GPC3-Unc5 receptor complex structure and role in cell migration

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration

The metastatic dissemination of neuroblastoma, an embryonal pediatric cancer : characterization and impact of the sympathetic microenvironment

Le neuroblastome (NB) est la tumeur solide extra-crâniale la plus fréquemment retrouvée chez les enfants. Cette pathologie dérive d’une population de cellules embryonnaires les cellules de la crête neurale troncales, à l’origine du lignage sympathico-adrénal. Les formes métastatiques du NB sont systématiquement diagnostiquées lorsque la maladie est à un stade avancé de dissémination expliquant le faible taux de survie ainsi qu’un manque d’informations cliniques quant aux étapes précoces de dissémination métastatique. Les précédents travaux de l’équipe ont mené à la conception d’un nouveau modèle animal de NB, basé sur l’embryon aviaire. Une première étude exploitant ce modèle a permis de mettre en évidence l’impact du microenvironnement embryonnaire physiologique sur le programme d’expression génique des cellules de NB (Delloye-Bourgeois et al., 2017). L’équipe a identifié une voie de signalisation induite dans les cellules de NB, favorisant leur départ en métastase. Mon travail de thèse a consisté à rechercher les signaux émanant du microenvironnement paraspinal et influant sur les étapes précoces du processus métastatique. Mon premier objectif a été de caractériser l’impact du microenvironnement paraspinal (ganglions sympathiques : GS et dorso-rachidiens : DRG) sur les propriétés de cohésion intercellulaires et migratoires des cellules de NB, en travaillant sur des milieux de culture conditionnés par les GS et les DRG embryonnaires. Le deuxième objectif de notre étude a été de décrire de façon exhaustive le sécrétome des GS et des DRG embryonnaires afin d’identifier des candidats clés du microenvironnement qui impacteraient le programme génique des cellules de NB signant leur départ en métastase. En parallèle, nous avons conduit une analyse transcriptomique des cellules de NB confrontées aux signaux émanant du microenvironnement paraspinal. Nous avons ainsi observé que les milieux conditionnés de GS et de DRG induisent une décohésion des cellules de NB ainsi qu’une augmentation significative du potentiel migratoire et invasif des cellules tumorales in vitro. L’analyse croisée des données de protéomique et transcriptomique a conduit à l’identification d’une signature moléculaire centrée sur les glycoprotéines OLFM, notamment l’OLFM1, dont les fonctions sur la génération et la migration des cellules de la crête neurale ont été précédemment décrites (Barembaum et al., 2000). La combinaison d’expérimentations in vitro et in vivo consistant à bloquer l’OLFM1 endogène dans le modèle aviaire de NB a confirmé le rôle majeur de cette protéine dans le processus de dissémination métastatique du NB. Les données générées nous ont par ailleurs permis de décrire une combinaison de gènes signant la phase d’échappement de la tumeur primaire des NBs métastatiques mais également d’autres cancers dérivés de la crête neurale. Mes travaux de thèse ont ainsi révélé un mécanisme clé du processus de dissémination métastatique du NB, qui consiste à détourner les signaux du microenvironnement embryonnaire favorisant l’échappement de la tumeur primaire. Cette étude ouvre la voie à la caractérisation des interactions entre les environnements embryonnaires, immatures et dynamiques, successivement rencontrés lors des étapes de dissémination du NB. A terme, ce champ de recherche permet d’envisager de nouvelles stratégies thérapeutiques dédiées aux NBs métastatiques.Neuroblastoma (NB) is the most common extracranial solid tumour found in children. This disease is derived from an embryonal cell population, the trunk neural crest cells that give rise to the sympathoadrenal cell lineage. The metastatic forms of NB are consistently diagnosed when the disease is already at a disseminated stage explaining a low survival rate and a lack of clinical information concerning the early steps of metastatic dissemination. Our team’s previous work led to the conception of a novel animal model of NB based on the avian embryo. A first study exploiting this model highlighted the physiological embryonic microenvironment’s impact on NB cells genes expression program (Delloye-Bourgeois et al., 2017). Our team identified a pathway induced in NB cells that promoted the onset of metastasis. My PhD work consisted in identifying the signals emanating from the paraspinal microenvironment and influencing the early steps of metastasis. My first aim was to characterize the paraspinal microenvironment’s impact (sympathetic ganglia: SG and dorsal root ganglia: DRG) on cell-cell cohesion and migratory properties of NB cells, working on conditioned media from embryonic SG and DRG. The second aim of our study was to described thoroughly the embryonic SG and DRG secretome in order to identify microenvironment key candidates that would impact the NB cells genetic program triggering the onset of metastasis. Simultaneously, we conducted a transcriptomic analysis of NB cells confronted to the paraspinal microenvironment signals. We thus observed that SG and DRG conditioned media induced a decohesion of NB cells and a significant increase of the migratory and invasive potential of tumour cells in vitro. Crossing the proteomic and transcriptomic data led to the identification of a molecular signature centered on OLFM glycoproteins, in particular OLFM1, whose functions on neural crest cells formation and migration were previously described (Barembaum et al., 2000). The combination of in vitro and in vivo experimentations consisting in blocking endogenous OLFM1 in our NB avian model confirmed the major role of this protein in NB metastatic dissemination. Moreover, the data obtained allowed us to describe a combination of genes attesting of an escape from the primary tumour of metastatic NBs but also of other cancers originating from neural crest. My PhD work highlighted a key mechanism of NB metastatic dissemination process consisting in hijacking the embryonic microenvironment signals to promote the escape from the primary tumour. Hence, this study paves the way for the characterization of the interactions between the different immature and dynamic embryonic microenvironments successively encountered throughout the NB dissemination steps. In the long term, this research field would allow to contemplate new therapeutic strategies dedicated to metastatic NBs

