins involved in AR signaling, cancer stem cells (CSC), and castration resistance in 1° CRPC reveal changes unique to each model Fig. . In LAPCBP5 Fig. , and no BP5 Fig. . In contBP5 Fig. . In the BP5 Fig. . The LAPBP5 Fig. . VCaP 1°-V7 Fig. . N-Cadhe-V7 Fig. . pERK1/2+/hi LNCaP model, 1° CRPC is sensitive to Enza for up to ~7 weeks after which Enza-resistant tumors emerge . In stark contrast, the AR−/lo LAPC9 1° CRPC  and AR knockout (AR-KO) LNCaP clonal cells to compare their intrinsic biological and tumorigenic properties, as well as responses to castration and Enza. Briefly, we adopt the zinc-finger nuclease mediated integration to insert an RFP reporter into the endogenous AR locus and have generated AR+ (AR-RFP+) LNCaP clones  treatment. Lower baseline , as well as DHT-stimulated reporter activities are observed in the AR-KO cells compared to AR+ cells  than AR-KO clones in intact NOD/SCID . AR+ cell-derived tumors all retain an AR+ phenotype  may preferentially suppress the clonogenic and tumorigenic capacities of AR-KO cells.Comparing the intrinsic biological properties, as well as tumor-regenerating potential in ARCID Fig.  and NOD/CID Fig.  mice. Whype Fig. . These s+ cells when implanted in castrated mice . Importantly, in Enza medium, 53% (107/201) of single AR+ LNCaP cells stay quiescent with no cell division  compared to 35% (85/244) of AR-KO cells in the same media . Also, AR+ LNCaP cells show a high propensity to undergo death at the first cell division in the presence of Enza . These results again suggest that androgens may exert inhibitory effects on AR-KO cells.Of interest, when we compare the cell division profiles of AR-KO LNCaP cells in DHT vs. Enza, 28% cells undergo cell-cycle progression in Enza-containing media, which is significantly higher than 11% of the AR-KO cells in the presence of DHT  and the results reveal a prominent competitive advantage for AR+ LNCaP cells in DHT and for AR-KO cells in Enza-containing media, respectively  tumor cells represent the majority in the tumors  in RNA-Seq 27, either alone or with Enza. Remarkably, although ABT-199 alone does not inhibit the growth of 1° LNCaP CRPC  and 6 CSC genes  in RNA-Seq  upregulation in CRPC whereas ALDH7A1 is decreased  and “Neurogenesis”  pathways , and c-MYC by therapeutically targeting them alone or in combination. The BCL-2 inhibitor ABT-199 alone significantly inhibits LAPC9 CRPC growth  and organoid expansion 31. As shown in Fig. Consistent with our earlier biochemical characterizations Fig. , LAPC9 Cion Fig. , g. Enzaion Fig. , g. On tice Fig. . Finally−/lo LAPC9-type of CRPC cells/clones.Collectively, these xenograft-based therapeutic experiments targeting 3 CSC signaling molecules, i.e., BCL-2, integrin α2β1, and c-MYC, offer novel proof-of-concept strategies for treating the AR14. In fact, AR−/lo PCa cells pre-exist in untreated PCa14. By performing AR IHC analysis in prostatectomy specimens, we have observed ~5-20% of AR−/lo PCa cells in a dozen of untreated Gleason 6-7 tumors with AR+ PCa cells representing the majority14. Heterogeneity in AR expression in CRPC tends to become accentuated such that AR+ PCa cells may evolve into AR+/hi cells and the AR-/lo cell population may expand14, leading to co-evolution of both subpopulation of PCa cells in the primary (prostate) tumor , as well as by recent positron emission tomography/computed tomography-based AR-axis imaging analysis33.Heterogeneity in AR expression levels in PCa has been noted for decades, from untreated tumors to CRPC to disseminated metastasesmor Fig. . PCa met+/hi, nuc/cyto-AR, and AR−/lo, which can all be explained by well-established knowledge of steroid hormone receptor functions. Thus, under persistent selective pressure of castration and antiandrogens, PCa cells may overexpress (AR+/hi), lose (AR−/lo) or redistribute (cyto-AR) the target protein, AR, in order to survive. As more specific and potent AR-targeting therapeutics are being used in the clinic, more AR+ adenocarcinomas are being turned (reprogrammed) to AR−/lo NE-like tumors called CRPC-NE34 or AR pathway-independent AR−NE− tumors called double-negative PCa (or DNPC35). The emergence and increasing prevalence of AR−/lo PCa cells/clones beg the important questions of whether they still respond to further antiandrogen treatments and how they can be therapeutically targeted.By analyzing 195 TMA cores and 8 whole-mount slides of CRPC specimens  obtained from 89 patients (most of whom were treated in the pre-Enza era), we have observed 3 distinct AR expression patterns, AR+ and AR−/lo PCa cells possess intrinsically different biological and tumorigenic properties and how they respond to androgen-targeting therapeutics. Xenograft modeling of CRPC by serially propagating 4 AR+ human PCa models in castrated mice  reveals strikingly distinct evolutionary trajectories: the LNCaP model gradually becomes AR+/hi and VCaP and LAPC4 become nuc/cyto-AR whereas the LAPC9 model evolves into AR−/lo. Therapeutic experiments demonstrate that the 3 AR-expressing CRPC models are transiently sensitive to Enza whereas the AR−/lo LAPC9 CRPC are refractory to Enza treatment de novo. These results represent among the first prospective experimental evidence linking AR expression status to distinct castration and Enza responses. Ongoing studies are investigating how heterogeneity in AR distribution, e.g., largely cytoplasmic AR in LAPC4 CRPC, impacts tumor response to antiandrogens.The present study focuses on addressing the critical question of whether AR+ and AR-KO LNCaP cell clones that allow us to perform side-by-side comparisons of their in vivo tumorigenic capabilities and relative competitive advantages in both androgen-intact and androgen-ablated (castration/Enza) hosts, as well as well-controlled cell biology experiments including single cell tracing studies. The results have demonstrated: (1) AR-KO cells possess intrinsically high tumor- and CRPC-regenerating activities in castrated mice  AR+ PCa cells, as expected, require androgen/AR signaling for their clonogenic and tumorigenic activities  AR-ice Fig.  and in vice Fig.  whereas to Enza;  AR+ PCa ies Fig.  althoughons Fig. . While o−/lo  incomplete chemical castration and AR blockage; (2) intracrine androgen production; (3) and inter-dependent symbiotic relationship between AR+/hi and