Biology subjects

Coscia, F.

Publications and source records attributed to Coscia, F..

At least 19 recordsLinked to original sources

bioRxiv2026

Recording proteome dynamics in the late-stage embryo at hour-timescale, spatial, and single-cell resolution with MEMBRYO

Cell fate during development is directed by new gene expression states that replace prior ones. When and where a protein is first translated, and how long it will remain in the cell, dictate the timing and extent of a gene's phenotypic effect. Accessing protein synthesis and turnover information in the rapidly developing embryo remains a major challenge. Here we present MEMBRYO: a method for continuous stable isotope labeling (SILAC) of newly synthesized proteins throughout the live embryo at the hour-timescales of developmental phenotypes. We extend embryo culture into the late stages of organogenesis, thereby accessing the proteome of neurogenesis in the forebrain ex utero. Variations in protein synthesis and turnover activity lead to expression patterns that diverge from the corresponding transcripts, impacting molecular pathways and key milestones of cortical neuron differentiation. The high labeling depth enabled both spatial and single-cell SILAC proteomics analysis of cortical cells, revealing distinct layers of post-transcriptional gene expression regulation in progenitors and neurons. By continuous metabolic labeling in the live embryo, MEMBRYO tracks the past, present, and future of the developmental proteome.

developmental biology↗

bioRxiv2026

FLRT cell adhesion molecules counteract protocadherin-10-dependent cell segregation during cortical neuron migration

The processes that underlie the folding of the cerebral cortex of large mammals and the evolution of the smooth cortex of modern rodents are incompletely understood. Recent evidence highlighted adhesion-controlled neuronal migration as an important mechanism. Genetic deletion in mice of FLRT1 and FLRT3 cell-adhesion proteins perturbed cortical neuron migration, lead to region-specific neuronal clustering, and introduced cortical folds into the normally smooth mouse cortex, suggesting that Increased expression of FLRT protein may have contributed to cortex smoothening. Here, we identify protocadherin-10 (PCDH10) as a key regulator of neuronal clustering in this context. Using single-cell transcriptomics and spatial proteomics, we describe a cortical neuron subtype in which loss of FLRT1/3 triggers local upregulation of PCDH10 in a specific cortical region. Elevated PCDH10 drives homophilic adhesion and the formation of cell patches that segregate from FLR1/3-deficient migrating neurons. This segregation leads to discrete clustering of Flrt1/3-mutant neurons and correlates with the emergence of cortical folds. Consistent with an evolutionary role, comparative expression analysis showed reciprocal expression of FLRT3 and PCDH10, with high FLRT3 and low PCDH10 expression in the smooth mouse cortex, and the opposite pattern in the developing human cortex. Together, our findings reveal how the antagonism between different classes of cell adhesion molecules affects cortical neuron migration patterns, contributes to region-specific cortical folding and may have promoted evolutionary smoothening of the rodent cortex.

neuroscience↗

bioRxiv2025

Unravelling drug resistant proteotypes through phenotype-resolved proteomics of single-cell derived colonies

Drug resistance remains a major cause of treatment failure and disease progression and is closely linked to intratumoral heterogeneity. Mass spectrometry (MS)-based single-cell proteomics (SCP) can resolve drug-resistant cell states, but current methods lack clonal and phenotype information and are often confounded by cell-cycle and cell-size effects. Here, we introduce PhenoSCoP, a microscopy-guided discovery proteomics framework that profiles single-cell-derived colonies to map proteomic heterogeneity while preserving clonal origin and image-defined phenotypes. In head and neck squamous cell carcinoma models, PhenoSCoP distinguished heritable clone-intrinsic cisplatin (CP) response programs from adaptive treatment-induced changes, uncovered pre-existing resistant-like proteotypes in treatment-naive populations, and identified an NDUFB11-associated mitochondrial vulnerability in a resistant clone. In patient-derived primary HNSCC cells, CP-tolerant cells resolved into two proteomic states with distinct phenotypes and high or low S100A7/A8/A9 expression. These programs were also detected in HNSCC patient tissues and, in an illustrative matched progression case, increased across metastatic sites. PhenoSCoP therefore provides a phenotype-resolved framework for discovering stable and adaptive proteotypes associated with therapeutic response.

