Patient self-selection of over-the-counter medicines for the management of allergic rhinitis is suboptimal. The mapping of the allergic rhinitis network demonstrates that patients’ decisions with regards to their allergic rhinitis management can be influenced by up to 11 individuals/resources . This study aimed to identify the role of alters within the allergic rhinitis network and identify the factors that determined their degree of influence as perceived by the patient. This research was a qualitative exploration embedded in an empirical framework and social network theory. People with allergic rhinitis were interviewed about their network and transcripts were analysed deductively and inductively. Transcripts were coded by researchers independently and then discussed until agreement was reached. Forty-one participants described the roles of 17 alters on their allergic rhinitis management. The roles of alters fell within five categories: diagnosis, medication prescription/supply/administration, medication recommendation, information about allergic rhinitis and emotional support. Participant interactions with these alters were often acute and had a long standing effect, with the participants often navigating the long-term management on their own. The significance of the influence of each alter on their allergic rhinitis management was dependent on the level of trust in their relationship, impact of the role made to the participants’ day-to-day management of allergic rhinitis and/or the participant’s beliefs. Allergic rhinitis management was fragmented and had opportunity to be improved by developing strategies, resources and policies to support self-management in collaboration with patients and health-care professionals. While this system may provide easier access to more affordable medications, it also presents many challenges.2 Research investigating purchase of AR medications in the Australian setting has demonstrated that nearly 70% of people with AR self-select medication from pharmacies, with only 15% of these individuals optimally managing their AR symptoms.2 While these figures are concerning from a health-care perspective, people with AR express confidence in their ability to manage their symptoms,7 often underestimate the severity of their condition,8 and continue to live with an avoidably high burden of disease associated with suboptimal AR management.6All over the world, people with allergic rhinitis (AR) manage their symptoms with medicines that are purchased over the counter (OTC) in community pharmacies.9 While there is evidence to demonstrate the benefits of self-management in other chronic diseases such as asthma and diabetes,11 the continued burden and suboptimal management of AR symptoms in the community demonstrate that AR self-management needs to be reviewed and better supported in practice.Medication management of a chronic disease is only a fraction of what is required for successful self-management.3 research utilising the principles of social network theory has identified that their decision-making is embedded within an identifiable network.12 The AR network was developed utilising the principles of egocentric social network theory, which identifies connections and relationships with people and resources that influence health behaviours.13 The previously published AR network appears in Fig. 12 The AR network map is a visual representation of the people and resources  that were identified by people with AR as having an influence on their AR management. Alters were plotted on the map to show that those perceived to have greatest influence are closest to the centre of the map and those with least influence are furthest from the centre.However, prior to developing and optimising AR management strategies, we need to understand the influences behind patients’ AR decision making and behaviours to identify where the opportunity to improve AR management lies. Recent research exploring patient perspectives of AR management provide some insights. Specifically, recent data show that, while on the surface people with AR seem to be making their own decisions with regards to AR medication selection in the pharmacy,3 Clearly, there is a gap in our understanding of what role AR networks play in management and how they drive patients’ decision making. Therefore, this study aims to use qualitative enquiry to explore the patients’ perceptions of the roles of alters within the AR network and to understand their reasons behind their perceived level of influence on their AR management.While this research uncovered a densely populated network identified by people with AR as being of influence on their AR management, care must be taken in its interpretation as the map alone does not reveal the particular role of each alter in the patients’ AR management and there are several inconsistencies between what the map suggests and what the empirical evidence of patient behaviour in the community pharmacy suggests. For example, while the AR network map shows that general practitioners (GPs) and pharmacists to be most influential in the patients’ AR decision making, only 14% of patients purchasing AR medication in the pharmacy stated that their decision was based on the advice of a health-care professional (HCP).Forty-seven people with AR who completed their AR network map were invited to take part in this study and provided informed written consent. Forty-one completed the interview, with the remaining seven not completing the interview citing time limitations. Following data collection, all forty-one interviews were coded. Following the 21st interview, no further original codes were identified. Participants were aged between 18 and 86 years  and 67% were female. Eighty-three percent were from metropolitan Sydney and 17% from regional New South Wales.The roles of alters within the AR network as described by participants can be grouped within five categories; diagnosis, medication prescription/supply/administration, medication advice, information about allergic rhinitis and emotional support. The fact that some alters were described to have multiple roles means that some appear in more than one category and that more than one type of alter can perform the same role with respect to AR management.GPs, allergists and immunologists, ear nose and throat surgeons, respiratory specialist, an optometrist, a dentist, family and friends and the participants’ own experience were mentioned to have a role in the ‘diagnosis’ of AR. In some instances, the HCP observation of the physical manifestations of AR served to confirm a previous ‘diagnosis’ or suspicion of allergic sensitivity. The process of diagnosis was not always straightforward or well articulated, and in most instances, the participants reported it to be based on history taking of symptoms and their patterns and observations of the physical manifestations associated with AR, most often during a consultation outside of the context of AR. The use of skin prick tests for diagnosis were only mentioned with respect to consultations with allergists and immunologists (terms that were used interchangeably by the participants). In addition to providing a diagnosis themselves, GPs were identified for their role in referral to a specialist when required. Participants who nominated respiratory physicians within their AR network as a diagnostician also had comorbid asthma and said the consultations with them were predominantly for their asthma.“In the first instance, I’ve got a GP who referred me to an allergist.” (P57)“When I was a child, my GP noticed I was always sniffling, coughing and its wasn’t just fixed with Ventolin.” (P42)“My oldest brother has the same sort of thing as me.” (P44)There were also a subset of participants who had never seen a HCP about their AR and had self-diagnosed their AR based on symptoms such as sneezing and watery eyes, particularly if in response to an identifiable trigger such as animal dander or during a particular time of year often associated with AR. Self-diagnosis was also more prevalent if a family member also had AR and participants had identified similarity in their symptoms and experiences. Similarly, family and friends with AR were likely to offer a ‘diagnosis’ where they had made observations of symptoms and patterns they were suffering themselves.“I think when I found out I didn’t need to see the GP anymore, I’ve just been to the pharmacist.” (P8)“It’s been a while since my GP recommended that. I’ve never gone back for a follow up.” (P31)“I had it on prescription when I was a child in [Europe] and since I came to Australia I just buy it.” (P55)The role of the GP in prescribing medication was commonly reported. This included prescriptions for intranasal corticosteroids and antihistamines. If participants were able to access the prescribed medications OTC, they would not return to their GP for repeat prescriptions. In most circumstances, participants reported that AR was not the primary reason they had visited their GP for a consultation. This was often the case where the participant had comorbidities and was visiting their GP to have those addressed and would mention their AR at the end of a consultation. This was especially the case where there was an existing, long-term relationship between the GP and their patient. Participants who did visit a GP for a prescription often visited a medical centre and consulted GPs who they had no prior relationship with. If repeat visits were required to obtain repeat prescriptions, they would not necessarily visit the same GP. Participants also reported isolated interactions with their GPs with regards to their AR. AR was not something that they revisited their GP for and there were no reports of review consultations. The participants did report that they held on to the advice they received at consultations for many years and did not question the need for a review or change in management strategy.“I was advised at the time to just do short courses of those and I didn’t end up going back for -he didn’t need to see me again. So I didn’t actually have it followed up. (P42)Allergists and immunologists were identified for their role in prescribing immunotherapy. Some of the participants described being prescribed multiple courses of immunotherapy. Apart from requiring repeated immunotherapy, few participants reported any long-term follow-up with their allergists/immunologists.“I like him[respiratory physician] because he doesn’t believe in operations, whereas most other guys want to do other big things to your nose.”……..“I don’t really trust anybody else. I trust [respiratory physician] with my life. He has helped me enormously over the years and I don’t even really trust the GP.” (P33)Respiratory physicians were identified as having a role in prescribing treatment for AR. This was particularly during circumstances where the participant had an established relationship with respect to their comorbid asthma.“I just go in there and buy the stuff half the time and don’t even talk to them. I go in and I’ll be getting a Seretide script made up and I’ll get some Zyrtec or something.” (P2)A practice nurses was nominated within the AR network for their role in the administration of immunotherapy injections.‘A practice nurse helped with the injections.’ (P6)Some participants reported that they view the role of the pharmacist as a medication supplier. They reported knowing exactly what medicines they want and that they do not need advice or recommendation from a pharmacist. Upon self-selecting their medicines and making the purchase, they report being asked about whether they have used the medicine before to which they reply ‘yes’ and have no interest in discussing it further.Pharmacists, family and friends and participants’ ‘own experience’ were most commonly reported for their role in providing medication advice for the management of AR symptoms. .Pharmacists were often consulted with regards to recommending medications for AR. Consultations with the pharmacist or their staff were done at regular intervals during their repeat visits to the pharmacy for AR medicines, most specifically about whether any new treatment had become available. Participants also reported that pharmacists’ advice on medication encompassed safety concerns with pharmacists checking for potential drug contraindications with AR medications purchased over the counter.Participants’ own experience involved experimenting with OTC nasal products. On the rare occasion, a participant would identify a non-medication-related strategy that they had discovered to ease their AR symptoms, such as swimming. Participants would also rely on recommendations from their family and friends, especially if they were also AR sufferers and had recommended a particular product. Family and friends were identified for recommending medicines to treat AR symptoms and organising medical investigations/consultations. Recommendations made by a family member were especially held in high regard as there was a belief that familial people would have the same response to treatments.“I just self-select and I don’t really consult anyone else. That’s just been working for me so far. It hasn’t really been 100% working, because it’ s still happening.” (P56)“It’s trial and error. I just know-you know what works for yourself.” (P36)“He[the pharmacist] was really good. He was quite concerned about things; if any drugs that contraindicated. He used to check them.” (P9)“[Hay fever] affects my eyes so badly and I wear contact lenses my optometrist is probably a big one[influence].” (P4)“[My husband] is an expert and he’s been able to just find a tablet for me that is non-drowsy and it does seem to work quite rapidly, even though it does take an hour and a half.” (P30)“I’ve got more faith in them [friends rather than health care professionals].” (P10)Participants reported exploring AR management with a variety of alternative therapists. In some instances, participants felt their AR symptoms improved while others did not. Participants reported consulting an alternative therapist when they felt traditional therapies for AR were not successful. Other HCPs identified on the AR network map included a neurologist, dermatologist and optometrists for making treatment recommendations for AR symptoms during consultations for their area of specialty.Information regarding AR management was both actively sought and passively received by participants. Participants reported receiving and searching for information from pharmacists, family and friends, internet and media. Pharmacists were described as having a role in providing information about AR management beyond recommendations for medication. Participants valued the pharmacist providing instruction on correct use of an intranasal inhaler device as well as discussing minimisation of exposure to allergens. Pharmacists were perceived to have time available to be able to provide information to their patients. Family and friends were also noted to be a source of information about AR management, most particularly about medications. Many participants reported being told by family and friends that they can develop a tolerance to one type of antihistamine and that they should alternate therapy with more than one type.The alter titled ‘Media’ encapsulates television news and current affairs programs, newspapers, pamphlets and advertisements for commercial products. Participants received information about AR from these sources both passively and actively. Participants acknowledged the difference between commercial advertisements and information provided by experts in the field but were always interested in hearing about ‘new’ information, irrespective of the source. Not all information received would always be acted upon immediately but sometimes reflected upon at a later time point.“The pharmacist [checked nasal spray technique] did and I think I’m using it right. The nasal spray-the chemist-actually the local chemist where I work was the first guy who said you’re supposed to point the tube between your eyes when you put it up your, at an angle.” (P25)“I ended up doing a search on hay fever on medications or something like that……….Well the reason I was looking at it because I was just looking and trying to pick up on-I was checking about the correct way to take medication.” (P21)“I’ll always keep a casual eye out in the press, in the media, the news like for pollen index or any new medical advances.” (P1)“Advertising [in response to why a particular medication was selected].” (P20)“Papers written by experts or persons in the field, the doctorate field, that I take any notice of. Anyone that has written stuff that is not associated or trained in that field, no. It’s with a grain of salt.” (P9)“I have seen some pamphlets [in the pharmacy] and I’ve taken them away and read them. It’s kind of like they all say the same thing whatever.” (P33)The internet was placed in the AR network as a source of information that participants used proactively to educate themselves about AR; however, they had varied perspectives of the reliability of information available. Participants had actively searched for information about AR treatments for instructions on how to use their medicines. Otherwise, very few participants reported actively searching for AR information on the internet.“[Mum] had the desensitisation. There was another girl in the office here … with me that - she went through the desensitisation process. I discussed it with her. An uncle that gets hay fever really bad as well. My wife, anybody that’ll listen on a snotty, [expletive] day.“ (P13)Overall, very few participants described needing emotional support from their AR network. In the few instances that this was mentioned, emotional support was provided by family and friends, . Emotional support involved the alters providing empathy when the participants were not feeling their best due to their AR or feeling frustrated during a lengthy course of immunotherapy.The factors that determined the participants’ perceived degree of influence within the AR network were unique to each participant and their experiences with AR. These determining factors were not necessarily mutually exclusive and more than one may have been a determining factor of influence within the individual participant’s AR network.“I would place the immunologist that recommended the injections at number one, because that helped.” (P31)The perceived impact of the role of the alter on the day-to-day AR management determined the degree of influence within the AR network. This was independent of when or how frequently the contribution was made. If the participants’ current AR management was impacted, they were regarded to have more influence within the AR network.“Yeah, I just find that they just have better overall knowledge about medications and stuff.” (P15) (pharmacist strongest influence)“It was actually my GP who brought it up because she suffers from asthma and hay fever and she just said she’s noticed when she gets a hay fever attack her asthma is aggravated, I guess. Yeah so she actually asked me to try the nasal spray to see if it was effective and if it helped me because its helps her and to see if there was any change in my asthma.” (P23) (GP second highest level of influence)Trust of HCPs was determined by the participants’ preconceived ideas of the role of that HCP and their competence, i.e. HCPs who met the participants’ expectation were trusted. Participants with existing long-term relationships with HCPs reported the highest level of influence within their AR network. HCPs who were seen as being proactive earned the trust of participants. In some instances, parents/partners and pamphlets were reported to be more influential than HCPs with whom the participant had had an unsatisfactory experience.If the participant believed in medicines and the role of the HCP, then HCPs had a stronger influence within the AR network than non-HCPs, such as their family and friends.“I do have a regular pharmacist but they are not particularly helpful. They’re not helpful but… they did offer me the Nasonex and I think my mum had some and she gave it to me at the time and then I started using it and it just wasn’t working fast enough.” (P25) (pharmacist lowest degree of influence)Non-HCPs such as family and friends had a stronger influence within the AR network if the participant did not believe in HCPs and the effectiveness of medicines. These participants expressed that they would implement a friend’s recommendation if they have had a positive experience from it because they feel they had similar values with regards to what they are looking for in terms of AR relief.The aim of this research was to qualitatively explore the patient’s perceptions of the roles of alters within their AR network and to understand what determines their perceived degree of influence. While we already knew that people with AR obtain advice from a wide range of sources, we now know that the advice being provided is suboptimal, fragmented and not supported with follow-up review. This research demonstrates that, although participants identified themselves as having AR, very few described an official diagnosis and were making medication management decisions based on information provided many years ago without follow-up review. Overall, it is apparent that people with AR continued to be burdened by their symptoms, as they venture widely for guidance on AR medication. Unfortunately, in this era of patient self-selection for AR medications, patients are clutching at fragments of information obtained from a range of sources and making decisions without taking opportunity for assessment and evaluation. Our research demonstrated that, in the current environment of medication self-selection, there is an urgent need for an AR self-management strategy to be implemented in primary care.17 and that inaccurate diagnosis may be a factor in patient disillusionment with the management of their symptoms. Looking beyond the issues of diagnosis, HCPs were readily dispensing medication advice, yet very few opportunities for follow-up were reported and participants often continued to implement this advice long term without review. Participant reports of using their ‘own experience’ to determine their AR management further confirms the limited amount of review consultations with HCPs. Stand out reports of follow-up included interactions with the pharmacist during repeat purchases of medications and requests for anything new, but these were missed opportunities in that extensive review and assessment of current management was not described.This qualitative exploration of the AR network map identifies several issues with the current management of AR in the community that were otherwise not apparent on the AR network map independently. Diagnosis of AR is suboptimal in practice with many people self-diagnosing their condition. While GPs were reported for their role in diagnosis, reports of comprehensive diagnosis of AR were not available. This data confirms that AR diagnosis is a continued challenge in primary care9 and with well-known behaviourist approaches, including Cognitive Theory and the Health Belief Model.19 However, the significance of this outcome for practice is to highlight that more comprehensive research into the impact of the health beliefs of people with AR on their clinical outcomes is required. Optimising the management of AR in the community has always been challenging due to