Background: People with neurological dysfunction have been significantly affected by the ongoing coronavirus disease 2019 (COVID-19) crisis in receiving adequate and quality rehabilitation services. There are no clear guidelines or recommendations for rehabilitation providers in dealing with patients with neurological dysfunction during a pandemic situation especially in low- and middle-income countries. The objective of this paper was to develop consensus-based expert recommendations for in-hospital based neurorehabilitation during the COVID-19 pandemic for low- and middle-income countries based on available evidence. Methods: A group of experts in neurorehabilitation consisting of neurologists, physiotherapists and occupational therapists were identified for the consensus groups. A scoping review was conducted to identify existing evidence and recommendations for neurorehabilitation during COVID-19. Specific statements with level 2b evidence from studies identified were developed. These statements were circulated to 13 experts for consensus. The statements that received ≥80% agreement were grouped in different themes and the recommendations were developed. Results: 75 statements for expert consensus were generated. 72 statements received consensus from 13 experts. These statements were thematically grouped as recommendations for neurorehabilitation service providers, patients, formal and informal caregivers of affected individuals, rehabilitation service organizations, and administrators. Conclusions: The development of this consensus statement is of fundamental significance to neurological rehabilitation service providers and people living with neurological disabilities. It is crucial that governments, health systems, clinicians and stakeholders involved in upholding the standard of neurorehabilitation practice in low- and middle-income countries consider conversion of the consensus statement to minimum standard requirements within the context of the pandemic as well as for the future. About 7.1% of the global burden of the diseases are shared by neurological disorders. Neurological disorders are the leading cause of disability-adjusted life years (DALY) contributing 276 million DALYs and the second leading cause of mortality with about 9 million deaths in 2016 globally. Neurological disorders such as stroke, headache disorders, epilepsy, dementia, Parkinson’s disease, traumatic brain injury and motor neuron disease amongst others can cause motor, sensory, cognitive, and emotional impairments, leading to disability and poor quality of life among those affected. The past three decades have seen a considerable rise in the absolute numbers of death and disability due to neurological diseases. In 2017, the worldwide prevalence (counts in thousands) of years lived with disabilities (YLD) caused by neurological disorders was 3,121,435  with an increase in YLD (percentage change in counts) by 35.1% (95% CI 31.9–38.1) from 1990 to 2007 and by a further 17.8% (95% CI 15.8–20.2) from 2007 to 2017.Neurological disorders remain one of the major contributors to death and disability globally. Evidence supporting the benefits of specialised rehabilitation services for a neurological disability is constantly growing. However, despite the benefits of specialised rehabilitation services for a neurological disability, inaccessibility, non-availability and lack of affordability of rehabilitation services for persons with disability in general, especially in low- and middle-income countries (LMICs) is a huge barrier. Lack of resources, limited awareness, ineffective health systems, lack of expertise , and low priority for chronic illnesses are some of the reasons for the challenges faced in optimal delivery of rehabilitation services in LMICs.Neurorehabilitation is a specialised form of rehabilitation that aims to effectively reduce impairments, improve function, and promote participation in patients with neurological dysfunction. Travel bans have restricted provision of rehabilitation service in the community/home too. People experiencing neurological disability are particularly more vulnerable in these contexts because the brain pathologies may impair their level of understanding about the pandemic situation and create more confusion and stress to effectively adhere to the restrictions imposed. This creates a double burden for persons with neurological disabilities to effectively manage their disability during the COVID-19 pandemic and other infectious diseases. The needs and the demand for rehabilitation services to meet the needs of people experiencing neurological disability could substantially increase if the situation is not mitigated.In addition to the pre-pandemic challenges, the ongoing coronavirus disease 2019 (COVID-19) pandemic has overwhelmed the effective delivery of healthcare and rehabilitation services globally. PWDs (persons with disabilities) who were previously accessing neurorehabilitation services are unable to access these services because of pandemic restrictions. Most of the institutions offering rehabilitation services have either closed or services have been disrupted. In addition, non-COVID-19 infected patients with other ailments continue to require optimal rehabilitation services. Infection with COVID-19 has also manifested various neurological associations affecting both the central and peripheral nervous systems  and could lead to potentially life-long disabling conditions without adequate and timely rehabilitative intervention.In the present circumstances, it is implicit that competent hospital-based rehabilitation services are all the more, an indispensable element of healthcare. Rehabilitation is crucial not only for optimising health outcomes in severe cases of COVID-19 with complicated respiratory involvements but also in facilitating early discharge and reducing the risk of readmission. Safety also remains a concern among rehabilitation professionals due to the need for prolonged and close contact with patients during most neurorehabilitation therapy and from aerosol-generating procedures. The lack of sufficient evidence-based data on the best practices in rehabilitation that minimize risks from COVID-19 has further impaired the optimal delivery of neurorehabilitation services.. Therefore, there is a need for rethinking the structures and processes for acute in-hospital neuro-rehabilitation. In this perspective, we aimed to develop the recommendations for in-hospital neurorehabilitation during and after the COVID-19 pandemic which could be a potential basis of reference and guidance for other similar conditions.However, the mismatch between demand and resources remains a challenge. For example, the lack or shortage of beds has led to rehabilitation facilities being utilised for other acute patient care; restriction of face-to-face treatment considered to be ‘non-urgent’ has translated into reduced access to vital rehabilitation. Such practices are thereby preventing patients with neurological disorders from regaining lost functional skillsThe objective of this study was to systematically develop consensus-based expert recommendations for hospital-based neurorehabilitation during the COVID-19 pandemic for low- and middle-income countries based on available evidence.This study was carried out in India between August 2020 and April 2021 and incorporated a phased approach with a mixed-methods design . There w1) Blinded rating from experts2) Inclusion of a multidisciplinary expert group to have a comprehensive inputDue to the nature of this study, i.e. consensus-based recommendation/guidelines, the authors were informed by the ethics committee (from the lead author’s institution) that ethical approval was not essential. The authors obtained individual written informed consent from each of the experts who were involved in the rating process.In this phase, a core group of experts in neurorehabilitation were identified by the lead author and by snowball contacting. The inclusion criteria to have them on board as subject experts and co-authors were as follows: (1) working experience in the field of stroke rehabilitation (2) working experience in or with stroke care/rehabilitation in LMICs having a minimum of ten years. All experts who co-authored this study were approached by the lead author via mail. There was no remuneration provided to any of the authors for their involvement in the study.st August 2020 was held among the core group members to discuss the purpose of the study, and the process for the development of the consensus-based recommendations was finalised. This meeting was conducted to determine the steps to be followed for consensus development. All core members were present, i.e. the 9 authors involved in this study. The first meeting included introductions and development of an overall draft of steps to be followed. The following meetings had specific agendas to assess and decide progress of the work. The core team was also divided into subgroups for each phase of the study and had a leader for each subgroup. The sessions were led by DG.The core group had multi-disciplinary expertise in neurorehabilitation and comprised of 1 neurologist, 3 physiotherapists, 3 occupational therapists, a postdoctoral fellow in stroke research, and a statistician. Initial consultation via video-conference on 21. The objective of the review was to identify available guidelines, position statements, consensus and recommendations related to neurological rehabilitation during the COVID-19 pandemic globally. This review aimed to explore the existing guidelines for acute neurorehabilitation globally during the context of COVID-19. A comprehensive search strategy was developed using MeSH terms for the concepts related to the aim and the search were run in MEDLINE and CINAHL. Searches were run on 12th September 2020. Studies to be included were screened and selected by two independent reviewers (MC and MA). Data were extracted, charted, and collated for expert consensus by four independent reviewers  from the included studies.A global scoping review was conducted to identify existing evidence and recommendations for neurorehabilitation during COVID-19. Given the extensive resources and processes involved, the detailed scoping review will be published separately. In brief, a six-stage scoping review methodology recommended by the Joanna Briggs Institute was carried out. A list of evidence-based statements for neurorehabilitation during COVID-19 was generated. These statements were converted to recommendations for consensus. The recommendations were thematised and presented under five themes. Coding was done by 3 authors . Themes were identified after the data was extracted to be able to extract as much data as possible that is relevant to the topic. Similar data were then grouped under specific themes. From the scoping review, literature related to the objectives were identified. Data related to in-hospital neurorehabilitation for any neurological condition during the COVID-19 pandemic were extracted from the included studies. Only those statements/data that had a level of evidence ≥2b according to the Oxford levels of evidence were synthesised to develop statements for consensus among the expert groupThemes identified were:Rehabilitation ProvidersTheme 1: Recommendations relevant toTele-rehabilitationTheme 2: Recommendations relevant toRehabilitation service Administration and ManagementTheme 3: Recommendations relevant toPatientsTheme 4: Recommendations relevant toInformal and Formal Caregivers, Awareness and Education of Patients and CaregiversTheme 5: Recommendations relevant to.Each theme of recommendations was further divided into two sub-themes based on whether the patients tested positive or negative for COVID-19 while receiving neurorehabilitation service in the hospital. The detailed list of all these recommendations is provided as extended data along with the additional four statements formed the basis for expert consensus.Four statements were further added to the first two themes (three to theme 1 and one to theme 2). There appeared to be specific gaps in the recommendation list, and hence these four statements were exclusively added by the authors. These 75-statement document). There was no remuneration provided to the experts and participation was completely voluntary.A concerted attempt was made to reach out to such experts to partake in this phase of consensus development through the authors’ contacts and snowball sampling strategies. The expert group was created in such a way to include various members of a multidisciplinary team. The experts were chosen if they had experience in the field of neurorehabilitation and had worked in