AR−/lo cells. On the other hand, it is conceivable that in mCRPC patients, some metastatic foci may be predominantly AR+/hi while others primarily AR−/lo (reviewed in 2), as reported recently in patient metastases35.Approximately 30% CRPC TMA cores are AR/lo Fig.  and 40-5/lo Fig. , which i−/lo LAPC9 CRPC implanted in castrated mice . Androgen suppression of AR−/lo PCa cells have previously been reported, for example, in ARCaP cells36, and this effect seems to be still mediated through AR37. Interestingly, androgens have also been shown to repress AR+/hi CRPC cells38. We are currently exploiting our novel AR-KO LNCaP and AR−/lo LAPC9 CRPC models to elucidate molecular mechanisms underlying androgen suppression of AR-deficient PCa and to determine whether the phenomenon of T suppression of AR−/lo CRPC in vivo might also involve host  mechanisms. Also ongoing are comprehensive RNA-Seq and biological studies that aim to elucidate the transcriptome differences between AR+ vs. AR−/lo (and AR-KO) PCa cells and to understand how some AR+ cells could develop tumors in castrated hosts.Intriguingly, androgen seems to exert preferential suppressive effects on AR-deficient PCa cells. The two AR-KO LNCaP clones, when implanted in intact male mice with or without exogenous DHT, hardly regenerated any tumors Fig. . Also, A+ and AR−/lo PCa cell subpopulations, which co-exist in untreated primary tumors14, may co-evolve under pressure from castration/Enza to generate AR+/hi (LNCaP-type), as well as AR−/lo (LAPC9-type) CRPC cells/clones  BCL-2, but not BCL-XL nor MCL-1, is exclusively upregulated in patient CRPC; (2) BCL-2 is directly induced by Enza treatment; and (3) BCL-2 is upregulated in both Enza-resistant AR+/hi and Enza-insensitive AR-/lo CRPC. These observations are consistent with early studies showing AR repression of BCL-241. Although BCL-2 has been a long-standing therapeutic target in PCa, early clinical trials using non-specific and toxic BCL-2 family inhibitors have all failed. ABT-199 represents a third-generation BCL-2-slective inhibitor42, has recently been FDA-approved for CLL patients27, and is quickly moving to clinical trials for lymphoma due to its anti-CSC activities43. Collectively, our CRPC modeling in LNCaP system suggests a proof-of-concept therapeutic regimen for AR+/hi CRPC clones, i.e., to combine steroid receptor  targeting therapeutics with inhibitors of ADT-induced CSC molecules such as BCL-2. Based on this rationale, we  have initiated a phase Ib/II clinical trial of combining Enza and Venetoclax  to treat Enza-naïve metastatic PCa patients aiming to delay/prevent CRPC. It should be noted that the VCaP CRPC do not upregulate BCL-2, suggesting that, due to genetic diversity , different CRPC clones may employ different survival strategies to evade ADT.Our studies suggest that ARnes Fig. . RNA-Seqres Fig. . Althoug−/lo CRPC clones. The LAPC9 CRPC, which phenotypically resembles AR−NE− DNPC35, is characterized by prominent upregulation of multiple stem cell/CSC molecules in addition to BCL-2 , led to dramatic inhibition of AI tumor growth  patient CRPC are also enriched in IFN-responsive gene signatures35. These new observations raise the intriguing possibility that AR-/lo CRPC cells might preferentially cross talk with the host immune system to escape immune surveillance.Xenograft modeling in LAPC9 system has also provided proof-of-concept therapeutic strategies for AR-2 Figs. ,  8c, d,wth Fig. . Similarwth Fig. , j. Of g+/hi and AR−/lo PCa cells and mCRPC patients harbor AR+/hi and AR−/lo metastatic clones34; therefore, curative anti-CRPC therapeutic strategies entail elimination of both PCa cell populations. Our observations that both AR+/hi LNCaP and AR−/lo LAPC9 CRPC are enriched in neurogenesis and lipid signaling gene signatures open the door for future exploration of novel regulators and therapeutic targets in CRPC. The availability of our novel CRPC xenograft and genetically matched AR+/AR-KO LNCaP clonal models should facilitate future studies on: (1) dynamic and reciprocal cooperation and interactions between the two PCa cell subpopulations during ADT and tumor relapse, (2) the causal functions of and novel signaling pathways in mediating Enza response by cytoplasmic AR , and (3) PCa cell reprogramming and lineage plasticity accompanying tumor progression and clinical treatments. These models and tools should also facilitate unbiased high-throughput small-molecule and genetic screens to identify novel AR-/lo CRPC cell-targeting therapeutics.Most prostate (primary) tumors in CRPC patients contain both AR+/hi, nuc/cyto-AR, and AR−/lo  and AR-knockout (AR-KO) LNCaP clones, which were then used in a battery of in vitro biological and in vivo tumorigenic and Enza response assays, and the results revealed many interesting and surprising differences in the two subpopulations of PCa cells with different AR expression status  were obtained from the Jackson Laboratory, and the breeding colonies were maintained in standard conditions in our animal facilities. All animal-related studies in this study have been approved by our Institutional Animal Care and Use Committee (IACUC) at the M.D. Anderson Cancer Center (ACUF#00000923-RN00) or the Roswell Park Comprehensive Cancer Center . Therapeutic reagents used in this study included enzalutamide , ABT-199 , Mifepristone/RU486 , JQ1 , docetaxel , and TC-I 15 or compound 15 .LNCaP and VCaP cells were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI (for LNCaP) or DMEM (for VCaP) plus 10% heat-inactivated fetal bovine serum (FBS) plus antibiotics. LAPC4 and LAPC9 xenograft lines were initially provided by Dr. Robert Reiter (UCLA) and have been used extensively in our previous studiesn = 40 cores with 2 cores per patient, provided by Dr. Jiaoti Huang now at Duke University) was made from 20 CRPC patients14; (2) Cornell TMA  was created from 33 CRPC patients; and (3) Roswell Park TMA  was acquired from 28 CRPC patients. In addition, WM slides from 8 CRPC patients were provided by Dr. Jiaoti Huang. Formalin-fixed paraffin-embedded (FFPE) sections were cut from these TMAs and WM samples and used for immunohistochemistry (IHC) of AR. Relevant information available on patient samples and treatment was summarized in Supplementary Data Three TMAs containing 195 patient CRPC cores were