Esther Wolfs

Programmed death‐ligand 1 (PD‐L1) expression, commonly quantified as tumour proportion score (TPS), is a key biomarker guiding immunotherapy in non‐small cell lung cancer (NSCLC). Manual scoring of PD‐L1 immunohistochemistry (IHC) is time‐consuming and challenged by preanalytical variability and interpretive subjectivity, which can hinder consistent treatment selection. Artificial intelligence (AI) tools aim to support more standardized PD‐L1 scoring. We compare publicly available evidence on PD‐L1 AI tools and identify barriers to routine implementation. We mapped publicly available evidence for seven commercial and 13 non‐commercial or research‐stage PD‐L1 AI tools across six interconnected levels: data characteristics and validation setting, data preparation and reference standard, supervision strategy, performance evaluation and agreement, generalizability and clinical relevance, and deployment traceability. Commercial sources more often described workflow integration, intended use, reader assistance and certification or product status, whereas non‐commercial studies more often detailed model architecture, supervision strategy, code/data availability and experimental design. Heterogeneity in cohorts, assay–scanner settings, reference standards, agreement metrics and decision‐threshold reporting limited direct comparison. Key gaps included scarce prospective multi‐centre validation, limited reporting at decision‐relevant TPS thresholds, and incomplete separation between peer‐reviewed validation evidence, vendor claims and deployment traceability. The resulting evidence map supports conditional appraisal and local verification rather than a universal ranking. Publicly available evidence for PD‐L1 TPS AI tools remains heterogeneous, and reported performance is not automatically portable between laboratories because it depends on the combined case mix, assay–scanner workflow, reference standard and TPS‐threshold setting. Therefore, current evidence does not support a universal ranking for routine practice. The six‐level comparison structure and end‐user crosswalk support shortlisting intended‐use‐compatible tools and, where relevant, comparing them locally using the same representative cases, assay–scanner workflow, reference standard and prespecified TPS‐threshold endpoints. More transparent reporting, robust external and prospective multi‐centre validation, and ongoing quality monitoring are needed for safe integration into routine human–AI pathology workflows.

Primary Schwann cell cultures derived from adult murine peripheral nerves are frequently compromised by fibroblast overgrowth, limiting their utility for in vitro studies. Existing enrichment strategies often rely on selective growth conditions, prolonged culture, or antibody-mediated positive selection, which may alter Schwann cell physiology, trigger unwanted signaling, or result in incomplete fibroblast removal. To address these limitations, we developed a reproducible negative selection protocol for the isolation and enrichment of primary adult murine Schwann cells using fluorescence-activated cell sorting (FACS). By specifically targeting contaminating fibroblasts using the marker CD90.2 (Thy-1.2), these cells are efficiently depleted while leaving the Schwann cell population unbound by antibody complexes. This approach enables the rapid generation of highly enriched Schwann cell populations without the need for extended in vitro expansion or antimitotic agents. By avoiding direct receptor-ligand interactions on Schwann cells, this method preserves their native physiological state and surface epitopes, supporting sensitive downstream applications. Successful Schwann cell enrichment was confirmed by quantitative PCR, western blot and immunocytochemistry, demonstrating the enrichment of Schwann cell-specific markers and minimal fibroblast contamination. Overall, this approach enables the rapid generation of highly enriched Schwann cell populations while preserving features relevant to mature Schwann cell biology. This protocol provides a robust and practical tool for researchers requiring physiologically representative adult Schwann cells for molecular, functional, and co-culture-based studies of the peripheral nervous system.

Vascularization remains a major challenge in tissue engineering, restricting both the functional integration of grafts as well as the physiological relevance of in vitro models. Inspired by the crucial role of Schwann cells (SCs) in guiding vascular development, we investigated their influence on the vascular network formation of human microvascular endothelial cells (HMVECs). Using melt electrowriting (MEW), we fabricated scaffolds consisting of a single layer of parallel fibers to mimic the oriented axons of the peripheral nerve. A suspended seeding approach was carried out to ensure rat-derived SCs adhered specifically to the fibers, creating parallel arrays with SCs exhibiting strong attachment, viability, and upregulation of myelination-, neurotrophic-, and pro-angiogenic-related genes. A customized system was built to co-culture SCs-laden scaffolds with HMVECs seeded on a hydrogel (2D) and in a hydrogel (3D). The parallel fiber distances were varied to assess the spacing of the SC arrays that impacted HMVEC organization. The results revealed that SCs on MEW scaffolds exhibited enhanced expression of key genes compared to a 2D monolayer control. Further, these SC-laden scaffolds significantly enhanced HMVEC network formation in both 2D and 3D environments, with reduced fiber distance showing stronger pro-angiogenic responses. To evaluate species differences, human dental pulp stem cell-derived SCs (HDPSC-SCs) were compared with rat primary SCs. HDPSC-SCs not only showed enhanced expression of pro-angiogenic genes on the scaffold but also promoted superior network formation compared to rat SCs. Collectively, our findings highlight the ability of MEW scaffolds to both pattern SC growth and stimulate a pro-regenerative SC phenotype as a strategy to modulate vascular network formation. This provides a foundation for using the patterning of SCs to drive neurovascular organization for in vitro models and more broadly as an approach of regenerative medicine.

