Lung 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.
The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The ...traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.
Dupuytren disease (DD) is a hand-localized fibrotic disorder characterized by a scar-like, collagen-rich cord. Treatment usually comprises surgical removal of the cord, but is associated with a high ...relapse rate, in some cases requiring finger amputation. There is currently no consensual medical approach for treating DD. Numerous preclinical studies have highlighted antifibrotic properties of metformin, and the aim of this study was to assess a potential antifibrotic role of metformin in DD. Fibroblasts from DD cords (DF) and phenotypically normal palmar fascia (PF) were extracted from surgical specimens and cultured. The fibrotic status of DF and PF was compared at baseline, and under profibrotic (TGF-β stimulation) and antifibrotic (metformin stimulation) conditions, using quantitative RT-PCR, western blot, immunocytochemistry, and a functional fibroblast contraction assay. At baseline, DF showed higher levels of fibrotic markers and contraction capacity compared with PF. Both types of fibroblasts responded to TGF-β stimulation. Treatment of DF and PF with metformin did not affect basal levels of fibrotic markers and contraction but largely prevented their induction by TGF-β. In conclusion, our data show that metformin inhibits TGF-β-induced expression of fibrotic markers and contraction in hand-derived fibroblasts. This supports the case for a clinical trial to assess the repurposing of metformin as an adjuvant to surgery, to prevent, reduce, or delay recurrence in at-risk DD patients.
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•Metformin cannot revert the fibrotic phenotype of myofibroblasts in DD patients.•Metformin can prevent TGF-β induced differentiation of hand fibroblast into myofibroblasts.•The role of metformin as an adjuvant treatment to surgery should be evaluated in clinical trials.
Lineage 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.
Synopsis
This study identifies a critical miRNA‐mediated regulatory axis controlling melanoma cell phenotypic adaptation and resistance to MAPK inhibitors (MAPKi) and targetable by the anti‐fibrotic drug nintedanib.
The fibrosis‐associated miR‐143/‐145 cluster is upregulated by targeted therapy and highly expressed in MAPKi‐resistant BRAF‐mutated melanoma displaying a pro‐fibrotic dedifferentiated mesenchymal‐like phenotype.
The two mature miRNAs miR‐143‐3p and miR‐145‐5p promote a dedifferentiated, slow‐cycling and invasive phenotype associated with altered ECM production and therapy resistance.
F‐acting bundling protein Fascin1 (FSCN1) appears as a key functional target of both miRNAs to promote transition towards this therapy‐resistant phenotype.
The miR‐143/‐145/FSCN1 axis regulates actin cytoskeleton dynamics and YAP/MRTF‐dependent mechanopathways.
Nintedanib prevents miR‐143/‐145 cluster upregulation and combination therapy with MAPKi and nintedanib normalizes the tumor ECM niche and delays relapse in an allograft melanoma model.
This study identifies a critical miRNA‐mediated regulatory axis controlling melanoma cell phenotypic adaptation and resistance to MAPK inhibitors (MAPKi) and targetable by the anti‐fibrotic drug Nintedanib.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ..."Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.
Homeostatic renal filtration relies on the integrity of podocytes, which function in glomerular filtration. These highly specialized cells are damaged in 90% of chronic kidney disease, representing ...the leading cause of end-stage renal failure. Although modest podocyte renewal has been documented in adult mice, the mechanisms regulating this process remain largely unknown and controversial. Using a mouse model of Adriamycin-induced nephropathy, we find that the recovery of filtration function requires up-regulation of the endogenous telomerase component TERT. Previous work has shown that transient overexpression of catalytically inactive TERT (i-TERT
mouse model) has an unexpected role in triggering dramatic podocyte proliferation and renewal. We therefore used this model to conduct specific and stochastic lineage-tracing strategies in combination with high throughput sequencing methods. These experiments provide evidence that TERT drives the activation and clonal expansion of podocyte progenitor cells. Our findings demonstrate that the adult kidney bears intrinsic regenerative capabilities involving the protein component of telomerase, paving the way for innovative research toward the development of chronic kidney disease therapeutics.
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.
Fibrosis is a deleterious invasion of tissues associated with many pathological conditions, such as Duchenne muscular dystrophy (DMD) for which no cure is at present available for its prevention or ...its treatment. Fibro-adipogenic progenitors (FAPs) are resident cells in the human skeletal muscle and can differentiate into myofibroblasts, which represent the key cell population responsible for fibrosis. In this study, we delineated the pool of microRNAs (miRNAs) that are specifically modulated by TGFβ1 in FAPs versus myogenic progenitors (MPs) by a global miRNome analysis. A subset of candidates, including several “FibromiRs”, was found differentially expressed between FAPs and MPs and was also deregulated in DMD versus healthy biopsies. Among them, the expression of the TGFβ1-induced miR-199a~214 cluster was strongly correlated with the fibrotic score in DMD biopsies. Loss-of-function experiments in FAPs indicated that a miR-214-3p inhibitor efficiently blocked expression of fibrogenic markers in both basal conditions and following TGFβ1 stimulation. We found that FGFR1 is a functional target of miR-214-3p, preventing the signaling of the anti-fibrotic FGF2 pathway during FAP fibrogenesis. Overall, our work demonstrates that the « FibromiR » miR-214-3p is a key activator of FAP fibrogenesis by modulating the FGF2/FGFR1/TGFβ axis, opening new avenues for the treatment of DMD.
Tetrafunctional block copolymers are molecules capable of complexing DNA. Although ineffective in vitro, studies in mice have shown that the tetrafunctional block copolymer 704 is a more efficient ...lung gene transfer agent than the cationic liposome GL67A, previously used in a phase II clinical trial in cystic fibrosis patients. In the present study, we compared the gene transfer capacity of the 704-DNA formulation and a cationic liposome-DNA formulation equivalent to GL67A in a larger-animal model, the newborn piglet. Our results indicate an efficacy of the 704-DNA formulation well above one order of magnitude higher than that of the cationic liposome-DNA formulation, with no elevated levels of interleukin-6 (IL-6), taken as a marker of inflammation. Transgene expression was heterogeneous within lung lobes, with expression levels that were below the detection threshold in some samples, while high in other samples. This heterogeneity is likely to be due to the bolus injection procedure as well as to the small volume of injection. The present study highlights the potential of tetrafunctional block copolymers as non-viral vectors for lung gene therapy.
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Abstract
Reliable measurement of cellular cytotoxicity is essential for the characterization of immune responses and for the monitoring of antibody treatment efficacy. Until now, the standard ...chromium 51Cr-release assay has remained the sole sensitive assay that measures cellular cytotoxicity. Alternative non-radioactive assays have been developed but they do not provide accurate measurement of target cell cytotoxicity. The cost and hazard of handling radioactivity are strong incentive to find alternative solutions to 51Cr. We took advantage of the recent development of cell-imaging multimode readers to develop a novel non-radioactive and real-time cytotoxic assay that demonstrates good reproducibility and sensitivity. The extent of target-cell cytotoxicity is monitored over time by imaging and quantifying live fluorescent target cells in 96-well plates using the cell-imaging multimode reader Cytation™ 5 from Biotek. We have developed classical NK cell assays in the presence or absence of blocking antibodies and antibody-dependent cell-mediated cytotoxicity (ADCC). We show that in these assays, cell killing occurs within the first two hours with a half maximum killing reached after 30 minutes. This technology has numerous applications such as NK and T cell cytotoxicity assays and can be extended to cell survival and apoptosis measurement assays.
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