Le séquençage de transcrits sur cellule unique appliqué à l'étude des mécanismes de formation et de résolution de la fibrose pulmonaire

Restoration of lung tissue integrity in response to injury depends in particular on the regenerative capacities of capillary endothelial cells (CEc), which may decline with ageing. Inadequate responses can lead to the development of chronic diseases such as idiopathic pulmonary fibrosis (IPF), which is characterised by the accumulation of extracellular matrix and remodelling of the alveolar environment, leading to progressive destruction of the parenchyma and culminating in respiratory failure. In mice, bleomycin-induced lung injury can be used to trigger a transient fibrotic response, which is resolved within a few weeks. However, preliminary data indicated a delay in fibrosis resolution in aged mice.To investigate the mechanisms of lung endothelial regeneration and the potential influence of ageing on resolution dynamics, we measured gene expression by single-cell RNA sequencing (scRNA-seq) in cells from whole lungs of young (7 weeks) and old (18 months) mice collected 14, 28 or 60 days after injection of bleomycin (2U.kg-1) or PBS. The count matrices from the 36 sequenced samples were integrated to obtain a single dataset of 44541 cells. Based on their transcriptomic profile, the cells were clustered and manually annotated on the basis of the expression of specific marker genes. In addition to the two subpopulations of CEc present in physiological conditions, called general capillaries (gCap) and aerocytes (aCap), three subpopulations almost exclusively present in bleomycin-treated mouse samples were identified. A first population of Col15a1-expressing cells, named SV EC, corresponds to systemic venous vessels normally restricted to the bronchi and which are described in IPF as invading the alveolar tissue. The other two populations, not described in the literature, are cells similar to gCap or aCap but expressing specific and angiogenesis-associated markers, such as Lrg1, a modulator of the TGF-β pathway. The transcriptomic signatures of these subpopulations suggested their involvement in the regenerative processes of the alveolar niche through intense signaling activity. Comparison of their observed dynamics during the evolution of fibrosis between young and old mice showed a shift in resolution in old mice, which at 28 days still showed a pathological signature in contrast to young mice. This delay was then confirmed by in situ hybridization approaches and by spatial transcriptomics data analysis. Furthermore, differential expression analyses between gCap from young and old mice revealed age-dependant signatures in both physiological and fibrotic conditions, with gCap from old control mice expressing genes of the bleomycin-induced signature. Finally, the similarities between the bleomycin-induced SV EC signature and that induced in human IPF suggest that the observed remodelling in the mouse model partially mimics that which characterizes the human pathology.Le rétablissement de l'intégrité du tissu pulmonaire en réponse aux blessures dépend notamment des capacités régénératives des cellules endothéliales capillaires (CEc), susceptibles de décliner au cours du vieillissement. Des réponses inadaptées peuvent entraîner le développement de pathologies chroniques telles que la fibrose pulmonaire idiopathique (FPI), qui se caractérise par l'accumulation de matrice extracellulaire et un remodelage de l'environnement alvéolaire, menant à une destruction progressive du parenchyme et aboutissant à une insuffisance respiratoire. Chez la souris, des lésions pulmonaires induites par la bléomycine peuvent être utilisées pour déclencher une réponse fibrotique transitoire dont les effets se résorbent en quelques semaines. Des données préliminaires indiquaient cependant un retard de résolution chez les souris âgées.Afin d'étudier les mécanismes de régénération de l'endothélium pulmonaire ainsi que l'influence potentielle de l'âge sur la dynamique de résolution, nous avons mesuré l'expression des gènes par séquençage de transcrit sur cellules uniques (scRNA-seq), dans les cellules issues de poumons entiers de souris jeunes (7 semaines) et âgées (18 mois) prélevés 14, 28 ou 60 jours après l'injection de bléomycine ou de PBS. Les matrices de comptes issues des 36 échantillons séquencées ont été intégrées de sorte à obtenir un unique jeu de données de 44541 cellules. En fonction de leur profil transcriptomique, les cellules ont été regroupées et annotées manuellement sur la base de l'expression de gènes marqueurs spécifiques. En plus des 2 sous populations de CEc présentes en condition physiologiques, appelées capillaires générales (gCap) et aerocytes (aCap), 3 autres sous-populations ont été identifiées dans les échantillons de souris traitées à la bléomycine. Une première population de cellules exprimant Col15a1, nommée SV EC, correspondant à des vaisseaux veineux systémiques normalement restreints aux bronches et qui sont décrits dans la FPI comme envahissant le tissu alvéolaire. Les deux autres populations, non décrites dans la littérature, sont des cellules similaires aux gCap ou aux aCap mais exprimant des marqueurs spécifiques et associés à l'angiogenèse, tels que Lrg1, un modulateur de la voie TGF-β. Les signatures transcriptomiques de ces sous populations suggèrent leur implication dans les processus de régénération de la niche alvéolaire via une intense activité de signalisation. La comparaison de leurs dynamiques observées au cours de l'évolution de la fibrose entre souris jeunes et âgées montre un décalage de la résolution chez les souris âgées, qui à 28 jours présentent encore une signature pathologique au contraire des souris jeunes. Ce décalage a ensuite été confirmé par des approches d'hybridation in situ et par des expériences de transcriptomiques spatiales. De plus, la comparaison des profils d'expression entre gCap de souris jeunes et vieilles a révélé des différences à la fois en condition physiologique et en condition de fibrose, les gCap de souris âgées contrôles exprimant notamment des gènes de la signature induite par la bléomycine. Enfin, les similitudes entre la signature SV EC induite par la bléomycine et celle induite par la FPI humaine suggèrent que la reprogrammation observée dans le modèle murin reproduit en partie celle qui caractérise la pathologie humaine