perceptions of triviality among the community and patients’ ‘lay expertise’20; however, most chronic diseases have patient management behaviour that is determined by their beliefs.9 The difference lies in the strategies developed to recognise and accommodate these beliefs within their management and interactions with HCPs. While AR management goal-setting strategies incorporating patient beliefs have been demonstrated to be successful,21 there is little evidence of their use in the community. HCPs supporting AR self-management need to be provided with tools to evaluate patient beliefs about AR and its management and adapt AR management strategies to accommodate these beliefs and we may need to look towards studies in asthma that explore patient beliefs and attitudinal clusters.23In addition to the identification of these systemic issues in the current management of AR in primary care, several patient factors were identified. Our research demonstrated that, there is no ‘role’ that is most significant in influencing a patient’s AR management. The degree of influence within the AR network is determined by three factors: the impact of the role of the alter on the patient’s day-to-day management of AR, the patient’s trust and confidence in the alter, and the patient’s health beliefs. These results are in line with established knowledge that patients’ beliefs influence behaviour25 and was chosen to explore AR management because the burden of AR on the community continues to be high, despite the availability of gold standard guidelines, pharmacological therapy and extensive research into HCP and patient perspectives.29 Similarly to the studies exploring asthma networks, this research identified patients accessing a broad range of resources to make decisions about their AR management, but unlike the asthma networks, people with AR reported more reliance on their own experience and isolated interactions with HCPs. Self-selection of medication is only one part of the dynamic self-management puzzle.9 Self-management requires supporting people to understand their condition, being able to monitor it and take appropriate action.9 Self-management requires continued collaboration among HCPs and the patient and must take into account the patients’ beliefs in order to be successful.9 Additionally, the HCPs providing the support for self-management must be supported with policy and protocols.31 This research has identified that several areas of AR management need to be targeted in order to optimise AR self-management and both HCPs and people with AR are in need of support.A qualitative exploration of the AR network from the perspective of the patient has not been conducted before. This research methodology has only relatively recently been used to explore patient-reported networks in asthmaAlthough this study has some limitations, they themselves have demonstrated the need for further research to target AR self-management in primary care. A significant limitation of this study is the self-reported diagnosis of AR among the participants, many who described their AR as self-diagnosed. The impact of this limitation is that the description of the roles of alters within the AR network are reflective of the population of people who manage their symptoms within the community setting believing they have AR and not those who have been formally diagnosed with AR. While future research into the AR network would seek to accurately recruit those diagnosed with AR, the population of this study has demonstrated that there are many people who believe they have AR and that provisions need to be made to ensure accessibility to diagnosis to optimise management of symptoms. A broader recruitment of future participants would address this study’s limitation associated with its sample size and recruitment pool. A larger and broader sample would add to the research to determine whether there is any variation with regards to geography, access to health care and socio-economic status and health beliefs.Further research into the AR network should explore possible variations among different AR phenotypes, existence of comorbidities, patient personalities and differing health beliefs.In conclusion, this qualitative exploration of the AR network has demonstrated that people managing their AR are not as well supported as the AR network map may suggest. AR management in the community has opportunity to be improved with the development of strategies, resources and policies to support AR self-management in collaboration with patients and HCPs.12  within the AR literature. The inductive analysis of the transcripts consisted of the identification of concepts within the transcripts that were not searched for but were found to emerge from the data. The researchers  read through the transcripts and identified statements that represented participant’s descriptions of ‘roles’, ‘importance’, ‘placement’ or ‘positioning’ of influences within the AR network as well as statements regarded as significant with regards to the role of alters that had not been pre-determined (coding). Following independent coding  of the transcripts, two meetings were held, with B.C., R.T., E.A., P.S., S.B.-A. and V.K., to discuss the findings. Consensus on the codes was established within these two meetings. B.C. noted the final discussion among the researchers and consolidated the agreed codes and supporting quotes from the transcripts. Three further meetings were held between B.C. and S.B.-A. to discuss representation of these results for this manuscript. All authors read and approved the final manuscript.This study utilised a combination of deductive and inductive qualitative analytical approaches. The deductive analytical approach involved searching for terms within the interview transcripts that represented pre-determined themes and concepts based on the previously documented AR networkATLAS.ti and nVivo (based on each researchers’ preference and familiarity) were used to facilitate the qualitative analysis by providing an organisational structure with which to document the themes and the evidentiary codes.Participants were not given the opportunity to provide feedback on the findings but were able to request a summary of the results.Further information on research design is available in the Reporting Summary


Background: We evaluated immunohistochemical AR expression and correlation with prognosis in a large series of homogeneously treated patients with primary TNBC.Material and Methods: Patients diagnosed with stage I-III TNBC between 2000 and 2015 at Istituto Oncologico Veneto who received treatment with surgery and neoadjuvant and/or adjuvant chemotherapy were included. Whole tissue slides were stained for AR. AR-positive expression was defined as >1% of positively stained tumor cells. Distant-disease-free survival (DDFS) was calculated from diagnosis to distant relapse or death. Late-DDFS was calculated from the landmark of 3 years after diagnosis until distant relapse or death.Results: We included 263 primary TNBC patients. Mean AR expression was 14% (range 0–100%), and 29.7% (n = 78) of patients were AR+. AR+ vs. AR- cases presented more frequently older age (p < 0.001), non-ductal histology (p < 0.001), G1-G2 (p = 0.003), lower Ki67 (p < 0.001) and lower TILs (p = 0.008). At a median follow up of 81 months, 23.6% of patients experienced a DDFS event: 33.3% of AR+ and 19.5% of AR- patients (p = 0.015). 5 years DDFS rates were 67.2% and 80.6% for AR+ and AR- patients . AR maintained an independent prognostic role beyond stage, but when TILs were added to the model only stage and TILs were independent prognostic factors. AR was the only factor significantly associated with late-DDFS: 16.4% of AR+ and 3.4% of AR- patients experienced a DDFS after the landmark of 3 years after diagnosis (p = 0.001). Late-DDFS rates at 5 years from the 3-year landmark were 75.8% for AR+ and 95.2% for AR- patients .Conclusions: AR expression is associated with worse outcome for patients with TNBC. In particular, AR+ TNBC patients are at increased risk of late DDFS events. These results reinforce the rationale of AR targeting in AR+ TNBC. Triple negative breast cancer (TNBC) represents the most lethal breast cancer subtype, accounting for around 15% of all breast cancer diagnoses and being associated with an increased risk of relapse at distant sites, mostly occurring within the first 3 years from diagnosis .In recent years, the application of genomic profiling techniques has allowed to dissect the heterogeneity of TNBC. At least four main TNBC subtypes have been defined , 4, inclAR is found to be expressed by immunohistochemistry in 60–80% of breast cancers, less frequently in estrogen receptor-negative as compared to estrogen-receptor positive tumors . In TNBCIn this study, we evaluated AR expression by immunohistochemistry and its correlation with distant disease-free survival in a large cohort of patients with non-metastatic TNBC homogeneously treated with surgery and systemic chemotherapy.We included 263 patients with non-metastatic TNBC  diagnosed from March 2000 to December 2015 at IRCCS Istituto Oncologico Veneto  who received treatment with surgery and neoadjuvant and/or adjuvant chemotherapy. Clinicopathological characteristics as well as treatment and follow up data were collected in a dedicated database. The study protocol was approved by the Ethical Committee of the Istituto Ocologico Veneto IRCCS . Written informed consent was obtained from patients.AR expression was evaluated on the following FFPE primary tumor samples for main analyses: surgical sample for patients treated with primary surgery followed by adjuvant chemotherapy and diagnostic core-biopsy for patients treated with neoadjuvant chemotherapy followed by surgery.In case of patients treated with neoadjuvant chemotherapy showing residual invasive breast cancer at the examination of the surgical sample, the FFPE surgical tumor block was also retrieved in order to conduct exploratory analysis of changes in AR expression from pre- to post-neoadjuvant chemotherapy.AR nuclear staining was evaluated on whole sections by immunohistochemistry with the Dako AR441 antibody. AR was scored by a dedicated pathologist, blinded for clinical data, and was considered positive in case of staining in at least 1% of tumor cells, consistently with most recent studies , 9.Tumor infiltrating lymphocytes (TILs) were evaluated according to consensus guidelines on hematoxylin and eosin-stained slides .Statistical analysis was carried out using IBM SPSS (version 24) software.2 test or the Mann-Whitney non-parametric test were used to study association between variables, according to their nature . The Wilcoxon signed-rank test was used to study the changes in AR expression before and after neoadjuvant chemotherapy in the subset of patients who received this type of treatment and showing residual invasive disease on the surgical sample.Descriptive statistics were performed for patient demographics and clinical characteristics. For continuous variables, median and quartiles were computed. The χp-values are two-sided, and significance level was set at p < 0.05.Distant-disease free survival (DDFS) was defined as the time from diagnosis to relapse at a distant site or death from any cause, whichever first. Late-DDFS analysis were performed from the landmark of 3 years after diagnosis until relapse at a distant site or death from any cause, whichever first. In late-DDFS analysis, patients with an event or censored before the landmark point were excluded. The landmark for late-DDFS was defined based on the pattern of relapse for TNBC that shows a peak in the hazard rate of recurrence in the first 3 years after diagnosis . OverallN = 78) showed a positive