Glioblastoma cell fate is differentially regulated by the microenvironments of the tumour bulk and infiltrative margin

Glioblastoma recurrence originates from invasive cells at the tumour margin that escape surgical debulking, but their biology remains poorly understood. Here we generated three somatic mouse models recapitulating the main glioblastoma driver mutations to characterise margin cells. We find that, regardless of genetics, tumours converge on a common set of neural- like cellular states. However, bulk and margin display distinct neurogenic patterns and immune microenvironments. The margin is immune-cold and preferentially follows developmental-like trajectories to produce astrocyte-like cells. In contrast, injury-like programmes dominate in the bulk, are associated with immune infiltration and generate lowly-proliferative injured neural progenitor-like (iNPCs) cells. In vivo label-retention approaches further demonstrate that iNPCs account for a significant proportion of dormant glioblastoma cells and are induced by interferon signalling within T-cell niches. These findings indicate that tumour region is a major determinant of glioblastoma cell fate and therapeutic vulnerabilities identified in bulk may not extend to the margin residuum

Author Guide for Addressing Animal Methods Bias in Publishing

There is growing recognition that animal methods bias, a preference for animal‐based methods where they are not necessary or where nonanimal‐based methods may already be suitable, can impact the likelihood or timeliness of a manuscript being accepted for publication. Following April 2022 workshop about animal methods bias in scientific publishing, a coalition of scientists and advocates formed a Coalition to Illuminate and Address Animal Methods Bias (COLAAB). The COLAAB has developed this guide to be used by authors who use nonanimal methods to avoid and respond to animal methods bias from manuscript reviewers. It contains information that researchers may use during 1) study design, including how to find and select appropriate nonanimal methods and preregister a research plan, 2) manuscript preparation and submission, including tips for discussing methods and choosing journals and reviewers that may be more receptive to nonanimal methods, and 3) the peer review process, providing suggested language and literature to aid authors in responding to biased reviews. The author's guide for addressing animal methods bias in publishing is a living resource also available online at animalmethodsbias.org, which aims to help ensure fair dissemination of research that uses nonanimal methods and prevent unnecessary experiments on animals

Proceedings of a workshop to address animal methods bias in scientific publishing

Animal methods bias in scientific publishing is a newly defined type of publishing bias describing a preference for animal-based methods where they may not be necessary or where nonanimal-based methods may already be suitable, which impacts the likelihood or timeliness of a manuscript being accepted for publication. This article covers the output from a workshop between stakeholders in publishing, academia, industry, government, and non-governmental organizations. The intent of the workshop was to exchange perspectives on the prevalence, causes, and impact of animal methods bias in scientific publishing, as well as to explore mitigation strategies. Output from the workshop includes summaries of presentations, breakout group discussions, participant polling results, and a synthesis of recommendations for mitigation. Overall, participants felt that animal methods bias has a meaningful impact on scientific publishing, though more evidence is needed to demonstrate its prevalence. Significant consequences of this bias that were identified include the unnecessary use of animals in scientific procedures, the continued reliance on animals in research—even where suitable nonanimal methods exist, poor rates of clinical translation, delays in publication, and negative impacts on career trajectories in science. Workshop participants offered recommendations for journals, publishers, funders, governments, and other policy makers, as well as the scientific community at large, to reduce the prevalence and impacts of animal methods bias. The workshop resulted in the creation of working groups committed to addressing animal methods bias and activities are ongoing

Glioblastoma cell fate is differentially regulated by the microenvironments of the tumor bulk and infiltrative margin

Glioblastoma (GBM) recurrence originates from invasive margin cells that escape surgical debulking, but to what extent these cells resemble their bulk counterparts remains unclear. Here, we generated three immunocompetent somatic GBM mouse models, driven by subtype-associated mutations, to compare matched bulk and margin cells. We find that, regardless of mutations, tumors converge on common sets of neural-like cellular states. However, bulk and margin have distinct biology. Injury-like programs associated with immune infiltration dominate in the bulk, leading to the generation of lowly proliferative injured neural progenitor-like cells (iNPCs). iNPCs account for a significant proportion of dormant GBM cells and are induced by interferon signaling within T cell niches. In contrast, developmental-like trajectories are favored within the immune-cold margin microenvironment resulting in differentiation toward invasive astrocyte-like cells. These findings suggest that the regional tumor microenvironment dominantly controls GBM cell fate and biological vulnerabilities identified in the bulk may not extend to the margin residuum

