Biology subjects

Conrad, T.

Publications and source records attributed to Conrad, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Spatiotemporal Mapping of Glomerular Endothelial Regeneration Identifies Apold1 as an Intrinsic Regulator of Repair

Abstract Endothelial cell (EC) injury is central to the pathogenesis of various renal disorders, highlighting the critical importance of vascular repair for restoring organ health. However, due to limited access to human tissue during active regeneration, the exact reparative processes remain largely unknown. This study aimed to investigate regeneration mechanisms specifically within the glomerular microvasculature by utilizing a selective endothelial cell injury (ECI) model in mice.Lineage tracing identified the origin of regenerating ECs. To uncover the transcriptional dynamics driving these reparative changes, we combined single-cell RNA sequencing and Xenium spatial transcriptomics (ST), mapping distinct glomerular EC (gEC) subpopulations across different phases of injury and recovery. Apold1 emerged as a key regulatory candidate within this process. To functionally validate this gene's specific role during the regenerative phase, Apold1-deficient (Apold1-/-) mice underwent the ECI model. After showing that gECs regenerate primarily from local, pre-existing renal ECs, our transcriptomic analysis identified 10 distinct gEC subpopulations and revealed a complex temporal pattern of regeneration. This process begins with the expression of genes associated with inflammation, followed sequentially by endothelial cell (EC) migration, the activation of immediate early genes, vascular remodeling and cellular proliferation. Within this dynamic process, the EC-specific gene Apold1 was identified as a key regulatory candidate. Apold1-/- mice exhibited impaired endothelial regeneration, characterized by increased endothelial activation and greater immune cell infiltration seven days post-ECI. To our knowledge, this is the first study to elucidate the spatiotemporal mechanisms of glomerular endothelial cell regeneration. By identifying local gECs as the primary source of repair, and stablishing the change in their expression pattern during regeneration, as well as identifying Apold1 as an intrinsic regulator, we have laid the groundwork for future research on renal microvascular disorders.

molecular biology↗

bioRxiv2026

Tumor-versus-non-tumor T cell enrichment supported by single-cell and spatial phenotyping enables antigen-agnostic prioritization of candidate tumor-reactive TCRs

Introduction: Tumor-reactive T cell receptors (TCRs) are promising candidates for the manufacturing of safe and effective adoptive TCR-T cell therapies, but their efficient identification remains a major challenge. We previously established an antigen-agnostic TCR-repertoire analysis workflow that prioritizes candidate TCRs based on intratumoral clonal expansion and high tumor-to-non-tumor (T/N) frequency ratios. Here, we investigated whether T/N-selected clonotypes show independent transcriptional and spatial features consistent with tumor association. Methods: Bulk and single-cell TCR repertoire sequencing of paired tumor and adjacent non-tumor tissues was used to identify clonotypes with high T/N ratios across 15 cancer cases, comprising six non-small cell lung cancers, five pancreatic cancers, two breast cancers, and two colorectal cancers. Candidate clonotypes were further characterized by paired single-cell TCR and gene expression profiling and, in two PDAC cases, by Xenium spatial transcriptomics to assess their cellular phenotype, spatial localization, and proximity to malignant cells. Results: High T/N ratios consistently marked T cell clonotypes with features of tumor reactivity across the studied solid tumor entities. The analysis included Cluster-A, a lead T cell cluster comprising highly similar TCRs shared among 14 HLA-A*02:01-positive patients in the present cohort. Cluster-A clonotypes were consistently enriched in tumor relative to matched non-tumor tissue, and previous functional studies demonstrated tumor recognition by Cluster-A members. In a focused analysis of a pancreatic cancer case, Cluster-A CD8 T cells were selectively detected in tumor tissue, preferentially localized near malignant epithelial cells, and displayed an antigen-experienced effector-memory phenotype with prominent expression of granzymes A and K (GZMA/GZMK). Importantly, in another PDAC case that lacked Cluster-A clonotypes, these features extended to independent clonotypes, revealing a continuous tumor-association axis in which increasing T/N ratios were associated with progressively closer localization to malignant cells and increased GZMA/GZMK expression. Thus, repertoire enrichment, spatial tumor proximity, and effector-associated transcriptional states converged across independent tumor-enriched clonotypes. Outlook: These findings support the T/N ratio as a robust primary criterion for identifying candidate tumor-reactive TCRs. Integration of single-cell and spatial transcriptomics provides supporting biological evidence of tumor association and a framework for prioritizing therapeutic TCR candidates when functional validation is limited.