AR expression. Images of representative slides are shown in p = 0.002), non-ductal histology (p < 0.001), Grade 1–2 tumors (p = 0.003), lower Ki67 (p < 0.001), lower TILs (p = 0.008). There was no difference in stage and treatment received according to AR. Considering neoadjuvant and adjuvant therapy combined, 73% of patients received both an anthracycline and a taxane as part of chemotherapy treatment.Mean AR expression level was 14% (range 0–100%). Of 263 TNBC patients, 29.7% (At a median follow up of 81 months (95% CI 74–87), 62 patients have experienced a DDFS event (23.6%). Type of DDFS event was: distant relapse in 56 patients (90%) and death in 6 patients . The rate of events was higher in AR+ as compared to AR- patients .p = 0.018). The HR for DDFS for the comparison of AR+ vs. AR- groups was 1.82 .As shown in p = 0.161). The HR for OS for the comparison of AR+ vs. AR- patients was 1.48 .Univariate and multivariate cox models for DDFS are reported in p = 0.024) and TILs . In multivariate analysis including AR and Stage, both factors maintained an independent prognostic role . When TILs were added to the multivariate model, only stage and TILs maintained an independent prognostic value. The HR for the association between AR status and DDFS in multivariate models including the three variables was 1.57 .In addition to AR, the other factors that were associated in univariate analysis with DDFS, were stage  were AR+ and n = 148 (73%) were AR-. At a median follow up of 47 months (95% CI 41-53) n = 14 DDFS events have occurred. The rate of event was higher in AR+  vs. AR- patients . Type of DDFS event included: 10 distant relapses and 4 deaths . AR+ patients showed more frequently distant relapses  as compared to AR- patients .Since Kaplan Meier curves showed that the prognostic effect of AR on DDFS appeared driven by the occurrence of late recurrences in AR+ patients, we performed a landmark survival analysis for late-DDFS to study the association between AR and late outcome. This analysis included 203 patients who were DDFS-free at 3 years from initial diagnosis and were not censored before the landmark point: p < 0.001). Univariate late-DDFS cox analysis for the comparison of AR+ vs. AR- patients showed HR = 5.67 . No other factor showed a significant association with late-DDFS including: age , histologic Grade , stage  and TILs . However, number of events was low.Kaplan Meier curves in A list of cases with DDFS event and matched clinicopathological features is provided as n = 30). The rate of pCR was similar in AR+ and AR- patients: 25.9 and 28.8%, respectively (p = 0.778). Tumor tissue sample from the surgical specimen obtained after neoadjuvant chemotherapy was available for AR evaluation for n = 60 patients without pCR (patients' flow diagram provided in p = 0.172). All those cases that were AR- on the diagnostic core-biopsy were also AR- on the surgical specimen (n = 43), whereas 41% of the 17 initially AR+ cases lost AR expression after neoadjuvant chemotherapy (χ2p < 0.001).Of the 108 patients who received neoadjuvant chemotherapy, information on pathological response was available for 107 cases. A pathological complete response (pCR), defined as the absence of invasive cancer cells in the breast and axillary lymphnodes on the surgical specimen, was observed in 28% of cases  is very limited.We found that 30% of TNBC cases were classified as AR+, which is in line with a number of other studies –8. The cn = 130 and n = 182, respectively) (n = 581) have reported, over a median follow up of 16.5 years, a significantly unfavorable breast cancer-specific survival in multivariable models for AR+ vs. AR- patients  . Multivactively) , 18. In patients . Indeed,PIK3CA and AKT1 mutations , the methods for AR assessment in line with the most recent studies and the length of follow up (median 81 months), allowing to uncover the impact of AR on late outcome.Limitations of our study include the retrospective nature and the low number of events in late-DDFS analysis that imposes caution in results interpretation and further validation in additional studies.In conclusion, our results show that the evaluation of AR in TNBC is able to identify a subgroup of patients at worse prognosis, especially for the occurrence of late events. Further validation in other studies is warranted. These data support the rationale for the ongoing evaluation of antiandrogen therapies in TNBC.MD planned and coordinated the manuscript. VT performed the pathology assessment. VG supervised the whole writing of the manuscript. Each author participated for appropriate portions of the content. All the authors conceived the review and approved of the final analysis and results.MD has received fees from EliLilly for consultancy role and participation on advisory boards; fees from Genomic Health for consultancy role; fees from Celgene for participation on advisory boards. VG has received honoraria from EliLilly and Roche for participation on advisory boards, and honoraria from AstraZeneca and Novartis. The remaining 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.


To compare the biomechanical properties of a novel height-adjustable nano-hydroxyapatite/polyamide-66 vertebral body (HAVB) with the titanium mesh cage (TMC) and artificial vertebral body (AVB), and evaluate its biomechanical efficacy in spinal stability reconstruction.A 3D nonliner FE model of the intact L1-sacrum was established and validated. Three FE models which instrumented HAVB, TMC, and AVB were constructed for surgical simulation. A pure moment of 7.5 Nm and a 400-N preload were applied to the three FE models in 3D motion. The peak von Mises stress upon each prosthesis and the interfaced endplate was recorded for analysis. In addition, the overall and intersegmental range of motion (ROM) of each model was investigated to assess the efficacy of each model in spinal stability reconstruction.AVB had the greatest stress concentration compared with TMC and HAVB in all motions . The peak stress on HAVB was 3.1–10.3% of TMC and 1.6–3.9% of AVB. The maximum stress values on L2 caudal and L4 cranial endplates are different between the three FE models: 0.9–1.9, 1.3–12.1, and 31.3–117.9 times of the intact model on L2 caudal endplates and 0.9–3.5, 7.2–31.5, and 10.3–56.4 times of the intact model on L4 cranial endplates in HAVB, TMC, and AVB, respectively, while the overall and segmental ROM reduction was similar between the three models, with AVB providing a relatively higher ROM reduction in all loading conditions .HAVB had similar biomechanical efficacy in spinal stability reconstruction as compared with TMC and AVB. The material used and the anatomic design of HAVB can help avoid stress concentration and the stress shielding effect, thus greatly reducing the implant-associated complications. HAVB exhibited some advantageous biomechanical properties over TMC and AVB and may prove to be a potentially viable option for spinal stability reconstruction. Further in vivo and vitro studies are still required to validate our findings and conclusions. Corpectomy is generally accepted as an effective surgical procedure for spinal cancer metastasis, infection, deformity, and traumatic injuries , 2. HoweAlthough some in vivo and in vitro studies have compared the biomechanical properties of the TMC and AVB systems and found no significant difference between them , 17, fewVarious types of bioceramics have been used for treating bone defects , 18–20, Nano-hydroxyapatite/polyamide66 (n-HA/PA66) as a biomimetic biomaterial has been approved for clinical application for more than 10 years, and many products made of this material have been used for spinal reconstruction –28. AddiTo construct the model geometry, a high-resolution CT scan was obtained at 1-mm intervals in a 36-year-old healthy male. The obtained CT images severed as a DICOM format and were imported into the software Mimics 16.0  to construct a three-dimensional (3D) model of the L1-sacrum. Software Hypermesh 13.0  was used to perform mesh generation, and Abaqus software  was used for FEM simulation. The FEM consists of the cortical bone, cancellous bone, endplates, intervertebral discs, articular cartilage, and seven ligamentous systems including the anterior longitudinal ligament (ALL), posterior longitudinal ligament (PLL), ligamentous flavum (LF), capsular ligaments (CL), intertransverse ligaments (ITL), interspinous ligaments (ISL), and supraspinous ligaments (SSL)   was compared in all models (Fig. Corpectomy has become a common surgical procedure for spinal tumors, deformity, infection, and trauma , 2. AccoAs TMC and AVB have been widely used in clinical practice, many in vitro cadaveric studies have been performed to verify the efficacy of TMC and AVB in spinal stability reconstruction . To compCappuccino et al.  reportedAlthough there is no significant difference in biomechanical properties between TMC and AVB, inconsistent clinical complications have been reported. Mark et al.  reportedA tremendous amount of force concentration was observed at one point of the spikes in AVB, and this may be useful in explaining the incidence of adjacent vertebral fracture and low back pain after surgery. While an astonishing force concentration was detected in TMC and AVB, the peak stress value on HAVB was much lower, probably due to two main reasons. One is that the material of n-HA/PA66 has a similar elasticity modulus with our human cortical bone, and this can effectively reduce the stress shielding effect. The other is the morphological design of HAVB that enlarges the contact surface with the endplate, which helps disperse the stress loaded on the prosthesis. Given some advantageous biomechanical properties over TMC and AVB, HAVB can be used as a viable option for spinal stability reconstruction.The result of biomechanical analysis in this study demonstrates that this novel prosthesis of HAVB made of n-HA/PA66 has the similar stress value and distribution compared with INTACT model in all motions, indicating that the material of n-HA/PA66 is a viable material for bone tissue implantations. In addition, some other new bioceramics have been studied recently and enhanced with different nanocomposties , 21, 37.There are several limitations in our study. First, as the two components of the HAVB system are rigidly fixed with no relative sliding in all loading conditions, it may not reflect the real clinical situation. In addition, we failed to consider the effect of bone grafting or bone cement filling in the prosthesis, knowing that they may also affect spinal stability. Finally, as the material properties applied to the element of the FE model do not exactly reflect the real behavior of the human lumbar spine, the result of FE analysis should be interpreted as a trend only, and further in vitro and vivo studies are required.The present study has demonstrated that HAVB has the similar biomechanical efficacy in spinal stability reconstruction as compared with TMC and AVB. The difference in clinical complications between TMC and AVB can be explained by the differential stress values and stress distributions on these prostheses. More attention should be paid to the protection of flexion and rotation during postoperative rehabilitation training. The material and configuration design of the prosthesis is closely related to the complications associated with the implant. Given some advantageous biomechanical properties over TMC and AVB, HAVB may prove to be a promising implant for spinal column reconstruction. Further in vivo and vitro studies are still required to validate our findings and conclusions.Additional file 1: Figure S1. Comparison of ROM between the current FE model and Shim’s model in six different working conditions.


