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The central nervous system (CNS) is protected by a network of specialised barrier tissues, including the blood–brain barrier (BBB), leptomeningeal barriers, the choroid plexus epithelial cells forming the blood cerebrospinal fluid barrier, and glia limitans that collectively maintain CNS homeostasis, regulate immune surveillance, and protect neural tissue from harmful insults. While BBB dysfunction is a well established feature of multiple sclerosis (MS), the molecular responses and coordinated contributions of the remaining CNS barriers to disease initiation and progression remain poorly understood. This project aims to define the molecular landscape of CNS barrier cells in health and during the onset of CD4⁺ and CD8⁺ T cell-mediated neuroinflammation at single-cell resolution. We hypothesize that individual barrier compartments undergo distinct yet coordinated transcriptional remodeling that regulates barrier integrity, immune cell trafficking, and inflammatory signaling during neuroinflammation.

We are currently establishing an optimized workflow for the simultaneous isolation of high-quality single barrier cell suspensions from the brain, spinal cord, dura mater, and choroid plexus from the same mouse. We are using our established fluorescent transgenic reporter mouse models, the VE-cadherin-GFP knock-in mice, which labels the endothelial junctions and fibroblast layers in the leptomeninges and the Aquaporin-4 -mRuby3 knock-in reporter mouse that labels the astrocyte endfeet forming the glia limitans. Additionally, we will use barrier cell specific markers to sort for vascular endothelial cells, meningeal fibroblasts, choroid plexus epithelial cells and glia limitans forming astrocytes while preserving transcriptomic integrity for single-cell RNA sequencing.

Our work focuses on optimising tissue dissociation conditions to maximise recovery and viability of these barrier cell populations. The resulting dataset will provide a high-resolution molecular atlas of CNS barrier compartments and identify compartment-specific transcriptional responses associated with immune surveillance and neuroinflammation.

These findings will improve our understanding of barrier biology and identify candidate pathways governing barrier dysfunction, and establish a foundation for developing therapeutic strategies aimed at restoring barrier integrity in MS.

Ageing is the leading cardiovascular risk factor, yet human microvascular ageing is challenging to model. As blood-borne factors can drive and reverse vascular ageing (Kiss et al., GeroScience 2020), we used a high-throughput perfusable 3D human microvascular platform to test whether aged serum induces microvascular-ageing features in endothelial cells (ECs), reproducing human EC-ageing signatures.

GFP-labelled dermal microvascular ECs and fibroblasts were co-seeded in fibrin within custom 3D-printed chambers embedded in 96-well plates. Samples were exposed to physiological interstitial flow for maturation. Sera from 12 young (<35 y) and 12 old (>60 y) healthy males were profiled by SomaScan proteomics and an ageing clock. Once perfusable, microvascular networks received pooled young or old serum. ECs were GFP-sorted and profiled with bulk SMART-seq (one pool per age; three donors each). Differential expression and concordance was tested against vessel-resolved (capillary, artery, vein) signatures from a human EC ageing atlas (HAECA; Dobner et al., bioRxiv 2025).

Networks were perfusable (diameters 5-60 µm) and stable ≥21 days. We confirmed EC identity, basement membrane formation and support by perivascular fibroblasts. Young and old sera were proteomically distinct (clock age ~59 vs ~78 y; 130 differential proteins). Aged serum reprogrammed the EC transcriptome (299 differentially expressed genes [DEGs]) through coordinated repression of a matrisome and mesenchymal/angiogenic program (epithelial–mesenchymal transition [EMT] strongest; collagens, core matrisome, angiogenesis). We also observed up-regulated proliferation, whereas senescence was not induced. This signature overlapped with genes down-regulated with ageing in HAECA capillaries, but not artery/vein arms, indicating a capillary rather than large-vessel response.

The model recapitulates microvascular identity hallmarks. Aged serum drives a capillary-restricted endothelial shift that is distinct from senescence. Two limitations remain: the comparison considers one serum pool per age group, relying on external concordance. Further, whether the response is EC-intrinsic or partly reflects stromal content is unresolved. Ongoing work repeats the screen with up to 12 individual donors to address pooling and refines the serum ageing clock using Global Neurodegeneration Proteomics Consortium (GNPC) data.

