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Around 25 staff and students from the Ludwig Institute for Cancer Research donned their running shoes and team t-shirts to run 10K in support of Muscular Dystrophy UK. The 2017 Oxford 'Town and Gown' race on Sunday 14th May attracted more than 4000 runners, from first-timers to serious athletes. The Ludwig team were rewarded for all their training efforts by beautiful sunshine and a fantastic crowd.
Mechanical confinement governs phenotypic plasticity in melanoma.
Phenotype switching is a form of cellular plasticity in which cancer cells reversibly move between two opposite extremes: proliferative versus invasive states1,2. Although it has long been hypothesized that such switching is triggered by external cues, the identity of these cues remains unclear. Here we demonstrate that mechanical confinement mediates phenotype switching through chromatin remodelling. Using a zebrafish model of melanoma coupled with human samples, we profiled tumour cells at the interface between the tumour and surrounding microenvironment. Morphological analysis of interface cells showed elliptical nuclei, suggestive of mechanical confinement by the adjacent tissue. Spatial and single-cell transcriptomics demonstrated that interface cells adopted a gene program of neuronal invasion, including the acquisition of an acetylated tubulin cage that protects the nucleus during migration. We identified the DNA-bending protein HMGB2 as a confinement-induced mediator of the neuronal state. HMGB2 is upregulated in confined cells, and quantitative modelling revealed that confinement prolongs the contact time between HMGB2 and chromatin, leading to changes in chromatin configuration that favour the neuronal phenotype. Genetic disruption of HMGB2 showed that it regulates the trade-off between proliferative and invasive states, in which confined HMGB2high tumour cells are less proliferative but more drug-resistant. Our results implicate the mechanical microenvironment as a mechanism that drives phenotype switching in melanoma.
IFNγ-induced PD-L1+MHC II+ macrophages and Tim-3+ tumor-reactive CD8+ T cells predict a response to anti-PD-1 therapy in tumor-bearing mice.
While immune checkpoint inhibitors have led to durable responses in various cancer types, a substantial proportion of patients do not respond to these interventions. To uncover potential factors associated with a positive response to immunotherapy, we used a bilateral tumor model using P815 mastocytoma implanted in DBA/2 mice. In this model, only a fraction of tumor-bearing mice responds to anti-PD-1 treatment. Thus, it provides a valuable model to explore the influence of the tumor microenvironment (TME) in determining the efficacy of immune checkpoint blockade (ICB)-based immunotherapies. It also allows for the analysis of a pretreatment tumor and inference of its treatment outcome based on the response observed in the contralateral tumor. Herein, we report that tumor-reactive CD8+ T-cell clones expressing high levels of Tim-3 were associated with a positive antitumor response following anti-PD-1 administration. Our study also revealed distinct differentiation dynamics in tumor-infiltrating myeloid cells in responding and non-responding mice. An IFNγ-enriched TME promoted the differentiation of monocytes into PD-L1posMHC IIhigh cells in mice responding to immunotherapy. Monocytes present in the TME of non-responding mice failed to reach the same final stage of differentiation trajectory, suggesting that an altered monocyte to macrophage differentiation route may hamper the response to ICB. These insights will direct future research towards a temporal analysis of tumor-associated macrophages (TAMs), aiming to identify factors responsible for transitions between differentiation states within the TME. This approach may pave the way to novel strategies to enhance the efficacy of PD-1 blockade.
Nitric oxide promotes cysteine N-degron proteolysis through control of oxygen availability
Selected proteins containing an N-terminal cysteine (Nt-Cys) are subjected to rapid, O 2 -dependent proteolysis via the Cys/Arg-branch of the N-degron pathway. Cysteine dioxygenation is catalyzed in mammalian cells by 2-aminoethanethiol dioxygenase (ADO), an enzyme that manifests extreme O 2 sensitivity. The canonical substrates of this pathway in mammalia are the regulators of G-protein signaling 4, 5, and 16, as well as interleukin-32. In addition to operating as an O 2 -sensing mechanism, this pathway has previously been described as a sensor of nitric oxide (NO), with robust effects on substrate stability upon modulation of NO bioavailability being widely demonstrated. Despite this, no mechanism to describe the action of NO on the Cys/Arg N-degron pathway has yet been substantiated. We demonstrate that NO can regulate the stability of Cys N-degron substrates indirectly via the regulation of ADO cosubstrate availability. Through competitive, O 2 -dependent inhibition of cytochrome C oxidase, NO can substantially modify cellular O 2 consumption rate and, in doing so, alter the availability of O 2 for Nt-Cys dioxygenation. We show that this increase in O 2 availability in response to NO exposure is sufficient to alter both dynamic and steady-state ADO substrate levels. It is likely that this mechanism operates to couple O 2 supply and mitochondrial respiration with responses to G-protein-coupled receptor stimulation.