Augmented reality (AR) has the potential to create compelling learning experiences. However, there are few research works exploring the design and evaluation of AR for educational settings. In our research, we treat AR as a type of multimedia that is situated in authentic environments and apply multimedia learning theory as a framework for developing our educational applications. We share our experiences in developing a handheld AR system and one specific use case, namely, situated vocabulary learning. Results of our evaluations show that we are able to create AR applications with good system usability. More importantly, our preliminary evaluations show that AR may lead to better retention of words and improve student attention and satisfaction. Augmented reality (AR) is the seamless integration of virtual objects and real environments Azuma . In otheResearch works argue that AR’s strengths and therefore its applicability to education are embodied cognition  develop an AR application, (2) test its usability, and (3) test its effects on learning. To these ends, we demonstrate our development and evaluation framework for prototyping AR learning experiences. We apply AR to the task of memorizing vocabulary words and test AR’s effect on both learning and student motivation. Finally, because there is little literature substantiating the benefits of AR to learning, we test AR’s effectiveness as a platform for a memorization task and examine its impact on student motivation.The general public is becoming more familiar with AR mainly because of AR browsers used for conveying a variety of location-based information  compared to those who used printed materials and audio recordings . The observed large effect (d = 1.0) is attributed to practicing English in real-life situations and encouraging the creativity of the students in handling conversations.Researchers explain that ubiquitous, context-aware systems are useful for facilitating situated cognition  gained more situated words  than words that were designed to appear more frequently .Similar to MicroMandarin, Vocabulary Wallpaper  system uses RFID to tag the objects in the environment to present vocabulary words relevant to an object. They equipped a PDA with an RFID reader which scans the environment. A question is presented to the users on the PDA and they answer by tapping their PDAs to the correct object. They evaluated the usability of TANGO in two user studies. In the first user study with six students . It has three assumptions, namely, dual-channels, limited capacity, and active processing. First, humans have two separate channels for perceiving visual and auditory information. Second, individuals can only attend to a limited amount of information at any given time. Lastly, learning only takes place if the learner actively processes incoming information by connecting it to prior knowledge. Multimedia learning identifies five processes Mayer  in learnSituated vocabulary learning leverages on the prior knowledge of places. Visualizing the information in context-rich environments using AR can aid students in creating meaningful associations between the content and the real environment. This promotes having a more elaborated knowledge and having more memory retrieval cues. Situated multimedia aids in the cognitive process of integrating incoming information with prior knowledge. This is consistent with the findings of Fujimoto et al. , 2012.However, AR is also prone to presenting too much information and too much context from the environment leading to cluttered displays  and the iPad mini . The system works with fiducial markers . The hypotheses are as follows:Students will perform worse on a delayed post-test with non-AR compared with the immediate post-test.Students will perform worse on a delayed post-test with AR compared with the immediate post-test.Students will perform better in an immediate post-test with non-AR.Students will perform better in a delayed post-test with AR.Students will rate AR as a more motivating instructional material.Students will maintain their attention better with AR.Students will find the contents presented through AR to be more relevant to them.Students will feel more confident with non-AR.Students will feel more satisfied with AR.For our experiments, we controlled both location and time constraints. All of our students were only allowed to use the applications inside their respective laboratories. However, the applications are available to them at any time they want to study on that day. Given these features, we had seven hypotheses which we tested for significance in the 0.05 level via Student’s We adapted a between-groups approach with 31 participants  to test our application for studying Filipino words. The first languages of the participants are Japanese (13), Chinese (5), Portuguese (3), German, English, Turkish, Bosnian, Indonesian, Finnish, Arabic, Spanish, Nepali, and Wolof. In our experiments, we divided the people into the treatment groups with consideration to the distribution balance of their first languages.Eighteen participants were recruited from one laboratory. We set up our system inside their laboratory Fig.  so that The participants used the assigned application for a recommended duration of 10–15 minutes per day for 5 days. The AR version was used inside a refreshment area with a maximum of four people using the application at the same time  and delayed post-test (24 items) consist of questions on recognizing the word in a multiple choice question, recalling the translation of the word, and guessing which word fits in different contexts.For further analysis of the usability of our AR application, we asked the participants in the AR group to answer the HARUS which measures general system usability, ease of handling the AR application, and ease of understanding the portrayed information. Lastly, both AR and non-AR applications logged time-stamped button pushes, words studied, and tablet acceleration and orientation based on the built-in sensors. We did not notice any burden on the application due to the system logging even after extended use.We adapted a within-subjects design with 14 participants  to test the application for learning 20 German words (10 for AR and 10 for non-AR). Each participant used the AR and non-AR versions for a maximum of 8 minutes. Seven used the AR version first, whereas the other seven used the non-AR version first to balance any effect of the ordering of the treatment. For the AR version, the learners viewed the content on a small area around a laboratory technician’s desk. The markers were placed near each other in a small area to minimize the time spent on transferring from one object to another. This was important because we wanted to observe the study time of the students. For the non-AR version, they used the application while sitting inside the same room.The students are then asked to answer 10 multiple choice questions that test their skill to recognize a word using a recognition game Fig. . Aside fOur experiments involved a small sample size, thus, the results should be interpreted with caution. These experiments should be replicated with a bigger sample size. Nevertheless, these results can guide future design of AR applications and experiments in situated vocabulary learning with AR. In our experiments, we observed significant decrease in immediate to delayed post-test scores with non-AR but not for AR, suggesting that students who learned via AR retained more vocabulary. No significant differences were observed in learning outcomes between using AR and non-AR applications for vocabulary learning. However, students report better attention and satisfaction in using our system. In summary, we found evidence that supports hypotheses 1, 3, 6, and 9 but not 2, 4, 5, 7, and 8.We computed the SUS score and its factors from the participant responses in experiment 1. The AR application scored 74 on the SUS, whereas the non-AR application scored 80, as shown in Table d = 0.63). Despite the differences in usability, using these applications for comparison is reasonable because both represented our best effort and had above average usability. We achieved a good usability score because we applied insights from previous research in multimedia learning. Furthermore, our current interface features were minimal, and the task was simple.We found a marginally significant difference between the two interfaces with a moderate effect size (d = 0.84), whereas the differences for AR is marginally significant, with a small effect size (d = 0.14). Thus, we found evidence supporting hypothesis 1 but not hypothesis 2.Table These results are consistent with the work of Fujimoto et al. , 2012 whd = 0.75) compared with the AR group, thus supporting hypothesis 3. The breakdown in Table Table In most practical cases, people do not usually apply their learning immediately after studying. Rather, they would use their knowledge after a few days, either for a test or to apply it to a new lesson. As such, the delayed post-test is a more important point of comparison for learning than the immediate post-test. After 12–14 days, the significant difference in learning disappeared Table . This isAssuming that implementation quality was a factor affecting the learning of the students, we could do fairer comparisons of post-test scores if both AR and non-AR applications have almost the same SUS score. However, we observed a difference of six SUS points between the AR and non-AR applications. We conducted ANCOVA to take into account this difference in usability.p > 0.05) in variances. The ANCOVA results in Table We can conduct ANCOVA because the difference in SUS score was not significant. We also checked the homogeneity of variances using Levene’s test. The results of Levene’s test showed that there are no significant differences p > 0.0 in variaTo gain further insight regarding the differences between studying with AR and non-AR applications, we calculated the total amount of time the application is open and the total number of button pushes for LISTEN, TRANSLATE, and DESCRIBE buttons. We found that the non-AR application was used significantly longer compared to the AR application Table , a findiIn order to study with the non-AR application, the students had to keep the application open for the entire study period. However, when studying with AR, the students could put the application down and rehearse the words by going through each object in the room and calling out the vocabulary. In this case, using the application becomes unnecessary because the room itself represents the learning material. We think this connection with digital content and the real environment is one important feature of AR that could be exploited in ubiquitous learning.We also found some differences in the amount of buttons pushed in the AR application compared with the non-AR counterpart. All the three buttons  where used more in general, with the TRANSLATE button being pushed significantly more. This could mean that AR may be more motivating for students, especially for maintaining attention as Di Serio et al. reported . In anotM = 94 %, SD = 8 %) and using non-AR  for vocabulary learning. On the average, the non-AR group answered our multiple questions faster  than the AR group  for each question. However, this difference was not significant.There was no significant difference between the recognition test between using AR . As a use case of the AR system, we filled the system with Filipino and German vocabulary contents, thereby creating two AR applications for situated vocabulary learning.We evaluated the AR applications by combining methods in human-computer interaction, usability engineering, and education technology. We observed differences in immediate post-tests wherein students who used the non-AR application performed better than those who used AR. This effect is only temporary as both AR and non-AR users have almost equal scores in the delayed post-tests. We observed a larger difference between immediate post-test and delayed post-test for the students who used the non-AR application. This suggests that using AR resulted in better retention. This result needs to be explored further because our evaluation involved a small sample size only.Aside from differences in post-tests, the potential of AR lies in the difference in the learning experience, more specifically, reducing cognitive load, improving attention, and increasing satisfaction. Although preliminary, our experiments suggest that AR as multimedia may lead to better attention and satisfaction.For future work, experiments with bigger sample size must be used to explore deeper into how students can learn better with AR. Moreover, aside from cross-sectional studies comparing AR with a more traditional interface, longitudinal studies are necessary to explore the evolution of students’ knowledge and skills over time.