or with LMIC settings. A total of 17 experts were identified and invited to take part via email. The communication to experts included an invitation letter, information leaflet and consent form . Missing responses were asked to be completed by the experts until a complete response was obtained for all the statements in the document.In total, 13 experts consented to participate. The expert group consisted of physiotherapists, occupational therapists, a clinical psychologist, speech & language therapist and nurses with minimum ten years of experience as clinicians, practitioners and researchers, especially in LMICs. Demographic details of the experts  are available as extended data. These statements were compiled as the expert consensus statements for the in-hospital neurorehabilitation in LMICs recommendations in all of the five themes mentioned above. These expert consensus statements are presented inRecommendations receiving a score of ≥3 were considered as strong agreement and thus considered for calculation of the percentage of consensus. Out of these, 72 recommendations received an agreement score of ≥3 by 80% or more of the expert participantsTo the four additional recommendations, experts were asked to respond with an explanation of their agreement/disagreement. Out of four, 80% or more consensus was received for two recommendations, namely,(1)  Rehabilitation providers (including COVID-19 ward nurses) refer to case history and details from the patient file before the therapy session to reduce the amount of time spent at bedside (92.3% consensus), and(2)  Develop protocols for safe, effective and feasible tele-rehabilitation implementation during COVID-19 (84.6% consensus).However, it was emphasised that such protocols should allow for therapy dosages to be customizable according to patient needs, approved by rehabilitation professionals and considerate of the safety and privacy issues of both rehabilitation providers and patients. Experts also suggested that in-person hands-on therapy should be initiated once a patient is tested negative for COVID-19..Expert consensus for key aspects of in-hospital neurological rehabilitation services was specific to the rehabilitation service providers, patients, formal and informal caregivers of affected individuals, rehabilitation service organization, and administrators. The consensus statements were also classified according to the levels of evidence. There were specific components that were considered important by the experts in each of these key aspects. For service providers, it was training, implementation, appropriate use of Personal Protective Equipment, adequate safety measures, prioritized therapeutic goal setting, patient safety and therapy effectiveness. For patients, this was related to comprehending symptoms of COVID-19, therapeutic exercises regime including intensity, use of assistive devices or equipment for therapeutic exercise, postural stabilisation and documentation of practice. To our knowledge, ours is the only consensus-based guideline developed to date, addressing the aspect of in-hospital based neurorehabilitation during the ongoing pandemic, and its transferability and application to other similar airborne outbreaks. Previous consensus guidelines have either addressed acute management of stroke in LMICs, neurorehabilitation in LMICs, not specific to in-hospital setting or post-COVID rehabilitation as a wholeThe consensus statements for caregivers of hospitalised individuals were related to education, training, use of tele-rehabilitation services and reassurance. The consensus statements for management were related to the use of hybrid models of care, organization of strategic pathways for care and rehabilitation, developing criteria as well as prioritization of patient safety and need-based therapeutic engagement with or without caregiver engagement. Lastly, for tele-rehabilitation, the consensus was predominantly related to developing and implementing of secure tele-consultation and tele-rehabilitation services for patients with neurological disability and educating the users and rehabilitation service providers about tele-rehabilitation.These aspects have to be considered highly crucial and essential during the provision of in-patient neurological rehabilitation services for patients affected by neurological disability who may or may not be tested positive for COVID-19 in the pandemic situation. Although the pandemic seems to be settling down globally, these consensus statements might prove useful during the subsequent waves of the pandemic and also in the post-pandemic future.The consensus statements need to be contextualised according to the settings. Though the consensus statements came from experts from and with experience working in low-resource settings, it may be useful in all the settings irrespective of the availability of resources. However, implementation of these statements requires contextualisation, especially with respect to resource availability. Highly developed health care systems with adequate resources might have to prioritize rigour in implementation, whereas low resource settings with poor health systems must prioritize relevance. Knowledge, skills and competencies of the rehabilitation professionals in infection control, personal safety and tele-rehabilitation needs to be tested and trained to ensure the appropriate delivery of the recommendations. Frequent faculty development programs could be organised to ensure capacity building and quality delivery of service. This study does have its strength and limitations. Firstly, expert consultations, focus group discussions and consensus meetings were conducted virtually as opposed to the in-person meetings, given the pandemic situation. The number of experts chosen for the consensus were representative of a limited geographical area. Both these limitations are considered to have reduced the number of recommendations. However, the expertise and experience of the expert group was diverse and hence it is expected that this would have not compromised the comprehensiveness and overall representation for the consensus. This study is one of the first to develop an in-hospital neurorehabilitation consensus during the COVID-19 pandemic. The expert recommendation was developed through a methodologically rigorous process (a systematic scoping review). The mix of methods for development of the recommendation and the Delphi process to arrive at consensus ensured that the recommendation statements were evidence-based, substantiated by expert consensus. This enhances generalizability and pragmatic implementation in clinical practice.Given the current experiences of combating the pandemic worldwide, there is paucity of evidence and guidelines for ensuring patient safety and effective rehabilitation service provision for neurologically disabled patients admitted in the hospitals with or without COVID-19. This consensus statement envisages to provide key recommendations that can be optimised to enhance patient safety and service effectiveness. Systematic implementation of the consensus statement is of utmost importance to empower neurological rehabilitation service providers and patients with neurological disability. It is crucial that governments and health systems in low- and middle-income countries consider inclusive planning and policy making to convert the consensus statements to minimum standards for neurorehabilitation practice in this pandemic context and in the future.https://doi.org/10.17605/OSF.IO/HCSX7.Open Science Framework: Expert Consensus for in-hospital neurorehabilitation during the COVID-19 pandemic in low-and-middle income countries.This project contains the following underlying data:- Consensus paper rating_Raw_Data.xlsxhttps://doi.org/10.17605/OSF.IO/39MF4.Open Science Framework: Expert Consensus for in-hospital neurorehabilitation during the COVID-19 pandemic in low-and-middle income countries.This project contains the following underlying data:- Supplementary File.docx (The information leaflet consisting of instructions as well as elaborate list of 75 statement recommendations which was sent out to the experts)- Invitation and Instructions for Experts.pdfCreative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the The paper had been submitted as Research Article and reports about the methods and results of a multidisciplinary expert consensus project for in-hospital neurorehabilitation during the COVID-19 pandemic and the healthcare context of low- and middle-income countries. It addresses aspects that are considered relevant for rehabilitation providers, administration and management, patients, and caregivers both for hospital-based neurorehabilitation of COVID-19 cases and non-COVID-19 cases in need for neurorehabilitation during the pandemic. The consensus project aimed to provide and agree on practice recommendations relevant for these stakeholders during the pandemic in low- and middle-income countries. Such guidance is important and can serve these stakeholders as orientation for healthcare provision and the development of regionally contextualized clinical pathways. Some comments might be given that could help to further improve the manuscript.Author and expert review group: The authors mention that the experts chosen for the consensus were representative of a limited geographical area; this might not only have reduced the number of recommendations, but might to some extent also affect their global applicability. (Neuro-)Psychological problems  are frequent sequelae of COVID-19. Psychology as a discipline could be considered as less strongly represented in the work as might have been ideal (i.e. a limitation). In addition, patient representatives seemed not to be involved.Healthcare question to be addressed: Recommendations for Neurorehabilitation of COVID-19 cases is strongly related to the specific Neuro-COVID presentations  seen, their severity, frequency, and any “clusters” of presentation, e.g. Long-/Post-COVID-19. Such information  is lacking in the manuscript and could be added indicating the major clinical problems faced and hence to be addressed by practice recommendations.Methodological issues: Scope of the review undertaken: A scoping review - as conducted in this project - can include any and all types of literature .existing evidence and recommendations”). The authors further state “The objective of the review was to identify available guidelines, position statements, consensus and recommendations related to neurological rehabilitation during the COVID-19 pandemic globally. This review aimed to explore the existing guidelines for acute neurorehabilitation globally during the context of COVID-19.” Explicit inclusion and exclusion criteria for entries and a complete search strategy for at least one major database could be included in the manuscript to clarify the matter. To some extent the manuscript remained unclear what the basis of data extraction for the scoping review was. And, there is uncertainty about any evidence on neurorehabilitation of COVID-19 cases and non-COVID-19 cases in need of neurorehabilitation during the pandemic that was searched for and used . Such restrictions of the scope of review might be mentioned. If the entries searched for had been guidelines, they might have been missed partially due to any non-publication in peer-review journals, but rather online publication by governmental or medical society websites. E.g., in Germany a guideline with consensus-based expert recommendations for in-hospital based rehabilitation during the COVID-19 pandemic (including neurorehabilitation) with 64 recommendations had been published . If not done, it might be more correct in the given context to speak of “consensus-based expert recommendations”. For the development of evidence-based recommendations - with the scope of recommendations aimed for as stated above - the evidence from clinical research  would have systematically been search for, critically appraised, and then practice recommendations would have been deduced within a evidence-to-decision framework .” “Recommendations receiving a score of ≥3 were considered as strong agreement and thus considered for calculation of the percentage of consensus.” The two constructs, i.e. “relevance” and “agreement” seem to be “mixed-up” here. E.g., the panellists might have had a high degree of agreement that a recommendation was of little relevance, and conversely a low agreement that another was of high relevance. Accordingly, it should be made clear in the manuscript, what the criteria for agreement (and degree of agreement) and methods to analyse agreement were, and how the recommendation selection process was defined a priori considering both factors “relevance” and “agreement” .Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?NoIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Neurorehabilitation, practice recommendation/guideline developmentI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. It is an important manuscript and a useful contribution to the global literature on the role of rehabilitation in COVID-19. This is based on an expert consensus of selected rehabilitation professionals mainly working in India (an LMIC) who gathered virtually to provide consensus recommendations on for in-hospital neurorehabilitation during the COVID-19 pandemic in low- and middle-income countries.  My concern is that many guidelines and recommendations have been published for Post COVID Rehabilitation. Although many of them are not specific to LIC/LMIC, it is important to cite them in order to provide the context to the global efforts being done by rehabilitation professionals in different parts of the world. Some notable examples are as follows:https://www.mdpi.com/2077-0383/10/8/1691https://www.europeanreview.org/article/24211https:/www.medicaljournals.se/jrm/content/abstract/10.2340/16501977-2776 Neurorehabilitation is a multidisciplinary team effort with a physiatrist/ Rehabilitation Medicine Physician as an integral and often the team leader. This is the global norm in the majority of the countries where neurorehabilitation services are well established. This consensus panel of 13 experts did not have even a single Rehabilitation Medicine Physician. This is a major limitation of the manuscript which must be mentioned and explained. The virtual meeting has been mentioned as one of the weaknesses of this manuscript. I tend to disagree. In fact, this is a strength of this manuscript that a diverse group of experts from different parts of the world was brought together virtually to share their expertise and give recommendations. Please amend. The literature search needs to be redone and additional relevant references based on the data and experience sharing from other LIC/LMIC need to be integrated. Keywords ideally should not be the same words used in the title.https://www.ncbi.nlm.nih.gov/meshConsult the MeSH database to choose appropriate keywords.  Other minor comments are as follows:Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Neurorehabilitation, Stroke rehabilitation, Spinal Cord Injury Rehabilitation, Pain ManagementI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This is well constructed, interesting and useful study. It is novel and adds to the understanding and practice of stroke rehabilitation in LMICs. It is largely well written and clear. There are some minor errors in writing style e.g. "Themes were identified after the data was extracted to be able to extract as much data as possible that is relevant to the topic." which require rewording to increase clarity. It would also be useful to know what qualitative methodology was adopted in the thematic analysis (was it inductive or deductive) and some consideration of the researcher's potential influence on these themes. A further unacknowledged limitation is that the recommendations for patients and carers were developed without input from carers or patients. This is important to acknowledge as they may have prioritised/agreed differently on the themes that pertained to them than the healthcare providers. Overall, this article adds to an understanding of practice during the pandemic and its authors should be commended on producing it during such a challenging time.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Neurological Rehabilitation, clinical research in stroke.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Antiandrogen therapy is a primary treatment for patients with metastasized prostate cancer. Whilst the biologic mechanisms of antiandrogens have been extensively studied, the operating protocols used for the characterization of these drugs were not identical, limiting their comparison. Here, the antiandrogens Bicalutamide, Enzalutamide, Apalutamide, and Darolutamide were systematically compared using identical experimental setups. Androgen-dependent LNCaP and LAPC4 cells as well as androgen-independent C4-2 cells were treated with distinct concentrations of antiandrogens. Androgen receptor (AR)-mediated gene transactivation was determined using qPCR. Cell viability was measured by WST1 assay. Protein stability and AR localization were determined using western blot. Response to the tested antiandrogens across cellular backgrounds differed primarily in AR-mediated gene transactivation and cell viability. Antiandrogen treatment in LNCaP and LAPC4 cells resulted in AR protein level reduction, whereas in C4-2 cells marginal decreased AR protein was observed after treatment. In addition, AR downregulation was already detectable after 4 h, whereas reduced AR-mediated gene transactivation was not observed before 6 h. None of the tested antiandrogens displayed an advantage on the tested parameters within one cell line as opposed to the cellular background, which seems to be the primary influence on antiandrogen efficacy. Moreover, the results revealed a prominent role in AR protein stability. It is one of the first events triggered by antiandrogens and correlated with antiandrogen efficiency. Therefore, AR stability may surrogate antiandrogen response and may be a possible target to reverse antiandrogen resistance. The ANubeqa®) ,7. Firstreatment ,7. All aIn the last 3 decades, several cell line models have been introduced to PC research. These include androgen-dependent cell lines  and androgen-independent cell lines  . In addiThe effects of antiandrogens on AR-signaling have been investigated extensively. However, a direct comparison of these drugs on AR-signaling is limited. The experimental procedures used for their initial characterization differ in used cell models, cellular assays, culture conditions, incubation times, and drug concentrations. For example, Darolutamide was tested with AR-HEK293 cells, and Bicalutamide was tested with LNCaP/AR cells ,13. FurtThe human PC cell lines LNCaP and PC3 were obtained from the American Type Culture Collection (ATCC). C4-2 cells were kindly provided by Prof. Thalmann  . Dr A. CMethyltrienolone (R1881) , Bicalutamide , Enzalutamide , Apalutamide , and Darolutamide  were dissolved in DMSO as a 100 mM stock solution and stored in aliquots at −80 °C. As R1881 is not metabolized as rapidly as the natural 5α-dihydrotestosterone, it was used as androgen for the studies . Competi24 h after cell seeding, the medium was changed to 5% dextran-coated charcoal treated FCS  for 24 h to deplete the medium of steroid hormones and growth factors. Subsequently, cells were treated with R1881 alone or with a combination of R1881 and antiandrogens. Treatment concentration and duration were chosen due to previous studies revealing a maximal response after 1 nM R1881 treatment at 16 h ,27.2, 340 mM Sucrose, 10% Glycerol, 1 mM DTT, protease inhibitor) before the addition of 0.1% Triton X-100 and incubation for 5 min on ice. Subsequently, the suspension was centrifuged at 1300× g for 4 min at 4 °C to separate the nuclei from the cytoplasmic proteins. After transferring the cytoplasmic fraction into a new reaction tube, the nuclei pellet was washed once in 500 µL cytoplasmic lysis buffer. After repeated centrifugation, the nuclei were lysed using 100 µL RIPA lysis buffer. According to the manufacturer’s protocol, protein concentrations were determined using the Quick Start™ Bradford Protein Assay . After separation, NuPAGE™ LDS Sample Buffer (4×) was added to the fractions, and the nuclei samples were sonicated. Then, 20 µg of the fractions was used for western blot analysis. Lamin A/C (nuclear fraction) and GAPDH (cytoplasmic fraction) detection controlled fraction quality : AR (Hs00171172_m1), PSA/KLK3 (Hs02576345_m1), TMPRSS2 (Hs01122322_m1), PROSTEIN/SLC45A3 (Hs01026319_g1), HPRT1 (Hs02800695_m1), TBP (Hs00427620_m1).Total RNA was isolated using the DIRECT-ZOL RNA MINIPREP  following the manufacturer’s instructions. Complementary DNA (cDNA) synthesis was performed with 500 ng total RNA using the Superscript II RNase H Reverse Transcriptase kit . The quantitative real-time polymerase chain reaction (qPCR) was performed with GoTaq Probe qPCR Master Mix  and appropriate primers for 45 cycles on a LightCycler 480 instrument . The geometric mean of Cell viability was assessed using the WST-1 cell proliferation reagent . Cells  were seeded into 96-well culture plates. After 24 h, the medium was changed to a 50 µL medium containing 5% FCSdcc  for 24 h. Subsequently, cells were treated with the synthetic androgen R1881 or with a combination of R1881 and antiandrogens diluted in 50 µL Medium containing 5% FCSdcc. After 72 h treatment, 10 µL WST-1 solution was added for an additional 2 h. Subsequently, absorbance was recorded at 450 nm (Reference 620 nm) for each well using Mithras LB 940 . After subtracting background absorbance, results were calculated as x-fold of 1 nM R1881 treated cells.p-values of ≤ 0.05 were considered statistically significant. All differences highlighted by asterisks were statistically significant as encoded in figure legends . All experiments have been performed in at least three biological replicates unless noted otherwise.Prism 9.1.2  was used for statistical analyses. Differences between treatment groups were analyzed using Two-Way ANOVA with Dunnett’s correction for multiple comparisons. PSA, TMPRSS2, PROSTEIN) were detected by qPCR and displayed as a mean change of 1 nM R1881 treated control [To determine the effects of antiandrogens on AR-mediated gene transactivation, the mRNA levels of three AR target genes  which was similar to the level of complete steroid hormone withdrawal (~0.53 fold of 1 nM R1881), starting at a concentration of 1 µM and peaking at 10 µM A. The otIn C4-2 cells, Bicalutamide treatment resulted in a significant decrease in cell viability at 10 µM to ~0.70 fold B. TherefSimilar to the results seen in LAPC4 cells on AR-activity, all anti-androgen treatments only caused a significant reduction in cell viability (~0.50 fold) when used at 10 µM. Except for Enzalutamide, a decrease in cell viability starts at a concentration of 0.1 µM and peaks at 10 µM C. Only AComparison of the influence of the tested antiandrogens on cell viability reveals that the cell line C4-2 responded with the lowest and LAPC4 with the highest sensitivity to antiandrogen treatment D–G.To assess the impact of antiandrogens on AR localization, LNCaP, C4-2, and LAPC4 cells were starved for 24 h followed by non-treatment, 1 nM R1881 treatment, and different concentrations of antiandrogens for 2 h. Subsequently, cells were fractionated, and AR levels were determined in nuclear and cytoplasmic fractions. Treatment with 1 nM R1881 resulted in a significant increase of nuclear AR levels in all cell lines A,B. AlthIn C4-2 cells D, the prIn LAPC4 cells E, all anConversely, no significant cytoplasmic AR was observed after antiandrogen treatment .AR mRNA levels, LNCaP, C4-2, and LAPC4 cells were starved for 24 h followed by treatment with 1 nM R1881 and different concentrations of antiandrogens for 16 h. None of the antiandrogens mediated any change in AR mRNA levels in the tested cell lines  complexes, receptor dimerization, nuclear translocation, DNA binding, reduction of coactivator binding, and promotion of corepressor interaction ,38. The Among other mechanisms, sensitivity to antiandrogens is mediated by AR regulation and genetic changes, including AR overexpression, AR amplification, and AR somatic point mutations . Point mInhibition of AR translocation to the nucleus by antiandrogens is essential to prevent DNA binding and coactivator recruitment of the ligand-receptor complex and thus the transcriptional activity of the AR . In contAR protein levels are influenced by antiandrogen treatment . In accoThis study compared the AR inhibitory efficacy of the antiandrogens Bicalutamide, Enzalutamide, Apalutamide, and Darolutamide. The data demonstrated that all tested antiandrogens inhibit AR signaling to a similar extent. Significant differences were not found in comparing antiandrogens but were evident in cell line-specific differences in cell responses. Therefore, the cellular background, especially AR mutations, may represent an essential factor in antiandrogen efficiency. In addition, this study was able to show a correlation between the level of antiandrogen-induced AR protein reduction and the response of the cell lines to antiandrogen treatment and therefore identifies AR turnover as a possible indicator for antiandrogen treatment response.