Platelets sequester extracellular DNA, capturing tumor-derived and free fetal DNA.
Platelets are anucleate blood cells vital for hemostasis and immunity. During cell death and aberrant mitosis, nucleated cells release DNA, resulting in "cell-free" DNA in plasma (cfDNA). An excess of cfDNA is deleterious. Given their ability to internalize pathogen-derived nucleic acids, we hypothesized that platelets may also clear endogenous cfDNA. We found that, despite lacking a nucleus, platelets contained a repertoire of DNA fragments mapping across the nuclear genome. We detected fetal DNA in maternal platelets and cancer-derived DNA in platelets from patients with premalignant and cancerous lesions. As current liquid biopsy approaches utilize platelet-depleted plasma, important genetic information contained within platelets is being missed. This study establishes a physiological role for platelets that has not previously been highlighted, with broad translational relevance.
The Ebola Data Platform: A prospective, standardised, clinical dataset collected during the 2013-2016 West African Ebola outbreak.
The Ebola Data Platform (EDP) was developed to strengthen knowledge and capacity across health, research, and humanitarian communities to reduce the impact of Ebola through responsible data use. This collaborative initiative was established by West African governments, NGOs, academic organisations, and intra-governmental health organisations directly involved in the 2013-2016 West African Ebola outbreak. The platform was established to provide a centralised, standardised dataset of individual patient data collected during the outbreak for the purpose of research to improve Ebola treatment and control, and includes over 13,600 patient records of individuals infected and treated from 22 different Ebola treatment centres across Guinea, Sierra Leone, Liberia, and Nigeria. Patient data are available from treatment centre triage and admission, inpatient clinical observations, and outcomes, with outpatient follow-up available for some datasets. Data include signs and symptoms, pre-existing comorbidities, vital signs, laboratory testing, treatments, complications, dates of admission and discharge, mortality, viral strains, and other data. This publication describes characteristics of the EDP dataset, its architecture, methods for data access and tools for utilising the dataset.
Temporo-spatial cellular atlas of the regenerating alveolar niche in idiopathic pulmonary fibrosis
Abstract Healthy alveolar repair relies on the ability of alveolar stem cells to differentiate into specialized epithelial cells for gas exchange. In chronic fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF), this regenerative process is abnormal but the underlying mechanisms remain unclear. Here, using human lung tissue that represents different stages of disease and a 33-plex single-cell imaging mass cytometry (IMC), we present a high-resolution, temporo-spatial cell atlas of the regenerating alveolar niche. With unbiased mathematical methods which quantify statistically enriched interactions, CD206himacrophage subtype and an alveolar basal intermediate epithelial cell emerge as the most statistically robust spatial association in the epithelial and immune cell interactome, found across all stages of disease. Spatially resolved receptor–ligand analysis further offers an in silico mechanism by which these macrophages may influence epithelial regeneration. These findings provide a foundational step toward understanding immune–epithelial dynamics in aberrant alveolar regeneration in IPF.
Blood Lipoproteins Shape the Phenotype and Lipid Content of Early Atherosclerotic Lesion Macrophages: A Dual-Structured Mathematical Model
AbstractMacrophages in atherosclerotic lesions exhibit a spectrum of behaviours or phenotypes. The phenotypic distribution of monocyte-derived macrophages (MDMs), its correlation with MDM lipid content, and relation to blood lipoprotein densities are not well understood. Of particular interest is the balance between low density lipoproteins (LDL) and high density lipoproteins (HDL), which carry bad and good cholesterol respectively. To address these issues, we have developed a mathematical model for early atherosclerosis in which the MDM population is structured by phenotype and lipid content. The model admits a simpler, closed subsystem whose analysis shows how lesion composition becomes more pathological as the blood density of LDL increases relative to the HDL capacity. We use asymptotic analysis to derive a power-law relationship between MDM phenotype and lipid content at steady-state. This relationship enables us to understand why, for example, lipid-laden MDMs have a more inflammatory phenotype than lipid-poor MDMs when blood LDL lipid density greatly exceeds HDL capacity. We show further that the MDM phenotype distribution always attains a local maximum, while the lipid content distribution may be unimodal, adopt a quasi-uniform profile or decrease monotonically. Pathological lesions exhibit a local maximum in both the phenotype and lipid content MDM distributions, with the maximum at an inflammatory phenotype and near the lipid content capacity respectively. These results illustrate how macrophage heterogeneity arises in early atherosclerosis and provide a framework for future model validation through comparison with single-cell RNA sequencing data.