In prostate carcinogenesis, androgens are known to control the expression of the transient receptor potential melastatin 8 (TRPM8) protein via activation of androgen receptor (AR). Overexpression and/or activity of TRPM8 channel was shown to suppress prostate cancer (PCa) cell migration. Here we report that at certain concentrations androgens facilitate PCa cell migration. We show that underlying mechanism is inhibition of TRPM8 by activated AR which interacts with the channel within lipid rafts microdomains of the plasma membrane. Thus, our study has identified an additional nongenomic mechanism of the TRPM8 channel regulation by androgens that should be taken into account upon the development of novel therapeutic strategies. PCa development starts from epithelial cells in the peripheral zone of the prostate and is androgen controlled2. Metastasis development at late PCa stages is the main cause of mortality. The main treatment is tumor ablation followed by hormone therapy and androgen suppression in particular. However, survival and proliferation of some PCa cells often become androgen-independent, allowing to escape the above treatment and giving rise to more aggressive forms of cancer3. This androgen insensitivity significantly increases PCa mortality rate and suggests that control of PCa progression by androgens have multiple modalities.In developed countries, prostate cancer (PCa) is the second most frequently diagnosed cancer and the third most common cause of death by cancer in men4. Its expression increases during the initial stages of PCa but is reduced after anti-androgen therapy5. TRPM8 expression is androgen-dependent with genomic regulation of TRPM8 by the androgen receptor (AR); once activated by androgens, the AR binds to the androgen response elements (AREs) located upstream of the trpm8 promoter gene6. Androgens could also affect the TRPM8 channel in a nongenomic manner8. However, possible recruitment of the AR to regulation of TRPM8 channel activity and its role in the effects androgens awaits elucidation.The transient receptor potential (TRP) melastatin 8 (TRPM8) channel is involved in PCa, and is one of the most promising clinical targets10. As the expression and/or activation of TRPM8 suppresses PCa cell migration12, TRPM8 was pinpointed as potential molecular target antagonizing metastatic transition of PCa.The key event in metastatic progression is cancer cell intravasation that depends on the two processes: cell migration and invasion. Several studies have suggested that TRPM8 plays a central role in the regulation of PCa cell migration and the transition to the androgen-independent aggressive stage of PCa has been shown to positively correlate with loss of TRPM8 expressionThe aims of this study were to investigate whether variations in androgen levels affect PCa cell migration and, if so, to identify the underlying mechanisms. To this end, we (1) studied the role of the AR in regulation of TRPM8 channel by androgens, (2) analyzed interaction between the AR and TRPM8 proteins and their localization in the plasma membrane (PM) lipid microdomains and (3) assessed the recruitment of this mechanism to control of PCa cell migration.2+ concentration ([Ca2+]c) induced by TRPM8 activation in PCa cells (PC3) overexpressing TRPM8 (PC3-M8). The TRPM8 specificity of the [Ca2+]c response was confirmed using several pharmacological agents targeting TRPM8. Using confocal x-y time series imaging of the fluo-4 – loaded cells, we found that stimulation of TRPM8 with 10 µM icilin triggered transient elevation of [Ca2+]c followed by [Ca2+]c oscillations  followed by [Ca2+]c oscillations. This pattern of response did not depend on AR bioavailability and the absence of AR was not generally altered following 15-min pretreatment of the cells (10 nM TST)  Fig.  & S2. Hoent Fig.  and abolity Fig. . These oTo investigate the effect of androgens on the PCa cell migration, we performed single cell video tracking using confocal microscopy on PC3 cells overexpressing either AR or TRPM8. Overexpression of either affected the migration speed in TST-independent manner: the former decreased it by 40.19 ± 1.29%, while addition of AR increased it by 15.97 ± 1.29% Fig. . TRPM8/A17. The second one includes a stop codon at position 640, leading to constitutive nuclear translocation  in lymph node carcinoma of prostate (LNCaP) cells demonstrated close proximity of the endogenous TRPM8 and AR, and revealed that their co-localization is reduced (by 50 ± 3.3%) following treatment with 100 nM TST Fig. . FurtherFurther, GST pull-down assay using the GST-tagged N- and C- termini of TRPM8 and in vitro translated AR protein showed that the AR protein interacted with both the N-terminal and C-terminal peptides of TRPM8  but had 48 ± 7.6% stronger affinity for M8-Nt Fig. . TST ind19. We thus evaluated the involvement of the AR NTD polyQ tracts in the TRPM8/AR interaction using several AR mutants characterized by sequential deletions as described in Material and Methods. The pull-down assay with either GST-M8-Nt or GST-M8-Ct revealed no difference between in vitro translated AR polyQ mutant proteins  the amino-terminal domain (AR-NTD) of the AR and (2) the fragment of the AR including the central DNA-binding domain (DBD) followed by the ligand-binding domain in the carboxy-terminal end (LBD) responsible for regulation of the transcription by the receptor (AR-DBD/LBD) Fig. . TRPM8 Gins Fig. .The functional interaction of TRPM8 and AR implies the presence of both proteins in the same cellular microdomain. We first determined the localization of both proteins at the cell surface with biotinylation of LNCaP cells endogenously expressing TRPM8 and AR Fig.  and PC3 20. We have therefore labeled the cholesterol-rich fractions using the mCherry-tag theta subunit of cholera toxin, toxin θ21 and performed the immunofluorescence confocal detection of TRPM8, AR and toxin θ in LNCaP cells . However, TRPM8 was expressed more frequently in well-differentiated PCa (ISUP group 1) than in less differentiated tumors (ISUP group 2 or higher) (P = 0.051). Moreover, positive TRPM8 expression strongly correlated with the AR score (AR score 197 in TRPM8-positive tumors versus 150 in TRPM8-negative tumors) (P = 0.002)  of 200 hormone-naive, clinically localized cancer samples, which included 158 pT2 tumors and 42 pT3 tumors. These samples represented 57 well-differentiated cases (ISUP group 1) and 143 less differentiated cases (ISUP group 2 and higher). In addition, 48 cases of castration-resistant PCa (CRPC) were selected from patients treated exclusively with androgen deprivation therapy. The TMA assay showed TRPM8-positive staining in 36% of the clinically localized, hormone-naive cases and only 4% of CRPC cases (2 cases of 48). Nevertheless, in clinically localized PCa, no correlation was found between TRPM8 expression and progression to the pTNM stage  concentrations (10 nM) decrease TRPM8-mediated Ca22. TRPM8 overexpression without any stimulation is sufficient to induce a decrease in cell migration, suggesting a role of basal TRPM8 activity in this process22. Moreover, activation of TRPM8 with icilin, also has been shown to decrease cell migration speed12. In PCa cells, this inhibition occurs through inactivation of focal adhesion kinase (FAK)22, a nonreceptor protein tyrosine kinase. FAK phosphorylation is critical for focal adhesion formation and, hence, for cellular processes such as migration or invasion23. In line with these observation inactivation of TRPM8 by 10 nM of testosterone increased phosphorylation of FAK with a consequent increase in cell migration. The results of this study provide new dimension to our understanding of the complexity of the processes underlying PCa progression and not only support the view on TRPM8 as a potential molecular target in anti-PCa therapy24, but also highlight the AR as a TRPM8 regulator protein that should be taken into the account upon development of novel anti-PCa therapeutic strategies.Our data show that TRPM8 expression in PC3 reduces cell migration speed in line with previous data9. PSA, generally accepted as a PCa marker, is secreted into the fluid of glandular ducts25 and acts via bradykinin 2 receptor signaling pathway to facilitate accumulation of the TRPM8 functional channels in the PM11. TRP channel-associated factor (TCAF1), not only promotes translocation of the TRPM8 protein to the PM, but also induces an additional open state of the channel, thus, facilitating Ca2+ influx into the cell24. While regulation of TRPM8 by PSA is mediated via G-protein coupled receptor(s) and downstream signaling cascade, TCAF1 exerts its effects via