Environmental cues from neural crest derivatives act as metastatic triggers in an embryonic neuroblastoma model

International audienceAbstract Embryonic malignant transformation is concomitant to organogenesis, often affecting multipotent and migratory progenitors. While lineage relationships between malignant cells and their physiological counterparts are extensively investigated, the contribution of exogenous embryonic signals is not fully known. Neuroblastoma (NB) is a childhood malignancy of the peripheral nervous system arising from the embryonic trunk neural crest (NC) and characterized by heterogeneous and interconvertible tumor cell identities. Here, using experimental models mimicking the embryonic context coupled to proteomic and transcriptomic analyses, we show that signals released by embryonic sympathetic ganglia, including Olfactomedin-1, induce NB cells to shift from a noradrenergic to mesenchymal identity, and to activate a gene program promoting NB metastatic onset and dissemination. From this gene program, we extract a core signature specifically shared by metastatic cancers with NC origin. This reveals non-cell autonomous embryonic contributions regulating the plasticity of NB identities and setting pro-dissemination gene programs common to NC-derived cancers

GPC3-Unc5 receptor complex structure and role in cell migration

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration

Thrombotic and hemorrhagic complications of COVID-19 in adults hospitalized in high-income countries compared with those in adults hospitalized in low- and middle-income countries in an international registry

Background: COVID-19 has been associated with a broad range of thromboembolic, ischemic, and hemorrhagic complications (coagulopathy complications). Most studies have focused on patients with severe disease from high-income countries (HICs). Objectives: The main aims were to compare the frequency of coagulopathy complications in developing countries (low- and middle-income countries [LMICs]) with those in HICs, delineate the frequency across a range of treatment levels, and determine associations with in-hospital mortality. Methods: Adult patients enrolled in an observational, multinational registry, the International Severe Acute Respiratory and Emerging Infections COVID-19 study, between January 1, 2020, and September 15, 2021, met inclusion criteria, including admission to a hospital for laboratory-confirmed, acute COVID-19 and data on complications and survival. The advanced-treatment cohort received care, such as admission to the intensive care unit, mechanical ventilation, or inotropes or vasopressors; the basic-treatment cohort did not receive any of these interventions. Results: The study population included 495,682 patients from 52 countries, with 63% from LMICs and 85% in the basic treatment cohort. The frequency of coagulopathy complications was higher in HICs (0.76%-3.4%) than in LMICs (0.09%-1.22%). Complications were more frequent in the advanced-treatment cohort than in the basic-treatment cohort. Coagulopathy complications were associated with increased in-hospital mortality (odds ratio, 1.58; 95% CI, 1.52-1.64). The increased mortality associated with these complications was higher in LMICs (58.5%) than in HICs (35.4%). After controlling for coagulopathy complications, treatment intensity, and multiple other factors, the mortality was higher among patients in LMICs than among patients in HICs (odds ratio, 1.45; 95% CI, 1.39-1.51). Conclusion: In a large, international registry of patients hospitalized for COVID-19, coagulopathy complications were more frequent in HICs than in LMICs (developing countries). Increased mortality associated with coagulopathy complications was of a greater magnitude among patients in LMICs. Additional research is needed regarding timely diagnosis of and intervention for coagulation derangements associated with COVID-19, particularly for limited-resource settings