systems biology↗

bioRxiv2025

Ultrasensitive proteomics uncovers nociceptor diversity and novel pain targets

The richness of our somatosensory experience is reflected in the functional diversity of somatic sensory neurons. Single-cell sequencing (scRNA) of sensory neurons has revealed a molecular basis for such diversity1-3. However, sensory neuron diversity has yet to be captured at the level of the proteome. Here, we combined electrophysiology with deep visual proteomics (DVP)4 to quantify over 6,000 proteins from phenotypically-defined sensory neurons and identified novel proteomic markers of sensory neuron subtypes. Comparative analysis revealed both concordance and meaningful divergence between transcriptomes and proteomes. We further show that up to 3,000 proteins can be quantified from one-fourth of a single neuron, demonstrating subset-specific protein signatures. In culture, nociceptive neurons can be acutely sensitized to mechanical stimuli by nerve growth factor (NGF) which normally drives inflammatory pain in vivo5. Indeed, overnight exposure of peptidergic nociceptors to NGF and a protein kinase C (PKC) activator produced functional sensitization associated with proteome changes. Functional knockdown experiments identified the up-regulated B3GNT2 enzyme as a potential effector of nociceptor sensitization. In summary, we present a high-resolution proteomic resource linking molecular identity to function, enabling the discovery of novel mechanisms underlying somatic sensation and pain sensitization.

systems biology↗

bioRxiv2025

OpenDVP: An experimental and computational framework for community-empowered deep visual proteomics

Deep visual proteomics (DVP) is an emerging approach for cell type-specific and spatially resolved proteomics. However, its broad adoption has been constrained by the lack of an open-source end-to-end workflow in a community-driven ecosystem. Here, we introduce openDVP, an experimental and computational framework for simplifying and democratizing DVP. OpenDVP integrates open-source software for image analysis, including MCMICRO, QuPath, and Napari, and uses the scverse data formats AnnData and SpatialData for multi-omics integration. It offers two workflows: a fast-track pipeline requiring no image analysis expertise and an artificial intelligence (AI)-powered pipeline with recent algorithms for image pre-processing, segmentation, and spatial analysis. We demonstrate openDVPs versatility in three archival tissue studies, profiling human placenta, early-stage lung cancer, and locally relapsed breast cancer. In each study, our framework provided insights into health and disease states by integrating spatial single-cell phenotypes with exploratory proteomic data. Finally, we introduce deep proteomic profiling of cellular neighborhoods as a scalable approach to accelerate spatial discovery proteomics across biological systems.

systems biology↗

bioRxiv2025

Maternal smoking in early pregnancy disrupts placental function through syncytiotrophoblast and macrophage dysregulation.

Smoking in pregnancy is the leading avoidable cause of gestational morbidity and mortality, causally linked to fetal growth restriction (FGR). The placenta, functional interface between mother and fetus is essential for healthy fetal development. For the first time, we studied cell type-resolved smoking effects on placental development at high molecular resolution using single-nucleus RNA sequencing and deep visual proteomics of matched tissues. We validated our findings through an independent cohort and in-vitro cigarette smoke exposure to primary human trophoblast cells. Our results show placental macrophages (Hofbauer cells; HBC) and the syncytiotrophoblast (STB) barrier are most affected by smoking, with dysregulation of cell-cell adhesion, extracellular matrix organization, and stress phenotype. STBs show moderate compositional increases in smokers and in-silico trophoblast differentiation modelling indicates a preferential shift towards the STB lineage in this group. The trophoblast displays a large upregulation of pro-angiogenic effectors, increases in xenobiotic detoxification, reduced mitochondrial function, and vastly altered transmembrane transport. These molecular changes affect placental development with important consequences for fetal growth. We provide insight into placental dysfunction contributing to FGR early in pregnancy, before clinical symptoms appear. We anticipate this data to advance diagnostics and therapies to improve FGR outcomes.

systems biology↗

bioRxiv2025

An ultrasensitive spatial tissue proteomics workflow exceeding 100 proteomes per day