Single cell RNA sequencing to study the mechanisms of pulmonary fibrosis formation and resolution

Le rétablissement de l'intégrité du tissu pulmonaire en réponse aux blessures dépend notamment des capacités régénératives des cellules endothéliales capillaires (CEc), susceptibles de décliner au cours du vieillissement. Des réponses inadaptées peuvent entraîner le développement de pathologies chroniques telles que la fibrose pulmonaire idiopathique (FPI), qui se caractérise par l'accumulation de matrice extracellulaire et un remodelage de l'environnement alvéolaire, menant à une destruction progressive du parenchyme et aboutissant à une insuffisance respiratoire. Chez la souris, des lésions pulmonaires induites par la bléomycine peuvent être utilisées pour déclencher une réponse fibrotique transitoire dont les effets se résorbent en quelques semaines. Des données préliminaires indiquaient cependant un retard de résolution chez les souris âgées.Afin d'étudier les mécanismes de régénération de l'endothélium pulmonaire ainsi que l'influence potentielle de l'âge sur la dynamique de résolution, nous avons mesuré l'expression des gènes par séquençage de transcrit sur cellules uniques (scRNA-seq), dans les cellules issues de poumons entiers de souris jeunes (7 semaines) et âgées (18 mois) prélevés 14, 28 ou 60 jours après l'injection de bléomycine ou de PBS. Les matrices de comptes issues des 36 échantillons séquencées ont été intégrées de sorte à obtenir un unique jeu de données de 44541 cellules. En fonction de leur profil transcriptomique, les cellules ont été regroupées et annotées manuellement sur la base de l'expression de gènes marqueurs spécifiques. En plus des 2 sous populations de CEc présentes en condition physiologiques, appelées capillaires générales (gCap) et aerocytes (aCap), 3 autres sous-populations ont été identifiées dans les échantillons de souris traitées à la bléomycine. Une première population de cellules exprimant Col15a1, nommée SV EC, correspondant à des vaisseaux veineux systémiques normalement restreints aux bronches et qui sont décrits dans la FPI comme envahissant le tissu alvéolaire. Les deux autres populations, non décrites dans la littérature, sont des cellules similaires aux gCap ou aux aCap mais exprimant des marqueurs spécifiques et associés à l'angiogenèse, tels que Lrg1, un modulateur de la voie TGF-β. Les signatures transcriptomiques de ces sous populations suggèrent leur implication dans les processus de régénération de la niche alvéolaire via une intense activité de signalisation. La comparaison de leurs dynamiques observées au cours de l'évolution de la fibrose entre souris jeunes et âgées montre un décalage de la résolution chez les souris âgées, qui à 28 jours présentent encore une signature pathologique au contraire des souris jeunes. Ce décalage a ensuite été confirmé par des approches d'hybridation in situ et par des expériences de transcriptomiques spatiales. De plus, la comparaison des profils d'expression entre gCap de souris jeunes et vieilles a révélé des différences à la fois en condition physiologique et en condition de fibrose, les gCap de souris âgées contrôles exprimant notamment des gènes de la signature induite par la bléomycine. Enfin, les similitudes entre la signature SV EC induite par la bléomycine et celle induite par la FPI humaine suggèrent que la reprogrammation observée dans le modèle murin reproduit en partie celle qui caractérise la pathologie humaine.Restoration of lung tissue integrity in response to injury depends in particular on the regenerative capacities of capillary endothelial cells (CEc), which may decline with ageing. Inadequate responses can lead to the development of chronic diseases such as idiopathic pulmonary fibrosis (IPF), which is characterised by the accumulation of extracellular matrix