• Enhanced hyperspectral tomography for bioimaging by spatiospectral reconstruction.

      Warr, Ryan; email: ryan.warr@postgrad.manchester.ac.uk; Ametova, Evelina; Cernik, Robert J; Fardell, Gemma; Handschuh, Stephan; orcid: 0000-0002-2140-7892; Jørgensen, Jakob S; Papoutsellis, Evangelos; Pasca, Edoardo; Withers, Philip J (2021-10-21)
      Here we apply hyperspectral bright field imaging to collect computed tomographic images with excellent energy resolution (~ 1 keV), applying it for the first time to map the distribution of stain in a fixed biological sample through its characteristic K-edge. Conventionally, because the photons detected at each pixel are distributed across as many as 200 energy channels, energy-selective images are characterised by low count-rates and poor signal-to-noise ratio. This means high X-ray exposures, long scan times and high doses are required to image unique spectral markers. Here, we achieve high quality energy-dispersive tomograms from low dose, noisy datasets using a dedicated iterative reconstruction algorithm. This exploits the spatial smoothness and inter-channel structural correlation in the spectral domain using two carefully chosen regularisation terms. For a multi-phase phantom, a reduction in scan time of 36 times is demonstrated. Spectral analysis methods including K-edge subtraction and absorption step-size fitting are evaluated for an ex vivo, single (iodine)-stained biological sample, where low chemical concentration and inhomogeneous distribution can affect soft tissue segmentation and visualisation. The reconstruction algorithms are available through the open-source Core Imaging Library. Taken together, these tools offer new capabilities for visualisation and elemental mapping, with promising applications for multiply-stained biological specimens.
    • Advancing Mental heaLth Provision In PHarmacY (AMPLIPHY): A feasibility study.

      Gorton, Hayley C; email: h.c.gorton@hud.ac.uk; Riste, Lisa; email: lisa.riste@manchester.ac.uk; Littlewood, Donna; email: donna.littlewood@manchester.ac.uk; Pickering, Gary; email: gary@cpcw.org.uk; Armitage, Christopher J; email: chris.armitage@manchester.ac.uk; Ashcroft, Darren M; email: darren.ashcroft@manchester.ac.uk (2021-11-09)
      Specifically-designed community pharmacy-based services represent opportunities to support people with their mental health. As few such services exist worldwide, the Advancing Mental HeaLth Provision In PharmacY (AMPLIPHY) was designed with stakeholders. The purpose was to support people with their mental health when initiated on new prescription or change in drug, dose or quantity of antidepressant, through a series of consultations (up to 3 months). The aim was to evaluate the feasibility of the AMPLIPHY service. The objectives were to: i) pilot the service; ii) examine anonymous consultation data to understand patients' characteristics, priorities and consultation focus and iii) Evaluate pharmacists' experiences. This service evaluation involved a concurrent mixed methods design. Patient characteristics were extracted from consultation notes and descriptive statistics applied. Content analysis was used to summarise consultation foci and comparisons between patients' priorities with consultation content were made. Pharmacists were interviewed at the start (n = 10) and end (n = 4) of the pilot with themes identified using thematic analysis. Seventy-six patients participated (63% of recruitment target). The median age was 39 (IQR 28-47) and 62% were female. Seventy percent of patients had one consultation, 26% had two and 4% had three. Prescription for new antidepressant was the most common reason for entry (74%) and sertraline was most prescribed (46%). Consultations commonly focussed on life experience (n = 51), medication (n = 47), health (n = 42), support (n = 36) and patients' expression of their feelings (n = 31). The pharmacists' experiences were summarised in three themes: i) Motivation, ii) Practicalities and iii) Experience and Outcomes. Pharmacists were motivated to deliver this novel service and some patients were willing to join the service. Analysis of patient demographics, consultation notes and interviews provide insight into the strengths and challenges of the service and provides a blueprint for future service developments. [Abstract copyright: Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.]
    • A mathematical model which examines age-related stochastic fluctuations in DNA maintenance methylation.