Achieving high-resolution spatial tissue proteomes requires careful balancing and integration of optimized sample processing, chromatography, and MS acquisition. Here, we present an advanced cellenONE protocol for loss-reduced tissue processing and compare all Evosep ONE Whisper Zoom gradients (20, 40, 80, and 120 samples per day), along with three common DIA acquisition schemes on a timsUltra AIP mass spectrometer. We found that tissue type was as important as gradient length and sample amount in determining proteome coverage. Moreover, the benefit of increased tissue sampling was gradient- and dynamic range-dependent. Analyzing mouse liver, a high dynamic range tissue, over tenfold more tissue sampling led to only ~30% gain in protein identification for short gradients (120 SPD and 80 SPD). However, even the lowest tested tissue amount (0.04 nL, 40,000 {micro}m3) yielded 3,200 reproducibly quantified proteins for the 120 SPD method. Longer gradients (40 SPD and 20 SPD) instead significantly benefited from more tissue sampling, quantifying over 7,500 proteins from 0.5 nL of tonsil T-cell niches. Finally, we applied our workflow to a rare squamous cell carcinoma of the oral cavity, uncovering disease-associated pathways and region-specific protein level changes. Our study demonstrates that more than 100 high-quality spatial tissue proteomes can be prepared and acquired daily, laying a strong foundation for cohort-size spatial tissue proteomics in translational research.

systems biology↗

bioRxiv2025

Design of combination therapeutics from protein response to drugs in ovarian cancer cells

High-grade serous ovarian cancer (HGSOC) remains the most lethal gynecologic malignancy, and novel treatment approaches are needed. Here, we used unbiased quantitative protein mass spectrometry to assess the cellular response profile to drug perturbations in ovarian cancer cells for the rational design of potential combination therapies. Analysis of the perturbation profiles revealed proteins responding across several drug perturbations (called frequently responsive below) as well as drug-specific protein responses. The frequently responsive proteins included proteins that reflected general drug resistance mechanisms, such as changes in drug efflux pumps. Network analysis of drug-specific protein responses revealed known and potential novel markers of resistance, which were used to rationalize the design of anti-resistance drug pairs. We experimentally tested the anti-proliferative effects of 12 of the proposed drug combinations in 6 HGSOC cell lines. While response typically varies across different cell lines, 10 of the 12 combinations tested have either an additive or synergistic CI index in at least one cell line and may therefore be plausible candidates for overcoming or preventing resistance to single agents. Serendipitously, we observed an unexpectedly strong 0.05-0.11 micromolar response to GPX4 inhibitors as single agents in the OVCAR-4 cell line. We propose several drug combinations as potential therapeutic candidates in ovarian cancer, as well as GPX4 inhibitors as single agents. HighlightsO_LI5000-protein response to 7 drugs in an ovarian cancer cell line profiled by protein mass spectrometry C_LIO_LINetwork analysis suggested potential pathways of drug resistance inferred from response profiles C_LIO_LIDemonstration of a general method for profiling adaptive response to therapeutic interventions with implications for the development of anti-resistance therapy C_LIO_LIPlausible anti-resistance drug combinations tested for antiproliferative effect in up to 6 ovarian cancer cell lines C_LIO_LIDrug combinations with additive or synergistic antiproliferative effects are plausible pre-clinical candidates for overcoming or preventing resistance to single agents C_LIO_LISeveral combinations were synergistic in some cell lines (with PARPi, MEKi, and SRCi) C_LIO_LIWe observed 0.05-0.11 micromolar response to GPX4 inhibitors as single agents in the OVCAR-4 cell line C_LI

cancer biology↗

bioRxiv2025

Deep spatial proteomics of ovarian cancer precursor lesions delineates early disease changes and cell-of-origin signatures