and remodelling of the alveolar environment, leading to progressive destruction of the parenchyma and culminating in respiratory failure. In mice, bleomycin-induced lung injury can be used to trigger a transient fibrotic response, which is resolved within a few weeks. However, preliminary data indicated a delay in fibrosis resolution in aged mice.To investigate the mechanisms of lung endothelial regeneration and the potential influence of ageing on resolution dynamics, we measured gene expression by single-cell RNA sequencing (scRNA-seq) in cells from whole lungs of young (7 weeks) and old (18 months) mice collected 14, 28 or 60 days after injection of bleomycin (2U.kg-1) or PBS. The count matrices from the 36 sequenced samples were integrated to obtain a single dataset of 44541 cells. Based on their transcriptomic profile, the cells were clustered and manually annotated on the basis of the expression of specific marker genes. In addition to the two subpopulations of CEc present in physiological conditions, called general capillaries (gCap) and aerocytes (aCap), three subpopulations almost exclusively present in bleomycin-treated mouse samples were identified. A first population of Col15a1-expressing cells, named SV EC, corresponds to systemic venous vessels normally restricted to the bronchi and which are described in IPF as invading the alveolar tissue. The other two populations, not described in the literature, are cells similar to gCap or aCap but expressing specific and angiogenesis-associated markers, such as Lrg1, a modulator of the TGF-β pathway. The transcriptomic signatures of these subpopulations suggested their involvement in the regenerative processes of the alveolar niche through intense signaling activity. Comparison of their observed dynamics during the evolution of fibrosis between young and old mice showed a shift in resolution in old mice, which at 28 days still showed a pathological signature in contrast to young mice. This delay was then confirmed by in situ hybridization approaches and by spatial transcriptomics data analysis. Furthermore, differential expression analyses between gCap from young and old mice revealed age-dependant signatures in both physiological and fibrotic conditions, with gCap from old control mice expressing genes of the bleomycin-induced signature. Finally, the similarities between the bleomycin-induced SV EC signature and that induced in human IPF suggest that the observed remodelling in the mouse model partially mimics that which characterizes the human pathology

Novel dynamics of human mucociliary differentiation revealed by single-cell RNA sequencing of nasal epithelial cultures

The upper airway epithelium, which is mainly composed of multiciliated, goblet, club and basal cells, ensures proper mucociliary function and can regenerate in response to assaults. In chronic airway diseases, defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs. Using single-cell transcriptomics and lineage inference, we have unraveled trajectories from basal to luminal cells, providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells, which we have entitled deuterosomal cells, is defined by specific markers, such as DEUP1, FOXN4, YPEL1, HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells, thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; and (3) a repertoire of molecules involved in the regeneration process, such as keratins or components of the Notch, Wnt or BMP/TGFβ pathways, can be identified. Confirmation of our results on fresh human and pig airway samples, and on mouse tracheal cells, extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation

Detecting subtle transcriptomic perturbations induced by lncRNAs knock-down in single-cell CRISPRi screening using a new sparse supervised autoencoder neural network