immunology↗

bioRxiv2026

Differential multi-omics analysis of pulmonary arterial hypertension microvascular endothelial cells for differential drug response

Pulmonary arterial hypertension (PAH) represents a heterogeneous group of disorders that involves complex molecular dysregulations, which are not fully captured by single-omics analyses. We apply our network-based multi-omics analysis framework, DrDimont, to transcriptomic, proteomic, phosphoproteomic, and kinase screening data from lung microvascular endothelial cells of PAH patients and controls. Thereby, we extend the functionality of DrDimont to incorporate kinase-kinase interactions during the construction of condition-specific multi-omics networks. Kinase interactions are inferred from phosphorylations of screened substrates that are weighted by kinase-substrate predictions. Differential interaction scores from the network-based analysis between PAH and control uncover alterations centered on kinases, in particular top hits relating to MAPK signaling, such as MAPK13, MAP2K, or upstream IRAK1, and other MAPK/MAP2K family members. Further highly differential nodes were ACADSB, GPX7, DSE (for proteins), and AIM1, LY96, CHSY3 (for mRNAs). When prioritizing drug candidates by mapping drug targets onto the differential network, we find high scores for the drug tacrolimus (FK506) and several anti-neoplastic MAPK inhibitors (e.g., selumetinib, trametinib), as well as agents acting on general proliferation via (mitochondrial) DNA transcription (e.g., epirubicin, topotecan). Integrating kinase activity screens into our explainable multi-omics network-based analyses reveals kinase-centered alterations and therapeutic hypotheses in PAH that complement single-layer classical differential expression analyses.

bioinformatics↗

bioRxiv2026

Distinct macrophage and T cell programs shape pancreatic inflammation during metabolic stress and aging

Type 2 diabetes is linked to systemic inflammation driven by metabolic stress and aging. Although pancreatic inflammation associated with these factors is well documented, the dynamics of immune cell populations and their molecular changes remain poorly understood. We characterized immune cell alterations in the pancreas and pancreatic islets during Western diet (WD) feeding and aging using imaging mass cytometry (IMC) and single-cell RNA sequencing (scRNA-seq). Spatial and transcriptional analyses were performed to define immune cell subtype composition, activation states, and inferred cell-cell communication programs under metabolic and age-related stress conditions. Our analyses identified expansion of an F4/80low macrophage subtype and activated effector-like CD8+ T cells throughout the pancreas during WD feeding and aging. Within pancreatic islets, single-cell RNA sequencing identified a type I interferon-responsive macrophage population with low F4/80 expression that expanded during overnutrition. Notably, the type I interferon responses elicited by these stressors diverged: aging was associated with a more canonical type I interferon response, whereas overnutrition induced a broader response that included STAT3-associated transcriptional programs. We further provide evidence for enhanced cytokine-mediated communication between macrophages and a CD8+ cytotoxic T-cell population under overnutrition and aging. These findings show that metabolic stress and aging remodel pancreatic inflammation through overlapping but distinct immune mechanisms, involving expansion of F4/80low macrophages, activation of divergent type I interferon programs, and enhanced macrophage-CD8+ T-cell communication. Together, these findings suggest that distinct therapeutic approaches may be required to preserve islet function in type 2 diabetes driven by metabolic stress versus aging. Article HighlightsO_LIMetabolic stress and aging remodel pancreatic inflammation through overlapping but distinct immune mechanisms. C_LIO_LISpatial and single-cell analyses identified conserved remodeling of pancreatic macrophage populations accompanied by activation of cytotoxic T-cell responses during metabolic stress and aging. C_LIO_LIIn pancreatic islets, macrophages exhibited distinct type I interferon-associated transcriptional programs, with aging showing a more canonical interferon response and metabolic stress eliciting a broader inflammatory program that included STAT3-associated transcriptional signatures. C_LIO_LIThese findings provide a framework for understanding how metabolic stress and aging differentially shape pancreatic inflammation. C_LI