Metastatic progression critically depends on remodeling of the tumor microenvironment, where the vascular endothelium plays a central role. Transcriptomic profiling of endothelial cells isolated from lung metastatic lesions of several mouse models identified TNFRSF9, encoding the co-stimulatory receptor CD137, among the upregulated genes during metastasis. Although CD137 is primarly known for its role in the immune compartment, the functional significance of its de novo endothelial expression remains unclear. This project aims to identify the CD137L-expressing cells engaging with endothelial CD137 and to determine how these interactions shape metastatic progression.

Primary tumor, spontaneous and experimental lung metastasis models were tested by subcutaneous or intravenous LLC1.1 cell injection, according to the experimental setting. Endothelial-specific CD137 knockout (ecCD137KO) mice and floxed littermate controls were used to assess the impact of endothelial CD137 deletion on tumor growth and metastatic burden. Vascular remodeling and immune cell infiltration were assessed by immunofluorescence (IF) and flow cytometry, respectively. CD137L expression was characterized by IF, flow cytometry and qPCR on cells sorted from metastatic foci. Endothelial CD137 induction was assessed following co-culture with either wild-type LLC1.1 or CD137L-deficient LLC1.1 cells by IF.

Protein analyses confirmed de novo CD137 expression in endothelial cells from primary tumors and lung metastases. Endothelial-specific CD137-deficient mice developed a distinct metastatic phenotype with altered primary tumor growth, metastatic burden, vascular remodeling, and immune cell infiltration. CD137L expression was detected in immune, stromal/perivascular and tumor cell populations, indicating that endothelial CD137 receives multiple signaling inputs within the metastatic niche. Co-culture assays further demonstrated the induction of de novo endothelial CD137 expression in vitro.

We have identified de novo endothelial CD137 as a novel regulator of metastatic progression, which supports the coordinating multicellular communication within the metastatic niche. Ongoing in vitro and in vivo studies are now focused on identification of CD137L-expressing cellular compartments that functionally engage endothelial CD137 and characterization of the signaling axis driving metastatic progression.

Aging is associated with profound molecular and functional alterations, which exhibit sex-specific differences contributing to divergent aging trajectories in males and females [33982659]. Evidence from heterochronic parabiosis suggests that systemic circulating factors can modulate vascular function and promote rejuvenation, although the contribution of sex in this process is unclear [39154047, 31806903]. Here, we hypothesize that human endothelial cells differentially respond to age- and sex-specific circulating cues present in human serum.

Human dermal blood endothelial cells (HDBECs) were isolated from skin biopsies of healthy donors (>60y.o.) using a FACS-based strategy, enabling the recovery of cells from the vascular niche. Cell identity was assessed by flow cytometry, immunofluorescence and 3D assays of vasculogenesis. HDBECs were exposed to human sera from healthy young (18-35 y.o) or old (>60 y.o.) male and female donors, followed by bulk RNA sequencing to assess differential responses induced by donor age and sex.

We successfully enriched HDBECs from skin biopsies (73.56±10.19% of the isolated endothelial cell fraction). Expanded HDBECs retained their endothelial identity (95.08±5.30% CD45⁻CD31⁺PDPN⁻), further confirmed by high ERG positivity (89.95±3.91%). HDBECs preserved vasculogenic capacity, forming 3D microvascular networks in vitro [42014446]. Transcriptomic analyses revealed distinct sex-specific molecular programs: young male serum selectively upregulated Protein Kinase B (AKT) signaling compared to old male serum, whereas young female serum primarily restored cholesterol metabolic and biosynthetic pathways.

These data suggest that youthful circulating factors support endothelial function through distinct, sex-specific mechanisms: enhancing AKT signaling pathways in males, while preserving metabolic and lipid homeostasis in females. To consolidate these findings, we are now focusing on the validation of the identified pathways through biochemical assays and proteomics. We are also increasing the complexity of the setup, including co-culture with other cells of the vascular niche in a 3D physiological setting. Overall, these results suggest that sex-specific responses of the endothelium should be carefully considered for the development of therapeutics targeting vascular aging.

Primary endothelial cells isolated from adult donors are essential for modeling vascular aging and age-related diseases (PMID: 35101902). However, their limited proliferative capacity, imposed by the Hayflick limit, represents a major bottleneck for generating the large cell numbers required for mechanistic studies, drug screening, and advanced in vitro models (PMID: 32987094). Here, we developed a transient immortalization strategy to enable the scalable expansion of Human Dermal Blood Endothelial Cells (HDBECs) from elderly donors, followed by reversion to a mortal state while preserving key hallmarks of aging.