originally derived from 81 CRPC patients  and incubated with primary antibodies (Supplementary Table Basic IHC and IF procedures in FFPE sections (4 μm) were detailed previously17. To establish the CR lines, parental AD tumor cells were purified, mixed with Matrigel, injected subcutaneously and serially passaged in surgically castrated immunodeficient mice. The LAPC4 and LAPC9 AD and CR xenograft lines were maintained and passaged in NOD/SCID mice, whereas LNCaP and VCaP AD and CR xenograft lines were passaged in NSG mice. Xenograft tumors that became CR were termed primary (1°) CRPC and CR tumors that became Enza-resistant were termed secondary (2°) CRPC  xenograft tumors, LNCaP, VCaP, LAPC4, and LAPC9, were routinely maintained in intact immunodeficient NOD/SCID or NSG miceRPC Fig. .17. In brief, xenograft tumors were harvested, chopped into pieces (~1 mm3), and digested with Accutase  for 30 min at room temperature (RT) under constant rotations. Single cells were collected via a pre-wetted 40-μm strainer and further purified on Histopaque-1077 (Sigma) gradient to deplete debris and dead cells.Basic xenograft tumor processing was described previously14. Single HPCa epithelial cells were purified into Trop2+CD45− cell population using flow cytometry (FACS), cultured in PureCol bovine collagen solution (Advanced BioMatrix) coated dishes16 in modified WIT medium25, and treated with DMSO (vehicle control) or Enza (10 or 20 μM).All primary HPCa samples were obtained with written informed patient consent in accordance with federal and institutional guidelines, which was approved by M.D. Anderson Cancer Center Institutional Review Board (IRB) (Protocol number LAB04-0498) and the Roswell Park Comprehensive Cancer Center IRB (BDR073416). Three HPCa samples were utilized in this study: HPCa229 , HPCa231 , and HPCa232 . Basic procedures for patient HPCa sample processing have been described previouslyTotal RNA was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. qRT-PCR was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad). qPCR data were normalized to 18 S RNA. Primers utilized were: 18 S, FWD: 5′-AAGTCCCTGCCCTTTGTACACA-3′ REV: 5′-GATCCGAGGGCCTCACTAAAC-3′; AR, FWD: 5′-TAGGGCTGGGAAGGGTCTAC-3′ REV: 5′-GGGAGGTGCTGCGCTC-3′; and PSA, FWD: 5′-GATGAAACAGGCTGTGCCG-3′ REV: 5′-CCTCACAGCTACCCACTGCA-3′.AR stop codon  and P2  , and the reporter gene in the vector, OFP (orange fluorescence protein) immediately after the cas9 nuclease was used to screen for the transfected cells via FACS. Four microgram of total gRNA1 and gRNA2 vectors were transfected into 500,000 LNCaP cells with Lipofectamine 2000. Three days after transfection, LNCaP single cell suspension was analyzed and sorted for OFP+ cells with BD FACSAria™ Fusion cell sorter. OFP+ cells were counted with hemocytometer after sorting and seeded into 96-well plates at one cell/well. Clonal cells were collected after four weeks and genotyped using primers: ARN FWD: 5′-GAGACAGACTGTGAGCCTAGC-3′ and ARN REV: 5′-GCTGTGAAGGTTGCTGTTCC-3′. Sequence data were analyzed with Seqman Pro. The resultant LNCaP clones with AR knocked out were called AR-KO clones.The general scheme for generating AR-KO LNCaP cell clones was presented in Supplementary Fig. 4 cells/well) and were co-transfected with 1 μg ARE (androgen-responsive element) or PSA reporters together with the Renilla luciferase internal normalization plasmid (phRL-CMV). As a control, one group was treated with 10 nM DHT (dihydrotestosterone) 12 h before measuring luciferase activities. The ratio of firefly to Renilla luciferase activity was determined with a dual luciferase assay (Promega) 48 h later. Unpaired two-tailed Studentʼs t-test was used to compare differences between groups, and P < 0.05 was considered statistically significant.LNCaP clonal cells were seeded in 24-well plates  for 5 h. Cells were fixed with 4% PFA for 5 min and permeabilized with 1% Triton-X100. DNA was denatured using 3 M hydrochloric acid in 1% Triton-X100 for 20 min and then treated with 0.1 M sodium borate for 15 min. Slides were blocked with background sniper  for 20 min and incubated with mouse monoclonal BrdU specific antibody  for 5 h at room temperature, followed by incubation with goat anti-mouse IgG conjugated to Alexafluor 488 and counterstaining with DAPI.+ LNCaP cells were suspended in Matrigel  and injected subcutaneously into castrated or T (testosterone)-implanted NSG male mice (8–10 weeks old). Tumor growth was measured twice weekly in two dimensions using a digital caliper. Tumor volume was calculated using 1/2 (length × width2). Animal body weight and health status were monitored during the experiments. At the end of experiments, tumors were harvested, and tumor incidence, weight, and gross images were recorded. For Enza therapeutic experiments  and Enza . In the mean time, 200,000 AR+ cells were injected subcutaneously into intact NSG male mice (~6 weeks old), which were castrated 1 week prior to initiation of Enza treatment at day 30, when animals were randomized into vehicle control (n = 8) and Enza  groups. Enza was dissolved in DMSO as 90 mg/ml stock solution, which was diluted in corn oil (Sigma) to make 2.5 mg/ml working solution. Mice were given 100 μL i.p.  injections 3 times a week.In general, 200,000 AR-KO and AR+ clonal cells were placed on the incubator stage of a JuLI™ Stage Real-Time microscope system, and maintained at 37 °C, 5% CO2 and >95% humidity in RPMI medium supplemented with 10% FBS, 1% Pen/Strep, and Enza (2 μM) or DHT (0.1 nM). Phase, RFP and GFP images were collected continuously with a ×20 objective lens at 2-h intervals for 96 h. Data analysis was performed using JuLI™ Stage Software.LNCaP AR-KO and AR+) or AR+ (RFP+) cells were plated into one well in 6-well plates. Cell images were captured with JuLI™ Stage Real-Time microscope at 2-h intervals. Three points of each group were recorded and cell confluence data was measured using ImageJ by analyzing the proportion of fluorescence positive area of the cell images. For in vivo competition assays, 200,000 each of AR-KO and AR+ cells mixed together were injected subcutaneously into castrated or T-supplemented male mice. Once palpable, tumors were harvested at different time points and analyzed using a BD LSR II flow cytometer. A minimum of 10,000 cells were analyzed for each time point.For in vitro competition assays, AR-KO cells were infected with pGIPZ non-silencing GFP control lentivirus at an MOI of 10 to mark the cells with GFP. For in vitro competition assays, 5000 of AR-KO  and/or LAPC9 (AD and AI) tumors were harvested and total RNA was extracted by RNeasy mini kit (Qiagen), which includes on-column DNA digestion to deplete genomic DNA. 1000 ng of RNA was used to synthesize cDNA libraries using the Illumina TruSeq Stranded Total RNA LT Sample Prep kit according to the manufacturer’s guidelines. The libraries were then sequenced via 75 nucleotide paired-end running on HiSeq 2000 instrument . 