Biology subjects

Carbone, F.

Publications and source records attributed to Carbone, F..

9 recordsLinked to original sources

bioRxiv2025

Multiplex imaging combined to machine learning enable automated profiling of cortical malformations applications in tuberous sclerosis complex

Malformations of cortical development such as tuberous sclerosis complex arise within a heterogeneous cellular landscape that conventional histopathology only partially resolves. Here, we combined a 19-marker multiplex immunofluorescence panel with a machine learning-driven image analysis pipeline to map and quantify over 365 000 cells from paediatric surgical cortex, defining the single-cell architecture of TSC lesions. Microtubers were objectively delineated by vimentin and detected in all TSC samples but absent from non-dysplastic controls. Within these structures, balloon cells exhibiting strong pS6 activation occupied lesion cores and were confined to microtuber boundaries, whereas dysmorphic neurons were more diffusely distributed into adjacent cortex. The microtuber niche was dominated by astroglial remodeling: immature and reactive vimentin-positive astrocytes, including Lamp5-positive subsets, accumulated at and around lesion rims, while mature GFAP-positive astrocytes showed only modest changes. Distance-based spatial analyses revealed neuronal exclusion from microtuber centres with gradual recovery in surrounding tissue, indicating local network disruption. Unsupervised clustering and niche modelling recapitulated these spatial gradients, identifying a glial-dominated ecosystem that concentrates balloon cells, increases inter-neuronal distances, and reduces cell-cell interactions. Together, these data support a model in which cortical tubers arise through the coalescence of microtubers orchestrated by balloon cells and reactive gliosis during corticogenesis. Beyond elucidating disease architecture, our automated framework enables reproducible lesion detection, quantitative cell-state mapping, and spatial readouts applicable across malformations of cortical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/690101v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@dc3258org.highwire.dtl.DTLVardef@6d4149org.highwire.dtl.DTLVardef@d93354org.highwire.dtl.DTLVardef@1b8f83d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Sexually dimorphic interzonal crosstalk reshapes the adrenal cortex in response to pathophysiological challenges

Structured Abstract BackgroundPrimary aldosteronism is the most common form of secondary arterial hypertension, due to autonomous aldosterone production from the adrenal cortex. Genome wide association studies discovered genetic risk loci associated with the disease, which may affect adrenal cortex renewal, differentiation and lineage conversion. Genetic susceptibility may be modulated by environmental challenges. MethodHere we investigate how environmental cues affecting mineralocorticoid output modulate adrenal cortex homeostasis, cell lineage conversion and zone-specific transcriptional landscape. We have used a newly developed Cyp11b2Cremouse model and characterised its adaptation to a high or low salt diet (HSD, LSD), as well as dexamethasone (DEX) treatment. To explore underlying mechanisms, deep functional and morphological phenotyping, lineage tracing and spatial transcriptomics of the adrenal cortex were performed. ResultsCyp11b2 expression was detected in Cyp11b2Cre-mTmG mice as early as day P1 in different areas of the ZG, associated with high plasma aldosterone levels, with lineage conversion of zona glomerulosa (ZG) into zona fasciculata (ZF) cells progressing between 2 and 9 weeks of age and a progressive reduction of ZG size and evolution of cell components of the adrenal cortex over time. Transdifferentiation progressed into the X-zone (ZX) in females, revealing a previously unrecognized connection between ZF and ZX cells in adult mice. A sexually dimorphic, reciprocal interaction between the ZG and the ZF in adapting to salt diets or DEX treatment was observed, involving changes in cell composition and transcriptional reprogramming of the three zones. ConclusionHSD, LSD and DEX induce a sexually dimorphic cellular and transcriptional response, involving all layers of the adrenal cortex, and the reciprocal contribution of ZG and ZF cells, indicating functional interaction between adrenocortical zones in adapting to external cues.

physiology↗

bioRxiv2025

CLONAL LYMPHOCYTE EXPANSIONS AND JAK-STAT PATHWAY MUTATIONS DEFINE A PATHOGENIC CONTINUUM DRIVING RESISTANCE TO GLUTEN-FREE DIET IN CELIAC DISEASE