HDBECs were isolated from skin biopsies of 48-, 60-, and 67-year-old donors (PMID: 42014446) and transiently immortalized using a lentiviral FLEX-SV40 Large T/iCasp9 system. Following cell expansion, immortalization was reversed by Cre-mediated recombination, and residual immortalized cells were eliminated with AP20187. Endothelial identity and the aging phenotype were assessed by immunofluorescence and RT-qPCR using a panel of endothelial and aging-associated markers representative of multiple hallmarks of aging. Epigenetic age was further assessed using a Horvath-based DNA methylation clock.

Transient immortalization markedly enhanced HDBEC proliferative capacity and enabled large-scale cell expansion. Following Cre-mediated de-immortalization and AP20187 selection, cells reacquired a physiological cell-cycle profile while preserving endothelial identity (ERG⁺ nuclei: 97.7±2.3%). Elevated γ-H2AX-positive nuclei (21.8±3.4%) were consistent with preservation of aging-associated DNA damage, while markers representative of cellular senescence (KLF6), nutrient sensing (SIRT1, SIRT6, Klotho), oxidative stress (MAFF), proteostasis (P4HB), endothelial function (NOS3 and KLF2), and mitochondrial homeostasis (LARS2) supported maintenance of multiple hallmarks of aging. DNA methylation clock analysis is underway to evaluate the preservation of epigenetic age.

This reversible immortalization strategy enables the scalable expansion of adult HDBECs while preserving endothelial identity and key hallmarks of aging. The resulting cells provide a robust platform for the generation of physiologically relevant 3D microvascular models of vascular aging and age-related diseases and are currently being applied to the biofabrication of engineered microvascular networks.

Central nervous system (CNS) border zones including the leptomeninges play a pivotal role in maintaining CNS immune privilege, by restricting immune cell entry into the CNS parenchyma. Effector T cells can access the subarachnoid space (SAS) bordered by fibroblasts forming the arachnoid and pia mater to perform CNS immune surveillance. Only upon antigen recognition on tissue-resident antigen-presenting cells in the SAS, T cells get locally reactivated, breach the glia limitans and reach the CNS parenchyma, leading to neuroinflammation. We have shown by in vivo live-cell imaging that effector T cells crawl on the pial fibroblast monolayer prior to crossing it. This resembles the post-arrest T cell behaviour during their multi-step extravasation across the endothelial blood-brain barrier (BBB). As endothelial ICAM-1 mediates T cell crawling on the BBB, we here asked if T cell crawling on pia mater fibroblasts is also mediated by ICAM-1.

We used VE-cadherin-GFP reporter mice to visualize vascular endothelial and leptomeningeal fibroblast junctions, and ODC-OVA mice as a model for CD8 T cell-driven neuroinflammation. We first investigated ICAM-1 protein expression on the pia mater with immunofluorescence stainings of decalcified vertebral column and head sections and with in vivo labelling of the cervical spinal cord. We next investigated the role of pial ICAM-1 in T cell crawling on the pia mater by two-photon intravital microscopy of the cervical spinal cord. We observed tdTomato+ CD8 T cells in the SAS crawling on VE-cadherin-GFP+ pia mater fibroblasts during CNS immune surveillance and neuroinflammation. To study the contribution of pial ICAM-1 in T cell crawling we administered intra-cisterna magna ICAM-1 function-blocking antibodies and analyzed behavioural changes including CD8 T cell detachment, crawling speed and crawling directionality.

We detected ICAM-1 on pia mater fibroblasts in the brain and spinal cord. Our preliminary observations show that ICAM-1 mediates CD8 T cell crawling on pia mater fibroblasts.

These findings suggest that molecular mechanisms associated with vascular endothelial leukocyte trafficking may also regulate CD8 T cell migration across the pia mater, providing new insights into T cell trafficking at leptomeningeal barriers and mechanisms that may contribute to CNS immune surveillance and to the initiation of CNS neuroinflammation.

