Biology subjects

Halbritter, F.

Publications and source records attributed to Halbritter, F..

16 recordsLinked to original sources

bioRxiv2026

A Conserved Chromatin-Driven Checkpoint Defines Late Macrophage Maturation Independent of Tissue Specialization

Macrophage identity is widely viewed as a product of ontogeny and tissue-specific imprinting. Here, we identify a conserved, chromatin-driven maturation checkpoint that operates independently of initial lineage commitment and broadly across tissue contexts. Using long-term bone marrow-derived macrophage cultures, we uncover a late-stage transition characterized by coordinated transcriptional and epigenomic remodeling. Integration with in vivo developmental, tissue-resident, and monocyte-repopulation datasets demonstrates that this program is conserved across ontogenies, tissues, species, and experimental systems, revealing a previously unrecognized stage of macrophage maturation. Functionally, late maturation preserves core macrophage activities while promoting lysosomal expansion and fundamentally rewiring innate immune responsiveness. Mature macrophages display enhanced stimulus-specific responses to interferons and microbial danger signals, coupled to increased metabolic and inflammatory competence while restricting interferon-induced transcriptional memory. Together, our findings identify late macrophage maturation as a conserved regulatory checkpoint that reprograms the logic of innate immune responsiveness through chromatin remodeling shaping innate immune function.

immunology↗

bioRxiv2026

A human lung organoid co-culture model of early bone sarcoma metastasis reveals contact-dependent epithelial remodeling at the metastatic interface

Lung metastasis drives mortality across cancer types, yet how infiltrating tumor cells remodel the lung epithelium to establish metastatic niches remains poorly understood. Here we establish MESCUL (MEtastatic Sarcoma Co-CULture), a co-culture platform combining human lung organoids with patient-derived bone sarcoma cells to model early tumor-lung epithelial interactions in a physiologically relevant 3D system. MESCUL reveals that direct tumor-epithelial contact induces rapid, reproducible lung epithelial remodeling across Ewing sarcoma (ES) and osteosarcoma (OS) models and multiple organoid donor backgrounds, which is contact-dependent and not recapitulated by paracrine signaling. Single-cell RNA sequencing identifies LIMES (Lung Interface Metastasis Signature), a shared transcriptional program encompassing focal adhesion assembly, matrix metalloprotease (MMP) upregulation, and emergence of a damage-associated transitional cell state, in both ES and OS. Mechanistically, tumor-derived fibronectin (FN1) engages epithelial integrin receptors to activate focal adhesion kinase (FAK), driving amphiregulin (AREG) induction and MMP-mediated remodeling; FN1 alone phenocopies this response, and FAK inhibition attenuates it, nominating the FN1-integrin-FAK-AREG axis as a candidate therapeutic vulnerability. The LIMES program, identified through the MESCUL co-culture model, is spatially confined to the tumor-lung interface in patient metastases of both ES and OS, as demonstrated by spatial transcriptomics across nine patients. Massons trichrome staining of matched patient sections reveals pronounced collagen deposition in the peri-tumoral lung parenchyma, consistent with LIMES acting upstream of a wound-healing cascade that proceeds to structural fibrotic remodeling in patient tissue. Together, these findings establish the lung epithelium as an active participant in metastatic colonization, characterize a pharmacologically targetable, spatially restricted epithelial remodeling response at the bone sarcoma-lung interface across OS and ES, and introduce MESCUL as a tractable 3D platform for investigating lung metastasis, with possible implications for tumor types beyond bone sarcomas.

cancer biology↗

bioRxiv2025

Mevalonate pathway activation in Ewing sarcoma reveals a 3D-specific synergy between statins and BCL-xL inhibition