Conséquences d’un faible stress réplicatif sur le programme de replication des cellules normales et cancéreuses

DNA replication is very well orchestrated in mammalian cells thanks to a tight regulation of the temporal order of replication origin activation, commonly called replication timing (RT). The replication timing of a given replication domain (RD) is very robust and depends on the cell type. Upon low replication stress, replication forks progress slower and it has been shown that some fragile regions are replicated later or even under-replicated. These replication delay leads to DNA damage and genetic instability, a common marker of cancers. Except for these fragile regions, the direct impact of low replication stress on the RT of the whole genome has not been explored yet. The aim of my thesis was to analyse and compare the replication timing of 6 human cell lines from different tissues (healthy or from tumours) in response to mild replication stress. Assessing this question, I have first observed heterogeneous response in between cell lines, some cancer cells were much more impacted by low replication stress. Strikingly, in some cancer cells, specific RD are undergoing a switch from late to early replication in response to replication stress. Very interestingly, this RT alteration was still detected in daughter cells. These findings disclosed a new mechanism mainly used by cancer cells in response to replication stress that brings another proof of their genome plasticity, allowing a quick response and adaptation to stress that, eventually, gives better resistance to genotoxic agents.La réplication de l'ADN est un processus finement orchestré dans les cellules eucaryotes grâce à la régulation temporelle de l'activation des origines de réplication, phénomène mieux connu sous le nom de timing de réplication. Le timing de réplication d'un domaine génomique est très robuste et dépend de l'identité cellulaire. Lors d'un faible stress réplicatif, la fourche de réplication est ralentie et certaines régions fragiles du génome voient leur timing de réplication retardé voire ne sont pas du tout dupliquées. Ainsi, le ralentissement du processus de réplication peut avoir pour conséquence des cassures de l'ADN et à fortiori de l'instabilité génomique, un marqueur bien connu des cancers. Hormis ces régions fragiles déjà identifiées, l'impact direct d'un faible stress sur le "timing" de réplication de l'ensemble du génome n'a pas encore été étudié. L'objectif de ma thèse était donc d'analyser et de comparer le timing de réplication de 6 lignées cellulaire de différents tissus humain (sain ou tumoral) en réponse à un faible stress réplicatif. J'ai ainsi pu observer une réponse hétérogène entre les différentes lignées et un timing de réplication plus impacté dans certaines lignées cancéreuses en réponse au stress. En particulier, certaines lignées cancéreuses présentent de très fortes avancées de timing dans certaines régions précises du génome en réponse au stress réplicatif. De surcroit, nous avons observé que ces avancées de timing qui sont retrouvées dans la génération cellulaire suivante. Ainsi, lors de ma thèse, j'ai mis en évidence un nouveau mécanisme mis en place surtout par les cellules cancéreuses en réponse au stress réplicatif qui apporte une preuve supplémentaire de la plasticité du génome de ces cellules, leur permettant de répondre et de s'adapter rapidement à des conditions de stress et, éventuellement, de mieux résister aux agents génotoxiques

Conséquences d'un faible stress réplicatif sur le programme de réplication des cellules normales et cancéreuses