Here, saturated and unsaturatedhomologues, including the oxidized forms 5-(2′-oxo) AR andtheir plant metabolites, were profiled by ultrahigh-performance liquidchromatography–ion mobility separation–high-resolutionmass spectrometry in 18 cultivars of einkorn, emmer, spelt, commonwheat, and tritordeum, cultivated in two consecutive years under uniformagronomic conditions. The average content of AR ranged between 672.5± 129.8 and 1408.9 ± 528.0 mg/kg, exceeding 2380 mg/kg insome samples and highlighting a superior content in tritordeum andin modern cultivars with respect to old wheat genotypes. By evaluatingthe effect of environmental and agronomic factors on the differentvariables, the harvest year resulted to be always significant, whilelocation and variety influenced AR abundance only for some homologues.Furthermore, the spatial distribution of AR was investigated by massspectrometry imaging using transversal cross sections of wheat kernels.Our results show that AR homologues are mainly localized in the testaand in the outer pericarp of wheat kernels.5- Among different Triticum spp., thetotal AR content may vary between hexaploid, tetraploid, and diploidspecies, suggesting a remarkable heritability of their content andan important role of the genetic background on their accumulation.14 Furthermore, as already reported for other phenolic compounds, ARcomposition is strongly affected by environmental and agronomic conditions.13 For instance, their content may range from 250to 1400 and from 200 to 950 μg/g dry matter in whole grainsrye and wheat, respectively.5 Hexaploidtritordeum, in particular, is an amphidiploid cereal derived fromthe cross between wild barley (Hordeum chilense Roem. et Schultz.) and cultivated durum wheat (Triticumturgidum (T. turgidum) L. ssp. durum Desf.), whose use in food processingis increasing.17 To our knowledge, the occurrence and profile of ARin tritordeum  (AABBHchHch) have not been previously reported in theliterature.18The main saturated homologues of AR arefound in wheat, rye, barley,triticale grains,11 while 5-(2-oxo)alkylresorcinols were reported for the first timein wheat19 and rye.20 Furthermore, conjugated forms of these phenolic lipids were reported in Cybianthusmagnus spp. monococcum), emmer (T. turgidum spp. dicoccum), spelt (Triticumaestivum (T. aestivum) spp. spelta), common wheat (Triticumaestivum (T. aestivum) spp. aestivum), and tritordeum, establishing thegenetic and environmental contribution to their variability. In additionto AR quantification, advanced analytical techniques were appliedto investigate the formation of AR metabolites and to finely locatethem within the cereal kernel.Therefore, in this work, we aimed at characterizing the AR homologueprofiles in different cultivars of einkorn , 2,5-dihydroxybenzoicacid (DHB), and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich(Steinheim). Gelatin used for embedding was obtained from VWR International. Glass microscope slides  were obtained from R. Langenbrinck .LC–MS-grade methanol, ethyl acetate, and 2-propanol were purchasedfrom Scharlab Italia Srl ; bidistilled water was obtainedusing a Milli-Q system . MS-grade ammoniumformate and formic acid from Fisher Chemical  were also used.Triticum spp. and tritordeumwere collected, including diploid,tetraploid, and hexaploid species .The same agronomic technique wasadopted for all cultivars . Aliquots of 2 kg of grainswere taken from each plot to determine the test weight (TW), the thousand-kernelweight (TKW), and the grain moisture content, using a GAC 2000 grainanalyzer . The TKW was determined ontwo 100-kernel sets for each sample (only whole seeds without huskswere considered) using an electronic balance. Kernels of each plotwere milled through a laboratory centrifugal mill  equipped with a 1 mm sieve and homogenized. Prior tochemical analyses, all the samples were ground to a fine powder (particlesize of <300 μm) with a Cyclotec 1093 sample mill  and stored for 2 weeks at −25 °C untilthe beginning of the analyses.The process was optimized considering(i) the solvent-to-solid ratio, where 1 g of the sample was extractedwith 20 mL or 30 mL of ethyl acetate; (ii) extraction cycle duration,where samples were extracted by shaking for 60, 90, and 120 min; and(iii) extraction repetition, where the procedure was repeated up to3 times until exhaustion.One gram of ground cereals was stirredfor 60 min at 240 strokes/min with 20 mL of ethyl acetate and thencentrifuged for 10 min at 14,000 rpm. The supernatant (1000 μL)was dried under nitrogen flow. After two repetitions, the supernatantswere pooled, reconstructed into 1 mL of mobile phase B, and injectedinto the UHPLC–TWIMS–QTOF.26 by using 5 mM ammonium formatein Milli-Q water/methanol  (solvent A) and 5 mM ammoniumformate in isopropanol/methanol/Milli-Q water  (solventB) both acidified with 0.1% formic acid. The following multistep elutiongradient was used: 0.0 min  to 1.0 min, subsequently 1–5 min , and 11.0 min . Aftera 4.5 min isocratic step, the system was re-equilibrated to initialconditions for 2.5 min . Samples were permanentlykept at 10 °C.An ACQUITYI-Class UPLC separation system coupled to a Vion IMS QTOF mass spectrometer equipped with an electrospray ionization (ESI)interface was employed for AR profiling. Samples were injected (1μL) and chromatographically separated using a reversed-phaseC18 BEH ACQUITY column . Gradient elution was performed as previouslyreported,m/z 100–1000. Source settings were maintainedusing a capillary voltage of 2.5 kV, a source temperature of 120 °C,a desolvation temperature of 500 °C, and a desolvation gas flowof 1000 L/h. A TOF analyzer was operated in sensitivity mode, anddata were acquired using HDMSE, which is a data-independent approach(DIA) coupled with ion mobility. The optimized ion mobility settingsincluded a nitrogen flow rate of 90 mL/min (3.2 mbar), a wave velocityof 650 m/s, and a wave height of 40 V. The device within the Vionwas calibrated using a Major Mix IMS calibration kit  to allow for CCS values to be determined in nitrogen. The calibrationcovered the CCS range from 130 to 306 Å2. The TOFwas also calibrated prior to data acquisition and covered the massrange from m/z 151 to 1013. TOFand CCS calibrations were performed for both positive- and negative-ionmode. Data acquisition was conducted using UNIFI 1.8 .Mass spectrometry data were collected innegative electrospray mode over the mass range of Supporting Information (Table S1).Alkylresorcinol identification was performed by comparisonof retentiontime, fragmentation patterns, and collision cross sections with thestandard collection in our UNIFI library, created by running a mixof standards with the same analytical method. Quantification of targetanalytes was performed using external standard calibration (rangeof 0.1–25 mg/kg). Quantification of unsaturated AR was based on the relative responseof an alkylresorcinol with the same chain length due to the lack ofanalytical standards. AR values were reported on a dry matter (DM)basis. The main validation parameters are briefly summarized in the Statisticalanalyses were performedusing IBM SPSS v.23.0 . Data were analyzedby ANOVA followed by Tukey’s post hoc test (α = 0.05).Principal component analysis was constructed with log-normalized andpareto-scaled data using SIMCA V.16.0.2 .27 Briefly, samples were embedded in 2% gelatin,and then, 20 μm-thick sections were cut from the middle of eachgrain at −20 °C using a cryomicrotome . To obtain uniform sections,due to the fragility of the seed, adhesive tape kept over the trimmedsample during cryosectioning was used. The sections were transferredto a glass slide and kept at −80 °C until the day of theanalysis. Before matrix application, optical images of the sectionswere captured using a digital microscope VHX-5000 . DHB (10 mg mL–1) in acetonitrile  was sprayed with a pneumatic sprayer 28 to ensure uniformcoating of tissue sections with the microcrystalline matrix. The sizeand uniformity of the deposited crystals were checked prior to AP-SMALDIMSI experiments.Samplepreparation for atmospheric-pressure scanning microprobe matrix-assistedlaser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI)was performed following the protocol previously optimized.29 The dried-dropletmethod was used to assess different matrices, by mixing 1 μLof an alkylresorcinol standard with 1 μL of matrix solutionand spotting 0.5 μL onto an 80-well stainless-steel plate.This combination of a matrix and a solventwas chosen because it gives rise to the highest signal intensitiesfor alkylresorcinol standards, following the protocol previously optimizedfor urushiol MALDI ionization.Imaging experiments of wheat seed sections were performed usinga high-spatial-resolution (≥5 μm step size) AP-SMALDIMSI ion source  coupledto a Q Exactive HF orbital trapping mass spectrometer .30 For the experiments describedbelow, a scanning step size of 20 μm was set. The mass spectrometerwas operated in positive-ion mode. The following parameters were set:scan range, m/z 250–1000;target voltage, +3 kV; capillary temperature, 250 °C; automaticgain control (AGC) was disabled; cycle time for one pixel, 1 s. Internalmass calibration was performed using known matrix ion signals as lockmass values (m/z 716.12462), providinga mass accuracy of better than 2 ppm root-mean-square error over theentire measurement.The smallest laser beam focusresulted in an ablation spot diameterof 5 μm.m/z valueswere generated using the MIRION software package31 with a bin width of Δ(m/z)/(m/z) = ±5 ppm.MS images were normalized to the highest intensity of each ion species.The METASPACE platform (https://metaspace2020.eu/) was employed for metabolite annotation of MSI data, selecting Metlinand LIPID MAPS as databases.Ion images of selected Extractionrecovery is a key step in the characterization of bioactivecompounds in plants. Here, we optimized the winter cereal sample preparationto obtain a complete