Bone sarcomas are rare and aggressive pediatric cancers with limited progress in targeted therapy development, in part due to the poor physiological relevance of conventional two-dimensional (2D) culture systems used for preclinical testing. To address this gap, we developed a standardized three-dimensional (3D) culture and drug-testing platform for Ewing sarcoma (ES) and osteosarcoma (OS) that more accurately recapitulates in vivo tumor biology. Across 3D spheroids, bioprinted constructs, and patient-derived xenograft (PDX) cultures, we observed a consistent activation and dependency on the mevalonate pathway in ES. Leveraging this platform, we identified a selective therapeutic synergy between statins, which inhibit mevalonate pathway flux, and BCL-xL inhibitors, a vulnerability that was not detectable in 2D cultures. These findings highlight the mevalonate pathway as a targetable metabolic dependency in ES and demonstrate how physiologically grounded 3D models can uncover clinically actionable treatment strategies that remain hidden in traditional 2D systems. Our findings show that 3D tumor models can expose actionable metabolic vulnerabilities obscured by traditional approaches, supporting their use in rational combination therapy discovery for aggressive pediatric sarcomas.

cancer biology↗

bioRxiv2025

Directing stem cell differentiation by chromatin state approximation

A prime goal of regenerative medicine is to replace dysfunctional cells in the body. To design protocols for producing target cells in the laboratory, one may need to consider exponentially large combinations of culture components. Here, we investigated the potential of iteratively approximating the target phenotype by quantifying the distance between chromatin profiles (ATAC-seq) of differentiating cells in vitro and their in-vivo counterparts. We tested this approach on the well-studied generation of erythroblasts from haematopoietic stem cells, evaluating a fixed number of components over two sequential differentiation rounds (8x8 protocols). We found that the most erythroblast-like cells upon the first round yielded the most erythroblast-like cells at the second round, suggesting that greedy selection by chromatin approximation can be a viable optimisation strategy. Furthermore, by analysing regulatory sequences in incompletely reprogrammed chromatin regions, we uncovered transcriptional regulators linked to roadblocks in differentiation and made a data-driven selection of ligands that further improved erythropoiesis. In future, our methodology can help craft notoriously difficult cells in vitro, such as B cells.

genomics↗

bioRxiv2025

Dynamic modelling of EWS::FLI1 fluctuations reveals molecular determinants of phenotypic tumor plasticity and prognosis in Ewing sarcoma

The mechanisms underlying tumor cell plasticity driving drug resistance and disease progression remain poorly understood. In Ewing sarcoma (EwS), variations in EWS::FLI1 (EF) activity have been associated with epithelial-mesenchymal plasticity (EMP). Using degron technology, we titrated endogenous EF in an EwS cell line and linked phenotypic states to distinct EF thresholds. Strikingly, modest EF depletion promoted a pro-metastatic phenotype, that diminished upon near-complete EF loss. Nascent RNA sequencing revealed distinct gene clusters with heterogenous response patterns to varying EF dosage. Target genes most sensitive to subtle EF depletion contained GGAA microsatellites in EF-bound enhancers. Furthermore, we identified Kruppel-like zinc-finger transcription factors associated with EF-repressed EMP genes. Transient EF depletion followed by rapid restoration to simulate oncoprotein fluctuations identified persistently dysregulated genes associated with poor prognosis. This study underscores the therapeutic challenge of insufficient EF inhibition and provides a foundation for exploiting oncoprotein dynamics to uncover therapeutic vulnerabilities in fusion-driven cancers. Beyond EwS, our results underscore the broader impact of oncoprotein dosage dynamics in cancers with otherwise quiet genomes. SIGNIFICANCEWe report EwS as a paradigm for the importance of oncogene fluctuations in tumor cell plasticity and disease progression. Effective therapeutic strategies must ensure complete EF depletion to prevent inadvertent metastasis.

cancer biology↗

bioRxiv2024

YAP1 is a key regulator of EWS::FLI1-dependent malignant transformation upon IGF-1 mediated reprogramming of bone mesenchymal stem cells