DNA replication is very well orchestrated in mammalian cells thanks to a tight regulation of the temporal order of replication origin activation, commonly called replication timing (RT). The replication timing of a given replication domain (RD) is very robust and depends on the cell type. Upon low replication stress, replication forks progress slower and it has been shown that some fragile regions are replicated later or even under-replicated. These replication delay leads to DNA damage and genetic instability, a common marker of cancers. Except for these fragile regions, the direct impact of low replication stress on the RT of the whole genome has not been explored yet. The aim of my thesis was to analyse and compare the replication timing of 6 human cell lines from different tissues (healthy or from tumours) in response to mild replication stress. Assessing this question, I have first observed heterogeneous response in between cell lines, some cancer cells were much more impacted by low replication stress. Strikingly, in some cancer cells, specific RD are undergoing a switch from late to early replication in response to replication stress. Very interestingly, this RT alteration was still detected in daughter cells. These findings disclosed a new mechanism mainly used by cancer cells in response to replication stress that brings another proof of their genome plasticity, allowing a quick response and adaptation to stress that, eventually, gives better resistance to genotoxic agents.La réplication de l'ADN est un processus finement orchestré dans les cellules eucaryotes grâce à la régulation temporelle de l'activation des origines de réplication, phénomène mieux connu sous le nom de timing de réplication. Le timing de réplication d'un domaine génomique est très robuste et dépend de l'identité cellulaire. Lors d'un faible stress réplicatif, la fourche de réplication est ralentie et certaines régions fragiles du génome voient leur timing de réplication retardé voire ne sont pas du tout dupliquées. Ainsi, le ralentissement du processus de réplication peut avoir pour conséquence des cassures de l'ADN et à fortiori de l'instabilité génomique, un marqueur bien connu des cancers. Hormis ces régions fragiles déjà identifiées, l'impact direct d'un faible stress sur le "timing" de réplication de l'ensemble du génome n'a pas encore été étudié. L'objectif de ma thèse était donc d'analyser et de comparer le timing de réplication de 6 lignées cellulaire de différents tissus humain (sain ou tumoral) en réponse à un faible stress réplicatif. J'ai ainsi pu observer une réponse hétérogène entre les différentes lignées et un timing de réplication plus impacté dans certaines lignées cancéreuses en réponse au stress. En particulier, certaines lignées cancéreuses présentent de très fortes avancées de timing dans certaines régions précises du génome en réponse au stress réplicatif. De surcroit, nous avons observé que ces avancées de timing qui sont retrouvées dans la génération cellulaire suivante. Ainsi, lors de ma thèse, j'ai mis en évidence un nouveau mécanisme mis en place surtout par les cellules cancéreuses en réponse au stress réplicatif qui apporte une preuve supplémentaire de la plasticité du génome de ces cellules, leur permettant de répondre et de s'adapter rapidement à des conditions de stress et, éventuellement, de mieux résister aux agents génotoxiques

Impact of low replication stress on the replication program of cancer and non-tumor cells

La réplication de l'ADN est un processus finement orchestré dans les cellules eucaryotes grâce à la régulation temporelle de l'activation des origines de réplication, phénomène mieux connu sous le nom de timing de réplication. Le timing de réplication d'un domaine génomique est très robuste et dépend de l'identité cellulaire. Lors d'un faible stress réplicatif, la fourche de réplication est ralentie et certaines régions fragiles du génome voient leur timing de réplication retardé voire ne sont pas du tout dupliquées. Ainsi, le ralentissement du processus de réplication peut avoir pour conséquence des cassures de l'ADN et à fortiori de l'instabilité génomique, un marqueur bien connu des cancers. Hormis ces régions fragiles déjà identifiées, l'impact direct d'un faible stress sur le "timing" de réplication de l'ensemble du génome n'a pas encore été étudié. L'objectif de ma thèse était donc d'analyser et de comparer le timing de réplication de 6 lignées cellulaire de différents tissus humain (sain ou tumoral) en réponse à un faible stress réplicatif. J'ai ainsi pu observer une réponse hétérogène entre les différentes lignées et un timing de réplication plus impacté dans certaines lignées cancéreuses en réponse au stress. En particulier, certaines lignées cancéreuses présentent de très fortes avancées de timing dans certaines régions précises du génome en réponse au stress réplicatif. De surcroit, nous avons observé que ces avancées de timing qui sont retrouvées dans la génération cellulaire suivante. Ainsi, lors de ma thèse, j'ai mis en évidence un nouveau mécanisme mis en place surtout par les cellules cancéreuses en réponse au stress réplicatif qui apporte une preuve supplémentaire de la plasticité du génome de ces cellules, leur permettant de répondre et de s'adapter rapidement à des conditions de stress et, éventuellement, de mieux résister aux agents génotoxiques.DNA replication is very well orchestrated in mammalian cells thanks to a tight regulation of the temporal order of replication origin activation, commonly called replication timing (RT). The replication timing of a given replication domain (RD) is very robust and depends on the cell type. Upon low replication stress, replication forks progress slower and it has been shown that some fragile regions are replicated later or even under-replicated. These replication delay leads to DNA damage and genetic instability, a common marker of cancers. Except for these fragile regions, the direct impact of low replication stress on the RT of the whole genome has not been explored yet. The aim of my thesis was to analyse and compare the replication timing of 6 human cell lines from different tissues (healthy or from tumours) in response to mild replication stress. Assessing this question, I have first observed heterogeneous response in between cell lines, some cancer cells were much more impacted by low replication stress. Strikingly, in some cancer cells, specific RD are undergoing a switch from late to early replication in response to replication stress. Very interestingly, this RT alteration was still detected in daughter cells. These findings disclosed a new mechanism mainly used by cancer cells in response to replication stress that brings another proof of their genome plasticity, allowing a quick response and adaptation to stress that, eventually, gives better resistance to genotoxic agents