High-grade serous ovarian cancer (HGSOC) is a devastating disease that is frequently detected at an incurable stage. Advances in ultrasensitive mass spectrometry-based spatial proteomics have provided a unique opportunity to uncover early molecular events in tumorigenesis and common dysregulated pathways with high therapeutic potential. Here, we present a comprehensive proteomic analysis of serous tubal intraepithelial carcinoma (STIC), the HGSOC precursor lesion, covering more than 10,000 proteins. We found that STICs and concurrent invasive carcinomas were indistinguishable at the global proteomic level, revealing a similar level of molecular heterogeneity. Using cell-type resolved tissue proteomics, we revealed strong cell-of-origin signatures preserved in STICs and invasive tumors and identified early dysregulated pathways of therapeutic relevance, such as an onco-metabolic increase in cholesterol biosynthesis. Finally, we uncovered substantial remodeling of the co-evolving tumor microenvironment, affecting approximately one-third of the stromal proteome, and derived a common signature associated with progressive immunosuppression and extracellular matrix restructuring. In summary, our study highlights the power of spatially resolved quantitative proteomics to dissect the molecular underpinnings of early carcinogenesis and provides a rich proteomic resource for future biomarker and drug target research in ovarian cancer.

systems biology↗

bioRxiv2024

Targeted disruption of linkage-specific ubiquitylation reveals a key role of K29-linked ubiquitylation in epigenome integrity

Linkage-specific ubiquitin chains dictate the functional outcome of numerous critical ubiquitin-dependent signaling processes. However, the functions and targets of several poly-ubiquitin topologies remain poorly defined due to a paucity of tools for their specific detection and manipulation. To remedy this knowledge gap, we applied a cell-based ubiquitin replacement strategy enabling targeted conditional abrogation of each of the seven lysine-based ubiquitin chain types in human cells to profile system-wide impacts of disabling formation of individual chain types. Focusing on K29-linked ubiquitylation, we reveal a strong association of this linkage type with chromatin-associated proteins and show that the H3K9me3 methyltransferase SUV39H1 is a prominent cellular target of this modification. We demonstrate that K29-linked ubiquitylation is essential for proteasomal degradation of SUV39H1 despite its extensive modification by K48-linked ubiquitylation, and that K29-linked ubiquitylation of SUV39H1 is catalyzed and reversed by TRIP12 and TRABID, respectively. Preventing K29-linked ubiquitylation-mediated control of SUV39H1 stability deregulates the H3K9me3 landscape, but not other histone marks. Collectively, our ubiquitin replacement cell line panel and datasets provide valuable resources for illuminating cellular functions of linkage-specific ubiquitin chains and establish a key role of K29-linked ubiquitylation in epigenome integrity.

cell biology↗

bioRxiv2024

Molecular mechanism of thyroxine transport by monocarboxylate transporters

Thyroid hormones (the common name for prohormone thyroxine and the bioactive form triiodothyronine) control major developmental and metabolic processes. Release of thyroid hormones from the thyroid gland into the bloodstream and their transport into target cells is facilitated by plasma membrane transporters, of which monocarboxylate transporter (MCT)8 and the highly homologous MCT10 are most important. Patients with MCT8 mutations suffer from a severe neurodevelopmental and metabolic disorder, however, the molecular mechanism underlying thyroid hormone transport is unknown. Using cryogenic-sample electron microscopy (cryo-EM), we determined the ligand-free and thyroxine-bound human MCT8 structures in the outward-open state and the thyroxine-bound human MCT10 in the inward-facing state. Our structural analysis revealed a network of conserved gate residues involved in conformational changes upon thyroxine binding, triggering ligand release on the opposite compartment. We then determined the structure of a folded, but inactive patient-derived MCT8 mutant, indicating a subtle conformational change which explains its reduced transport activity. In addition, we determined the structure of MCT8 bound to its inhibitor silychristin, revealing an interaction with residues essential to drive transition to the inward-facing state, thereby locking the protein in the outward-facing state. This study provides molecular and structural insights into normal and disordered thyroid hormone transport.

biochemistry↗

bioRxiv2024

Spatial omics of acute myocardial infarction reveals a novel mode of immune cell infiltration

