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Metastatic dissemination is driven by genetic, biochemical, and biophysical cues that favor the distant colonization of organs and the formation of life-threatening secondary tumors. We have previously demonstrated that endothelial cells (ECs) actively remodel during extravasation by enwrapping arrested tumor cells (TCs) and extruding them from the vascular lumen while maintaining perfusion. In this work, we dissect the cellular and molecular mechanisms driving endothelial remodeling. Using high-resolution intravital imaging in zebrafish embryos, we demonstrate that the actomyosin network of ECs controls tissue remodeling and subsequent TC extravasation. Furthermore, we uncovered that this cytoskeletal remodeling is driven by altered endothelial-calcium (Ca 2+ ) signaling caused by arrested TCs. Accordingly, we demonstrated that the inhibition of voltage-dependent calcium L-type channels impairs extravasation. Lastly, we identified P2X4, TRP, and Piezo1 mechano-gated Ca 2+ channels as key mediators of the process. These results further highlight the central role of endothelial remodeling during the extravasation of TCs and open avenues for successful therapeutic targeting., Competing Interests: The authors declare no competing interests., (© 2025 The Authors. Published by Elsevier Inc.)

Inositol 1,4,5-trisphosphate (IP3) receptor type 1 ( ITPR1 ), 2 ( ITPR2 ), and 3 ( ITPR3 ) encode the IP3 receptor (IP3R), a key player in intracellular calcium release. In four unrelated patients, we report that an identical ITPR3 de novo variant-NM_002224.3:c.7570C>T, p.Arg2524Cys-causes, through a dominant-negative effect, a complex multisystemic disorder with immunodeficiency. This leads to defective calcium homeostasis, mitochondrial malfunction, CD4 + lymphopenia, a quasi-absence of naïve CD4 + and CD8 + cells, an increase in memory cells, and a distinct TCR repertoire. The calcium defect was recapitulated in Jurkat knock-in. Site-directed mutagenesis displayed the exquisite sensitivity of Arg 2524 to any amino acid change. Despite the fact that all patients had severe immunodeficiency, they also displayed variable multisystemic involvements, including ectodermal dysplasia, Charcot-Marie-Tooth disease, short stature, and bone marrow failure. In conclusion, unlike previously reported ITPR1-3 deficiencies leading to narrow, mainly neurological phenotypes, a recurrent dominant ITPR3 variant leads to a multisystemic disease, defining a unique role for IP3R3 in the tetrameric IP3R complex.

Extracellular vesicles released by tumors (tEVs) disseminate via circulatory networks and promote microenvironmental changes in distant organs favoring metastatic seeding. Despite their abundance in the bloodstream, how hemodynamics affect the function of circulating tEVs remains unsolved. We demonstrated that efficient uptake of tEVs occurs in venous endothelial cells that are subjected to hemodynamics. Low flow regimes observed in veins partially reroute internalized tEVs toward non-acidic and non-degradative Rab14-positive endosomes, at the expense of lysosomes, suggesting that endothelial mechanosensing diverts tEVs from degradation. Subsequently, tEVs promote the expression of pro-angiogenic transcription factors in low flow-stimulated endothelial cells and favor vessel sprouting in zebrafish. Altogether, we demonstrate that low flow regimes potentiate the pro-tumoral function of circulating tEVs by promoting their uptake and rerouting their trafficking. We propose that tEVs contribute to pre-metastatic niche formation by exploiting endothelial mechanosensing in specific vascular regions with permissive hemodynamics., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Adipose tissue plays a central role in age-related diseases. While RNAseq protocols exist for many tissues, few data have been generated with this technology to explore gene expression in adipocytes, particularly during aging. Here, we present a protocol to analyze the transcriptional changes that occur in adipose tissue during normal and accelerated aging in mouse models. We describe steps for genotyping, diet control, euthanasia, and dissection. We then detail RNA purification and genome-wide data generation and analysis. For complete details on the use and execution of this protocol, please refer to De Cauwer et al. (2022) iScience. Sep 16;25(10):105149., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Following chemotherapy, a mediastinal germ cell tumor can lead to a mature teratoma that is composed of tissues derived from all three germ layers. Although teratoma is usually curable, in rare cases it can give rise to various somatic tumors and exceptionally it undergoes melanocytic neuroectodermal tumor (MNT) transformation, a process that is not well-described. We report a patient with a postchemotherapy thymic teratoma associated with an MNT component who, 10 years later, additionally presented a vertebral metastasis corresponding to an anaplastic MNT. Using exome sequencing of the mature teratoma, the MNT and its metastatic vertebral anaplastic MNT components, we identified 19 somatic mutations shared by at least two components. Six mutations were common to all three components, and three of them were located in the known cancer-related genes KRAS (p.E63K), TP53 (p.P222X), and POLQ (p.S447P). Gene set enrichment analysis revealed that the melanoma tumorigenesis pathway was enriched in mutated genes including the four major driver genes KRAS , TP53 , ERBB4 , and KDR , indicating that these genes may be involved in the development of the anaplastic MNT transformation of the teratoma. To our knowledge, this is the first molecular study realized on MNT. Understanding the clinicopathological and molecular characteristics of these tumors is essential to better understand their development and to improve therapeutics., (© 2023 Mayeur et al.; Published by Cold Spring Harbor Laboratory Press.)