recovery of AR.14 and no derivatization steps1 are required.Starting from the solvent/matrixratio, a greater volume (1 g with 30 mL) did not provide a higherAR recovery or other advantages if compared to 20 mL. The extractionefficiency did not increase with time , suggestingthat the equilibrium of the solute inside and outside the plant matrixwas reached after 60 min. On the other hand, repetition of the extractionprocess with a fresh solvent increased the content of AR, suggestinga saturation of the extraction solvent during the first cycle. Theoptimized sample preparation finally included two extraction cyclesof 60 min using 20 mL of ethyl acetate. The first cycle extracted92.7% (±1.05) of the total AR, while the second cycle extracted7.3% (±1.14). The procedure was repeated up to 3 times, but inthe third extraction, no AR were detected, indicating that the matrixwas exhausted after 2 cycles. Compared to previously developed extractionprocedures, a shorter extraction timeTriticum spp. andtritordeum  were identified in winter cereals, which agreed withprevious reports.33 In particular, thepredominant homologue in all five species was AR 21:0, representingalmost half of the total AR content, with a percentage ranging from48.1 (tritordeum) to 58.3% (emmer). AR 19:0 was the second most abundanthomologue in the hexaploid species T. aestivum spp. aestivum (25.3%) and spp. spelta (26.1%) followed by AR 23:0, AR 19:1, AR 17:0, AR 21:1, and AR 25:0.In contrast, tritordeum, emmer, and einkorn contained higher portionsof AR 23:0, that is, 32.9, 28.4, and 28.3%, respectively, followedby the homologues AR 19:0, AR 25:0, AR 21:1, and AR 19:1 (34The presenceof AR homologues (AR 17:0–AR 25:0) in selected grains was measuredby UHPLC–IMS–HRMS. The average content of AR rangedbetween 672.5 ± 129.8 and 1408.9 ± 528.0 mg/kg of dry matter(DM) among different cultivars of itordeum 2. These  AR 19:1 2. This tTriticum speciesindicated that the accumulation of AR is influenced both by the geneticbackground and the environmental factors.36 Therefore, MANOVA was carried out on ARhomologues, considering their accumulation over the growing locations,varieties, and harvesting years. By evaluating the effect of eachfactor on the different variables, the year resulted to be alwayssignificant (p < 0.01) while location and varietyonly for some homologues as shown in Recent studies involving different Table S2 of the Supporting Information).As a consequence, in both sites, the severity of foliar  and head (Fusarium head blight) disease in 2017–2018was higher, leading to a quicker senescence process and a clearlylower test weight (TW) and thousand-kernel weight (TKW). On average,grain yields were 4.2 and 3.3 t ha–1 in 2017 and2018, respectively (see Table S3 of theSupporting Information), as a consequence of the lower TW (72.1 vs67.2 kg hl–1) and TKW (45.3 vs 38.7 g). A low starchaccumulation in the grain during ripening in 2018 may have determineda lower dilution of the AR contents, concentrated mainly in the outerlayer.37Similarly, the PCA in –1 DM) and common wheat(956.5 ± 356.1 mg kg–1 DM) contained the highestAR concentration followed by the tetraploid emmer (843.3 ± 106.4mg kg–1 DM) and hexaploid spelt (817.9 ± 174.7mg kg–1 DM). Diploid einkorn (770.0 ± 251.9mg kg–1 DM) exhibited the lowest AR concentrations and Aubusson (1307.7 ± 391.3 mg kg–1 DM) modern wheats (see –1 DM) andAndriolo (772.4 ± 89.5 mg kg–1 DM) reportedthe lowest AR content. Previous reports on modern and old wheat cultivarswere mainly focused on the comparison of the total phenolic content,39 while no data on AR have been reported so far.The higher accumulation of AR in modern genotypes may be explainedconsidering the differences in kernel size among genotypes. Indeed,modern cultivars  are characterizedby a small kernel size and a higher pericarp/endosperm ratio comparedto old genotypes that are considerably larger .Focusing on common wheat,11 cultivars were analyzed includingold, modern, and pigmented varieties see 1. The AReats see 2A. On th25 but no difference in thephenolic lipid was found. This may be explained by the involvementof separate biosynthetic pathways or at least by an independent regulationof alkylresorcinol synthases, as in the case of anthocyanins whereboth classes share the involvement of type III PKS enzymes.40The AR contents of pigmented wheatswere in line with those ofthe other modern varieties. These grains are characterized by a higherconcentration of carotenoids (cv. Bonavita) or anthocyanins (cv. Rossoand Skorpion),p < 0.05) over the two harvesting yearsonly for modern common wheat varieties  and notfor old ones  and pigmented (−20%) than old genotypes (−6%)(see Table S3 of the Supporting Information).Since old wheat cultivars have a lower capacity to respond to productionfactors with a modification in the yield components and the graintraits , a greater stability oftheir composition is expected also within different production situations,as far as the intensity of the cropping system is concerned .25Regarding the environmental effects, thecontents of AR significantlychanged (old ones 2B. This Triticum species.42 The ratios for common wheat (0.09 ± 0.04), spelt (0.05± 0.02), and emmer (0.04 ± 0.00) were in line with thosereported in the literature,14 while foreinkorn, this evaluation was not possible with AR 17:0 < LOQ. AR17:0 is known to be present in einkorn,38 even if in a small amount, but we could not detect it probably dueto its low ionization efficiency obtainable with an ESI source, comparedto other sources such as APCI.43Furthermore, the ratio of some AR homologues wascalculated anddiscussed 2.In parTriticum species.44The indicator significantly decreased according to hexaploid >tetraploid ≥ diploid. This confirmed the important role ofthe genetic background on the accumulation of AR in wheat, in agreementwith evidence in the literature reporting on the remarkable heritabilityof the AR content, and the differences in AR profiles between hexaploid,tetraploid, and diploid 6 As reportedin 14 suggestingthe stability of this indicator despite the great variability of environmentalconditions. Indeed, even though the absolute concentration of AR 21:0and AR 23:0 varied significantly .Conversely to the trend observed for commonwheat varieties, theAR content in tritordeum varieties did not change significantly overthe two harvesting years, suggesting a lower susceptibility to environmentalconditions  compared to the other species consideredin the present study. This can be explained considering the ratioof this durum wheat parent,Figure S2 of the Supporting Information). Thesederivatives were reported for the first time in rye, and their presencein all the evaluated samples suggests a higher diversity in the ARprofile than previously suggested.48Five 5-(2-oxo)AR were putatively identified, as summarized in Since analytical standardsare not available, we tentatively identifiedthem using HRMS checking the exact mass (mass error less than 3 ppm),the match of experimental and theoretical isotope pattern in termsof spacing and relative intensities, comparing their retention timewith the corresponding nonoxidized homologue and by investigatingtheir fragmentation pattern.m/z 417.3364, correspondingto the [M – H]− ion with a putative formulaC27H46O3 . The other two fragment ionswere identified corresponding to the α-cleavage (m/z 123.0446) and its rearrangement (m/z 81.0341). Indeed, as previously described,20 the cleavage arose from the α-positionof carbonyl, suggesting that the oxidation occurred in position 2′.Furthermore, the earlier elution (6.03 min) of such a form from theC18 column compared with its nonoxidized homologue AR 21:0 (7.0 min)was consistent with the putative identification. The fragmentationmass spectra of the other AR oxo are show in the Supporting Information.As an example, the AR 21:0 oxowas identified in the full scanmass spectrum at C27H46O3 3. To verC27H46O3 3 shows t48 but there was no informationabout their relative abundance in different cereal species. Differentfrom saturated AR in which the most abundant homologue was AR 21:0,the most abundant oxo homologue was AR 23:0 oxo in all five speciesfollowed by AR 21:0 oxo, AR 25:0 oxo, AR 23:1 oxo, and AR 21:1 oxoin common wheat and spelt. On the other hand, for einkorn, emmer,and tritordeum, AR 23:0 oxo was followed by AR 25:0 oxo, AR 21:0 oxo,AR 23:1 oxo, and AR 21:1 oxo .5-(2-Oxo)AR were found in rye and in other winter cerealsas reportedby Suzuki,49 caused by several bioticand abiotic stressors. Indeed, considering the ratio AR 21:0/AR 23:0previously described, these two species can be considered more susceptibleto fungal infestation, and thus, they may be exposed to higher oxidativestress.Einkorn and emmer showed thehighest contents of all the 5-(2-oxo)ARhomologues. This might be due to the accumulation of reactive oxygenspecies (ROS)50 This could explain why the major5-(2-oxo)AR homologue detected in the present study was AR 23:0 oxo,while the saturated one was AR 21:0. Indeed, it may be postulatedthat the lower accumulation of AR 23:0 was due to the preferred synthesisof the corresponding oxo homologue, resulting in a lower accumulationof saturated AR 23:0.Few publications reported that 5-(2-oxo)AR derives froma commonintermediate of AR, which is a β fatty acid, and not directlyfrom oxidation of AR.Further studies are required to understandthe role in plant ofthese AR and the modulation of their biosynthetic route, but theirwidespread presence in different species and cultivar is confirmedby our data.Table S4 of the Supporting Information. However,we could not find any of them. So far, conjugated derivatives werealready reported for Cybianthus magnus,while in cereal AR, homologues from 15 C are present.Furthermore, conjugated derivatives of AR weresearched. The completedatabase of glucoside and methyl derivatives is reported in us magnus and GrevFigure S5).AR werealso