Background&AimsDespite recent advances, refractory celiac disease (RCD) poses challenging questions. In type 2 RCD (RCD2), the lack of response to the gluten-free diet is attributed to an intestinal intraepithelial lymphoma carrying driver JAK1 or STAT3 mutations. However, it remains unclear whether these can be safely targeted for therapy. In RCD1, pathogenic insights are still lacking. MethodsDuodenal biopsies and peripheral blood mononuclear cells (PBMCs) from patients with RCD1, RCD2, active CeD, CeD in remission, and controls were analyzed. Lymphocyte populations were characterized using single-cell transcriptomic, genomic, and TCR repertoire profiling. Functional and exome sequencing analyses were performed on patient-derived RCD2 cell lines exposed to JAK inhibitors. ResultsWe show that clonal malignant RCD2 lymphocytes exhibit interpatient similarities but substantial intratumoral heterogeneity, and provide in vitro evidence that JAK inhibitors can select drug-resistant tumor cells, arguing against their use as monotherapy. In RCD1, we identified clonal T-cell expansions harboring mutations that enhance the JAK-STAT pathway. The detection of both RCD2 and a CD4 lymphoproliferation in a patient initially diagnosed with RCD1 further illustrates the diversity of lymphoproliferative outcomes in celiac disease. ConclusionsThese findings suggest that RCD subtypes may share underlying mechanisms driven by clonal evolution and JAK-STAT dysregulation. They also highlight the potential limitations of JAK inhibitor monotherapy and the importance of molecularly informed therapeutic strategies. What You Need to KnowO_ST_ABSBACKGROUND AND CONTEXTC_ST_ABSRefractory celiac disease (RCD) can lead to intestinal lymphoma, but the biological processes driving immune cell transformation and therapy resistance remain incompletely understood. NEW FINDINGSSingle-cell analyses reveal clonal evolution, JAK-STAT pathway dysregulation, and shared molecular features between RCD subtypes, with implications for disease progression and treatment response. LIMITATIONSSample size and reliance on ex vivo models limit generalizability; further in vivo validation of resistance mechanisms is needed to confirm therapeutic implications. CLINICAL RESEARCH RELEVANCEThese findings suggest that RCD subtypes may share underlying mechanisms driven by clonal evolution and JAK-STAT dysregulation. They also highlight the potential limitations of JAK inhibitor monotherapy and the importance of molecularly informed therapeutic strategies. BASIC RESEARCH RELEVANCEThis work uncovers mechanisms of immune cell transformation in chronic intestinal inflammation and provides insight into how tumor heterogeneity and somatic mutations drive disease progression and therapeutic resistance. Lay summaryThis study reveals shared mechanisms in refractory celiac disease subtypes driven by clonal evolution and JAK-STAT activation, highlighting limits of JAK inhibitor monotherapy and the need for personalized treatments.

cancer biology↗

bioRxiv2024

Induction of the ISR by AB5 subtilase cytotoxin drives IFN-I expression in pDCs via STING activation

We demonstrate that exposure to the AB5 subtilase cytotoxin (SubAB) induces the unfolded protein response (UPR) in human peripheral blood mononuclear cells, concomitant with a pro-inflammatory response across distinct cell subsets. Notably, SubAB selectively induces type-I interferon (IFN) expression in plasmacytoid dendritic cells, acting synergistically with Toll-like receptor 7 stimulation. The induction of type-I IFN in response to SubAB relies on stimulator of interferon genes (STING) activation, coupled with protein synthesis inhibition mediated by protein kinase R-like endoplasmic reticulum kinase and phosphorylation of the eukaryotic translation initiation factor 2 subunit-alpha. By impeding mRNA translation through the integrated stress response, SubAB precipitates the downregulation of the negative innate signaling feedback regulator Tax1-binding protein 1. This downregulation is necessary to unleash TANK-binding kinase 1 signaling associated with STING activation. These findings shed new light on how UPR-inducing conditions may regulate the immune system during infection or pathogenesis.

immunology↗

bioRxiv2023

Evolutionary fingerprints of EMT in pancreatic cancers

Mesenchymal plasticity has been extensively described in advanced and metastatic epithelial cancers; however, its functional role in malignant progression, metastatic dissemination and therapy response is controversial. More importantly, the role of epithelial mesenchymal transition (EMT) and cell plasticity in tumor heterogeneity, clonal selection and clonal evolution is poorly understood. Functionally, our work clarifies the contribution of EMT to malignant progression and metastasis in pancreatic cancer. We leveraged ad hoc somatic mosaic genome engineering, lineage tracing and ablation technologies and dynamic genetic reporters to trace and ablate tumor-specific lineages along the phenotypic spectrum of epithelial to mesenchymal plasticity. The experimental evidences clarify the essential contribution of mesenchymal lineages to pancreatic cancer evolution and metastatic dissemination. Spatial genomic analysis combined with single cell transcriptomic and epigenomic profiling of epithelial and mesenchymal lineages reveals that EMT promotes with the emergence of chromosomal instability (CIN). Specifically tumor lineages with mesenchymal features display highly conserved patterns of genomic evolution including complex structural genomic rearrangements and chromotriptic events. Genetic ablation of mesenchymal lineages robustly abolished these mutational processes and evolutionary patterns, as confirmed by cross species analysis of pancreatic and other human epithelial cancers. Mechanistically, we discovered that malignant cells with mesenchymal features display increased chromatin accessibility, particularly in the pericentromeric and centromeric regions, which in turn results in delayed mitosis and catastrophic cell division. Therefore, EMT favors the emergence of high-fitness tumor cells, strongly supporting the concept of a cell-state, lineage-restricted patterns of evolution, where cancer cell sub-clonal speciation is propagated to progenies only through restricted functional compartments. Restraining those evolutionary routes through genetic ablation of clones capable of mesenchymal plasticity and extinction of the derived lineages completely abrogates the malignant potential of one of the most aggressive form of human cancer.