      Zagkos, Loukas; email: l.zagkos@imperial.ac.uk; Roberts, Jason; Auley, Mark Mc (2021-11-11)
      Due to its complexity and its ubiquitous nature the ageing process remains an enduring biological puzzle. Many molecular mechanisms and biochemical process have become synonymous with ageing. However, recent findings have pinpointed epigenetics as having a key role in ageing and healthspan. In particular age related changes to DNA methylation offer the possibility of monitoring the trajectory of biological ageing and could even be used to predict the onset of diseases such as cancer, Alzheimer's disease and cardiovascular disease. At the molecular level emerging evidence strongly suggests the regulatory processes which govern DNA methylation are subject to intracellular stochasticity. It is challenging to fully understand the impact of stochasticity on DNA methylation levels at the molecular level experimentally. An ideal solution is to use mathematical models to capture the essence of the stochasticity and its outcomes. In this paper we present a novel stochastic model which accounts for specific methylation levels within a gene promoter. Uncertainty of the eventual site-specific methylation levels for different values of methylation age, depending on the initial methylation levels were analysed. Our model predicts the observed bistable levels in CpG islands. In addition, simulations with various levels of noise indicate that uncertainty predominantly spreads through the hypermethylated region of stability, especially for large values of input noise. A key outcome of the model is that CpG islands with high to intermediate methylation levels tend to be more susceptible to dramatic DNA methylation changes due to increasing methylation age. [Abstract copyright: Copyright © 2021 Elsevier Inc. All rights reserved.]
    • Co-designing new tools for collecting, analysing and presenting patient experience data in NHS services: working in partnership with patients and carers

      Small, Nicola; orcid: 0000-0002-7879-7967; email: nicola.small@manchester.ac.uk; Ong, Bie Nio; orcid: 0000-0001-8138-8139; Lewis, Annmarie; Allen, Dawn; Bagshaw, Nigel; Nahar, Papreen; orcid: 0000-0002-5817-8093; Sanders, Caroline; orcid: 0000-0002-0539-928X; Hodgson, Damian; Dehghan, Azad; Sharp, Charlotte; et al. (BioMed Central, 2021-11-27)
      Abstract: Background: The way we collect and use patient experience data is vital to optimise the quality and safety of health services. Yet, some patients and carers do not give feedback because of the limited ways data is collected, analysed and presented. In this study, we worked together with researchers, staff, patient and carer participants, and patient and public involvement and engagement (PPIE) contributors, to co-design new tools for the collection and use of patient experience data in multiple health settings. This paper outlines how the range of PPIE and research activities enabled the co-design of new tools to collect patient experience data. Methods: Eight public contributors represented a range of relevant patient and carer experiences in specialist services with varied levels of PPIE experience, and eleven members of Patient and Participation Groups (PPGs) from two general practices formed our PPIE group at the start of the study. Slide sets were used to trigger co-design discussions with staff, patient and carer research participants, and PPIE contributors. Feedback from PPIE contributors alongside verbatim quotes from staff, patient and carer research participants is presented in relation to the themes from the research data. Results: PPIE insights from four themes: capturing experience data; adopting digital or non-digital tools; ensuring privacy and confidentiality; and co-design of a suite of new tools with guidance, informed joint decisions on the shaping of the tools and how these were implemented. Our PPIE contributors took different roles during co-design and testing of the new tools, which supported co-production of the study. Conclusions: Our experiences of developing multiple components of PPIE work for this complex study demonstrates the importance of tailoring PPIE to suit different settings, and to maximise individual strengths and capacity. Our study shows the value of bringing diverse experiences together, putting patients and carers at the heart of improving NHS services, and a shared approach to managing involvement in co-design, with the effects shown through the research process, outcomes and the partnership. We reflect on how we worked together to create a supportive environment when unforeseen challenges emerged (such as, sudden bereavement).
    • Structure of ABCB1/P-Glycoprotein in the Presence of the CFTR Potentiator Ivacaftor