Background/Objectives: Oral squamous cell carcinoma (OSCC) is the most common oral cancer, progressing from hyperplasia to dysplasia, carcinoma in situ (CIS), and finally invasive squamous cell carcinoma (ISCC). Developing an animal model that mimics both early and advanced OSCC stages has been challenging. The 4-Nitroquinoline 1-oxide (4NQO) model is considered one of the most suitable, as it represents all stages of OSCC. Nevertheless, thoroughly understanding the properties of the 4NQO model is essential for preclinical testing of novel therapeutics. Methods: We aimed to characterize the 4NQO rat model using two application methods—drinking water and topical application—over eight months. Monthly sacrifices allowed histopathological analysis and ex vivo magnetic resonance imaging (MRI) to track tumor progression. Results: CIS was observed at three months in the drinking water group, evolving into ISCC by six months, while topical application induced CIS at eight months without ISCC formation. The tongue was divided into three regions and histological properties, lesion size, and invasion depth were analyzed. In the drinking water group, particularly in the body of the tongue, we saw earlier CIS development, larger lesions, and deeper invasion. Additionally, assessment of proliferative properties showed an increased cell division in dysplastic lesions that reduced upon invasion. MRI was able to show macroscopic tumoral lesions, in concordance with histology. Conclusions: Overall, the drinking water method closely mimics human OSCC, validating the 4NQO model for translational OSCC research.

Cancer remains a leading cause of mortality, emphasizing the need for innovative therapies. Plasma-treated liquids, containing reactive oxygen and nitrogen species, have demonstrated therapeutic potential. This study investigates the physicochemical properties and anti-cancer efficacy of phosphate-buffered saline (PBS) treated using a novel liquid-submerged plasma jet, which enhances interactions between plasma species and the liquid for a more uniform treatment. Operational parameters including voltage, gas flow, and treatment time, were optimized concurrently. Notably, the submerged configuration produced significantly higher H2O2 concentrations in PBS (up to 2000 μM) compared to the above-liquid plasma set-ups reported in literature. However, NO2 concentrations remained low (6-18 μM). Voltage variations influenced H.O. production but had a minimal effect on NO2, while gas flow rates did not impact their concentrations. PBS maintained a stable pH, demonstrating its effective buffering capacity. Stability tests showed H.O. remained stable at 21∘C, slightly increased at 4∘C, and decreased at 37∘C; nitrites were stable below 21∘C but slightly decreased at 37∘C. Plasma-treated PBS selectively reduced oral squamous cell carcinoma (OSCC) cell viability while sparing healthy keratinocytes (HaCaT), with H.O. identified as the primary anti-cancer agent. These findings suggest that PBS plasma-treated using a new liquid-submerged set-up shows potential as selective OSCC therapy.

Digital pathology has become increasingly popular for research and clinical applications. Using high-quality microscopes to produce Whole Slide Images of tumor tissue enables the discovery of insights into biological aspects invisible to the human eye. These are acquired through downstream analyses using spatial statistics and artificial intelligence. Determination of the quality and consistency of these images is needed to ensure accurate outcomes when identifying clinical and subclinical image features. Additionally, the time-intensive process of generating high-volume images results in a trade-off that needs to be carefully balanced. This study aims to determine optimal instrument settings to generate representative images of pathological tissue using digital microscopy. Using various settings, an H&E stained sample was scanned using the ZEISS Axio Scan.Z1. Next, nucleus segmentation was performed on resulting images using StarDist. Subsequently, detections were compared between scans using a matching algorithm. Finally, nucleus-level information was compared between scans. Results indicated that while general matching percentages were high, similarity between information from replicates was relatively low. Additionally, settings resulting in longer scanning times and increased data volume did not increase similarity between replicates. In conclusion, the scan setting ultimately deemed optimal combined consistent and qualitative performance with low throughput time.