4 (for LNCaP) or 5 (for LAPC9) biological replicates were prepared for each condition. The reads were mapped to the reference human genome sequence (hg38) using TopHat version 2.0.10 (ref.45) and Bowtie version 2.1.0 (ref.46) with parameters “-r 50 --mate-std-dev 50 --library-type fr-unstranded –G”. More than 80% fragments were mapped to human genome. The number of fragments in each known gene from GENCODE Release 21 (ref.47) was enumerated using htseq-count from HTSeq package (version 0.6.1) (ref.48) with parameters “-f bam -m union -s reverse”. Genes with fewer than 10 fragments in all the samples were removed before differential expression analysis. There were a total of 60,119 genes, and after the removal of genes with low number of fragments, there remained 23,933 genes for LNCaP and 20,842 genes for LAPC9 models. DEseq (version 1.14.0) (ref.49) was used to call differentially expressed genes (DEGs) in our samples.Basic procedures have been previously describedhttp://bioinfogp.cnb.csic.es/tools/venny/ using lists of DEGs from two differential expression comparisons. Heatmaps were plotted by the free downloaded software of Cluster 3.0 and Java Tree View. To generate the landscape profile of RNA-Seq, for the fragments that have both ends mapped, the first reads were kept. Together with the reads from the fragments that have only one end mapped, every read was extended to its 3′ end by 200 bp in exon regions. For each read, a weight of 1/n was assigned, where n is the number of positions the read was mapped to. The sum of weights for all the reads that cover each genomic position was rescaled to normalize the total number of fragments to 1 M and averaged over 10 bp resolution. The normalized values were displayed using UCSC genome browser (http://genome.ucsc.edu/).To define DEGs, we set up a stringent statistic cutoff of fold change (FC) of ≥2 and the false discovery rate (FDR) <0.05. A total of 2451 DEGs was identified between LNCaP AD and 1° CRPC and 3254 DEGs between LNCaP AD and LNCaP 2° CRPC. Also, 601 DEGs were identified between 2° CRPC vs. 1° CRPC using the criteria of FC ≥ 1.5 and FDR < 0.05, which were specific for Enza resistance. In LAPC9, 3929 DEGs were identified between AD tumors and CRPC. The Venn diagram was generated at www.ingenuity.com. Basic procedures have been described in our earlier report25. In brief, we uploaded our DEG lists with FC ≥ 2 and FDR < 0.05 into the IPA software, and ran the Core Analysis to find the enriched signaling pathways. The FC and FDR were estimated by DESeq. IPA used Fisher’s exact test to determine if the genes in each pathway significantly overlap our DEG lists.Ingenuity Pathway Analysis  was used to analyze pathways that are preferentially enriched in our LNCaP and LAPC9 CRPC models compared to the corresponding AD tumors using an IPA software downloaded from 50 to correlate our LNCaP and LAPC9 CRPC models (vs. AD tumors) to known gene expression signatures. The normalized count table estimated by DESeq for all genes  was taken as the input for GSEA and the genes were ranked in GSEA by the Signal/Noise metric. The sample permutation was performed by 1000 times to assess the statistical significance. We followed the GSEA user’s guide (http://www.broadinstitute.org/gsea/doc/GSEA UserGuideFrame.html) using default parameters to run the software. The FDR in GSEA is the estimated probability that a gene set with a given “normalized enrichment score/NES” represents a false positive finding, and an FDR < 0.25 for GSEA is considered statistically significant. For GSEA, our genes  are used as the ranked gene list  to interrogate GSEA database.We performed GSEA 22 ; (3) an RNA-Seq analysis comparing AD LNCaP vs. androgen-independent LNCaP-AI cells52 ; (5) an in-house RNA-Seq data set comparing the PSA−/lo vs. PSA+ LNCaP cells  a m55 identified gene signatures upregulated in primary B lymphocytes isolated from transgenic mice overexpressing Bcl-2  vs. non-stem cells (CD31−) from adipose tissue  with their human counterparts, and showed genes upregulated in the stem cell subpopulation  version 6.0 to determine correlations between our gene set and gene sets in the MSigDB. Representative data sets presented were also summarized in Supplementary Table l-2 Fig. . Boquest60. The first method24 is based on a list of 70 genes and the score was the Pearson correlation coefficient of normalized FPKM values of each sample with the mean value of the NEPC samples in the paper. The score is called integrated NEPC score. The second method60 is based on a smaller list of 50 genes and the NE_score was the weighted sum of the FPKM value of those 50 genes. The raw data were obtained from Beltran et al.24. We determined the relative NE_scores in the LNCaP and LAPC9 CRPC vs. AD using the second method by Lee et al.60. Briefly, the list of 50 genes was selected to best distinguish NEPC and ADPC samples from Beltran et al24 using a logistic regression model and elastic net regularization. The weighted sum of the 50 genes was treated as NE_score and results were presented as relative NE_scores in AD tumors vs. CRPC  and drugs were administered intraperitoneally 3 times a week till the end. For targeted therapeutic experiments in AR+/hi LNCaP-type of CRPC ; (2) Enza (30 mg/kg) + RU486/Mifepristone  (n = 12); (3) Enza (30 mg/kg) + ABT-199  (n = 12); (4) Combo: Enza (30 mg/kg) + RU486 (20 mg/kg) + ABT-199 (50 mg/kg) (n = 12). For experiments in AR-/loLAPC9-type of CRPC ; (2) JQ1 30 (n = 10); (3) α2β1 inhibitor /compound 15  (n = 12); (4) Combo: JQ1 (50 mg/kg) + α2β1-i (20 mg/kg) (n = 12). For additional experiments in AR-/lo LAPC9 CRPC, once tumors grew up, castrated mice were randomly divided and drugs