Implementation of Recommendations on the Use of Corticosteroids in Severe COVID-19

Importance: Research diversity and representativeness are paramount in building trust, generating valid biomedical knowledge, and possibly in implementing clinical guidelines. Objectives: To compare variations over time and across World Health Organization (WHO) geographic regions of corticosteroid use for treatment of severe COVID-19; secondary objectives were to evaluate the association between the timing of publication of the RECOVERY (Randomised Evaluation of COVID-19 Therapy) trial (June 2020) and the WHO guidelines for corticosteroids (September 2020) and the temporal trends observed in corticosteroid use by region and to describe the geographic distribution of the recruitment in clinical trials that informed the WHO recommendation. Design, setting, and participants: This prospective cohort study of 434 851 patients was conducted between January 31, 2020, and September 2, 2022, in 63 countries worldwide. The data were collected under the auspices of the International Severe Acute Respiratory and Emerging Infections Consortium (ISARIC)-WHO Clinical Characterisation Protocol for Severe Emerging Infections. Analyses were restricted to patients hospitalized for severe COVID-19 (a subset of the ISARIC data set). Exposure: Corticosteroid use as reported to the ISARIC-WHO Clinical Characterisation Protocol for Severe Emerging Infections. Main outcomes and measures: Number and percentage of patients hospitalized with severe COVID-19 who received corticosteroids by time period and by WHO geographic region. Results: Among 434 851 patients with confirmed severe or critical COVID-19 for whom receipt of corticosteroids could be ascertained (median [IQR] age, 61.0 [48.0-74.0] years; 53.0% male), 174 307 (40.1%) received corticosteroids during the study period. Of the participants in clinical trials that informed the guideline, 91.6% were recruited from the United Kingdom. In all regions, corticosteroid use for severe COVID-19 increased, but this increase corresponded to the timing of the RECOVERY trial (time-interruption coefficient 1.0 [95% CI, 0.9-1.2]) and WHO guideline (time-interruption coefficient 1.9 [95% CI, 1.7-2.0]) publications only in Europe. At the end of the study period, corticosteroid use for treatment of severe COVID-19 was highest in the Americas (5421 of 6095 [88.9%]; 95% CI, 87.7-90.2) and lowest in Africa (31 588 of 185 191 [17.1%]; 95% CI, 16.8-17.3). Conclusions and relevance: The results of this cohort study showed that implementation of the guidelines for use of corticosteroids in the treatment of severe COVID-19 varied geographically. Uptake of corticosteroid treatment was lower in regions with limited clinical trial involvement. Improving research diversity and representativeness may facilitate timely knowledge uptake and guideline implementation

Characteristics and outcomes of an international cohort of 600 000 hospitalized patients with COVID-19

Background: We describe demographic features, treatments and clinical outcomes in the International Severe Acute Respiratory and emerging Infection Consortium (ISARIC) COVID-19 cohort, one of the world's largest international, standardized data sets concerning hospitalized patients. Methods: The data set analysed includes COVID-19 patients hospitalized between January 2020 and January 2022 in 52 countries. We investigated how symptoms on admission, co-morbidities, risk factors and treatments varied by age, sex and other characteristics. We used Cox regression models to investigate associations between demographics, symptoms, co-morbidities and other factors with risk of death, admission to an intensive care unit (ICU) and invasive mechanical ventilation (IMV). Results: Data were available for 689 572 patients with laboratory-confirmed (91.1%) or clinically diagnosed (8.9%) SARS-CoV-2 infection from 52 countries. Age [adjusted hazard ratio per 10 years 1.49 (95% CI 1.48, 1.49)] and male sex [1.23 (1.21, 1.24)] were associated with a higher risk of death. Rates of admission to an ICU and use of IMV increased with age up to age 60 years then dropped. Symptoms, co-morbidities and treatments varied by age and had varied associations with clinical outcomes. The case-fatality ratio varied by country partly due to differences in the clinical characteristics of recruited patients and was on average 21.5%. Conclusions: Age was the strongest determinant of risk of death, with a ∼30-fold difference between the oldest and youngest groups; each of the co-morbidities included was associated with up to an almost 2-fold increase in risk. Smoking and obesity were also associated with a higher risk of death. The size of our international database and the standardized data collection method make this study a comprehensive international description of COVID-19 clinical features. Our findings may inform strategies that involve prioritization of patients hospitalized with COVID-19 who have a higher risk of death

The value of open-source clinical science in pandemic response: lessons from ISARIC

International audienc