cell biology↗

bioRxiv2026

The role of stridulations during the mating of Nicrophorus vespilloides

In many insect species, mating is coordinated through multimodal signaling, yet less obvious channels are often overlooked. In the burying beetle Nicrophorus vespilloides, chemical communication is well-documented, but the role of substrate-borne vibrational signals (stridulations) during courtship remains unknown. We investigated whether stridulation is essential for mating success through two sets of experiments. First, we found a positive correlation between the frequency of stridulations and both the number and duration of copulation events. Second, we employed a silencing experiment to test the necessity of these signals by silencing males, females, or both partners. We found no significant differences between silenced and control groups regarding the frequency or duration of physical contact and mounting events, suggesting that stridulation is not required for mate recognition or the initiation of courtship. However, the proportion of successful copulations relative to mounting events was significantly lower when females were silenced. These results suggest that while N. vespilloides relies on a redundant multimodal system that likely utilizes chemical cues to initiate mating, vibrational signals, particularly from the female, may play a critical role in facilitating successful copulation. This study provides the first evidence for the role of stridulation in the mating behavior of N. vespilloides and highlights the potential for female-mediated vibrational signaling in burying beetle courtship.

animal behavior and cognition↗

bioRxiv2026

Epithelial mesenchymal transition initiates precancer states in BRCA1 mutation carriers

Epithelial-to-mesenchymal transition (EMT) is activated to equip cells with the capacity to adapt to and escape hostile conditions. While EMT is required for cancer progression, its role in breast cancer initiation remains elusive. Given the basal-like phenotype of breast cancers arising in female carriers of germline BRCA1 pathogenic variants (BRCA1 carriers), we hypothesized that enhanced EMT susceptibility underlies precancerous initiation in mammary epithelium. Perturbation of patient-derived normal mammary organoids from BRCA1 carriers and non-carriers with inflammatory cytokines induced copy number variations (CNV) and the acquisition of oncogenic mutations in both groups. However, in organoids derived from BRCA1 carriers, cytokine exposure induced morphological, transcriptomic, and functional EMT, accompanied by a transition to basal-like phenotype. Concomitant DNA damage accumulation in organoids from BRCA1-carriers demonstrated PARP inhibitor sensitivity. EMT-primed states were identified in a subpopulation of normal mammary epithelium from BRCA1 carriers. We demonstrate the utility of patient-derived normal BRCA1 heterozygous mammary organoids to reveal a plastic, high-risk epithelial state that is associated with a transient, targetable vulnerability.

cancer biology↗

bioRxiv2025

Cross-fostering reveals that acoustic communication during parental care alleviates prenatal disadvantages in beetle offspring

Acoustic communication plays an important role in social interactions for many animals. It is used in a broad range of behaviours, such as parental care. Acoustic communication is also involved in the brood care of Nicrophorus vespilloides (HERBST 1783), which, together with the other burying beetles, are one of the rare exceptions among insects in that they practice biparental brood care. Previous studies showed that a lack of acoustic communication in burying beetles negatively affects the weight and survival rate of their offspring (Conrad et al. 2024). However, it remained unclear whether these results were mainly based on prenatal or postnatal effects of silencing. Therefore, in this study we wanted to find out whether negative effects on N. vespilloides larvae, which are due to a lack of prenatal acoustic communication between the parents, can be compensated by the postnatal brood care of stridulating foster parents. For this purpose, we applied parafilm on the pars stridens of some beetle pairs so that they could no longer stridulate. In a 2x2 cross-fostering experiment, larvae originating from silenced or stridulating parents were reared by either silenced or stridulating foster parents. The larvae of silenced parents showed a lower hatching weight than the larvae of control parents. At dispersal, larvae with stridulating original and foster parents were significantly heavier than those reared by silenced foster parents, from either origin. At dispersal, larvae with stridulating original and foster parents were significantly heavier than those reared by silenced foster parents, from either origin. Thus, the stridulating foster parents were apparently able to partially compensate the initial weight deficits of the larvae from silenced original parents. There were no effects of silencing on offspring survival. Our experiment demonstrates that post-hatching acoustic communication during brood care of N. vespilloides is of greater importance for larval growth than pre-hatching acoustic communication. Still, the inability to stridulate compromises the quality of both prenatal and postnatal care. It is conceivable that the acoustic communication of the parents provides orientation for the larvae when feeding, or that the adults coordinate their tasks and negotiate their parental investment. Our findings contribute further to our understanding of the function of parental communication during brood care.