were delivered as follows: (1) vehicle control (n = 12); (2) ABT-199 (50 mg/kg) (n = 12); (3) Combo: JQ1 (50 mg/kg) + ABT-199 (50 mg/kg) (n = 12). ABT-199 (5 mg/ml) was dissolved in ethanol, and prepared in Phosal-50 PG (Lipoid): polyethylene glycol-400 (Spectrum): ethanol (6:3:1)26, whereas other drugs were dissolved in 3–5% DMSO or ethanol and prepared in corn oil (Sigma). In another experiment, castrated NSG males bearing LNCaP 1° CRPC were treated with ABT-199  or vehicle . Tumor growth was measured weekly in two dimensions using a caliper and tumor volumes were determined using 1/2 (length × width2). Animal body weights and health status were monitored. At the end, tumors were harvested, and tumor incidence, weight, and gross images were recorded.NOD/SCID or NSG male mice (8–10 weeks old) were castrated on day 0. After 7–10 days, 100,000–500,000 CRPC cells in different models were injected subcutaneously in Matrigel. For Enza treatment Fig. , once tuRPC Fig. , drugs wRPC Fig. , drugs wTo overexpress BCL-2 in LNCaP cells, we first produced the Precision LentiORF BCL-2 lentivirus in 293 T packaging cells (ATCC), which was titered using GFP positivity in HT1080 cells. LNCaP cells were infected at MOI 10–15 for 72 h at 37 °C. Precision LentiORF Bcl-2 lentiviral vectors (Clone ID: PLOHS_100004100) and the control vector (Precision LentiORF RFP positive control) were purchased from GE Dharmacon. The overexpression effect was assessed using Western blotting and GAPDH was used as a loading control. For clonal assays, BCL-2 overexpressing LNCaP cells were plated at a clonal density  under both regular serum-containing (RPMI-1640 medium + 10% FBS + 1% penicillin/streptomycin) and AR-inhibited (RPMI-1640 medium + 10% FBS + 1% penicillin/streptomycin + 10 μM Enza) conditions. Holoclones14 were counted and imaged 2 weeks after plating. For tumor assays, control and BCL-2 overexpressing LNCaP cells were mixed with Matrigel and injected s.c. at 100,000 cells/injection in intact NSG mice (8–10 weeks). Tumor volume was measured at weekly basis and the median tumor volume was shown in Fig. To examine if BCL-2 can promote tumorigenicity in castrated hosts, tumor cells were obtained from AD tumors and injected into castrated NSG mice at 20,000 cells/injection.17 and single cells were obtained using a 40-μm cell strainer and purified using Histopaque-1077 . Cells were plated at a density of 25,000 cells/cm2 in a 384 well plate. Cells were plated in 5% Matrigel diluted with organoid media63 without androgens. Three days after plating, organoids were treated with various doses of Enza, ABT-199, or an equimolar combination of the 2 drugs in a constant-ratio combination. Cell viability was analyzed 6 days post treatment using CellTiter-Blue Cell Viability Assay (Promega).LAPC9 CRPC (AI) xenografts were digested to single cells using Accumaxt-test was used to compare significance in tumor weight and tumor volume, and χ2 test was employed to compare tumor incidence. The results were presented as mean ± S.D. P < 0.05 was considered statistically significant.Unpaired two-tailed Student’s Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


The anti-platelet effects of AR agonists were evaluated in healthy subjects , using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). Compared to P2Y12 antagonists, AR agonists were much less or not effective under static conditions, but demonstrated similar antiplatelet activity in flow. In most cases, AR agonists significantly enhanced the anti-platelet effect of P2Y12 antagonists, despite possessing different selectivity profiles and antiplatelet activities. Importantly, their inhibitory effects in combination with P2Y12 antagonists were similar in high- and low-responders to P2Y12 inhibitors. In conclusion, a combination of anti-platelet agents acting via the P1 and P2 purinergic receptors represents a promising alternative to existing antithrombotic therapy.Large inter-individual variation in platelet response to endogenous agonists and pharmacological agents, including resistance to antiplatelet therapy, prompts a search for novel platelet inhibitors and development new antithrombotic strategies. The present in vitro study evaluates the beneficial effects of three adenosine receptor (AR) agonists , different in terms of their selectivity for platelet adenosine receptors, when used alone and in combination with P2Y The leading causes of morbidity and mortality in developed countries are cardiovascular disease and stroke, resulting predominantly from arterial thrombosis dependent on blood platelet hyperreactivity. Anti-platelet therapy would appear to be an obvious solution for the treatment and management of such disorders ,2. Sever1 receptor initiates platelet aggregation, while the P2Y12 receptor enhances this process, eventually leading to the formation of a clot. Due to this fact, the P2Y12 receptor is the main therapeutic target in anti-platelet therapy targeted at the ADP-dependent activation pathway [12 inhibitors include the thienopyridine-class inhibitors , the ATP analogue cangrelor, and the cyclo-pentyl-triazolo-pyrimidine derivative ticagrelor [12 receptor inhibitor that reversibly and non-competitively blocks ADP signalling [ADP is one of the key mediators of both physiological haemostasis and thrombosis, being not only a direct agonist of platelets, but also an important factor released from platelet intracellular structures, enhancing the platelet response initially induced by other activators. Platelets have two ADP receptors on their surface: the P2Y pathway . Generalcagrelor ,5. Thiengnalling .1, A2A, A2B and A3) only A2A and A2B are expressed in platelets [Adenosine is an important purine metabolite, serving not only as a component of nucleic acids and ATP, the most important energy carrier in the cell, but also as a signalling molecule regulating many cell processes ,8. Adenolatelets ,9. Activlatelets .2A or A2B receptors, and others are non-selective compounds activating more than one type of adenosine receptor. One of the oldest known anti-platelet adenosine analogues is 2-chloroadenosine [Apart from the natural agonist adenosine, a group of synthetic, long-lasting agonists were developed ; of thesdenosine . Other Adenosine ,13,14.12 receptor). Unfortunately, such treatment is still beset by the