Myocardial infarction (MI) continues to be a leading cause of death worldwide. Even though it is well-established that the complex interplay between different cell types determines the overall healing response after MI, the precise changes in the tissue architecture are still poorly understood. Here we generated an integrative cellular map of the acute phase after murine MI using a combination of imaging-based transcriptomics (Molecular Cartography) and antibody-based highly multiplexed imaging (Sequential Immunofluorescence), which enabled us to evaluate cell-type compositions and changes at subcellular resolution over time. One striking finding of these analyses was the identification of a novel mode of leukocyte accumulation to the infarcted heart via the endocardium - the inner layer of the heart. To investigate the underlying mechanisms driving this previously unknown infiltration route, we performed unbiased spatial proteomic analysis using Deep Visual Proteomics (DVP). When comparing endocardial cells of homeostatic hearts and infarcted hearts, DVP identified von Willebrand Factor (vWF) as an upregulated mediator of inflammation 24 hours post-MI. To further explore the immune mediating capabilities of vWF and its effect on tissue repair, we performed functional blocking of vWF during acute murine MI. This resulted in a reduced amount of infiltration by CCR2+ monocytes and worse cardiac function post-MI. Our study provides the first spatial map of acute murine MI with subcellular resolution and subsequently discovers a novel route of immune infiltration. Furthermore, we identified vWF as a critical immune mediating agent for endocardial immune cell infiltration.

systems biology↗

bioRxiv2023

An automated and fast sample preparation workflow for laser microdissection guided ultrasensitive proteomics

Spatial tissue proteomics integrating whole-slide imaging, laser microdissection and ultrasensitive mass spectrometry is a powerful approach to link cellular phenotypes to functional proteome states in (patho)physiology. To be applicable to large patient cohorts and low sample input amounts, including single-cell applications, loss-minimized and streamlined end-to-end workflows are key. We here introduce an automated sample preparation protocol for laser microdissected samples utilizing the cellenONE(R) robotic system, which has the capacity to process 192 samples in three hours. Following laser microdissection collection directly into the proteoCHIP LF 48 or EVO 96 chip, our optimized protocol facilitates lysis, formalin de-crosslinking and tryptic digest of low-input archival tissue samples. The seamless integration with the Evosep ONE LC system by centrifugation allows on-the-fly sample clean-up, particularly pertinent for laser microdissection workflows. We validate our method in human tonsil archival tissue, where we profile proteomes of spatially-defined B-cell, T-cell and epithelial microregions of 4,000 {micro}m2 to a depth of [~]2,000 proteins and with high cell type specificity. We finally provide detailed equipment templates and experimental guidelines for broad accessibility.

systems biology↗

bioRxiv2023

SARS-CoV-2 hijacks fragile X mental retardation proteins for efficient infection

Viruses interact with numerous host factors to facilitate viral replication and to dampen antiviral defense mechanisms. We currently have a limited mechanistic understanding of how SARS-CoV-2 binds host factors and the functional role of these interactions. Here, we uncover a novel interaction between the viral NSP3 protein and the fragile X mental retardation proteins (FMRPs: FMR1 and FXR1-2). SARS-CoV-2 NSP3 mutant viruses preventing FMRP binding have attenuated replication in vitro and have delayed disease onset in vivo. We show that a unique peptide motif in NSP3 binds directly to the two central KH domains of FMRPs and that this interaction is disrupted by the I304N mutation found in a patient with fragile X syndrome. NSP3 binding to FMRPs disrupts their interaction with the stress granule component UBAP2L through direct competition with a peptide motif in UBAP2L to prevent FMRP incorporation into stress granules. Collectively, our results provide novel insight into how SARS-CoV-2 hijacks host cell proteins for efficient infection and provides molecular insight to the possible underlying molecular defects in fragile X syndrome.

microbiology↗

bioRxiv2023

Serialized On-grid Lift-In Sectioning for Tomography (SOLIST)

Cryo-focused ion beam milling has enabled groundbreaking structural discoveries in native cells. Progress toward medically relevant applications, however, has been slow. We here present an adaptation of the cryo-lift out procedure for Serialized On-grid Lift-In Sectioning for Tomography (SOLIST), which increases throughput, reduces ice contamination, and enhances sample stability. With these improvements, new specimens, ranging from high-pressure frozen reconstituted LLPS droplets to human forebrain organoids, are accessible to cryo-electron tomography.