AbstractEwing sarcoma (EwS) is an aggressive cancer of adolescents in need of effective treatments. Insulin like growth factor (IGF) 1 was previously reported an autocrine growth factor for EwS, but only 10% of patients responded to IGF-1 receptor blockade. Although presumed to originate from mesenchymal progenitors during bone development, targeting of the EwS driver oncogene EWS::FLI1 to the mesenchymal lineage in a conditional mouse model did not result in tumor formation but led to skeletal malformations and perinatal death. We report that transient exposure to IGF-1 concentrations mimicking serum levels during puberty reprogrammed limb-derived mesenchymal cells of EWS::FLI1-mutant mice to stable transformation and tumorigenicity. We identified a modular mechanism of IGF-1-driven tumor promotion in the early steps of EwS pathogenesis, in which Yap1 plays a central role. Pharmacologic Yap1/Tead inhibition reversed the transformed phenotype of EWS::FLI1 expressing cells. Our data provide a rationale for combined IGF-1R and YAP/TEAD inhibition in the treatment of EwS patients. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/603565v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@a6a298org.highwire.dtl.DTLVardef@1fcab8aorg.highwire.dtl.DTLVardef@1621e27org.highwire.dtl.DTLVardef@a9994e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

NK cells shape the clonal evolution of B-ALL cells by IFN-γ production

The term cancer immunoediting describes the dual role by which the immune system can suppress and promote tumour growth and is divided into three phases: elimination, equilibrium and escape. The role of NK cells has mainly been attributed to the elimination phase. Here we show that NK cells play a role in all three phases of cancer immunoediting. Extended co-culturing of DNA barcoded mouse BCR/ABLp185+ B acute lymphoblastic leukaemia (B-ALL) cells with NK cells allowed for a quantitative measure of NK cell-mediated immunoediting. Whereas most tumour cell clones were efficiently eliminated by NK cells, a certain fraction of tumour cells harboured an intrinsic primary resistance. Furthermore, DNA barcoding revealed tumour cell clones with secondary resistance, which stochastically acquired resistance to NK cells. NK cell cytotoxicity put a selective pressure on B-ALL cells, which led to an outgrowth of primary and secondary resistant tumour cell clones, which were characterised by an IFN-{gamma} signature. Besides well-known regulators of immune evasion, our analysis of NK cell resistant tumour cells revealed the upregulation of genes, including Ly6a, which we found to promote NK cell resistance in leukaemic cells. Translation of our findings to the human system showed that high expression of LY6E on tumour cells impaired the physical interaction with NK cells and led to worse prognosis in leukaemia patients. Our results demonstrate that tumour cells are actively edited by NK cells during the equilibrium phase and use different avenues to escape NK cell-mediated eradication.

cancer biology↗

bioRxiv2023

Single-cell RNA-seq differential expression tests within a sample should use pseudo-bulk data of pseudo-replicates

Single-cell RNA sequencing (scRNA-seq) has become a standard approach to investigate molecular differences between cell states. Comparisons of bioinformatics methods for the count matrix transformation (normalization) and differential expression (DE) analysis of these data have already highlighted recommendations for effective between-sample comparisons and visualization. Here, we examine two remaining open questions: (i) What are the best combinations of data transformations and statistical test methods, and (ii) how do pseudo-bulk approaches perform in single-sample designs? We evaluated the performance of 343 DE pipelines (combinations of eight types of count matrix transformations and ten statistical tests) on simulated and real-world data, in terms of precision, sensitivity, and false discovery rate. We confirm superior performance of pseudo-bulk approaches without prior transformation. For within-sample comparisons, we advise the use of three pseudo-replicates, and provide a simple R package DElegate to facilitate application of this approach.

bioinformatics↗

bioRxiv2022

Cross-species analysis identifies conserved transcriptional mechanisms of neutrophil maturation

Neutrophils are evolutionarily conserved innate defense cells implicated in diverse pathological processes. Zebrafish models have contributed substantially to our understanding of neutrophil functions, but similarities to human neutrophil maturation have not been characterized limiting applicability to study human disease. We generated transgenic zebrafish strains to distinguish neutrophil maturation grades in vivo and established a high-resolution transcriptional profile of neutrophil maturation. We linked gene expression at each stage to characteristic transcription factors, including C/ebp{beta}, important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human confirmed high molecular similarity in immature stages and discriminated zebrafish-specific from pan-species gene signatures. Applying pan-species neutrophil maturation signatures in RNA-seq data from neuroblastoma patients revealed an association of metastatic tumor cell infiltration in the bone marrow with an increase in mature neutrophils. Our detailed neutrophil maturation atlas provides a valuable resource for studying neutrophil function at different stages across species in health and disease. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/518146v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@d46deaorg.highwire.dtl.DTLVardef@7c2647org.highwire.dtl.DTLVardef@b535eeorg.highwire.dtl.DTLVardef@e7dc38_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Neuroblastoma-associated chromosomal aberrations drive cell identity loss in human neural crest via disruption of developmental regulators