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the CNS. While blood-brain-barrier breakdown (BBB) and immune cell infiltration have been recognized as major hallmarks of MS, deposition of extracellular matrix (ECM) proteins, e.g. collagen, laminin and fibronectin (Fn), are often identified in chronic active MS lesions. There are two forms of fibronectin, plasma fibronectin (pFn) and cellular fibronectin (cFn), both of which have been suggested to contribute to the upregulation of Fn in MS lesions. pFn was proposed to leak into the CNS parenchyma upon BBB breakdown. Previously, using the experimental autoimmune encephalomyelitis (EAE) model in CX3CR1-GFP/CCR2-RFP reporter mice, we have demonstrated leakage of fibrinogen from leptomeningeal vessels into the subarachnoid space (SAS) of the spinal cord. In this study, we aim to investigate the deposition of fibronectin in the subarachnoid space across the EAE disease course and how fibronectin clears out of the CNS

EAE animal model, immunofluorescence staining, confocal imaging, near-infrared imaging

We found significant increase of Fn deposition in the mouse SAS of spinal cord at the chronic stage, but not the EAE peak stage. Surprisingly, above the cribriform plate (CP), a major cerebrospinal fluid (CSF) efflux pathway in mice, there was significant deposition of Fn at the EAE peak stage, with a tendency of reduction at the chronic stage. In the meantime, CCR2+ monocytes accumulated in the CP region showed upregulation of the integrin α5 subunit of the Fn receptor. In addition, Fn deposition was found on the wall of the CSF-draining lymphatic vessels, while some immune cells decorated with surface Fn staining were found in the lumen of lymphatic vessels.

These results suggest that in EAE animal model, leptomeningeal vessel breakdown leads to the leakage of Fn into the SAS. Along with the CSF outflow, soluble Fn may clear out of the CNS through CP-lymphatic vessels. Aggregation of Fn in the CNS has been shown to hamper remyelination process, therefore timely and efficient clearance of soluble Fn may be beneficial to CNS demyelinating disease.

In recent years, increasing attention has been devoted to the gut–brain axis, highlighting the role of gut-derived metabolites in regulating intercellular communication within the brain under both physiological and pathological conditions. Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults, with a 5-year survival rate lower than 10%. Recent evidence from a murine model of intestinal dysbiosis demonstrated that alterations in the gut microbiota can increase glycine levels in the brain parenchyma. Notably, GBM metabolic reprogramming has been shown to rely on glycine availability to sustain tumor growth and proliferation. With our research we aim to investigate the potential impact of glycine on blood–brain barrier (BBB) function in the context of GBM, in terms of permeability and expression of endothelial trafficking molecules.

We developed an in vitro two-chamber BBB model consisting of primary brain microvascular endothelial cells exposed on the abluminal side to GBM-conditioned medium, thereby reproducing key features of the tumor microenvironment. Using this experimental setup, glycine was luminally administered, under both basal and pro-inflammatory conditions. Moreover, to mimic the tumor-associated increase in glycine levels observed within the peritumoral brain parenchyma, the effects of the abluminal administration of glycine were also tested.

Luminally administered glycine exerted anti-inflammatory effects, significantly reducing the expression of the adhesion molecules ICAM-1 and VCAM-1. However, abluminally administered glycine modulated BBB properties by reducing endothelial permeability while simultaneously inducing a modest increase in ICAM-1 and VCAM-1 expression, suggesting a complex regulatory effect on endothelial activation.

Overall, our findings indicate that microbiota-driven alterations in glycine availability within the GBM microenvironment may significantly influence BBB function, with potential implications for immune cell transmigration across the BBB. These observations support the existence of a gut microbiota–glycine–BBB axis and suggest that dysbiosis-associated metabolic changes may contribute to tumor progression through modulation of BBB properties.

The rupture of a cerebral blood vessel deprives the tissue of essential oxygen and nutrients and represents a critical medical emergency known as haemorrhagic stroke. While initial interventions secure the bleed and reduce elevated intracranial pressure, secondary complications persist. These arise from the contamination of cerebrospinal fluid (CSF) and subsequent haemoglobin degradation in the subarachnoid space, driving early brain injury and delayed cerebral ischaemia. Enhancing the clearance of these toxic blood products via CSF drainage offers a promising therapeutic strategy to mitigate secondary complications. Although CSF drainage pathways remain debated, lymphatic vessels traversing the cribriform plate alongside olfactory nerve bundles represent a major drainage route, which our group demonstrated can drain intact erythrocytes. Here, we aim to target lymphatic CSF drainage following haemorrhagic stroke to mitigate secondary complications.