biophysics↗

bioRxiv2023

A framework for ultra-low input spatial tissue proteomics

Spatial tissue proteomics combining microscopy-based cell phenotyping with ultra-sensitive mass spectrometry (MS)-based proteomics is an emerging and powerful concept for the study of cell function and heterogeneity in health and disease. However, optimized workflows that preserve morphological information for image-based phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected archival tissue, are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low input formalin-fixed, paraffin-embedded (FFPE) material. Benchmarking in the murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions with high quantitative reproducibility. Applied to human tonsil, we profiled 146 microregions including spatially defined T and B lymphocyte niches and quantified cell type specific markers, cytokines, immune cell regulators and transcription factors. These rich data also highlighted proteome dynamics in spatially defined zones of activated germinal centers, illuminating sites undergoing active B-cell proliferation and somatic hypermutation. Our results demonstrate the power of spatially-resolved proteomics for tissue phenotyping by integrating high-content imaging, laser microdissection, and ultra-sensitive mass spectrometry. This approach has broad implications for a wide range of biomedical applications, including early disease profiling, drug target discovery and biomarker research.

systems biology↗

bioRxiv2023

High-resolution molecular atlas of a lung tumor in 3D

Cells live and interact in three-dimensional (3D) cellular neighborhoods. However, histology and spatial omics methods mostly focus on 2D tissue sections. Here we present a 3D spatial atlas of a routine clinical sample, an aggressive human lung carcinoma, by combining in situ quantification of 960 cancer-related genes across [~]340,000 cells with measurements of tissue-mechanical components. 3D cellular neighborhoods subdivided the tumor microenvironment into tumor, stromal, and immune multicellular niches. Interestingly, pseudotime analysis suggested that pro-invasive epithelial-to-mesenchymal transition (EMT), detected in stroma-infiltrating tumor cells, already occurred in one region at the tumor surface. There, myofibroblasts and macrophages specifically co-localized with pre-invasive tumor cells and their multicellular molecular signature identified patients with shorter survival. Moreover, cytotoxic T-cells did not infiltrate this niche but colocalized with inhibitory dendritic and regulatory T cells. Importantly, systematic scoring of cell-cell interactions in 3D neighborhoods highlighted niche-specific signaling networks accompanying tumor invasion and immune escape. Compared to 2D, 3D neighborhoods improved the characterization of immune niches by identifying dendritic niches, capturing the 3D extension of T-cell niches and boosting the quantification of niche-specific cell-cell interactions, including druggable immune checkpoints. We believe that 3D communication analyses can improve the design of clinical studies investigating personalized, combination immuno-oncology therapies.

systems biology↗

bioRxiv2022

Disturbed trophoblast transition links preeclampsia progression from placenta to the maternal syndrome

Pre-eclampsia (PE) is a syndrome that affects multiple organ systems and is the most severe hypertensive disorder in pregnancy. It frequently leads to preterm delivery, maternal and fetal morbidity and mortality and life-long complications1. We currently lack efficient screening tools2, 3 and early therapies4, 5 to address PE. To investigate the early stages of early onset PE, and identify candidate markers and pathways, we performed spatio-temporal multi-omics profiling of human PE placentae and healthy controls and validated targets in early gestation in a longitudinal clinical cohort. We used a single-nuclei RNA-seq approach combined with spatial proteo- and transcriptomics and mechanistic in vitro signalling analyses to bridge the gap from late pregnancy disease to early pregnancy pathomechanisms. We discovered a key disruption in villous trophoblast differentiation, which is driven by the increase of transcriptional coactivator p300, that ultimately ends with a senescence-associated secretory phenotype (SASP) of trophoblasts. We found a significant increase in the senescence marker activin A in preeclamptic maternal serum in early gestation, before the development of clinical symptoms, indicating a translation of the placental syndrome to the maternal side. Our work describes a new disease progression, starting with a disturbed transition in villous trophoblast differentiation. Our study identifies potential pathophysiology-relevant biomarkers for the early diagnosis of the disease as well as possible targets for interventions, which would be crucial steps toward protecting the mother and child from gestational mortality and morbidity and an increased risk of cardiovascular disease later in life.

systems biology↗