Charcot–Marie–Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy caused by a 1.5 Mb tandem duplication of chromosome 17 harbouring the PMP22 gene. This dose-dependent overexpression of PMP22 results in disrupted Schwann cell myelination of peripheral nerves. To obtain better insights into the underlying pathogenic mechanisms in CMT1A, we investigated the role of PMP22 duplication in cellular homeostasis in CMT1A mouse models and in patient-derived induced pluripotent stem cells differentiated into Schwann cell precursors (iPSC-SCPs). We performed lipidomic profiling and bulk RNA sequencing (RNA-seq) on sciatic nerves of two developing CMT1A mouse models and on CMT1A patient-derived iPSC-SCPs. For the sciatic nerves of the CMT1A mice, cholesterol and lipid metabolism was downregulated in a dose-dependent manner throughout development. For the CMT1A iPSC-SCPs, transcriptional analysis unveiled a strong suppression of genes related to autophagy and lipid metabolism. Gene ontology enrichment analysis identified disturbances in pathways related to plasma membrane components and cell receptor signalling. Lipidomic analysis confirmed the severe dysregulation in plasma membrane lipids, particularly sphingolipids, in CMT1A iPSC-SCPs. Furthermore, we identified reduced lipid raft dynamics, disturbed plasma membrane fluidity and impaired cholesterol incorporation and storage, all of which could result from altered lipid storage homeostasis in the patient-derived CMT1A iPSC-SCPs. Importantly, this phenotype could be rescued by stimulating autophagy and lipolysis. We conclude that PMP22 duplication disturbs intracellular lipid storage and leads to a more disordered plasma membrane owing to an alteration in the lipid composition, which might ultimately lead to impaired axo-glial interactions. Moreover, targeting lipid handling and metabolism could hold promise for the treatment of patients with CMT1A.

Wolfram syndrome (WS) is a rare childhood disease characterized by diabetes mellitus, diabetes insipidus, blindness, deafness, neurodegeneration and eventually early death, due to autosomal recessive mutations in the WFS1 (and WFS2 ) gene. While it is categorized as a neurodegenerative disease, it is increasingly becoming clear that other cell types besides neurons may be affected and contribute to the pathogenesis. MRI studies in patients and phenotyping studies in WS rodent models indicate white matter/myelin loss, implicating a role for oligodendroglia in WS-associated neurodegeneration. In this study, we sought to determine if oligodendroglia are affected in WS and whether their dysfunction may be the primary cause of the observed optic neuropathy and brain neurodegeneration. We demonstrate that 7.5-month-old Wfs1 ^Δexon8 mice display signs of abnormal myelination and a reduced number of oligodendrocyte precursor cells (OPCs) as well as abnormal axonal conduction in the optic nerve. An MRI study of the brain furthermore revealed grey and white matter loss in the cerebellum, brainstem, and superior colliculus, as is seen in WS patients. To further dissect the role of oligodendroglia in WS, we performed a transcriptomics study of WS patient iPSC-derived OPCs and pre-myelinating oligodendrocytes. Transcriptional changes compared to isogenic control cells were found for genes with a role in ER function. However, a deep phenotyping study of these WS patient iPSC-derived oligodendroglia unveiled normal differentiation, mitochondria-associated endoplasmic reticulum (ER) membrane interactions and mitochondrial function, and no overt signs of ER stress. Overall, the current study indicates that oligodendroglia functions are largely preserved in the WS mouse and patient iPSC-derived models used in this study. These findings do not support a major defect in oligodendroglia function as the primary cause of WS, and warrant further investigation of neurons and neuron-oligodendroglia interactions as a target for future neuroprotective or -restorative treatments for WS.

Background: Dysregulation of the endo-lysosomal–autophagy pathway has been identified as a critical factor in the pathology of various demyelinating neurodegenerative diseases, including peripheral neuropathies. This pathway plays a crucial role in transporting newly synthesized myelin proteins to the plasma membrane in myelinating Schwann cells, making these cells susceptible to lysosome-related dysfunctions. Nevertheless, the specific impact of lysosomal dysfunction in Schwann cells and its contribution to neurodegeneration remain poorly understood. Methods: We aim to mimic lysosomal dysfunction in Schwann cells using chloroquine, a lysosomal dysfunction inducer, and to monitor lysosomal leakiness, Schwann cell viability, and apoptosis over time. Additionally, due to the ethical and experimental issues associated with cell isolation and the culturing of human Schwann cells, we use human dental pulp stem cell-derived Schwann cells (DPSC-SCs) as a model in our study. Results: Chloroquine incubation boosts lysosomal presence as demonstrated by an increased Lysotracker signal. Further in-depth lysosomal analysis demonstrated an increased lysosomal size and permeability as illustrated by a TEM analysis and GAL3-LAMP1 staining. Moreover, an Alamar blue assay and Caspase-3 staining demonstrates a reduced viability and increased apoptosis, respectively. Conclusions: Our data indicate that prolonged lysosomal dysfunction leads to lysosomal permeability, reduced viability, and eventually apoptosis in human DPSC-SCs.