investigated by MSI to unveil their spatial distributionwithin the common wheat kernel. Localizations of AR were studied usingtransversal cross sections made from the middle of the grain. SixAR were detected and localized at the tissue level, among other alsounsaturated side-chain homologues, including AR 19:0, AR 19:1, AR21:0, AR 21:1, AR 23:0, and AR 25:0. Their MSI images are reportedin Homologues ranging from C19 to C25 were detectedas protonatedand/or sodiated ion species, while AR 17:0 was not found. This mightbe due to its low concentration and to its lower ionization efficiencycompared to the longer homologues.Figure S5). No AR were found in the endosperm or in the germ layers.A distinct pattern of accumulationwas noticed for separate homologues.In particular, AR 21:0 was found to be accumulated mainly in the cuticleof the testa, while AR 21:1 was located both in the inner and outercuticle of the pericarp see 4. Also, 51 Here, the exact location of differentAR homologues has been made possible by the use of high-resolutionAP-SMALDI MS imaging.AR distribution has been traditionally investigated by analyzingtheir content in pearling fractions, and microscopy has targeted themto the pericarp layer.6 thus, their localization is consistent with their biologicalrole as antifungal compounds. Indeed, their amphiphilic structuresuggests their involvement in plant defense, and the concentrationwithin the outer layers seems to be sufficient to act as a chemicalbarrier against the fungal spread. These data complement, with theaccuracy of the details allowed by MS imaging, previous reports onthe distribution of AR in wheat kernels.In winter cereals, these secondary metabolitesare reported toinhibit the growth and spread of fungal infections;


The identification and quantification of this imaging biomarker allows for estimating the level and possibly the amount of intraretinal inflammation in major degenerative retinal disorders, whose inflammatory component has already been demonstrated . Currently, diabetic retinopathy (DR) probably represents the best clinical model to apply this analysis in the definition of this clinical biomarker. However, the main limitation to the clinical use of HRF is related to the technical difficulty of counting them: a time-consuming methodology, which also needs trained examiners. To contribute to solve this limitation, we developed and validated a new method for the semi-automatic detection of HRF in OCT scans. OCT scans of patients affected by DR, were analyzed. HRF were manually counted in High Resolution spectral domain OCT images. Then, the same OCT scans underwent semi-automatic HRF counting, using an ImageJ software with four different settings profiles. Statistical analysis showed an excellent intraclass correlation coefficient (ICC) between the manual count and each of the four semi-automated methods. The use of the second setting profile allows to obtain at the Bland–Altman graph a bias of −0.2 foci and a limit of agreement of ±16.3 foci. This validation approach opens the way not only to the reliable and daily clinical applicable quantification of HRF, but also to a better knowledge of the inflammatory component—including its progression and regression changes—of diabetic retinopathy.Optical coherence tomography (OCT) allows us to identify, into retinal layers, new morphological entities, which can be considered clinical biomarkers of retinal diseases. According to the literature, solitary, small (<30 µm), medium level hyperreflective  retinal foci (HRF) may represent aggregates of activated microglial cells and an  HRF are described as small, punctiform hyperreflective lesions, visible on linear OCT scans in the retina, either in normal subjects and in patients with signs of chorio-retinal diseases –8. They ammation , 9, 10. ammation .One of the main issues in the clinical application of HRF is that that their manual counting on OCT scans depends on the learning curve of the examiner, and it takes time to be reliable and reproducible. For this reason, it can be understood the importance of a new approach able to carry out the HRF count in a semi-automated, standardized, reproducible, fast and objective way.In this study we report the experience of the development of a method that allows semi-automatic HRF count, validating it versus the manual reference count made by an expert operator.This is a retrospective evaluation of OCT images obtained in eyes affected by DR and collected from February to June 2020 (during COVID pandemic). This research was carried out in accordance with the Declaration of Helsinki. Because images were completely anonymous, a communication to the Local Review Board was performed, according to the current Italian and Regional legislation. A horizontal 180° linear OCT scan of 8.8 mm centered onto the fovea acquired in High Resolution modality with a 100 ART resolution was carried out using Spectralis HRA + OCT .Inclusion criteria for the OCT scans were: correct centering of the scan; image quality >30 . Each OCT scan was cropped at 1,500 µm from the fovea, both on the nasal and temporal side, with the aim of highlighting the central 3 mm of the retina, as previously reported , 11  was applied. The “denoised image” obtained was used as mask for applying the CLAHE  tool setting . On the resulting image the Spot Counter plugin was applied with different setting for each of the four methods  n (whole number of spots counted); (b) spot mean ; (c) image mean ; (d) list of each single spot intensity. The semi-automated analysis procedure is fully described in HRF were counted from the boundary between RNFL and ganglion cell layer (GCL) to the external limiting membrane (ELM). The reflectivity of RNFL, in fact, is too similar to that of the HRF to allow for any reliable distinction. Similarly, in retinal layers external to the ELM, any photoreceptor debris or pigmentary material may confound the exact HRF count. Subsequently, the same OCT images underwent a semi-automated analysis using an open-source available ImageJ software . Four different profiles of settings were applied to the software for the analysis of each image and the results were then compared to those obtained with the manual count. Briefly, the cropped images were imported in the software and the 8-bit format was settled. For the first and second method the plugin “ methods Table 1 The settled parameters have been specifically designed to prevent the software from counting any hard exudates or retinal vessels as HRF. However, a further correction factor was included to avoid the influence of the average intensity of the entire OCT image on HRF count. Then, the average image intensity value was multiplied by 2 and all HRF with an intensity greater than this value were excluded. This allowed for a greater precision in counting only those foci that really represent aggregates of activated microglia.The ability of the method to reproduce the manual count was assessed using ICC and the Bland–Altman diagram . The folWe collected 194 OCT linear scans of 97 diabetic patients. Forty-eight scans were excluded because of image quality <30, and six additional scans were excluded because of uncorrected centering of the scan. Thus, 140 OCT linear scans of 89 diabetic patients were processed. The mean age was 65.8 ± 9.5 years: 40 were female and 49 were male. Five patients were affected by type 1 diabetes (4.5%) and 84 by type 2 diabetes. All patients were affected by non-proliferative DR.The mean number of HRF at manual count was 58.9 ± 36.9 foci. After comparison of the four semi-automatic counting method, the second method showed the best results in terms of mean count of HRF (58.6 ± 39.7 foci) compared to the manual counting method, with a ICC of 0.98, a bias of −0.2 foci, and a range of ±16.3 foci at the Bland–Altman diagram Figure 3The use of objective, quantifiable medical signs, named as biomarkers, has become of greater relevance in clinical practice and research. In fact, they not only allow the identification of a normal biological or pathological process but also the quantification and follow-up of the process itself, becoming clinical and diagnostic tools in the common practice but also surrogate endpoints in clinical research . MoreoveSeveral steps forward have been made since Coscas et al.  first noMoreover, some Authors agree that the presence and number of HRF is a reliable biomarker of retinal response to treatment and even hypothesized an independent predictor of visual outcome, in DME but also in other retinal vascular diseases , 27.The quantification of HRF may be useful not only in DR. Several studies showed the importance of the number and location of HRF, from a diagnostic and prognostic point of view, in diseases such as age-related macular degeneration , multiplIn conclusion, this study provides a semi-automated, objective, reproducible and reliable method to count HRF using an open-source software in DR. This allows, even without the contribution of an expert physician, to semi-automatically count HRF, which may represent an important biomarker of inflammation in DR. This will allow to identify and grade a specific phenotype of DR and DME and offer a contribute to clinicians in quantifying the change of this phenotype after treatment.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee for Clinical Practice of the Azienda Ospedaliera di Padova. The patients/participants provided their written informed consent to participate in this study.Conceptualization, EM, LF, and EP. Methodology, TT, LF, RP, and EP. Validation, EP and EM. Formal analysis, TT, LF, RP, and EV. Investigation, TT, LF, EV, and EP. Resources, EP and EM. Data curation, TT, RP, and LF. Writing—original draft preparation, TT and EV. Writing—review and editing, LF, RP, and EM. Supervision, EM. All authors contributed to the article and approved the submitted version.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.