problem of resistance, especially among patients with type 2 diabetes, i.e., a group at higher risk of thromboembolic events [12 antagonist and an AR agonist, which was found to deepen the action of P2Y12 antagonist [In clinical practice, efficient anti-platelet treatment is often hindered by reduced sensitivity to many anti-platelet agents. To avoid higher drug doses, and therefore a higher risk of bleeding, combined therapy based on the administration of two or more drugs acting on different platelet activation pathways is often used as an alternative. An example of such an approach is the combined administration of acetylsalicylic acid (an inhibitor of thromboxane A2 formation) and clopidogrel , while the addition of AR agonization would provide adequate anti-platelet effect, and therefore excessive clot formation prevention. We used in vitro methods  to evaluate anti-platelet effects of P2Y12 inhibitors: cangrelor and prasugrel (active metabolite R-138727), as well as AR agonists: NECA , regadenoson , and LUF5835  in single  and dual (P2Y12 antagonist and AR agonist applied simultaneously) systems.The aim of this study is to further explore the potential of combined anti-platelet therapy consisting of simultaneous P2Y12 antagonists and effect was on the same level in high- and low-responders to P2Y12 inhibitors, which suggests that AR agonists have the potential to overcome resistance to P2Y12 blockers.In the present study, we found that the use of AR agonists can lead to significantly higher inhibition of platelet function caused by P2Yp < 0.01), while none of the AR agonists exhibited cytotoxic effect (viable cells fraction was not decreased in comparison to the control of non-treated platelets)  decreased the fraction of viable cells by 81% on average (atelets) . This en50). NECA yielded a curve with the maximal inhibition value of 79.1 ± 4.0%, and IC50 of 0.5 µM  with a coefficient of determination (2R) equal to 0.876 , and 2R equal to 0.201 : cangrelor and prasugrel metabolite R-138727 (PM). Each compound was used in its IC50, with the values taken from our previous work [50 value; therefore, a concentration of 100 µM was used.AR agonists were used in a combination with two P2Yous work : NECA 0.12 antagonists significantly reduced platelet aggregation: cangrelor by a mean value of 41% and PM by 46%; however, the percentage inhibition between subjects displayed high coefficients of variation: 54% and 38%, respectively, (n = 15). Among the AR agonists, NECA caused a statistically significant decrease in aggregation, whereas regadenoson and LUF5835 did not  combinations; however, the coefficients of variation were also high . The anti-aggregatory effect of AR agonists on the P2Y12 inhibited platelets was then evaluated in the established subpopulations.Since platelet sensitivity to P2Y12 antagonist + AR agonist group and the P2Y12 antagonist group, for both low- and high-responder groups, for all (six) combinations  and by 47% (PAC-1 binding); PM by 42% (P-selectin expression) and by 30% (PAC-1 binding). Observed coefficients of variation in the percentage inhibition by P2Y12 antagonists or AR agonists between subjects ranged from 23% to 41% for P-selectin expression and from 14% to 35% for PAC-1 binding (AR agonists were used in combination with two P2Y binding . Among A12 antagonist and an AR agonist caused significantly greater inhibition of platelet reactivity for cangrelor pairings with NECA and regadenoson (P-selectin only) (The simultaneous application of a P2Yin only) A., and Pin only) A,B. Coefin only) .12 inhibitors on thrombus formation under flow conditions, all compounds were used in concentrations corresponding to the previously established IC50 for platelet aggregation, apart from LUF5835, which was applied at 100 µM.In the experiments assessing the effects of AR agonists and P2Y12 antagonists alone decreased clot formation by cangrelor (by 68%) and PM (by 77%); however, the obtained variance coefficients for P2Y12 inhibitors were high: 103% and 104% respectively. PM treatment did achieve significant inhibition  or given orally (prasugrel) during the anti-platelet therapy [2A and A2B adenosine receptors [Adenosine receptor (AR) ligands, developed in recent decades, represent a group of agents with anti-inflammatory activity, which can be useful in the prevention and treatment of human diseases ,20,21. P therapy . More preceptors .12 antagonist (cangrelor or prasugrel metabolite) and an AR agonist  leads to a greater inhibition of platelet function than the P2Y12 antagonist alone. What is important is the effect was specific, as the examined AR agonists were not cytotoxic for the cells and their action was much more pronounced in individuals with poor response to P2Y12 inhibitors. In a certain sense, this study supports and complements our recent in vitro work, where the anti-aggregatory activity of other AR agonists was examined in the absence or presence of the P2Y12 antagonists given above [Our findings demonstrate that the use of combination of a P2Yen above .12 antagonists on the effects of the AR agonists, i.e., by comparing groups of high- and low-responders to P2Y12 inhibitors. To more closely reflect the in vivo environment, the anti-platelet effects of AR agonists were evaluated using whole blood techniques, under flow (thrombus formation) and static conditions (study of platelet activation and aggregation). However, whole blood aggregometry was used to estimate the inhibitory strength of AR agonists and assess the half maximal inhibitory concentration (IC50) values of examined compounds; they were also used to evaluate the anti-aggregatory effects of AR agonists in combination with P2Y12 antagonists, which were monitored in a whole population of healthy individuals and in subpopulations of high- and low-responders to P2Y12 inhibitors. It is important to note that all AR agonists chosen for the study were tested in a dual model with P2Y12 inhibitors, irrespective of the degree of anti-aggregatory activity they exhibited in preliminary experiments. However, the concentration of the AR agonist used for testing with the P2Y12 antagonist was chosen based on its anti-aggregatory activity: i.e., regardless of the parameter being estimated, the anti-aggregatory compound was applied at a concentration corresponding to