      Barbieri, Alessandro; orcid: 0000-0002-0135-9977; email: alessandro.barbieri@postgrad.manchester.ac.uk; Thonghin, Nopnithi; orcid: 0000-0001-5656-5973; email: nopnithi@g.swu.ac.th; Shafi, Talha; email: talha.shafi@manchester.ac.uk; Prince, Stephen M.; email: steve.prince@manchester.ac.uk; Collins, Richard F.; email: Richard.Collins@manchester.ac.uk; Ford, Robert C.; orcid: 0000-0002-0958-1505; email: robert.ford@manchester.ac.uk (MDPI, 2021-11-25)
      ABCB1/P-glycoprotein is an ATP binding cassette transporter that is involved in the clearance of xenobiotics, and it affects the disposition of many drugs in the body. Conformational flexibility of the protein within the membrane is an intrinsic part of its mechanism of action, but this has made structural studies challenging. Here, we have studied different conformations of P-glycoprotein simultaneously in the presence of ivacaftor, a known competitive inhibitor. In order to conduct this, we used high contrast cryo-electron microscopy imaging with a Volta phase plate. We associate the presence of ivacaftor with the appearance of an additional density in one of the conformational states detected. The additional density is in the central aqueous cavity and is associated with a wider separation of the two halves of the transporter in the inward-facing state. Conformational changes to the nucleotide-binding domains are also observed and may help to explain the stimulation of ATPase activity that occurs when transported substrate is bound in many ATP binding cassette transporters.
    • Renal hemofiltration prevents metabolic acidosis and reduces inflammation during normothermic machine perfusion of the vascularized composite allograft—A preclinical study

      Stone, John P.; orcid: 0000-0001-9452-2843; Amin, Kavit R.; Geraghty, Abbey; Kerr, Jak; Shaw, Matthew; Dabare, Dilan; Wong, Jason K.; Brough, David; Entwistle, Timothy R.; Montero‐Fernandez, Angeles; et al. (2021-11-26)
      Abstract: Introduction: Recent experimental evidence suggests normothermic machine perfusion of the vascularized composite allograft results in improved preservation compared to static cold storage, with less reperfusion injury in the immediate post‐operative period. However, metabolic acidosis is a common feature of vascularized composite allograft perfusion, primarily due to the inability to process metabolic by‐products. We evaluated the impact of combined limb‐kidney perfusion on markers of metabolic acidosis and inflammation in a porcine model. Methods: Ten paired pig forelimbs were used for this study, grouped as either limb‐only (LO, n = 5) perfusion, or limb‐kidney (LK, n = 5) perfusion. Infrared thermal imaging was used to determine homogeneity of perfusion. Lactate, bicarbonate, base, pH, and electrolytes, along with an inflammatory profile generated via the quantification of cytokines and cell‐free DNA in the perfusate were recorded. Results: The addition of a kidney to a limb perfusion circuit resulted in the rapid stabilization of lactate, bicarbonate, base, and pH. Conversely, the LO circuit became progressively acidotic, correlating in a significant increase in pro‐inflammatory cytokines. Global perfusion across the limb was more homogenous with LK compared to LO. Conclusion: The addition of a kidney during limb perfusion results in significant improvements in perfusate biochemistry, with no evidence of metabolic acidosis.
    • Heartburn as a Marker of the Success of Acid Suppression Therapy in Chronic Cough