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

cancer biology↗

bioRxiv2021

Early anteroposterior regionalisation of human neural crest is shaped by a pro-mesodermal factor

The neural crest (NC) is an important multipotent embryonic cell population and its impaired specification leads to various developmental defects, often in an anteroposterior (A-P) axial level-specific manner. The mechanisms underlying the correct A-P regionalisation of human NC cells remain elusive. Recent studies have indicated that trunk NC cells, the presumed precursors of the childhood tumour neuroblastoma, are derived from neuromesodermal-potent progenitors of the postcranial body (NMPs). Here we employ human embryonic stem cell differentiation to define how NMP-derived NC cells acquire a posterior axial identity. We show that TBXT, a pro-mesodermal transcription factor, mediates early posterior NC regionalisation together with WNT signalling effectors. This occurs by TBXT-driven chromatin remodelling via its binding in key enhancers within HOX gene clusters and other posterior regulator-associated loci. In contrast, posteriorisation of NMP-derived spinal cord cells is TBXT/WNT-independent and takes place under the influence of FGF signalling. Our work reveals a previously unknown role of TBXT in influencing posterior NC fate and points to the existence of temporally discrete, cell type-dependent modes of posterior axial identity control.

developmental biology↗

bioRxiv2021

Automated compound testing in zebrafish xenografts identifies combined MCL-1 and BCL-XL inhibition to be effective against Ewing sarcoma

Ewing sarcoma is a pediatric bone and soft tissue cancer for which new therapies to improve disease outcome and to reduce adverse effects of current standard treatments are urgently needed. To identify new and effective drugs, phenotypic drug screening has proven to be a powerful method and a cancer model ideally suited for this approach is the larval zebrafish xenograft system. Complementing mouse xenografts, zebrafish offer high-througput screening possibilities in an intact complex vertebrate organism. Here, we generated Ewing sarcoma xenografts in zebrafish larvae and established a workflow for automated imaging of xenografts, tumor cell recognition within transplanted zebrafish and quantitative tumor size analysis over consecutive days by high-content imaging. The increased throughput of our in vivo screening setup allowed us to identify combination therapies effective against Ewing sarcoma cells. Especially, combined inhibition of MCL-1 and BCL-XL, two anti-apoptotic proteins, was highly efficient at eradicating tumor cells in our zebrafish xenograft assays with two Ewing sarcoma cell lines and with patient-derived cells. Transcriptional analysis across Ewing sarcoma cell lines and tumors revealed that MCL-1 and BCL2L1, coding for BCL-XL, are the most abundantly expressed anti-apoptotic genes, suggesting that combined MCL-1/BCL-XL inhibition might be a broadly applicable strategy for Ewing sarcoma treatment.

cancer biology↗

bioRxiv2020

Novel imprints in mouse blastocysts are predominantly DNA methylation independent

In mammals, chromatin marks at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. This control is thought predominantly to involve parent-specific differentially methylated regions (DMR) in genomic DNA. However, neither parent-of-origin-specific transcription nor DMRs have been comprehensively mapped. We here address this by integrating transcriptomic and epigenomic approaches in mouse preimplantation embryos (blastocysts). Transcriptome-analysis identified 71 genes expressed with previously unknown parent-of-origin-specific expression in blastocysts (nBiX: novel blastocyst-imprinted expression). Uniparental expression of nBiX genes disappeared soon after implantation. Micro-whole-genome bisulfite sequencing (WGBS) of individual uniparental blastocysts detected 859 DMRs. Only 18% of nBiXs were associated with a DMR, whereas 60% were associated with parentally-biased H3K27me3. This suggests a major role for Polycomb-mediated imprinting in blastocysts. Five nBiX-clusters contained at least one known imprinted gene, and five novel clusters contained exclusively nBiX-genes. These data suggest a complex program of stage-specific imprinting involving different tiers of regulation.