To study haemorrhagic stroke, we utilised a subarachnoid haemorrhage (SAH) endovascular perforation model in transgenic PROX-1-EGFP mice. Histological analysis on decalcified skulls was performed at 2 hours to 3 days post-SAH to analyse changes in lymphatic drainage pathways. CSF dynamics were also evaluated non-invasively at 3 days post-SAH using near-infrared (NIR) imaging of tracer drainage to the superficial cervical lymph nodes following intraventricular injection.

Histology showed a significant increase of lymphatic intraluminal erythrocytes (TER-119+) at 2 hours post-SAH compared to sham, representing peak accumulation before signal returned to baseline by day 3. Conversely, lymphatic intraluminal fibrin(ogen) levels were comparable to sham at 2 hours post-SAH but significantly elevated by day 3 compared to earlier timepoints and sham mice. Preliminary assessment of CSF dynamics suggests that lymphatic drainage remains preserved despite these intraluminal changes.

At 3 days following SAH, CSF drainage lymphatic vessels appear to shift from erythrocyte draining to a possibly procoagulant state. While further investigation is required into the functionality of this pathway at this timepoint, it could provide a novel target to reduce secondary complications following haemorrhagic stroke.

The meninges form specialized layers that shape the subarachnoid space (SAS) anatomy at the brain surface. However, the arrangement of the leptomeninges and how they guide CSF flow remains incompletely understood, as much of our current knowledge derives from post-mortem tissue analysis or in vivo imaging modalities that lack the capacity to accurately delineate the leptomeninges.

We used intravital two-photon microscopy in vascular and leptomeningeal reporter mice to examine the architecture of the SAS and tracer distribution in the CSF under physiological conditions and during controlled perturbations. Our data confirmed the absence of trabeculae in the superficial SAS of the mouse brain and show that over most of the cortex, the arachnoid lies directly on top of the pia without leaving a vertical space.

After injection into the cisterna magna, tracers were initially distributed to spaces alongside pial arteries. By visualization of VE-Cadherin-GFP signals in arachnoid and pial fibroblasts, we determined that these para-arterial spaces are created by the arachnoid tenting over the pial arteries, creating a vertical widening of the SAS on either side of the artery, although the expansion is not always symmetrical. At artery-vein crossover points, tracers were found to access spaces alongside veins, indicating a continuation of the SAS at this region.
Strikingly, when tracers were infused into the cisterna magna at higher rates, we observed disturbances in the natural states of the SAS structure resulting in widespread tracer distribution throughout the pia-arachnoid interface and along penetrating vessels in the brain, underscoring the sensitivity of superficial meningeal compartments to experimental manipulation. Finally, terminal imaging revealed a rapid collapse of these vessel-associated spaces and redistribution of tracer to the perivascular spaces of arteries and veins entering and exiting the brain parenchyma, indicating post-mortem changes of CSF tracer distribution.

Together, these findings advance our understanding of the anatomy of the superficial CSF-accessible spaces in the mouse brain.

Multiple sclerosis (MS) is traditionally defined as an autoimmune disease of the central nervous system (CNS), with blood-brain barrier (BBB) breakdown considered a secondary consequence of neuroinflammation. Building on our previous work, we have challenged this view by showing that human induced pluripotent stem cell (hiPSC)-derived brain microvascular endothelial cells (BMECs) from persons with MS (pwMS) display intrinsic barrier impairment and a pro-inflammatory phenotype compared to their counterparts from healthy controls (HC). This study aims to determine whether these cell-autonomous defects are present at the earliest, preclinical stage of MS. To this end, we expanded our cohort to include individuals with radiologically isolated syndrome (RIS), who subsequently converted to clinical MS, alongside an increased number of MS and HC donors.

We established hiPSCs from 3 HCs, 2 persons with RIS, and 7 pwMS. Lines were differentiated into BMECs using the extended endothelial cell culture method (EECM). Phenotypic and functional properties were characterized using immunostaining, western blotting, flow cytometry, and permeability measurements. Their transcriptional profiles were further investigated by bulk RNA sequencing.

RIS- and MS-derived EECM-BMECs display impaired barrier properties compared to HC-derived BMECs. Transcriptional profiling distinguished MS- and RIS-derived BMECs from HC controls, revealing a downregulation of key junctional components that was further confirmed at both the RNA and protein levels. Specifically, CLDN5/claudin-5 expression was markedly reduced, accompanied by interrupted junctional localization of claudin-5. Additionally, CDH5/VE-cadherin expression was decreased, correlating with disorganized adherens junctions.