its IC50 value. In addition, the AR agonist which did not display antiaggregatory properties was analysed further at the highest used concentration: 100 μM. Cangrelor and prasugrel, being strong P2Y12 inhibitors, were used at the IC50 values, which were previously reported [However, while the two studies have similar aims and target populations, their experimental designs are quite different: they employ different AR agonists and use different methods to determine anti-platelet properties. In addition, the present work was extended with further analyses of the influence of P2Yreported .i = 290 nM), short acting A2A AR agonist with low affinity for the remaining adenosine receptors [2B adenosine receptor (EC50 = 10 nM) but, on the other, it can also interact with human A1 (Ki = 4.4 nM), A2A (Ki = 21 nM) and A3 (Ki = 104 nM) adenosine receptors [1 (Ki = 14 nM), A2A (Ki = 20 nM) and A3 (Ki = 25 nM) adenosine receptors and lower affinity at A2B AR (EC50 = 140 nM), was shown to be over 20,000 times more potent as a vasodilator and 5-10 more effective as an inhibitor of platelet aggregation in response to ADP and adrenaline [The anti-aggregatory potency of the tested compounds was examined at the beginning of the study. Apart from NECA, the anti-platelet effects of regadenoson and LUF5835 were not obvious. It is simply because the anti-platelet properties of regadenoson or LUF5835 have not been explored so far. Regadenoson  is a synthetic AR agonist currently approved for clinical use as a pharmacologic stress agent for myocardial perfusion imaging (MPI). It demonstrates non-inferiority to adenosine for detecting reversible myocardial perfusion defects, although it is a moderately selective  determined for the A2A adenosine receptor (high affinity receptor), which is preferential over the A2B AR (low affinity receptor) for adenosine [2A adenosine receptor was overestimated. This may be possible, as while the selectivity profile of LUF5835 is only given in one report of Beukers et al. [2A adenosine receptor than regadenoson: the Ki values were calculated to be within the range of 2.2‒60 nM for NECA [2A-selective AR agonist UK 432097 [2A receptor is not predictive of those effects [As with NECA, but unlike LUF5835, regadenoson caused significant inhibition of platelet aggregation within the concentration range 1‒100 μM. However, at a concentration of 1 µM or higher, NECA was approximately twice as effective as regadenoson in reducing platelet aggregation . Assumindenosine , regadens et al. , many mofor NECA ,35,36 anfor NECA ,38,39. TK 432097 . Interes50 values (determined in aggregation) and LUF5835 at 100 μM. At the indicated concentrations, the inhibitory effect of AR agonists on platelet activation (5%–19% inhibition) or aggregation (4%–35% inhibition) was considerably lower than observed in studies of platelet thrombus formation on collagen, which reported very high reduction of thrombus volume, i.e., by 75%–84%. NECA appeared to be the most effective AR agonist as it significantly diminished ADP-induced platelet activation, expressed by the fraction of CD62P-positive platelets, ADP-stimulated aggregation, and thrombus formation, compared to controls (DMSO). Regadenoson and LUF5835 only exerted significant inhibitory action against thrombus formation. These observations are mostly in agreement with the study of aggregation by shear stress that showed that some adenosine derivatives, including NECA, increased the blood flow and platelet cAMP level, reduced platelet activation and retention of white blood cells; however, the differences in platelet retention between samples and control were not statistically significant [In the light of these observations, further experiments were carried out with NECA and regadenoson at their ICnificant .12 antagonists, all examined parameters were strongly decreased as compared to controls; the degree of inhibition of platelet activation, aggregation and thrombus formation by P2Y12 inhibitors reached respectively 52%, 46% and 81%; insignificant changes were only observed in one case, i.e., formation of platelet thrombus in the presence of PM. Overall, the results indicate that compared to P2Y12 antagonists, AR agonists were much less effective under static conditions (in models with exogenous ADP), but demonstrated similar anti-platelet activity in flow (without ADP).As regards P2Y12 inhibitor, AR agonist), depending on the mode of treatment. A desirable cell response was obtained after incubation of whole blood with 10 µM ADP in the aggregation experiments and with 20 µM ADP when studying platelet activation. Such concentrations of ADP may appear high, but they are nevertheless used in studies of platelet function, particularly those concerning aggregation [Although it is possible that AR agonists may be able to inhibit platelet activation and aggregation more effectively at lower agonist concentration, the present study did not explore the agonist dose-response relationship for obvious reasons: our aim was to obtain high and reproducible cell reaction to ADP; the experimental protocol included five compounds, each of which was tested in various combinations, and more importantly, the samples contained from one to three platelet inhibitors , although the changes did not reach statistical significance. There are at least two reasons why this might be the case: P2Y12 antagonists significantly decreased thrombus volume when applied alone; in addition, very high inter-assay variation was observed. Indeed, the CV values for thrombus volume ranged from 62% to 184% and were substantially higher than the CVs obtained in the remaining assays. In comparison, Ranjit et al. report inter-assay CVs varying from 8% to 638% with regard to blood clot parameters measured by thromboelastography in whole blood, demonstrating the high variability between haemostatic parameters [In most cases, the combination of AR agonists with P2YPSB 0777 . In addirameters .12 receptor inhibitors. Adenosine receptor agonists seem to be an attractive alternative to GPIIbIIIa or P2Y1 receptor antagonists, which were demonstrated to have favorable effects on platelet function in combined therapy with thienopyridines [Taken together, our findings provide further support for the potential therapeutic use of AR agonists in dual anti-platelet therapy in