genomics↗

bioRxiv2020

Single-cell landscape of bone marrow metastases in human neuroblastoma unraveled by deep multiplex imaging

Bone marrow commonly serves as a metastatic niche for disseminated tumor cells (DTCs) of solid cancers in patients with unfavorable clinical outcome. Single-cell assessment of bone marrow metastases is essential to decipher the entire spectrum of tumor heterogeneity in these cancers, however, has previously not been performed. Here we used multi-epitope-ligand cartography (MELC) to spatially profile 20 biomarkers and assess morphology in DTCs as well as hematopoietic and mesenchymal cells of eight bone marrow metastases from neuroblastoma patients. We developed DeepFLEX, a single-cell image analysis pipeline for MELC data that combines deep learning-based cell and nucleus segmentation and overcomes frequent challenges of multiplex imaging methods including autofluorescence and unspecific antibody binding. Using DeepFLEX, we built a single-cell atlas of bone marrow metastases comprising more than 35,000 single cells. Comparisons of cell type proportions between samples indicated that microenvironmental changes in the metastatic bone marrow are associated with tumor cell infiltration and therapy response. Hierarchical clustering of DTCs revealed multiple phenotypes with highly diverse expression of markers such as FAIM2, an inhibitory protein in the Fas apoptotic pathway, which we propose as a complementary marker to capture DTC heterogeneity in neuroblastoma. The presented single-cell atlas provides first insights into the heterogeneity of human bone marrow metastases and is an important step towards a deeper understanding of DTCs and their interactions with the bone marrow niche.

cancer biology↗

bioRxiv2017

Distinct SoxB1 networks are required for naïve and primed pluripotency

Deletion of Sox2 from embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here we show that Sox2 can be deleted from EpiSCs with impunity. This is due to a shift in the balance of SoxB1 expression in EpiSCs, which have decreased Sox2 and increased Sox3 compared to ESCs. Consistent with functional redundancy, Sox3 can also be deleted from EpiSCs without eliminating self-renewal. However, deletion of both Sox2 and Sox3 prevents self-renewal. The overall SOXB1 levels in ESCs affect differentiation choices: neural differentiation of Sox2 heterozygous ESCs is compromised, while increased SOXB1 levels divert the ESC to EpiSC transition towards neural differentiation. Therefore, optimal SOXB1 levels are critical for each pluripotent state and for cell fate decisions during exit from naive pluripotency.

cell biology↗

bioRxiv2017

Functional dissection of the enhancer repertoire in human embryonic stem cells

Enhancers are genetic elements that regulate spatiotemporal gene expression. Enhancer function requires transcription factor (TF) binding and correlates with histone modifications. However, the extent to which TF binding and histone modifications can functionally define active enhancers remains unclear. Here we combine chromatin immunoprecipitation with a massively parallel reporter assay to identify functional enhancers in human embryonic stem cells (hESCs) genome-wide in a quantitative unbiased manner. While active enhancers associate with TFs, only a minority of regions marked by NANOG, OCT4, H3K27ac and H3K4me1 function as enhancers, with activity changing markedly with culture conditions. Our analysis also reveals a novel enhancer set associated with housekeeping genes. Moreover, while transposable elements associate with putative enhancers only some exhibit activity. Similarly, within super-enhancers, large tracts are non-functional, with activity restricted to small sub-domains. This catalogue of validated enhancers provides a valuable resource for further functional dissection of the regulatory genome.\n\nHighlightsO_LIA catalog of functional enhancers in hESCs including a novel housekeeping class\nC_LIO_LIActive enhancers feature specific transcription factors and transposable elements\nC_LIO_LIMajor shifts in enhancer activity occur during induction of naive pluripotency\nC_LIO_LISuper-enhancers consist of small units with enhancer function\nC_LI

genomics↗