cancer biology↗

bioRxiv2023

Enhanced inflammatory signaling driven by metabolic switch in Aicardi-Goutieres syndrome

Aicardi-Goutieres syndrome (AGS) is a genetic type I interferon (IFN)-mediated disease characterised by neurological involvement with onset in childhood. Chronic inflammation in response to uncontrolled type I IFN production is, among other things, associated with IP-10 secretion. We analysed, at the single-cell transcriptomic levels, peripheral blood samples from patients bearing AGS-causing mutations in SAMHD1, RNASEH2B or ADAR1 genes. Using machine-learning approaches and differential gene expression we identified a drastic loss of transcription factor hypoxia induced factor 1 (HIF-1) expression and activity associated with features of a metabolic switch and mitochondrial stress in monocytes/dendritic cells. Chemical stabilization of HIF-1, with a synthetic drug in an in vitro model of AGS, allowed us to reverse the energy metabolic switch, attenuate mitochondrial stress and markedly reduce IP-10 production. We therefore propose that energy metabolic switch contributes to exacerbated chronic inflammation in AGS, and that targeting this pathway might represent a promising therapeutic approach.

immunology↗

bioRxiv2021

A monocyte/dendritic cell molecular signature of SARS-CoV2-related multisystem inflammatory syndrome in children (MIS-C) with severe myocarditis

SARS-CoV-2 infection in children is generally milder than in adults, yet a proportion of cases result in hyperinflammatory conditions often including myocarditis. To better understand these cases, we applied a multi-parametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. The most severe forms of MIS-C (multisystem inflammatory syndrome in children related to SARS-CoV-2), that resulted in myocarditis, were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomic analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis, characterized by sustained NF-{kappa}B activity, TNF- signaling, associated with decreased gene expression of NF-{kappa}B inhibitors. We also found a weak response to type-I and type-II interferons, hyperinflammation and response to oxidative stress related to increased HIF-1 and VEGF signaling. These results provide potential for a better understanding of disease pathophysiology.

immunology↗

bioRxiv2020

9p21 Loss Defines the Evolutionary Patterns of Aggressive Renal Cell Carcinomas

Dedifferentiation and acquisition of chromosomal instability in renal cell carcinoma portends dismal prognosis and aggressive clinical behavior. However, the absence of reliable experimental models dramatically impacts the understanding of mechanisms underlying malignant progression. Here we established an in vivo genetic platform to rapidly generate somatic mosaic genetically engineerd immune-competent mouse models of renal tumors, recapitulating the genomic and phenotypic features of these malignancies. Leveraging somatic chromosomal engineering, we demonstrated that ablation of the murine locus syntenic to human 9p21 drives the rapid expansion of aggressive mesenchymal clones with prominent metastatic behavior, characterized by early emergence of chromosomal instability, whole-genome duplication, and conserved patterns of aneuploidy. This model of punctuated equilibrium provides a remarkable example of cross-species convergent evolution. SignificanceTo better understand the role of 9p21 in malignant progression, we generated a somatic mosaic GEMM of renal cancer, capturing the histological, genomic and evolutionary features of human disease. With this technology we demonstrated a critica role of 9p21 loss in metastatic evolution of RCC and provide a unique tool for testing new therapeutic treatments.

cancer biology↗

bioRxiv2020

Medium-chain acyl-CoA dehydrogenase, a gatekeeper of mitochondrial function in glioblastoma multiforme

Glioblastoma (GBM) is among the deadliest of human cancers. Despite extensive efforts, it has proven to be highly resistant to chemo- and immune-based therapeutic strategies, and little headway has been made with targeted inhibitors. Like many cancers, metabolism is dysregulated in GBM. Thus, to identify new vulnerabilities and drug targets in GBM, we conducted genetic screens using pooled RNAi libraries targeting metabolic enzymes. We screened multiple glioma stem cell-derived (GSC) xenograft models, which revealed that several enzymes involved in the mitochondrial metabolism of fatty acids were required for tumor cell proliferation. From among these, we focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids, due to its consistently high score across all of our screens, as well as its high expression level in multiple GSC models and its upregulation in GBM compared to normal brain. In this manuscript, we describe the dependence of GBM on sustained fatty acid metabolism to actively catabolize lipid species that would otherwise damage the mitochondrial structure. The uptake of mediumchain fatty acids lacks negative feedback regulation; therefore, in the absence of MCAD, medium-chain fatty acids accumulate to toxic levels, inducing reactive oxygen species (ROS), mitochondrial damage and failure, and apoptosis. Taken together, our findings uncover a previously unappreciated protective role exerted by MCAD in GBM cells, making it a unique and therapeutically exploitable vulnerability.

cancer biology↗