      Badri, H.; orcid: 0000-0002-6134-4347; email: huda.badri@manchester.ac.uk; Satia, I.; Bansal, V.; Mangi, M. A.; Tangaroonsanti, A.; DeVault, K. R.; Lee, A. S.; Houghton, L. A.; Smith, J. A. (Springer US, 2021-11-19)
      Abstract: Purpose: Gastro-oesophageal reflux disease (GORD) is commonly thought to play an important role in chronic cough and patients are often empirically treated with acid suppression therapy. We sought to investigate the response rate to acid suppression treatment in patients with and without heartburn attending two specialist cough clinics. Methods: A retrospective review of 558 consecutive patients referred to two specialist cough clinics was performed (UK and USA). Patients who were treated with acid suppression were included and their documented response to treatment was collected. Binary logistic regression was used to ascertain the value of reported heartburn in predicting the response of chronic cough to acid suppression therapy. Results: Of 558 consecutive referrals, 238 patients were excluded due to missing data or cough duration of < 8 weeks. The remaining 320 patients were predominantly female (76%), with mean age 61 yrs (± 13) and 96.8% non-smokers, with chronic cough for 36 (18–117) months. Of 72 patients with heartburn, 20 (28%) noted improvement in their cough with acid suppression, whereas of 248 without heartburn, only 35 (14%) responded. Patients reporting heartburn were 2.7 (95% C.I. 1.3–5.6) times more likely to respond to acid suppression therapy (p = 0.007). Conclusion: In specialist cough clinics, few patients report a response of their chronic cough to acid suppression therapy. Nonetheless, heartburn is a useful predictor substantially increasing the likelihood of benefit.
    • Whole-genome analysis of Nigerian patients with breast cancer reveals ethnic-driven somatic evolution and distinct genomic subtypes

      Ansari-Pour, Naser; orcid: 0000-0003-0908-0484; Zheng, Yonglan; orcid: 0000-0001-6597-7072; Yoshimatsu, Toshio F.; orcid: 0000-0003-2674-8159; Sanni, Ayodele; Ajani, Mustapha; orcid: 0000-0001-5758-5773; Reynier, Jean-Baptiste; Tapinos, Avraam; Pitt, Jason J.; Dentro, Stefan; Woodard, Anna; et al. (Nature Publishing Group UK, 2021-11-26)
      Abstract: Black women across the African diaspora experience more aggressive breast cancer with higher mortality rates than white women of European ancestry. Although inter-ethnic germline variation is known, differential somatic evolution has not been investigated in detail. Analysis of deep whole genomes of 97 breast cancers, with RNA-seq in a subset, from women in Nigeria in comparison with The Cancer Genome Atlas (n = 76) reveal a higher rate of genomic instability and increased intra-tumoral heterogeneity as well as a unique genomic subtype defined by early clonal GATA3 mutations with a 10.5-year younger age at diagnosis. We also find non-coding mutations in bona fide drivers (ZNF217 and SYPL1) and a previously unreported INDEL signature strongly associated with African ancestry proportion, underscoring the need to expand inclusion of diverse populations in biomedical research. Finally, we demonstrate that characterizing tumors for homologous recombination deficiency has significant clinical relevance in stratifying patients for potentially life-saving therapies.
    • What influences people's responses to public health messages for managing risks and preventing infectious diseases? A rapid systematic review of the evidence and recommendations.

      Ghio, Daniela; orcid: 0000-0002-0580-0205; Lawes-Wickwar, Sadie; Tang, Mei Yee; Epton, Tracy; email: tracy.epton@manchester.ac.uk; Howlett, Neil; orcid: 0000-0002-6502-9969; Jenkinson, Elizabeth; Stanescu, Sabina; Westbrook, Juliette; Kassianos, Angelos P; Watson, Daniella; et al. (2021-11-11)
      Individual behaviour changes, such as hand hygiene and physical distancing, are required on a population scale to reduce transmission of infectious diseases such as COVID-19. However, little is known about effective methods of communicating risk reducing information, and how populations might respond. To synthesise evidence relating to what (1) characterises effective public health messages for managing risk and preventing infectious disease and (2) influences people's responses to messages. A rapid systematic review was conducted. Protocol is published on Prospero CRD42020188704. Electronic databases were searched: Ovid Medline, Ovid PsycINFO and Healthevidence.org, and grey literature (PsyarXiv, OSF Preprints) up to May 2020. All study designs that (1) evaluated public health messaging interventions targeted at adults and (2) concerned a communicable disease spread via primary route of transmission of respiratory and/or touch were included. Outcomes included preventative behaviours, perceptions/awareness and intentions. Non-English language papers were excluded. Due to high heterogeneity studies were synthesised narratively focusing on determinants of intentions in the absence of measured adherence/preventative behaviours. Themes were developed independently by two researchers and discussed within team to reach consensus. Recommendations were translated from narrative synthesis to provide evidence-based methods in providing effective messaging. Sixty-eight eligible papers were identified. Characteristics of effective messaging include delivery by credible sources, community engagement, increasing awareness/knowledge, mapping to stage of epidemic/pandemic. To influence intent effectively, public health messages need to be acceptable, increase understanding/perceptions of health threat and perceived susceptibility. There are four key recommendations: (1) engage communities in development of messaging, (2) address uncertainty immediately and with transparency, (3) focus on unifying messages from sources and (4) frame messages aimed at increasing understanding, social responsibility and personal control. Embedding principles of behavioural science into public health messaging is an important step towards more effective health-risk communication during epidemics/pandemics. [Abstract copyright: © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.]
    • Estimating the late-life effects of social and emotional skills in childhood using midlife mediators.