Our findings underscore that intrinsic alterations in brain endothelial cells, manifested at transcriptional, protein, and functional levels, cause or contribute to altered BBB function as early as the RIS stage. This pre-existing barrier dysfunction, alongside additional risk factors, may play a crucial role for immune cell entry into the CNS and clinical MS onset.

Aging is a key determinant of metastatic susceptibility (1), yet its impact on the human blood-brain barrier (BBB) during melanoma brain metastasis (MBM) remains poorly understood (2). This project aims to develop a high-throughput, biofabricated human BBB platform to investigate how vascular aging influences melanoma extravasation.

(1) PMID: 41813904
(2) PMID: 23344048

Custom microchambers compatible with 96-well plates were designed in CAD software and 3D-printed using a biocompatible resin. Primary human brain microvascular endothelial cells, human astrocytes and human brain vascular pericytes were embedded in 3D fibrin hydrogel and seeded with an automated liquid handler. Constructs were cultured under unidirectional flow for 7–10 days, monitored by high-content imaging and tested for perfusion with fluorescent dextran. Aging was pre-induced by serial passaging and characterized by population doubling time, immunofluorescence for p16, Ki-67, SIRT1 and cell identity markers, plus targeted gene expression analysis. For co-culture experiments, melanoma cells from lymph node and brain metastases obtained from the same patient were perfused through the BBB constructs. Tumor–BBB interactions and extravasation were analyzed through real time high-content imaging.

The BBB model showed perfusable microvessel-like network formation covering approximately 60% of the hydrogel, with average vessel diameters ranging 30-35 um. Vessels were physiologically wrapped by pericytes and connected with astrocytic end-feet. Serial passaging increased population doubling time and p16 expression, while reducing Ki-67, SIRT1 and cell identity markers, consistent with an aging-like phenotype. Protein expression was correlated with gene expression analyses. Co-culture experiments showed that metastatic melanoma cells can be perfused through the BBB and that their vascular interactions and extravasation into the hydrogel compartment can be quantified over time.

We established a high-throughput BBB platform coupled with an in vitro aging strategy for each primary cell type composing the BBB. The model was successfully used to perfuse patient-derived melanoma cells from distinct metastatic sites. Current experiments are focused on the study of the extravasation dynamics of lymph node vs. brain metastatic melanoma cells and on the analysis of how vascular aging modulates MBM formation.

The lymphatic system has emerged as an active player in various disease conditions, such as chronic inflammation, lymphedema, and cancer, by sprouting into heavily branched lymphatic networks or dilating, which can serve as a marker of disease severity.
Using two-dimensional (2D) histochemistry, human dermal lymphatic vessels (LVs) have been shown to be strongly affected in pathology; however, only recently did our group demonstrate the heterogeneity of the lymphatic system in three-dimensional (3D) healthy human dermis, providing deeper insights into vessel architecture and remodelling. Although machine-learning tools for characterising 3D blood vascular systems have demonstrated great potential, automated quantitative analysis of human 3D LV networks remains limited due to the lack of segmentation tools capable of handling their complexity and heterogeneity.

We have developed a fully automated deep learning pipeline, called LymphNet, for 3D LV segmentation, trained on human dermal light-sheet microscopy datasets, based on an nnUNet architecture, using the open-source 3D Slicer software to provide a user-friendly graphical user interface (GUI) and enable easy morphometric analysis, including vessel diameter, branching points and sprouts, vessel volume, and length.

LymphNet demonstrates the ability to capture non-uniform vessel shapes and discontinuous junction patterns with high accuracy, providing unbiased quantification of the lymphatic network. Extensive data augmentation enables robust performance across donor samples and imaging variability from different microscopes, while its intuitive interface makes it accessible to non-technical users. Using LymphNet, we could quantify differences in morphological parameters across anatomical locations in human skin and between patients, demonstrating strong potential for future personalised diagnostic applications. In addition, LymphNet has strong precision with other imaging modalities, enabling quantification of human and mouse confocal data and clearly recognising differences between disease mouse models.

Our work thus enables high-throughput morphometric studies of 3D lymphatics and opens avenues for applying fully automated, user-friendly computational methods to a variety of lymphatic-related applications.