In chronic obstructive pulmonary disease (COPD), rehabilitation is recommended, but attendance rates are low. Tele-rehabilitation may be key. We evaluate the effect of a tele-rehabilitation program vs. standard rehabilitation on COPD. A randomized, non-inferiority study comparing eight weeks of tele-rehabilitation ) and standard rehabilitation in stable patients with COPD. At baseline, after 8 weeks and 3 and 6 months of follow-up, 6 min walk test distance (6MWTD), 7-day pedometry, quality of life, exercise tolerance, adherence, patient satisfaction and safety were assessed. Fifty-four patients  were included. Twenty-seven patients were randomized to tele-rehabilitation. Non-inferiority in Δ6MWTD at 8 weeks (47.4 ± 31.4), and at 3 (56.0 ± 38.0) and 6 (95.2 ± 47.1) months follow-up, was observed. No significant difference was observed in 7-day pedometry or quality of life. In the intervention group, 6MWTD increased by 25% and 66% at 3 and 6 months, respectively; adherence was 81%; and patient satisfaction was 4.27 ± 0.77 . Non-inferiority between groups and high adherence, patient satisfaction and safety in the intervention group were found after rehabilitation and at 3 and 6 months of follow-up. Tele-rehabilitation with VAPA seems to be a promising alternative. Rehabilitation forms part of the standard treatment in COPD as it improves the quality of life and exercise capacity and reduces symptoms and mortality , as wellwww.randomization.com (accessed on 19 January 2017) [The study was performed as a prospective, single-center, non-inferiority randomized, prospective clinical study comparing standard rehabilitation in patients with COPD with tele-rehabilitation. Randomization was performed digitally at ry 2017)  with subPatients with spirometry and physician-verified COPD from the outpatient clinic of the Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Denmark, were randomized if they: (1) were >18 years of age, (2) had been referred for standard rehabilitation and (3) signed an informed consent form. Musculoskeletal abnormalities, dizziness, substantial sensory or motor impairments, dementia and/or severe comorbidities that impeded training were all exclusion factors . Oxygen Patients were randomized 1:1 to 8 weeks of standard or tele-rehabilitation . During In the standard rehabilitation group, patients with COPD underwent a conventional standardized rehabilitation program, as implemented at Aarhus University Hospital. Patients attended 2 weekly group training sessions of 1 h at the hospital with instruction by a physiotherapist and 6 h of education about COPD and its treatment for 8 weeks .The primary endpoint was the difference between groups in the 6 min walk test distance (6MWTD)  from bast-test to analyze the secondary endpoints because 1. It is a robust test even with no normal distribution; 2. We did not find any outliers; 3. There was no previous hypothesis on the direction of the effects for the secondary endpoints; and 4. We expected a substantial number of dropouts potentially preventing the use of an analysis of variance (ANOVA). R and IBM SPSS Statistics version 25 were used to conduct the statistical analysis. According to their randomized treatment group, all patients’ results were analyzed, regardless of their adherence to or the received intended treatment, characterizing the use of the intention-to-treat approach [The primary endpoint is based on a minimally important clinical difference of 35 m in the 6 MWT distance . A 30% dapproach .A total of 95 COPD patients were screened, with 54 of them being enrolled in the study. The baseline demographics of eligible patients who declined to participate were similar to those who were included. Fifty-four patients with COPD were included from March 2017 to March 2019; 27 patients were randomized to standard rehabilitation and 27 to tele-rehabilitation with VAPA . Reasonsp = 0.14). Mean differences between groups and confidence intervals were calculated  to 3 months  and 10.5 min (+66%) from 8 weeks to 6 months . Overall, adherence was 82%. Patient satisfaction using the 5-point Likert scale scored 4.27 . No adverse events were reported by participants in the tele-rehabilitation with VAPA group during the trial (The exercise set mean time showed a difference of a 4 min (+25%) positive increment from 8 weeks (he trial .Results regarding exclusion criteria ; reasonsThe current study assessed VAPA, a new digital platform for tele-rehabilitation in COPD patients as an alternative to hospital-based rehabilitation. VAPA tele-rehabilitation was found to be non-inferior to traditional hospital rehabilitation in terms of sustained exercise capacity, and patient exercise tolerance, adherence, and satisfaction, and there were no safety issues. With respect to exercise capacity, tele-rehabilitation was non-inferior compared to standard rehabilitation based on the minimal important clinical difference of 35 m in the 6MWT endpoints . During Previous tele-rehabilitation studies in COPD ,16 have We found no statistical difference in 7-day pedometry or VMCPM between groups or over time in each group, although we saw a trend towards better performance with standard rehabilitation. This may in part be inherent in that patients in the control group had to visit the hospital twice a week during the first part of the trial. However, their pedometry data decreased at a similar pace compared to VAPA tele-rehabilitation. The large standard deviation in both groups may be explained in part by the number of patients and the fact that patients began the research at different seasons of the year, since patients entered during the summer may have walked more than those enrolled during the winter.We tried to digitize as much of the rehabilitation program’s information as feasible at VAPA and combined exercise training with empowering e-learning packages on how to live with COPD. There was no statistically significant difference between the groups in terms of QoL, i.e., the impact of digital e-learning and training was non-inferior to standard rehabilitation. These results suggest that the approach executed with e-learning was successful.There are a variety of technologies and tele-rehabilitation platforms to choose from. The majority rely on videoconferencing, with one or more patients practicing in real time with a therapist, or virtual reality technologies, with agents demonstrating the exercises. ,25. PatiThe aim of this technique is to allow patients to engage in pulmonary rehabilitation in the hospital rehabilitation unit over the internet. The second aim is to maintain telemediated training as a social event similar to meeting face-to-face at the hospital. The intention is to support the social needs of patients training in a virtual group similar to group training in standard rehabilitation programs. Patients benefit from this setup since they can train at home, but training is still planned at specific times, exactly like in the hospital setting, making it less flexible than the VAPA platform. In contrast to VAPA, where patients can train whenever they choose, patients must still schedule their day and energies around the training appointment . It is uThe patient drop-out rate in tele-rehabilitation with VAPA was lower than in the standard rehabilitation group but similar to the dropout rate that Sohanpal et al. found in their review on self-management support programs for COPD patients .There are several strengths of our study, including the randomized design and the long-term follow-up. Our study also has several limitations. The power calculations based on 6MWT do not allow the calculation of non-inferiority for the secondary parameters. The high drop-out rate, which is potentially attributable to a lack of commitment and 9transportation issues in standard rehabilitation and the small number of randomized participants that may have caused participants receiving tele-rehabilitation with VAPA to be younger, may have impacted the results. Patients with severe and advanced disease, on the other hand, are more likely to drop out, and our drop-out percentage is comparable to that reported in rehabilitation programs for patients with other chronic respiratory disorders ,51.Tele-rehabilitation with VAPA is non-inferior with respect to exercise capacity in patients with COPD when compared to a standard 8-week rehabilitation program. Tele-rehabilitation patients had a relatively high exercise time, high adherence and high patient satisfaction, and participation was without risk. Tele-rehabilitation with VAPA is a viable alternative rehabilitation approach for COPD patients, as well as a potentially effective tool for increasing COPD positive behavioral change toward a more physically active lifestyle.


Targeting the AR therefore remains the cornerstone for mPCa treatment. We have previously reported that activation of AR signalling affects taxane chemo-sensitivity in preclinical models of castration resistant PCa (CRPC). Here, we explored the anti-tumour efficacy of the AR targeted inhibitor enzalutamide combined with cabazitaxel.: We used the AR positive CRPC model PC346C-DCC-K to assess the in vitro and in vivo activity of combining enzalutamide with cabazitaxel. Subsequent validation studies were performed using an enzalutamide resistant VCaP model. To investigate the impact of AR signalling on cabazitaxel activity we used quantitative live-cell imaging of tubulin stabilization and apoptosis related nuclear fragmentation.: Enzalutamide strongly amplified cabazitaxel anti-tumour activity in the patient-derived xenograft models PC346C-DCC-K  and VCaP-Enza-B . Although enzalutamide treatment by itself was ineffective in reducing tumour growth, it significantly suppressed AR signalling in PC346C-DCC-K tumours as shown by AR target gene expression. The addition of enzalutamide enhanced cabazitaxel induced apoptosis as shown by live-cell imaging (P<0.001).: Our study demonstrates that cabazitaxel efficacy can be improved by simultaneous blocking of AR signalling by enzalutamide, even if AR targeted treatment no longer affects tumour growth. These findings support clinical studies that combine AR targeted inhibitors with cabazitaxel in CRPC. Androgen receptor (AR) signalling remains a key driver of neoplastic behaviour and therapeutic resistance in castration resistant prostate cancer (CRPC). We have previously reported that AR signalling drives taxane resistance in models of CRPC.Here we show that concomitant targeting of AR signalling by enzalutamide improves cabazitaxel activity even in tumours that have developed enzalutamide resistance. Here enzalutamide suppressed AR signalling, although tumour growth was not impacted, which reflected the treatment resistant phenotype of the CRPC models. Targeting the AR by enzalutamide enhanced cabazitaxel induced apoptosis and further suppressed proliferation. Overall, our study shows that cabazitaxel treatment efficacy is strongly enhanced by targeting AR signalling in models of CRPC.Our study paves the way for combining taxane chemotherapeutics with AR targeted agents, even for CRPC patients that have progressed on AR targeted compounds.Alt-text: Unlabelled box1in vivo [The androgen receptor (AR) pathway is a major driver of neoplastic behaviour in the different stages of metastatic prostate cancer (mPCa). Hence, androgen deprivation therapy (ADT), which blocks the production of the AR ligands testosterone and dihydrotestosterone levels, is the cornerstone of mPCa treatment. Although ADT effectively induces disease regression in the vast majority of patients, disease recurrence is inevitable and is classified as castration resistant prostate cancer (CRPC). Multiple studies have shown that the AR continues to play a major role in ADT resistance and progression to CRPC, as AR amplifications and mutations conferring androgen hypersensitivity and ligand promiscuity are frequently identified in samples from CRPC patients ,2. Largein vivo ,7. Moreo22.13 cells per well in 100 µl medium. VCaP-Enza-B cells were maintained in RPMI containing 10% steroid stripped (DCC) serum, while PC346C-DCC-K cells were maintained in PGM-DCC medium [AR, AR-V7 and several AR target genes. In short, RNA was isolated using the RNeasy mini kit  and qRT-PCR was performed as described previously AR, AR-V7, KLK3 (PSA) (using custom assays), FKBP5 and intact TMPRSS2 . PBGD and HPRT1 were measured by SYBER Green assay (SensiMix Syber Lo-ROX). The custom TaqMan assays and primer sequences used for SYBER Green have been described previously [The AR expressing CRPC model PC346C-DCC-K was obtained by long-term exposure to steroid stripped cell culture