The Protective Role of Dormant Origins in Response to Replicative Stress

Genome stability requires tight regulation of DNA replication to ensure that the entire genome of the cell is duplicated once and only once per cell cycle. In mammalian cells, origin activation is controlled in space and time by a cell-specific and robust program called replication timing. About 100,000 potential replication origins form on the chromatin in the gap 1 (G1) phase but only 20⁻30% of them are active during the DNA replication of a given cell in the synthesis (S) phase. When the progress of replication forks is slowed by exogenous or endogenous impediments, the cell must activate some of the inactive or “dormant„ origins to complete replication on time. Thus, the many origins that may be activated are probably key to protect the genome against replication stress. This review aims to discuss the role of these dormant origins as safeguards of the human genome during replicative stress

Exploring selective autophagy events in multiple biologic models using LC3-interacting regions (LIR)-based molecular traps

International audienceAutophagy is an essential cellular pathway that ensures degradation of a wide range of substrates including damaged organelles or large protein aggregates. Understanding how this proteolytic pathway is regulated would increase our comprehension on its role in cellular physiology and contribute to identify biomarkers or potential drug targets to develop more specific treatments for disease in which autophagy is dysregulated. Here, we report the development of molecular traps based in the tandem disposition of LC3-interacting regions (LIR). The estimated affinity of LC3-traps for distinct recombinant LC3/GABARAP proteins is in the low nanomolar range and allows the capture of these proteins from distinct mammalian cell lines , S. cerevisiae and C. elegans . LC3-traps show preferences for GABARAP/LGG1 or LC3/LGG2 and pull-down substrates targeted to proteaphagy and mitophagy. Therefore, LC3-traps are versatile tools that can be adapted to multiple applications to monitor selective autophagy events in distinct physiologic and pathologic circumstances

Low replication stress leads to specific replication timing advances associated to chromatin remodelling in cancer cells

DNA replication is well orchestrated in mammalian cells through a tight regulation of the temporal order of replication origin activation, named the replication timing, a robust and conserved process in each cell type. Upon low replication stress, the slowing of replication forks induces delayed replication of fragile regions leading to genetic instability. The impact of low replication stress on the replication timing in different cellular backgrounds has not been explored yet. Here we analysed the whole genome replication timing in a panel of 6 human cell lines under low replication stress. We first demonstrated that cancer cells were more impacted than non-tumour cells. Strikingly, we unveiled an enrichment of specific replication domains undergoing a switch from late to early replication in some cancer cells. We found that advances in replication timing correlate with heterochromatin regions poorly sensitive to DNA damage signalling while being subject to an increase of chromatin accessibility. Finally, our data indicate that, following release from replication stress conditions, replication timing advances can be inherited by the next cellular generation, suggesting a new mechanism by which cancer cells would adapt to cellular or environmental stress.DNA replication is well orchestrated in mammalian cells through a tight regulation of the temporal order of replication origin activation, named the replication timing, a robust and conserved process in each cell type. Upon low replication stress, the slowing of replication forks induces delayed replication of fragile regions leading to genetic instability. The impact of low replication stress on the replication timing in different cellular backgrounds has not been explored yet. Here we analysed the whole genome replication timing in a panel of 6 human cell lines under low replication stress. We first demonstrated that cancer cells were more impacted than non-tumour cells. Strikingly, we unveiled an enrichment of specific replication domains undergoing a switch from late to early replication in some cancer cells. We found that advances in replication timing correlate with heterochromatin regions poorly sensitive to DNA damage signalling while being subject to an increase of chromatin accessibility. Finally, our data indicate that, following release from replication stress conditions, replication timing advances can be inherited by the next cellular generation, suggesting a new mechanism by which cancer cells would adapt to cellular or environmental stress