combination with P2Yyridines ,47.12 inhibitors using aggregometry: a simple method which allows rapid assessment of platelet response.High degrees of inter-individual variability were previously observed for the platelet response to agonists (particularly ADP) and anti-platelet drugs ,49,50,5112 inhibitors, allowing their effects to be analysed in the established subpopulations. All the agonists also significantly improved the observed anti-aggregatory effects of P2Y12 inhibitors in both high- and low-responders, although the inhibition ratio indicated considerably higher inhibition of platelet aggregation by AR agonists among the P2Y12 low-responders than the high-responders  (NECA (CAS № 35920-39-9)), and Cayman  (regadenoson (CAS № 313348-27-5)). LUF5835 (2-amino-6-(1H-imidazol-2-ylmethylsulfanyl)-4-(3-hydroxy-phenyl) pyridine-3,5dicarbonitrile) was synthesized at Laboratory of Molecular Virology and Biological Chemistry, Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland. Cangrelor (AR-C69931MX) was from Cayman Chemical . Prasugrel metabolite (R-138727) was obtained from BOC Sciences . Calcein AM was obtained from Molecular Probes . Antibodies anti-human CD61/PerCP, CD61/PE, CD62/PE, PAC-1/FITC, mouse IgG1/PE isotype control, mouse IgG1/FITC isotype control, Cellfix, buffered sodium citrate was purchased from Becton-Dickinson . Phosphate buffered saline pH 7.4 (PBS) was obtained from Corning . Dimethyl sulfoxide (DMSO), adenosine diphosphate (ADP), and bovine serum albumin (BSA) were obtained from Sigma . All other chemicals, unless otherwise stated, were purchased from Avantor Performance Materials Poland S.A. .The stock and working solutions of cangrelor and prasugrel metabolite were prepared in distilled water. The 100 mM stock solutions of NECA, regadenoson and LUF5835 were prepared in DMSO. Stock solutions were then diluted with PBS to working concentrations not exhibiting precipitates, as noted by Boncler et al. , and addv/v. All individuals stated that they had not taken medications known to influence platelet function for at least two weeks prior to the study.Experiments were approved by the Ethics of Research in Human Experimentation Committee at the Medical University of Lodz, approval number (RNN/43/17/KE). After having received written consent from volunteers, blood was collected from healthy donors  into a vacuum tube containing 0.105 mol/L buffered sodium citrate, with a the final citrate: blood ratio of 1:9 12 inhibitors was assessed in resting platelets according to Rywaniak et al. [Platelet viability in the presence of AR agonist and P2Yk et al. . Samples12 inhibitors for 3 (AR agonists and cangrelor) or 15 (prasugrel metabolite) minutes at 37 °C, then 300 µL of blood was transferred into the measurement cell and diluted with 300 μL saline (0.9%) and preheated to 37 °C for another 3 min. Then, 10 μM ADP  was added and platelet aggregation was recorded continuously for 10 min using a Multiplate analyser . Area under the curve (AUC) was analysed. All measurements were completed within three hours of blood collection.The measurements was performed according to the manufacturer’s instructions. Briefly, whole blood was preincubated with an AR agonist and/or P2Y12 inhibitor for 3 (AR agonists and cangrelor) or 15 (prasugrel metabolite) minutes at 37 °C then platelets were activated with 20 µM ADP . Subsequently, a sample was diluted 10-fold with PBS, and labelled with anti-CD61/PerCP, anti-CD62P/PE and PAC-1/FITC antibodies , then fixed with CellFix for 1 h at RT. Directly before measurement, the samples were diluted 1:1 with PBS and the assay was performed, gathering 10,000 events (CD61/PerCP-positive objects), using FACSCanto II flow cytometer .Whole blood was preincubated with an AR agonist and/or a P2Y12 inhibitors on thrombus formation was assayed with the use of the Venaflux system  according to the protocol based on studies published elsewhere [12 inhibitor for three (AR agonists and cangrelor) or 15 (prasugrel metabolite) minutes at 37 °C. Samples were recalcified with CaCl2  immediately before measurement. The samples were then perfused through the channels of the chip using a shear force of 60 dyne/cm2 for 4 min. The thrombi were stained in channels by washing with 10 µg/mL fluorescein dissolved in PBS for two minutes at 5 dyne/cm2. Following this, the samples were perfused with CellFix for 4 min at 5 dyne/cm2. Such prepared channels were imaged by confocal microscopy.The effect of the AR agonists and P2Ylsewhere  using Ve3 . Volumes of separated thrombi were acquired for further analysis, and subsequently summarized to obtain total clot volume per sample.Confocal imaging of thrombi was performed with a Leica TCS SP8 confocal microscope with LAS X 2.0.2.15022 software  using the objective HC PL IR APO 40×/1.10 (water immersion). The 488 nm supercontinuum white light laser (WLL) (12% intensity) was used to excite the fluorescein-stained thrombi. The emission was collected by a photomultiplier tube detector in the range of 492–564 nm. Confocal Z-stack scans were performed at a rate of 400 Hz, zoom 1.0, pinhole 1.0 and line averaging set at 3 to improve image quality. In each field of a view 70 focal planes were acquired  . For theThe results are expressed as median with interquartile range. The Shapiro-Wilk test and Mauchley’s test were used to test the normality of data distribution and sphericity of variances, respectively. Normally distributed data was analysed with the pairwise Student t-test or two-way analysis of variance for repeated measures with the post hoc Bonferroni’s multiple comparisons test or repeated measures ANOVA with Geisser-Greenhouse correction and Holm-Sidak’s corrections for multiple comparisons test. Data departing from normality, variance sphericity and/or variance homogeneity were assessed with the Wilcoxon’s signed ranks test or Friedman’s test with Dunn’s correction for multiple comparisons. Coefficient of variation (CV) was used to compare the variability across the variables. Variables with extremely high values of CV (thrombus volume) were bootstrapped to ensure that the revealed differences between groups were not observed due to pure chance. The statistical analysis was performed using the following software packages: Statistica v.13 , StatsDirect v.2.8.0  and GraphPad Prism v.5.