Single-cell CRISPR-based transcriptome screens are potent genetic tools for concomitantly assessing the expression profiles of cells targeted by a set of guides RNA (gRNA), and inferring target gene functions from the observed perturbations. However, due to various limitations, this approach lacks sensitivity in detecting weak perturbations and is essentially reliable when studying master regulators such as transcription factors. To overcome the challenge of detecting subtle gRNA induced transcriptomic perturbations and classifying the most responsive cells, we developed a new supervised autoencoder neural network method. Our Sparse supervised autoencoder (SSAE) neural network provides selection of both relevant features (genes) and actual perturbed cells. We applied this method on an in-house single-cell CRISPR-interference-based (CRISPRi) transcriptome screening (CROP-Seq) focusing on a subset of long non-coding RNAs (lncRNAs) regulated by hypoxia, a condition that promote tumor aggressiveness and drug resistance, in the context of lung adenocarcinoma (LUAD). The CROP-seq library of validated gRNA against a subset of lncRNAs and, as positive controls, HIF1A and HIF2A, the 2 main transcription factors of the hypoxic response, was transduced in A549 LUAD cells cultured in normoxia or exposed to hypoxic conditions during 3, 6 or 24 h. We first validated the SSAE approach on HIF1A and HIF2 by confirming the specific effect of their knock-down during the temporal switch of the hypoxic response. Next, the SSAE method was able to detect stable short hypoxia-dependent transcriptomic signatures induced by the knock-down of some lncRNAs candidates, outperforming previously published machine learning approaches. This proof of concept demonstrates the relevance of the SSAE approach for deciphering weak perturbations in single-cell transcriptomic data readout as part of CRISPR-based screening

Blockade of the pro‐fibrotic reaction mediated by the miR‐143/‐145 cluster enhances the responses to targeted therapy in melanoma

International audienceLineage dedifferentiation toward a mesenchymal-like state displaying myofibroblast and fibrotic features is a common mechanism of adaptive and acquired resistance to targeted therapy in melanoma. Here, we show that the anti-fibrotic drug nintedanib is active to normalize the fibrous ECM network, enhance the efficacy of MAPK-targeted therapy, and delay tumor relapse in a preclinical model of melanoma. Acquisition of this resistant phenotype and its reversion by nintedanib pointed to miR-143/-145 pro-fibrotic cluster as a driver of this mesenchymal-like phenotype. Upregulation of the miR-143/-145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile. The 2 mature miRNAs generated from this cluster, miR-143-3p and miR-145-5p, collaborated to mediate transition toward a drug-resistant undifferentiated mesenchymal-like state by targeting Fascin actin-bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics and mechanotransduction pathways. Our study brings insights into a novel miRNA-mediated regulatory network that contributes to non-genetic adaptive drug resistance and provides proof of principle that preventing MAPKi-induced pro-fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy

Blockade of pro-fibrotic response mediated by the miR-143/-145 cluster prevents targeted therapy-induced phenotypic plasticity and resistance in melanoma

Abstract Lineage dedifferentiation towards a mesenchymal-like state is a common mechanism of adaptive response and resistance to targeted therapy in melanoma. Yet, the transcriptional network driving this phenotypic plasticity remains elusive. Remarkably, this cellular state displays myofibroblast and fibrotic features and escapes MAPK inhibitors (MAPKi) through extracellular matrix (ECM) remodeling activities. Here we show that the anti-fibrotic drug Nintedanib/BIBF1120 is active to normalize the fibrous ECM network, enhance the efficacy of MAPK-targeted therapy and delay tumor relapse in a pre-clinical model of melanoma. We also uncovered the molecular networks that regulate the acquisition of this resistant phenotype and its reversion by Nintedanib, pointing the miR-143/-145 pro-fibrotic cluster as a driver of the therapy-resistant mesenchymal-like phenotype. Upregulation of the miR-143/-145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile of resistant cells. The 2 mature miRNAs generated from this cluster, miR-143-3p and miR-145-5p collaborated to mediate phenotypic transition towards a drug resistant undifferentiated mesenchymal-like state by targeting Fascin actin-bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics as well as contributing to a fine-tuning of mechanotransduction pathways. Our study brings insights into a novel miRNA-mediated regulatory network that contributes to non-genetic adaptive drug resistance and provides proof-of-principle that preventing MAPKi-induced pro-fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy

Highlighting fibroblast plasticity in lung fibrosis: the WI-38 cell line as a model for investigating the myofibroblast and lipofibroblast switch