conditions as described previously . VCaP-EnC medium ,9. The f3in vivoAll animal experiments were approved by the Animal experiment committee under the Dutch experiments on Animal Act, with the reference number AVD101002017867. The current study is in compliance with the Arrive guidelines. Group size and experimental set-up were based on pilot experiments or previous studies using taxane treatment 3.16 PC346C-DCC-K cells. Tumour growth was observed after 2-5 weeks in 41 (∼80%) mice, to adhere to the predefined group size we added five mice from the RNA-sequencing experiment described below, as they were inoculated on the same day. Once tumour size surpassed a volume of 150 mm3, mice were stratified based on tumour size to receive daily oral enzalutamide treatment at 60 mg/kg or vehicle control  3 in size, mice were again stratified based on tumour volume to receive a single intraperitoneal administration of 33 mg/kg cabazitaxel or placebo control (NaCl). Overall, we had four treatment groups, cabazitaxel and enzalutamide mono-treatment, the combination treatment and placebo control, with 9-12 mice each. Tumour volume was monitored every 3-4 days by callipers and mice were followed until tumours exceeded a volume of 1500 mm3, or a maximum follow-up of 60 days after cabazitaxel treatment and subsequently euthanized by cervical dislocation. Other reasons for euthanizing the mice included continued weight loss, >15% loss in bodyweight in two days or >20% compared to start, and abnormal behaviour. Mice were maintained in an individually ventilated cage at 2-4 mice per cage, on a 12h dark/light cycle and cage enrichment was provided. Treatments and other procedures were initiated in the morning and food and water were provided ad libitum. Blood samples for PSA analysis were obtained at first tumour measurement, when mice were stratified to receive treatment and at the end of the experiment. For VCaP-Enza-B, we performed a pilot study to assess tumorigenicity, response to enzalutamide and cabazitaxel treatment. Sixteen mice were inoculated with 5*106 VCaP-Enza-B cells mixed with 100 µl Matrigel , tumour take was observed in all mice. Once tumours surpassed 150 mm3, mice were stratified based on tumour volume to receive daily enzalutamide treatment (60 mg/kg) or vehicle control for a period of 14 days. Impact of enzalutamide treatment on tumour growth during treatment was assessed using the log10-cell kill method 6 VCaP-Enza-B cells mixed with Matrigel , tumour take was observed in 19 out of 20 mice. Once tumours surpassed 150 mm3, mice were stratified to receive daily enzalutamide (60 mg/kg) treatment or vehicle control, then all mice received a single intravenous administration of cabazitaxel at tumour volume 750 mm3 at 10 mg/kg. Histology of the VCaP-Enza-B tumours and expression of the AR and cell cycle marker Ki67 were subsequently investigated by immunohistochemistry as described previously Fifty-two, six weeks old, NMRI nu/nu male mice  were subcutaneously inoculated with 5*103.2Twenty-one, six-week-old, NMRI nu/nu male mice were subcutaneously inoculated with PC346C-DCC-K cells, tumour take was observed in 20 mice. We estimated that we required 15 samples to perform differential gene expression analysis and therefore transferred five mice to the treatment efficacy experiment described above. The experimental set-up was similar as described above with the following adaptations: mice were stratified to a 2:1 ratio to receive daily enzalutamide treatment or vehicle control for seven days and were subsequently sacrificed and tumour xenografts snap-frozen. Total RNA was isolated from tumour xenografts as described previously 3.3H2B gene fused to red fluorescent protein (H2B-RFP). The EYFP-β-tubulin expression was used to monitor taxane target engagement and the histone marker for visualizing treatment induced perturbations of mitosis, proliferation and apoptosis. PC346C-DCC-K cells were first transduced to stably express H2B-RFP and selected based on RFP expression. Next cells were transfected with EYFP-β-tubulin and again selected based on fluorescent expression of both markers. Large scale lentiviral production was performed by calcium phosphate transfection of Hek293T (RRID: CVCL_1926) cells with the LV-RFP construct expressing H2B-RFP, pMD2.G plasmid for the viral envelope and psPAX2 for packaging 4 PC346C-DCC-K cells co-expressing H2B-RFP and EYFP-β-tubulin were plated with 100 µl PGM-DCC medium in a 96-wells Cell Carrier Ultra plates suited for live cell imaging . Cells were incubated for 72 hours to ensure optimal attachment and cell spreading. One hour before imaging, cells were pre-treated with 1 µM enzalutamide and/or 0.1 nM R1881. After imaging an initial pre-treatment time point (t-1), cells were exposed to 3 nM cabazitaxel and image acquisition was continued for 100 hours with an interval of 150 minutes. During intervals, the cells were stored in an integrated cell culture incubator to optimize cell viability. Sixteen images covering 103714 µm2  were acquired using an Opera Phenix spinning-disk confocal high-content screening system (PerkinElmer), equipped with a 40x water immersion object and a 16-bit sCMOS 4 Megapixel camera. EYFP-β-tubulin and H2B-RFP were sequentially excited with 488 nm and 561 nm solid state laser lines, detected at 500-550 nm and 570-630 nm wavelength ranges, respectively. Both analyses, apoptosis induction and microtubule stabilization, were performed using custom image analysis protocols in the Harmony analysis software . First, total area covered by cells was selected using the EYFP-β-tubulin signal. The level of microtubule stabilization was determined in the total area covered by EYFP-β-tubulin expressing cells, using the Haralick Contrast parameter [To study the impact of AR pathway inhibition on cellular response to cabazitaxel treatment, PC346C-DCC-K cells were engineered to overexpress enhanced yellow fluorescent protein (EYFP) labelled beta-tubulin (EYFP-β-tubulin) and the human histone arameter ,25. To q3.4in vivo we performed a Kaplan Meier analysis in which we defined survival as tumour volumes ≥1500 mm3 during the maximum follow-up of 60 days after treatment. Subsequent statistical comparison was performed using a log-rank test with a Bonferroni corrected pairwise comparison, as previously described The cell viability data obtained from cabazitaxel treated PC346C-DCC-K and VCaP(-Enza-B) cells was assessed using a non-linear regression analysis in GraphPad prism . The best-fit values were used to interpolate the IC50 value and compared by an extra-sum-of-squares F-test to determine the impact of the combined treatment with enzalutamide. To determine the impact of cabazitaxel treatment and the combination with enzalutamide 3.5This study was financially supported by an unrestricted grant by Sanofi, however Sanofi was not involved in the design, analysis and interpretation of this study or in writing the manuscript.44.1in vitro setting. We used the AR expressing CRPC cell line PC346C-DCC-K, for which we previously reported an interaction between AR signalling and taxane resistance in vivo anti-tumour efficacy of combining continuous enzalutamide treatment with a single administration of cabazitaxel, as compared to both mono-treatments and placebo . The combination treatment of enzalutamide and cabazitaxel was able to induce a complete tumour regression in 4/12 mice and substantially delayed the onset of progression in the remaining mice. Of note, the addition of enzalutamide did not result in greater toxicity, as bodyweight loss following cabazitaxel administration was comparable in mice receiving the combination or mono-treatment (Supplementary figure 1b). Overall, combining enzalutamide with cabazitaxel significantly improved tumour response compared to cabazitaxel monotherapy with median time to humane endpoint 77 days and 48 days respectively . Low dose cabazitaxel treatment was found to induce a partial tumour response, thus creating a therapeutic window to study the addition of enzalutamide. Combining enzalutamide with cabazitaxel treatment improved overall activity compared to cabazitaxel monotherapy, with median time to humane endpoint 80 and 53 days respectively . Principle component analysis of gene expression data clearly separated tumours obtained from enzalutamide treated from placebo control mice a. Subseq4.4Taxane chemotherapeutics exert their function by targeting tubulins leading to microtubule stabilization and cell cycle stalling, which can trigger cell death by mitotic catastrophe. To further unravel the impact of AR signalling on cabazitaxel efficacy, we used live-cell imaging. PC346C-DCC-K cells were transfected to dually express fluorescently labelled H2B c. Either5The present study explored whether cabazitaxel treatment efficacy could be enhanced by targeting the AR. In two PDX models we showed that adding enzalutamide to cabazitaxel induced greater anti-tumour activity, compared to both single treatments. Importantly, the combination treatment was found to remain active in enzalutamide resistant CRPC models where AR downstream signalling was significantly impacted by enzalutamide treatment, although failed to impact tumour growth by itself. Importantly, targeting the AR increased the rate of cabazitaxel induced apoptosis. Overall, our study shows that cabazitaxel efficacy is directly affected by AR signalling, which paves the way for promising combination treatments in CRPC.in vivo.Taxane chemotherapeutics exert their function by targeting the β-tubulin subunit of the tubulin polymer, which is part of the microtubule network, and induces stabilization. During mitosis, taxane treatment interferes with chromatid separation and eventually leads to cell cycle stalling in the G2/M phase. Arrest in mitosis can directly induce cell death by mitotic catastrophe, although cancer cell lines have shown to display variable response to taxane treatment in vivo setting, as the in vitro cell viability assays showed an additive interaction of enzalutamide combined with cabazitaxel. Overall, the underlying mechanisms resulting in greater cabazitaxel sensitivity in enzalutamide treated PCa cells remain to be clarified, although several metabolic pathways were significantly impacted by enzalutamide treatment. Potentially, multinucleated cells may become sensitive to enzalutamide induced suppression of metabolic activity leading to increased rates of apoptosis. Clearly further investigation is warranted to unravel if these pathways contribute to the enhanced cabazitaxel responsiveness.Sustained AR signalling plays a major role in CRPC, as exemplified by the frequency of genetic aberrations and splice-variant expression driving resistance to AR targeted treatment ,27. The in vivo experiments, the greatest impact of the combination treatment was on long-term suppression of tumour-growth after cabazitaxel treatment. Martin et al. did identify an impact of the combination treatment on the frequency of proliferating and apoptotic cells upon further inspection of the 22Rv1 tumours. Overall, we argue that a premature endpoint potentially compromised the assessment of the combination treatment.A preclinical study by Martin et al., also reported an added benefit of combining cabazitaxel with AR signalling targeted treatment in AR positive PCa models The addition of enzalutamide to cabazitaxel in CRPC patients, has been investigated in a phase I clinical trial performed at our institute 6The manuscript was written by LM, RM, RdW, ML, and RdW and reviewed by all authors. The study was designed by LM, RdW, ML and WvW. The VCaP-Enza-B cell line was obtained and characterized by RM, the PC346C-DCC-K H2B/tubulin expressing cell line was obtained and characterized by LM. In vitro experiments were performed by LM and SB. All animal experiment were designed by LM and WvW and performed by CdR and DS. The RNA-sequencing experiment was designed by LM, ML and HvdW and analysed by WvdG and LM. Experimental set-up and data analysis of live-cell imaging experiment was performed by LM and MvR.RDW—Advisory role/speaker fees; Sanofi, Merck, Lilly, Roche, Bayer, Janssen, Clovis and research funding ; Sanofi, Bayer. MPL—Advisory role/speaker fees; Incyte, Amgen, Janssen Cilag B.V., Bayer, Servier, Roche, Pfizer Sanofi Aventis Netherlands BV, Astellas and has received research funding  from Sanofi, JnJ, Merck and Astellas. RHJM—has received research funding  from Sanofi and Astellas. WvW—has received research funding  from Sanofi, Millennium, Janssen Pharmaceuticals, and Servier. LM—Advisory role/speaker fees from Sanofi. HvdW — Advisory role/speaker fees from Bayer.