direct interaction with TRPM8 channel protein, thus, resembling the action of the AR described here.Indeed, we have demonstrated that in the presence of AR, low doses of TST inhibit TRPM8 basal activity and promote the PCa cell migration Fig. . Other mtrpm8 promoter and (2) modulation of TRPM8 activity by nongenomic pathway via protein-protein interaction and/or TRPM8 phosphorylation28. Nongenomic regulation of TRP channels by steroid hormones is well appreciated. For example, it was reported that TRPV5 and TRPM6 are regulated by estrogens30, TRPM3 and TRPV1 are regulated by pregnenolone, and the activity of TRPC3, TRPC4, TRPC5 and TRPC6 is reduced by progesterone32. Here, using cytosolic (AR23) and nuclear (ARQ640X) AR mutants, we demonstrated that, in similarity to other TRP channels, the TRPM8 channel activity affecting cell migration is regulated via nongenomic pathway. By demonstrating that AR is necessary for the testosterone-mediated TRPM8 regulation affecting [Ca2+]c and cell migration, we validate that binding of testosterone to the AR protein occurs near the PM18. Indeed biotinylation assays demonstrated localization of AR in accessible vicinity to PM, what, as have been previously demonstrated, is necessary for nongenomic action of androgens in PCa34.It was reported previously that androgens may affect TRPM8 in two principal ways: (1) modulation of TRPM8 expression by genomic pathway via AREs upstream of the 35. Variations in the number of the repeats beyond this range were associated with pathology: increased in Kennedy’s disease36 and decreased in PCa high risk37. However, TRPM8 interacts with the N-terminal domain but not within the polyQ tracts of the AR. This pattern of interaction is favored by tetrameric structure of functional TRPM8, where the C- and N-terminal domains of TRPM8 are in close proximity38.AR comprises three distinct domains among which polyQ repeats (9 to 36) which are involved in the transactivation of the AR protein by a regulated interaction between the NTD and the C-terminal part of the AR39. Here we show further that stimulation with 10 nM TST facilitates accumulation of both, TRPM8 and AR, proteins in the cholesterol-rich fractions, thus promoting AR-TRPM8 interaction and TRPM8 inhibition. TST has molecular structure similar to that of cholesterol (characterized by sterol core) and, hence, like other steroids, may affect the membrane composition, fluidity and interaction between proteins via modulation of their microenvironment40. This may partially explain an escape of TRPM8 and AR from the cholesterol-rich fractions observed at 100 nM TST.The TRPM8 activity can be modulated via its lipid environment since accumulation of TRPM8 in lipid rafts reduces its activity41, comprises a CRAC-like motif ((L/V)-X1–5-(Y)-X1–5-(K/R))42. This CRAC-like motif is located in cytosolic NTD of TRPM8 near its first transmembrane domain and, perhaps, can be engaged upon accumulation of TRPM8 in cholesterol-rich regions of the PM. For example, it was demonstrated that TRPC1 comprises a caveolin1-binding motif facilitating its accumulation in lipid rafts43. However, the functional relevance of the TRPM8 CRAC-like motif remains to be elucidated and it would be interesting to investigate in which extend the TRPM8/AR interaction affects or not the recruitment of vesicle-like structures, characteristic of the channel trafficking45.The potential mechanism of TRPM8 channels relocation within PM towards lipid rafts may involve the dynamic interaction between TRPM8/AR and possible changes in structural conformation of both proteins. We hypothesize that the strength of TRPM8/AR interaction is a determinant of the two proteins conformation. The conformational changes, in turn, may expose lipid raft localization structure patterns of these proteins, such as putative CRAC (cholesterol recognition amino acid consensus) motif on TRPM8 and caveolin binding domain on AR. Indeed, bioinformatics assay suggested that TRPM8, not unlike TRPV18 suggest direct agonist action of TST on TRPM8, based on lipid bilayers or cell models not expressing AR. TST was applied at picomolar concentrations, which are 103 times lower than physiological levels in men (723.8 ± 221.1 ng/dl = 26.7 ± 8.1 nM)46. In our study, we focused on the role of androgens in PCa progression, we utilized cells expressing both AR and TRPM8  and demonstrated that TST mediates its inhibitory effect on TRPM8 via AR. This inhibitory effect, accelerating the PCa cell migration, is in agreement with the reports of correlation between low serum TST (<230 ng/dl or <8 nM) and tumor aggressiveness, poor prognosis and PCa metastasis47.Finally, previous studies by Asuthkar et al.We used human Prostate cancer cells (PC3) with stable TRPM8 overexpression (PC3-TRPM8) and human prostate cancer cells LNCaP. PC3-TRPM8 cells were obtained by a stable transfection of pcdna4-TRPM8 vector. PC3, PC3-TRPM8 and LNCaP cells were grown in RPMI (Invitrogen) supplemented with 10% of fetal bovine serum (Pan Biotech), L-glutamine  and PenStrep® .6 cells with lipofectamine 3000® (Thermo Fischer Scientific). The constructs used in this study were plasmids carrying the sequences of peGP-AR (addgene), peGFP-AR23  and peGFP-ARQ640X . We transfected 8 µg per dish for the immunoprecipitation assay and 2 µg per well for migration assay.Gene overexpression in PC3 and PC3-M8 cells were obtained by lipofection of 1.0 × 1024. The AR gene, the DNA and ligand binding domain region  of AR gene and the N-terminal domain  were cloned in the pGEM-T Easy vector  from pEGFP-AR vector (addgene #28235) using the following primers: for AR, 5′ ATGGAAGTGCAGTTAGGGCTGGG 3′ and 5′ TCACTGGGTGTGGAAATAGATG 3′; for AR-DBD/LBD, 5′ GCTCTAGAATGCCA CCCCAGAAGACCTGC 3′and 5′ CACTGGGTGTGGAAATAGATG 3′; for AR-NTD, 5′ ATGGAAGTGCAGTTAGGGCTGG 3′ and 5′ TCAGTGGAAAGTAATAGTCAAT GGGCA 3′. AR, AR-DBD/LBD and AR-NTD were both subcloned as EcoRI fragments into the pCMV-TnT vector  for in vitro translation.The coding sequence of the N-terminal tail of TRPM8 (690 aa) and C-terminal tail of TRPM8 was amplified from pGEX6p-2 TRPM8 Nt and pGEX6p-2 TRPM8 Ct vectors2 in an incubator chamber for time-lapse video recording (Okolab). Cell movement was monitored with an inverted microscope  using a 10×/0.25 NA Plan objective lens.Cells were seeded at low density and kept at 37 °C under 5% COImages were acquired every 10 min for a time lapse of 10 h with a CCD video camera using NIS-Element software (Nikon). Image stacks were analyzed with ImageJ software and at least 100 cells per condition were manually tracked using the MtrackJ plugin. Dividing cells as well as cells that exited the field of view during the acquisition period were excluded from the analysis of the cell movement speed. Cells were treated with testosterone at different concentration by adding testosterone in growth medium just before the acquisition was commenced. At least six fields for each condition were analyzed in each independent experiment. At least three independent experiments were done for each experimental condition.2+ imaging was performed with LSM 510 META confocal workstation using a Plan-Neofluar 40 × 1.3 NA objective . Fluo-4 was excited by the 488 nm line of 500 mW Argon ion laser  and the fluorescence was captured at wavelengths 505–545 nm. Image processing was carried out using LSM 5 software  and with custom routines written in IDL . Statistical analysis was performed using MicroCal Origin .Confocal Ca2+ concentrations were measured using the ratiometric dye Fura-2/AM  and quantified according to Grynkiewicz et al.48. Cells were loaded with 2.5 μM of Fura-2/AM  for 45 min then washed and immersed in the extracellular solution containing 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM N-(2-hydroxyethyl)-piperazine-N’-ethanesulfonic acid (HEPES), 10 mM glucose (NaOH to pH 7.35). Observations were performed at 37 °C on an Eclipse Ti microscope using an S Fluor 20×/0.75 NA objective lens (both from Nikon). Images were collected through a Rolera EM-C2 charge-coupled device (CCD) camera (QImaging) controlled with Metafluor software (Molecular Devices). Data were then analyzed with GraphPad Prism 6 software (GraphPad Corporation).Basal cytosolic Ca3 cells per confocal FluoroDish® (World Precision Instruments). Cells were treated with 100 nM of testosterone or vehicle only for 20 min at 37 °C before fixation and permeabilized with 4% PAF during 10 min. After permeabilization, cells were incubated in the blocking buffer (provided with the kit) for 30 min at 37 °C in a humidified chamber. Cells were incubated with the primary antibodies for 2 h at room temperature: goat anti-TRPM8 antibody  and rabbit anti-AR . Spatial distribution of the antibody-labeled proteins was examined using confocal z-sectioning (Zeiss LSM700) and analyzed with Zen 2010 software. The adequacy of the imaging protocol applied to the double-labeled LNCaPs was confirmed by control experiments on the single-labeled cells. Each experiment was repeated at least 3 times.Proximity ligation assay was performed using Duolink® In situ red starter kit goat/rabbit (Sigma-Aldrich). LNCaP were seeded at 20 × 10g. Protein concentrations were determined using a Bicinchoninic Acid Kit . 