Background: Myofibroblasts (MYFs) are generally considered the principal culprits in excessive extracellular matrix deposition and scar formation in the pathogenesis of lung fibrosis. Lipofibroblasts (LIFs), on the other hand, are defined by their lipid-storing capacity and are predominantly found in the alveolar regions of the lung. They have been proposed to play a protective role in lung fibrosis. We previously reported that a LIF to MYF reversible differentiation switch occurred during fibrosis formation and resolution. In this study, we tested whether WI-38 cells, a human embryonic lung fibroblast cell line, could be used to study fibroblast differentiation towards the LIF or MYF phenotype and whether this could be relevant for idiopathic pulmonary fibrosis (IPF). Methods: Using WI-38 cells, Fibroblast (FIB) to MYF differentiation was triggered using TGF-β1 treatment and FIB to LIF differentiation using Metformin treatment. We also analyzed the MYF to LIF and LIF to MYF differentiation by pre-treating the WI-38 cells with TGF-β1 or Metformin respectively. We used IF, qPCR and bulk RNA-Seq to analyze the phenotypic and transcriptomic changes in the cells. We correlated our in vitro transcriptome data from WI-38 cells (obtained via bulk RNA sequencing) with the transcriptomic signature of LIFs and MYFs derived from the IPF cell atlas as well as with our own single-cell transcriptomic data from IPF patients-derived lung fibroblasts (LF-IPF) cultured in vitro. We also carried out alveolosphere assays to evaluate the ability of the proposed LIF and MYF cells to support the growth of alveolar epithelial type 2 cells. Results: WI-38 cells and LF-IPF display similar phenotypical and gene expression responses to TGF-β1 and Metformin treatment. Bulk RNA-Seq analysis of WI-38 cells and LF-IPF treated with TGF-β1, or Metformin indicate similar transcriptomic changes. We also show the partial conservation of the LIF and MYF signature extracted from the Habermann et al. scRNA-seq dataset in WI-38 cells treated with Metformin or TGF-β1, respectively. Alveolosphere assays indicate that LIFs enhance organoid growth, while MYFs inhibit organoid growth. Finally, we provide evidence supporting the MYF to LIF and LIF to MYF reversible switch using WI-38 cells. Conclusions: WI-38 cells represent a versatile and reliable model to study the intricate dynamics of fibroblast differentiation towards the MYF or LIF phenotype associated with lung fibrosis formation and resolution, providing valuable insights to drive future research.Deutsche Forschungsgemeinschaft (DFG); ROR-ID:018mejw6

The nuclear hypoxia-regulated NLUCAT1 long non-coding RNA contributes to an aggressive phenotype in lung adenocarcinoma through regulation of oxidative stress

International audienceLung cancer is the leading cause of cancer death worldwide, with poor prognosis and a high rate of recurrence despite early surgical removal. Hypoxic regions within tumors represent sources of aggressiveness and resistance to therapy. Although long non-coding RNAs (lncRNAs) are increasingly recognized as major gene expression regulators, their regulation and function following hypoxic stress are still largely unexplored. Combining profiling studies on early-stage lung adenocarcinoma (LUAD) biopsies and on A549 LUAD cell lines cultured in normoxic or hypoxic conditions, we identified a subset of lncRNAs that are both correlated with the hypoxic status of tumors and regulated by hypoxia in vitro. We focused on a new transcript, NLUCAT1, which is strongly upregulated by hypoxia in vitro and correlated with hypoxic markers and poor prognosis in LUADs. Full molecular characterization showed that NLUCAT1 is a large nuclear transcript composed of six exons and mainly regulated by NF-κB and NRF2 transcription factors. CRISPR-Cas9-mediated invalidation of NLUCAT1 revealed a decrease in proliferative and invasive properties, an increase in oxidative stress and a higher sensitivity to cisplatin-induced apoptosis. Transcriptome analysis of NLUCAT1-deficient cells showed repressed genes within the antioxidant and/or cisplatin-response networks. We demonstrated that the concomitant knockdown of four of these genes products, GPX2, GLRX, ALDH3A1, and PDK4, significantly increased ROS-dependent caspase activation, thus partially mimicking the consequences of NLUCAT1 inactivation in LUAD cells. Overall, we demonstrate that NLUCAT1 contributes to an aggressive phenotype in early-stage hypoxic tumors, suggesting it may represent a new potential therapeutic target in LUADs