animal behavior and cognition↗

bioRxiv2025

circVDJ-seq for T cell clonotype detection in single-cell and spatial multi-omics

Monitoring T cell repertoires in human tissues provides important insights into immune response mechanisms in cancer, infectious diseases, and autoimmunity. However, retrieving VDJ information from single-cell and spatial transcriptomics workflows with 3-barcoding of cDNA remains resource-intensive or requires specialized sequencing equipment. Here, we introduce circVDJ-seq for simplified and cost-efficient T cell receptor (TCR) profiling from 3-directed workflows like single-cell or single-nucleus RNA sequencing, ATAC+RNA multi-omics, and spatial transcriptomics. Application of circVDJ-seq to freshly resected neuroblastomas and postmortem lymph nodes affected by pneumonia or COVID-19 reveals distinct immune microenvironments and T cell clonality patterns, highlighting broad utility across diverse clinical contexts.

genomics↗

bioRxiv2025

Aberrant brain-type neuronal programs in large-cell pancreatic neuroendocrine carcinoma

High-grade pancreatic neuroendocrine carcinoma (panNEC) is a rare, aggressive cancer with limited tissue availability. Single-nucleus transcriptomics of five large-cell panNECs revealed two clinically relevant neuroendocrine cell states: one highly proliferative with an aberrant brain-specific neuronal differentiation program and another stress-responsive state enriched for heat stress, hypoxia, and glycolysis. Our findings suggest potentially new therapeutic vulnerabilities, highlighting the need to evaluate the efficacy of combination therapies.

cancer biology↗

bioRxiv2025

Epiproteomic control of the epitranscriptome drives cortical neurogenesis

Neurological conditions are the leading cause of ill health worldwide. Here, we show that the neurodevelopmental disorder-associated ubiquitin ligase UBE3C regulates the cellular composition of the murine cerebral cortex and human brain organoids, with its loss favoring neurogenesis and suppressing glial fate. Using genetic complementation, we demonstrate that disease-associated UBE3C mutations alter its autoubiquitination activity and disrupt cortical lamination. Proteomic profiling of UBE3C-deficient forebrains and organoids identifies Cbll1 as a UBE3C substrate, and we show that the UBE3C-Cbll1 duo drives N6-methyladenosine (m6A) mRNA methylation. Hyperactivation of m6A writers in UBE3C-deficient neural progenitors impairs cell cycle exit, a defect reversible in vivo by the METTL3 inhibitor STM2457. Our findings uncover an epiproteomic mechanism controlling m6A-mediated gene expression and define a regulatory axis linking ubiquitin signaling to epitranscriptomic control of neural fate. This work provides a mechanistic framework for understanding neurodevelopmental disorders and highlights potential therapeutic strategies.

cell biology↗

bioRxiv2025

Complex Human Hair Bearing Skin Organoids as Model for Herpes Simplex Virus 1 Infection in the Skin

Epithelial surfaces are the initial site of Herpes Simplex Virus 1 (HSV-1) infection. After latency establishment in the peripheral nervous system, reactivation can lead to skin pathologies. To reflect the complexity of skin we used complex human hair bearing skin organoids as a new model system for HSV-1 infection. We analyzed spread of the infection through the three-dimensional tissue and the cell type specific host response in a spatial temporal manner. Single-cell-resolution spatial transcriptomics and imaging shows a restricted viral infection in keratinocytes of the epidermis and hair follicles. The host factor IER3 is upregulated in papillary fibroblasts of the dermis as the infection progresses through the tissue. In keratinocytes of the stratum basale, we find IRF3 upregulated in infected but also virus negative cells, suggesting a signaling mechanism to initiate antiviral responses. Furthermore, we find a cell type specific inflammatory response with an upregulation of TNFSF9 in dermal fibroblasts and TNF in keratinocytes. TNF target genes are induced in neighboring uninfected cells suggesting the induction of a localized inflammatory reaction to suppress viral infection. In summary, our findings position human hair-bearing skin organoids as a highly physiological model system for studying HSV-1 pathogenesis and reveal spatially organized host responses.