      Turner, Alex J; Fichera, Eleonora; Sutton, Matt; email: matt.sutton@manchester.ac.uk (2021-11-01)
      Social and emotional skills are known to affect health and non-health outcomes, but there is limited evidence on whether these skills in childhood affect late life outcomes because of a shortage of long-running datasets containing this information. We develop a three-stage procedure and use it to estimate the effect of childhood social and emotional skills on health and labour market outcomes in late-life. This procedure makes use of mediators in midlife which are shown to be predicted by childhood skills in one dataset and to predict late-life outcomes in another dataset. We use this method to combine estimates from the National Child Development Survey and the British Household Panel Survey. We find that childhood skills predict marital status, education, home ownership, income and health at age 46 years and these midlife variables predict levels of quality-adjusted life years and labour income accumulated by age 63 years. The combined estimates suggest a standard deviation increase in average Bristol Social Adjustment Guide total score at ages 7 and 11 is associated with 4.2% (standard error = 0.6%) additional quality-adjusted life years and more than 9.9% (£14,539, standard error = £2072) additional accumulated pre-tax earnings by age 63 years. Therefore, childhood interventions to increase social and emotional skills would be expected to reduce future healthcare costs and increase wealth. Our three-stage methodology can be used to predict the life-course effects of investments in childhood skills by combining results from datasets across population cohorts. [Abstract copyright: Copyright © 2021 Elsevier Ltd. All rights reserved.]
    • Neoadjuvant therapy or upfront surgery in advanced endometrial cancer: a systematic review protocol.

      McCarthy, Amy; Balfour, Katharine; El Sayed, Iman; Edmondson, Richard; Wan, Yee-Loi Louise; orcid: 0000-0003-1441-6050; email: louise.wan@manchester.ac.uk (2021-11-11)
      There is no consensus on the optimal treatment strategy for people with advanced endometrial cancer. Neoadjuvant therapies such as chemotherapy and radiotherapy have been employed to try to reduce the morbidity of surgery, improve its feasibility and/or improve functional performance in people considered unfit for primary surgery. The objective of this review is to assess whether neoadjuvant chemotherapy or radiotherapy improves health outcomes in people with advanced endometrial cancer when compared with upfront surgery. This review will consider both randomised and non-randomised studies that compare health outcomes associated with the neoadjuvant therapy and upfront surgery in advanced endometrial cancer. Potential studies for inclusion will be collated from electronic searches of OVID Medline, Embase, international trial registries and conference abstract lists. Data collection and extraction will be performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The methodological quality of the studies will be assessed using the Risk of Bias 2 and Risk of Bias in Non-randomised Studies of Interventions tools. If appropriate, we will perform a meta-analysis and provide summary statistics for each outcome. Ethics approval was not required for this study. Once complete, we will publish our findings in peer-reviewed publications, via conference presentations and to update relevant practice guidelines. [Abstract copyright: © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.]
    • Use of long-acting injectable antiretroviral agents for Human Immunodeficiency Virus: A review