Age-related diseases are major concern in developed countries. To avoid disabilities that accompany increased lifespan, pharmaceutical approaches are considered. Therefore, appropriate animal models are required for a better understanding of aging processes and potential in vivo assays to evaluate the impact of molecules that may delay the occurrence of age-related diseases. Few mouse models exhibiting pathological aging exist, but currently, none of them reproducibly mimics human diseases like osteoporosis, cognitive dysfunctions or sarcopenia that can be seen in some, but not all, elders. Here, we describe the premature aging phenotypes of Dicer-deficient mature animals, which exhibit an overall deterioration of many organs and tissues (skin, heart, and adipose tissue) ultimately leading to a significant reduction of their lifespan. Molecular characterization of transcriptional responses focused on the adipose tissue suggested that both canonical and non-canonical functions of DICER are involved in this process and highlight potential actionable pathways to revert it., Competing Interests: The authors declare no competing interest., (© 2022 The Author(s).)

The drivers of critical coronavirus disease 2019 (COVID-19) remain unknown. Given major confounding factors such as age and comorbidities, true mediators of this condition have remained elusive. We used a multi-omics analysis combined with artificial intelligence in a young patient cohort where major comorbidities were excluded at the onset. The cohort included 47 “critical” (in the intensive care unit under mechanical ventilation) and 25 “non-critical” (in a non-critical care ward) patients with COVID-19 and 22 healthy individuals. The analyses included whole-genome sequencing, whole-blood RNA sequencing, plasma and blood mononuclear cell proteomics, cytokine profiling, and high-throughput immunophenotyping. An ensemble of machine learning, deep learning, quantum annealing, and structural causal modeling were used. Patients with critical COVID-19 were characterized by exacerbated inflammation, perturbed lymphoid and myeloid compartments, increased coagulation, and viral cell biology. Among differentially expressed genes, we observed up-regulation of the metalloprotease ADAM9 . This gene signature was validated in a second independent cohort of 81 critical and 73 recovered patients with COVID-19 and was further confirmed at the transcriptional and protein level and by proteolytic activity. Ex vivo ADAM9 inhibition decreased severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake and replication in human lung epithelial cells. In conclusion, within a young, otherwise healthy, cohort of individuals with COVID-19, we provide the landscape of biological perturbations in vivo where a unique gene signature differentiated critical from non-critical patients. We further identified ADAM9 as a driver of disease severity and a candidate therapeutic target.