microbiology↗

bioRxiv2023

Osteopontin in colitis-associated carcinoma

Patients with ulcerative colitis (UC) and Crohns disease (CD) face a lifelong risk of developing colitis-associated carcinoma (CAC). Current insights into CAC development originate from murine CAC models, while human CAC studies primarily focus on mutational analysis of patient samples. Although the mutational landscape reveals distinct patterns and frequencies compared to colorectal cancer, it falls short in elucidating the inflammatory mechanisms of CAC development. Consequently, we adopted a multi-omics approach to unravel CAC carcinogenesis from an immunological perspective. Our data revealed a robust upregulation of SPP1 gene in CAC at both RNA and protein levels, expressed by CD68+ macrophages. In vitro OPN stimulation demonstrated no direct effect on intestinal epithelial organoids. However, the mutually exclusive spatial location of SPP1/OPN+ macrophages and CD8+ T cells suggests a crucial indirect role of SPP1/OPN in mediating an immunosuppressive tumour microenvironment.

cancer biology↗

bioRxiv2023

VoltRon: A Spatial Omics Analysis Platform for Multi-Resolution and Multi-omics Integration using Image Registration

The growing number of spatial omic technologies have created a demand for computational tools capable of managing, storing, and analyzing spatial datasets with multiple modalities and spatial resolutions. Meanwhile, computer vision is becoming an integral part of processing spatial data readouts where image registration and spatial data alignment of tissue sections are essential prior to data integration. Hence, there is a need for computational platforms that analyze data across spatial datasets with diverse resolutions as well as those that manipulate and process images of microanatomical tissue structures. To this end, we have developed VoltRon, a novel R package for spatial omics analysis with a unique data structure that accommodates data readouts with many levels of spatial resolutions (i.e., multi-resolution) including regions of interest (ROIs), spots, single cells, and even subcellular entities such as molecules. To connect and integrate these spatially diverse omic profiles, VoltRon accounts for spatial organization of tissue blocks (samples), layers (sections) and assays given a multi-resolution collection of spatial data readouts. An easy-to-use computer vision toolbox, OpenCV, is fully embedded in VoltRon that allows users to both automatically and manually register spatial coordinates across adjacent layers for data transfer without the need for external software tools. VoltRon is implemented in the R programming language and is freely available at https://github.com/BIMSBbioinfo/VoltRon.

bioinformatics↗

bioRxiv2023

Multimodal profiling of peripheral blood identifies proliferating circulating effector CD4+ T cells as predictors for response to integrin α4β7-blocking therapy in patients with inflammatory bowel disease

Despite the success of biological therapies in inflammatory bowel disease (IBD), patient management remains challenging due to a lack of therapy response predictors. Here we prospectively sampled two cohorts of IBD patient cohorts receiving the anti-integrin 4{beta}7 antibody vedolizumab. Samples were subjected to mass cytometry, single-cell RNA sequencing, single-cell V(D)J sequencing, serum proteomics, and multidimensional flow cytometry to comprehensively assess vedolizumab-induced immunological changes in the peripheral blood and their potential associations with treatment response. Vedolizumab induced changes in the abundance of both circulating innate and adaptive immune cell compartments and modified the T cell receptor diversity of circulating gut-homing CD4+ memory T cells. Through integration of multimodal parameters and machine learning, we identify that pretreatment activated proliferating CD4+ memory T cell abundance is associated with treatment failure, independent of clinical variables, thereby providing a reliable predictive classifier with significant implications for the personalized management of IBD patients.

immunology↗

bioRxiv2023

Sorting receptor SORCS2 facilitates a protective stress response in pancreatic islets