Advection-dominated models of atherosclerotic plaque composition: The impacts of cell death and cholesterol toxicity.
Advanced atherosclerotic plaques are characterised by a large necrotic core containing highly inflammatory lipids and debris from dead cells. In large plaques, newly recruited macrophages fail to penetrate this core, and instead push existing material deeper inside the plaque. In this paper, we consider two multiphase models for early atherosclerotic plaque growth, and we analyse their behaviour in the limiting regime where bulk advection drives mass transport of cells and lipids. In this regime, the dynamics of the deep plaque can be approximated by a system of advection-reaction equations. By applying the method of characteristics to these equations, we derive a set of ODEs that describes the evolution of individual segments of plaque tissue. We apply this approximation to a simple 1D three-phase model comprising macrophage foam cells, dead cells, and modified LDL, and we investigate how plaque tissue composition depends on the relative rates of cell death and efferocytosis (cell recycling). We also consider a six-phase model in which death rates depend on intracellular cholesterol content. We use this model to study the effects of cholesterol-induced toxicity, and the beneficial effects of high density lipoproteins (HDL), which can remove excess cholesterol from macrophages. We show that for both multiphase models, the advection-reaction approximations capture key structural features of the full model solutions, including the relative proportions of live and dead cells, and persistent spatial heterogeneities that arise from time-varying boundary influxes of LDL and HDL.
Proneural-Mesenchymal hybrid glioblastoma cells are resistant to therapy and dependent on nuclear import.
BackgroundDespite extensive research efforts, glioblastoma (GBM) remains a deadly disease with poor prognosis. Although previous studies have identified various cell states within GBM tumors, the molecular mechanism underlying adaptive GBM cell plasticity induced by conventional therapy remains unclear.MethodsWe used fluorescent reporters for proneural (PN) and mesenchymal (MES) subtypes to monitor GBM cell plasticity in real-time across multiple patient-derived cell lines. This approach revealed cells that concurrently expressed both proneural and mesenchymal markers. To investigate this unique hybrid population, we implemented a comprehensive methodological approach encompassing bulk and single-cell RNA sequencing, single-cell ChIP sequencing, nuclear proteomics, high-resolution imaging, orthotopic mouse models, clinical dataset analysis, and pharmacological and genetic techniques. This multifaceted strategy allowed us to gain functional and molecular insights into this distinct cellular population.ResultsWe showed that these hybrid cells are increased by conventional therapies, and are resistant to these therapies. At the molecular level, hybrid cells display significant alterations in chromatin structure and nuclear protein composition, elevated transcriptional activity, Myc activation, and improved transport between the nucleus and cytoplasm. Genetic and pharmaceutical inhibition of the nuclear import/export shuttling machinery, increased in hybrid cells, effectively suppressed adaptive GBM cell plasticity and hybrid identity, thereby enhancing the sensitivity of GBM cells to therapies.ConclusionOur results indicate that GBM hybrid cells play a crucial role in chemoradiation resistance. The nuclear transport machinery presents a potential therapeutic target for hybrid cells, offering a way to counteract the typical resistance to treatment observed in GBM.
BRN2 is a non-canonical melanoma tumor-suppressor
AbstractWhile the major drivers of melanoma initiation, including activation of NRAS/BRAF and loss of PTEN or CDKN2A, have been identified, the role of key transcription factors that impose altered transcriptional states in response to deregulated signaling is not well understood. The POU domain transcription factor BRN2 is a key regulator of melanoma invasion, yet its role in melanoma initiation remains unknown. Here, in a BrafV600EPtenF/+ context, we show that BRN2 haplo-insufficiency promotes melanoma initiation and metastasis. However, metastatic colonization is less efficient in the absence of Brn2. Mechanistically, BRN2 directly induces PTEN expression and in consequence represses PI3K signaling. Moreover, MITF, a BRN2 target, represses PTEN transcription. Collectively, our results suggest that on a PTEN heterozygous background somatic deletion of one BRN2 allele and temporal regulation of the other allele elicits melanoma initiation and progression.