      Ariyo, Olumuyiwa E; Jones, Christopher E
      The development of potent antiretroviral drugs has significantly reduced morbidity and mortality associated with human immunodeficiency virus infection, however, the effectiveness of these medications depends upon consistent daily oral intake. Non-adherence can lead to the emergence of resistance, treatment failure and disease progression. This has necessitated the development of long-acting antiretroviral formulations administrable via an infrequent dosing regimen. Long-acting injectable forms of cabotegravir and rilpivirine have reached advanced stages in clinical trials both for the treatment and prevention of HIV. Other long-acting agents are at various stages of development. This review evaluates the current research on the development of long-acting injectable antiretroviral agents for the treatment and prevention of HIV.
    • Changes in the Management of Patients having Radical Radiotherapy for Lung Cancer during the First Wave of the COVID-19 Pandemic in the UK.

      Banfill, K; email: kathryn.banfill@manchester.ac.uk; Croxford, W; Fornacon-Wood, I; Wicks, K; Ahmad, S; Britten, A; Carson, C; Dorey, N; Hatton, M; Hiley, C; et al. (2021-10-28)
      In response to the COVID-19 pandemic, guidelines on reduced fractionation for patients treated with curative-intent radiotherapy were published, aimed at reducing the number of hospital attendances and potential exposure of vulnerable patients to minimise the risk of COVID-19 infection. We describe the changes that took place in the management of patients with stage I-III lung cancer from April to October 2020. Lung Radiotherapy during the COVID-19 Pandemic (COVID-RT Lung) is a prospective multicentre UK cohort study. The inclusion criteria were: patients with stage I-III lung cancer referred for and/or treated with radical radiotherapy between 2nd April and 2nd October 2020. Patients who had had a change in their management and those who continued with standard management were included. Data on demographics, COVID-19 diagnosis, diagnostic work-up, radiotherapy and systemic treatment were collected and reported as counts and percentages. Patient characteristics associated with a change in treatment were analysed using multivariable binary logistic regression. In total, 1553 patients were included (median age 72 years, 49% female); 93 (12%) had a change to their diagnostic investigation and 528 (34%) had a change to their treatment from their centre's standard of care as a result of the COVID-19 pandemic. Age ≥70 years, male gender and stage III disease were associated with a change in treatment on multivariable analysis. Patients who had their treatment changed had a median of 15 fractions of radiotherapy compared with a median of 20 fractions in those who did not have their treatment changed. Low rates of COVID-19 infection were seen during or after radiotherapy, with only 21 patients (1.4%) developing the disease. The COVID-19 pandemic resulted in changes to patient treatment in line with national recommendations. The main change was an increase in hypofractionation. Further work is ongoing to analyse the impact of these changes on patient outcomes. [Abstract copyright: Copyright © 2021. Published by Elsevier Ltd.]
    • Terahertz Characterization of Lead-Free Dielectrics for Different Applications

      Zhang, Man; Zhang, Hangfeng; Jiang, Qinghui; orcid: 0000-0001-7851-2653; Gao, Feng; orcid: 0000-0002-5075-4076; Chen, Riqing; Zhang, Dou; orcid: 0000-0001-8555-2784; Reece, Michael John; orcid: 0000-0002-2293-7123; Yang, Bin; Viola, Giuseppe; Yan, Haixue; orcid: 0000-0002-4563-1100 (American Chemical Society (ACS), 2021-11-02)
    • Metacognitive Beliefs and Suicidal Ideation: An Experience Sampling Study