ObjectiveSORCS2 is an intracellular sorting receptor genetically associated with body mass index (BMI) in humans, yet its mode of action remains unknown. Elucidating the receptor function that defines its role in metabolic health is the objective of this work. MethodsCombining in vivo metabolic studies in SORCS2-deficient mouse models with ex vivo structural and functional analyses as well as single-cell transcriptomics of murine pancreatic tissues, we studied the pathophysiological consequences of receptor dysfunction for metabolism. ResultsOur studies identified an important role for SORCS2 in islet stress response essential to sustain glucose-stimulated insulin release. In detail, we show that SORCS2 is predominantly expressed in islet alpha cells. Loss of receptor expression coincides with the inability of these cells to produce osteopontin, a secreted factor that facilitates insulin release from beta cells under stress. In line with diminished osteopontin levels, beta cells in SORCS2- deficient islets show changes in gene expression patterns related to aggravated ER stress, protein misfolding, as well as mitochondrial dysfunction; and they exhibit defects in insulin granule maturation and a blunted response to glucose stimulation in vivo and ex vivo. Impaired glucose tolerance in receptor mutant mice coincides with alterations in body weight and composition. ConclusionOur data identified a novel concept in protective islet stress response involving the alpha cell receptor SORCS2 and provide experimental support for association of SORCS2 with metabolic control in humans.

molecular biology↗

bioRxiv2023

In situ Patch-seq analysis of microglia reveals a lack of stress genes as found in FACS-isolated microglia

We applied the patch-seq technique to harvest transcripts from individual microglial cells from cortex, hippocampus and corpus callosum of acute brain slices from adult mice. After recording membrane currents with the patch-clamp technique, the cytoplasm was collected via the pipette and underwent adapted SMART-seq2 preparation with subsequent sequencing. On average, 4138 genes were detected in 113 cells from hippocampus, corpus callosum and cortex, including microglia markers such as Tmem119, P2ry12 and Siglec-H. Comparing our dataset to previously published single cell mRNA sequencing data from FACS-isolated microglia indicated that two clusters of cells were absent in our patch-seq dataset. Pathway analysis of marker genes in FACS-specific clusters revealed association with microglial activation and stress response. This indicates that under normal conditions microglia in situ lack transcripts associated with a stress-response, and that the microglia-isolation procedure by mechanical dissociation and FACS triggers the expression of genes related to activation and stress.

immunology↗

bioRxiv2023

Intercellular extrachromosomal DNA copy number heterogeneity drives cancer cell state diversity

Neuroblastoma is characterised by extensive inter- and intra-tumour genetic heterogeneity and varying clinical outcomes. One possible driver for this heterogeneity are extrachromosomal DNAs (ecDNA), which segregate independently to the daughter cells during cell division and can lead to rapid amplification of oncogenes. While ecDNA-mediated oncogene amplification has been shown to be associated with poor prognosis in many cancer entities, the effects of ecDNA copy number heterogeneity on intermediate phenotypes are still poorly understood. Here, we leverage DNA and RNA sequencing data from the same single cells in cell lines and neuroblastoma patients to investigate these effects. We utilise ecDNA amplicon structures to determine precise ecDNA copy numbers and reveal extensive intercellular ecDNA copy number heterogeneity. We further provide direct evidence for the effects of this heterogeneity on gene expression of cargo genes, including MYCN and its downstream targets, and the overall transcriptional state of neuroblastoma cells. These results highlight the potential for rapid adaptability of cellular states within a tumour cell population mediated by ecDNA copy number, emphasising the need for ecDNA-specific treatment strategies to tackle tumour formation and adaptation.

cancer biology↗

bioRxiv2022

Tongue immune compartment analysis reveals spatial macrophage heterogeneity

The tongue is a unique muscular organ situated in the oral cavity where it is involved in taste sensation, mastication and articulation. As a barrier organ, which is constantly exposed to environmental pathogens, the tongue is expected to host an immune cell network ensuring local immune defence. However, the composition and the transcriptional landscape of the tongue immune system are currently not completely defined. Here we characterised the tissue-resident immune compartment of the murine tongue during development, health and disease, combining single cell RNA-sequencing with in situ immunophenotyping. We identified distinct local immune cell populations and described two specific subsets of tongue-resident macrophages occupying discrete anatomical niches. Cx3cr1+ macrophages were located specifically in the highly innervated lamina propria beneath the tongue epidermis and at times in close proximity to fungiform papillae. Folr2+ macrophages were detected in deeper muscular tissue. The two macrophage subsets originate from a common proliferative precursor during early postnatal development and responded differently to systemic LPS in vivo. Our description of the under-investigated tongue immune system sets a starting point to facilitate research on tongue immune-physiology and pathology including cancer and taste disorders.

immunology↗