The Nck-associated protein 1-like (NCKAP1L) gene, alternatively called hematopoietic protein 1 (HEM-1), encodes a hematopoietic lineage-specific regulator of the actin cytoskeleton. Nckap1l-deficient mice have anomalies in lymphocyte development, phagocytosis, and neutrophil migration. Here we report, for the first time, NCKAP1L deficiency cases in humans. In two unrelated patients of Middle Eastern origin, recessive mutations in NCKAP1L abolishing protein expression led to immunodeficiency, lymphoproliferation, and hyperinflammation with features of hemophagocytic lymphohistiocytosis. Immunophenotyping showed an inverted CD4/CD8 ratio with a major shift of both CD4+ and CD8+ cells toward memory compartments, in line with combined RNA-seq/proteomics analyses revealing a T cell exhaustion signature. Consistent with the core function of NCKAP1L in the reorganization of the actin cytoskeleton, patients' T cells displayed impaired early activation, immune synapse morphology, and leading edge formation. Moreover, knockdown of nckap1l in zebrafish led to defects in neutrophil migration. Hence, NCKAP1L mutations lead to broad immune dysregulation in humans, which could be classified within actinopathies., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Castro et al.)

Importance: Movement disorders are characterized by a marked genotypic and phenotypic heterogeneity, complicating diagnostic work in clinical practice and molecular diagnosis., Objective: To develop and evaluate a targeted sequencing approach using a customized panel of genes involved in movement disorders., Design, Setting and Participants: We selected 127 genes associated with movement disorders to create a customized enrichment in solution capture array. Targeted high-coverage sequencing was applied to DNA samples taken from 378 eligible patients at 1 Luxembourgian, 1 Algerian, and 25 French tertiary movement disorder centers between September 2014 and July 2016. Patients were suspected of having inherited movement disorders because of early onset, family history, and/or complex phenotypes. They were divided in 5 main movement disorder groups: parkinsonism, dystonia, chorea, paroxysmal movement disorder, and myoclonus. To compare approaches, 23 additional patients suspected of having inherited cerebellar ataxia were included, on whom whole-exome sequencing (WES) was done. Data analysis occurred from November 2015 to October 2016., Main Outcomes and Measures: Percentages of individuals with positive diagnosis, variants of unknown significance, and negative cases; mutational frequencies and clinical phenotyping of genes associated with movement disorders., Results: Of the 378 patients (of whom 208 were male [55.0%]), and with a median (range) age at disease onset of 31 (0-84) years, probable pathogenic variants were identified in 83 cases (22.0%): 46 patients with parkinsonism (55% of 83 patients), 21 patients (25.3%) with dystonia, 7 patients (8.4%) with chorea, 7 patients (8.4%) with paroxysmal movement disorders, and 2 patients (2.4%) with myoclonus as the predominant phenotype. Some genes were mutated in several cases in the cohort. Patients with pathogenic variants were significantly younger (median age, 27 years; interquartile range [IQR], 5-36 years]) than the patients without diagnosis (median age, 35 years; IQR, 15-46 years; P = .04). Diagnostic yield was significantly lower in patients with dystonia (21 of 135; 15.6%; P = .03) than in the overall cohort. Unexpected genotype-phenotype correlations in patients with pathogenic variants deviating from the classic phenotype were highlighted, and 49 novel probable pathogenic variants were identified. The WES analysis of the cohort of 23 patients with cerebellar ataxia led to an overall diagnostic yield of 26%, similar to panel analysis but at a cost 6 to 7 times greater., Conclusions and Relevance: High-coverage sequencing panel for the delineation of genes associated with movement disorders was efficient and provided a cost-effective diagnostic alternative to whole-exome and whole-genome sequencing.

Neurodevelopmental disorders with periventricular nodular heterotopia (PNH) are etiologically heterogeneous, and their genetic causes remain in many cases unknown. Here we show that missense mutations in NEDD4L mapping to the HECT domain of the encoded E3 ubiquitin ligase lead to PNH associated with toe syndactyly, cleft palate and neurodevelopmental delay. Cellular and expression data showed sensitivity of PNH-associated mutants to proteasome degradation. Moreover, an in utero electroporation approach showed that PNH-related mutants and excess wild-type NEDD4L affect neurogenesis, neuronal positioning and terminal translocation. Further investigations, including rapamycin-based experiments, found differential deregulation of pathways involved. Excess wild-type NEDD4L leads to disruption of Dab1 and mTORC1 pathways, while PNH-related mutations are associated with deregulation of mTORC1 and AKT activities. Altogether, these data provide insights into the critical role of NEDD4L in the regulation of mTOR pathways and their contributions in cortical development., Competing Interests: The authors declare no competing financial interests.