      Aadahl, Vikki; email: vikkiaadahl@gmail.com; Wells, Adrian; orcid: 0000-0001-7713-1592; email: adrian.wells@manchester.ac.uk; Hallard, Robert; email: Robert.Hallard@cntw.nhs.uk; Pratt, Daniel; orcid: 0000-0001-8843-1224; email: daniel.pratt@manchester.ac.uk (MDPI, 2021-11-24)
      The current study aimed to examine the relationship between metacognitive beliefs about suicidal ideation and the content and process of suicidal ideation. This was to examine the potential contribution of the Self-Regulatory Executive Function (S-REF) model (Wells and Matthew, 2015) to suicidal ideation. Twenty-seven participants completed both trait and state-level measures of suicidal ideation, negative affect, defeat, hopelessness, entrapment and metacognitive beliefs. Experience Sampling Methodology (ESM) was adopted to measure state-level measurements with participants invited to complete an online diary up to seven times a day for six days. Multi-level modelling enabled a detailed examination of the relationships between metacognitive beliefs and suicidal ideation. Positive (β = 0.241, p 0.001) and negative (β = 0.167, p 0.001) metacognitive beliefs about suicidal ideation were positively associated with concurrent suicidal ideation even when known cognitive correlates of suicide were controlled for. The results have important clinical implications for the assessment, formulation and treatment of suicidal ideation. Novel meta-cognitive treatments targeting beliefs about suicidal ideation are now indicated. A limited range of characteristics reported by participants affects the generalizability of findings. Future research is recommended to advance understanding of metacognition and suicide but results demonstrate an important contribution of the S-REF model.
    • The effect of hypoxia on PD-L1 expression in bladder cancer

      Smith, Vicky; email: victoria.smith-15@postgrad.manchester.ac.uk; Mukherjee, Debayan; Lunj, Sapna; Choudhury, Ananya; Hoskin, Peter; West, Catharine; Illidge, Tim (BioMed Central, 2021-11-25)
      Abstract: Introduction: Recent data has demonstrated that hypoxia drives an immunosuppressive tumour microenvironment (TME) via various mechanisms including hypoxia inducible factor (HIF)-dependent upregulation of programmed death ligand 1 (PD-L1). Both hypoxia and an immunosuppressive TME are targetable independent negative prognostic factors for bladder cancer. Therefore we sought to investigate whether hypoxia is associated with upregulation of PD-L1 in the disease. Materials and methods: Three human muscle-invasive bladder cancer cell lines (T24, J82, UMUC3) were cultured in normoxia (20% oxygen) or hypoxia (1 and 0.1% oxygen) for 24 h. Differences in PD-L1 expression were measured using Western blotting, quantitative polymerase chain reaction (qPCR) and flow cytometry (≥3 independent experiments). Statistical tests performed were unpaired t tests and ANOVA. For in silico work an hypoxia signature was used to apply hypoxia scores to muscle-invasive bladder cancers from a clinical trial (BCON; n = 142) and TCGA (n = 404). Analyses were carried out using R and RStudio and statistical tests performed were linear models and one-way ANOVA. Results: When T24 cells were seeded at < 70% confluence, there was decreased PD-L1 protein (p = 0.009) and mRNA (p < 0.001) expression after culture in 0.1% oxygen. PD-L1 protein expression decreased in both 0.1% oxygen and 1% oxygen in a panel of muscle-invasive bladder cancer cells: T24 (p = 0.009 and 0.001), J82 (p = 0.008 and 0.013) and UMUC3 (p = 0.003 and 0.289). Increasing seeding density decreased PD-L1 protein (p < 0.001) and mRNA (p = 0.001) expression in T24 cells grown in both 20 and 1% oxygen. Only when cells were 100% confluent, were PD-L1 protein and mRNA levels higher in 1% versus 20% oxygen (p = 0.056 and p = 0.037). In silico analyses showed a positive correlation between hypoxia signature scores and PD-L1 expression in both BCON (p = 0.003) and TCGA (p < 0.001) cohorts, and between hypoxia and IFNγ signature scores (p < 0.001 for both). Conclusion: Tumour hypoxia correlates with increased PD-L1 expression in patient derived bladder cancer tumours. In vitro PD-L1 expression was affected by cell density and decreased PD-L1 expression was observed after culture in hypoxia in muscle-invasive bladder cancer cell lines. As cell density has such an important effect on PD-L1 expression, it should be considered when investigating PD-L1 expression in vitro.