50 µg of sample were resuspended in SDS sample buffer, heated at 95 °C for 5 min and separated on 10% pre-cast SDS gel (Biorad). Polyvinylidene fluoride membranes were properly blocked and then incubated overnight with rabbit anti-TRPM8  and with rabbit anti-AR . The membrane was then washed with TBS containing 0.1% Tween 20 and incubated with the appropriate horseradish-peroxidase-conjugated antibodies (SantaCruz). Chemiluminescence assays were conducted using the SuperSignal West Dura chemiluminescent substrate (Thermo Fischer Scientific).Cells were seeded in Petri dishes with the appropriate medium and grown to a confluency of 80%. Before cell lysis, Petri dishes were kept on ice and the cells were washed twice with ice-cold PBS. Cells were lysated in RIPA Buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with the following protease inhibitors: 2 mg/ml aprotinin, 1 mM Na orthovanadate, 0.1 mM PMSF and 10 mM Sodium Fluoride. Lysates were centrifuged at 4 °C for 10 min at 12,000 × 4, pH 7.2, supplemented with anti-protease cocktail; Sigma-Aldrich). After lysate centrifugation , the protein concentration was determined using BCA assay (Thermo Fischer Scientific). An equal amount of supernatants was incubated overnight at 4 °C with 40 µl of His-tag beads  in IP buffer . The pellet was washed three times in IP buffer, resuspended in SDS sample buffer, heated at 95 °C for 5 min and separated on 10% pre-cast SDS–PAGE gels (Biorad). SDS-PAGE gels were analyzed by immunoblotting using rabbit anti-androgen receptor (AR)  and rabbit anti-TRPM8  antibodies. Each experiment was repeated at least three times. TRPM8 antibody specificity was validated using siRNA against the TRPM8 channel . After 48 h of transfection, the cells were incubated with different concentration of testosterone  during 20 min at 37 °C and washed twice with PBS. Cells were incubated on ice in lysis buffer  deletion of the longest tract (hAR ΔpolyQ), (2) deletion of the tract of 5 glutamine repetitions (hAR ΔQ5), (3) simultaneous deletion of the longest tract and the one with 6 glutamine repetitions (hAR ΔpolyQQ6), (4) simultaneous deletion of the longest tract and the one with 5 glutamine repetitions (hAR ΔpolyQQ5) and (5) simultaneous deletion of all three tracts (hAR ΔpolyQQ5Q6) 20. For the direct interaction assay, PCMV-TNT AR, PCMV-TNT DBD/LBD or PCMV-TNT NBD vector were translated in vitro using the TNT Quick Coupled Transcription/Translation Systems kit (Promega) and the FluoroTectTM GreenLys in vitro Translation Labeling System (Promega). In vitro translated proteins were incubated overnight at 4 °C together with the purified GST-fusion proteins in the presence or absence of testosterone. Subsequently, beads were washed extensively and bound proteins were eluted with SDS-PAGE loading buffer, separated on 10% SDS-PAGE gels and visualized by fluorescence imaging or immunoblotting using anti-AR  and anti-GST (1:1000 dilution) antibodies . Each experiment was repeated at least three times.TRPM8 N- and C-terminal tail GST-fusion proteins were produced and purified as described previously24. Biotinylated proteins were precipitated using neutravidin-agarose beads (Pierce). TRPM8 and AR expression was analyzed by immunoblotting of the precipitates (PM fraction) or of total cell lysates using the anti-TRPM8  and the anti-AR antibodies . Each experiment was repeated at least three times.The biotinylation assay was performed using the NHS-LC-LC-biotin kit  after homogenizing the cells in 600 µl of lysis buffer, as described previously49 and purified by Ni-Nta affinity column. Cells were incubated for 10 min with 1.25 µM of theta toxin at room temperature in 0% SVF medium supplemented with 1.25 µM of fatty acid-free BSA (Sigma-Aldrich) and cleared from aggregates by centrifugation at 14,000 × g for 5 min. Following cholesterol labeling, the cells were washed 4 times with 0% SVF medium and kept on 0 steroid medium for confocal imaging (Zeiss LSM780).Cholesterol was labeled with the theta subunit of cholera toxin provided by Dr. Donatienne Tyteca. Theta toxin was produced in bacteria with the pET28/his-mCherry-theta-D4 vector, as previously described50. Briefly, cells were homogenized in lysis buffer A  supplemented with 0.5% Triton X-100 mixed with complete protease inhibitors (Roche Applied Science) and solubilized by agitation during 30 min at 4 °C. The lysates were passed 10 times through a 21-gauge needle, and 500 µl of lysate were mixed with 700 µl of a 60 % OptiPrepTM solution  and applied to the bottom of a centrifugation tube. A discontinuous OptiPrepTM gradient was prepared by diluting 10 ml of 30% OptiPrepTM in 0.5% Triton X-100 containing lysis buffer A and 1 ml of buffer alone on the top. Gradients were centrifuged at 178,000 × g for 4 h at 4 °C in a Beckman Optima XPN-80 ultracentrifuge using a SW40-Ti rotor. After centrifugation, nine fractions (1.3 ml each) were collected from the top, and equal volumes of each fraction were analyzed by dot blot.Lipid rafts from LNCaP cells or TRPM8/AR overexpressing PC3 cells were obtained, as described previouslyn = 200) were obtained from patients treated with radical prostatectomy for localized PCa, including 158 pT2 tumors and 42 pT3. Fifty-seven PCa were well differentiated (ISUP group 1), and 143 less differentiated (ISUP group 2 and more).Hormone naive clinically localized cancer samples (HNCLC) (Forty-eight cases of castration resistant prostate cancers (CRPC) were selected from patients treated exclusively with androgen deprivation therapy (ADT). Tissues were collected by transurethral resection, performed due to the lower urinary tract symptoms associated with local tumor progression.Written informed consents were obtained from patients in accordance with the requirements of the medical ethic committee, Comité de Protection des Personnes of Tours Universitary Hospital of the 18th of February (DC-2014-2045).TMAs were constructed using formalin-fixed paraffin-embedded tissue samples. Original slides stained with hematoxylin-eosin were reviewed using the 2017 TNM classification and the 2014 modified “Gleason” system. For each case, 3 cores 0.6 diameter were transferred from the selected areas to the recipient block, using a TMA workstation . Serial 3 µm sections of the TMA blocks were used for immunohistochemistry. One section out of ten was stained with hematoxylin-eosin to check that the cores adequately represented diagnostic areas.Slides were deparaffinized, rehydrated, and heated in citrate buffer pH 6 for antigenic retrieval. After block of endogenous peroxidase with 3% hydrogen peroxide, the slides were exposed to the primary antibodies: anti-TRPM8  and anti-AR . Immunohistochemistry was performed using the streptavidin-biotin-peroxydase method with diaminobenzidine as the chromogen . Slides were finally counterstained with haematoxylin. Negative controls were performed by either omission of the primary antibody from staining protocol or by incubation with an irrelevant antibody.Staining for TRPM8 was scored as “–“ (staining not detected) and “+” (staining detected). AR-positive cells were expressed as a percentage of total epithelial cells. Moreover, intensity of AR staining was also scored as moderate “1” or high “2”. AR expression level was then defined as the product of percentage and intensity (range: 0–200).U test for continuous data.Values are expressed as means ± SEM. The statistical significance of differences between groups was determined by analysis of variance (ANOVA) followed by pairwise comparison using Tukey’s method for migration assays, immunoprecipitation or GST-pull down quantification. Student t-test was used for calcium imaging, PLA and biotinylation assays. The threshold for statistical significance was set at the 0.05 level. Statistical analysis was performed with GraphPad Prism 6 software (GraphPad Corporation) or, in the case of TMA, with StatView 5.0 software . Comparison between groups was performed using the χ2 test for categorical data and nonparametric Mann-Whitney Supplemental Movie S1Supplemental Movie S2Supplemental Figures 1 to 4 and their legends