Biology subjects

Bileck, A.

Publications and source records attributed to Bileck, A..

9 recordsLinked to original sources

bioRxiv2025

Integrated tissue proteomics and lipidomics from ovine organs suggests novel functional implications for fatty acids and lipid mediators.

Lipid mediators are potent biomolecules that may help to gain specific information about biological processes. However, due to incomplete functional annotations and hence interpretive challenges these molecules are hardly considered in molecular profiling experiments. We hypothesized that correlating lipid mediators extracted from tissues with corresponding proteomics data and the corresponding Gene Ontology terms characteristic of specific tissue types could enable functional annotation of these lipids. Thirteen organs and tissues from sheep (Ovis aries) were analyzed using mass spectrometry-based untargeted proteomics and lipidomics. In total, 4717 proteins and 166 free fatty acids, oxylipins, lysolipids, endocannabinoids, and bile acids were catalogued. This included the previously uncharacterized oxylipin 4-hydroxy-eicosatetraenoic acid (4-HETE), whose identity was confirmed by chemical synthesis. Co-expression and clustering analyses validated our hypothesis, successfully reproducing known and suggesting novel lipid mediator functions. Tissue levels of polyunsaturated fatty acids (PUFAs) correlated with pro-resolving mediators and, unexpectedly, with the cellular protein synthesis and folding machinery. These findings suggest that PUFAs and their derivatives support protein synthesis fidelity and efferocytosis, both essential contributors for the effective resolution of inflammation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/685055v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@126b41org.highwire.dtl.DTLVardef@13fa9adorg.highwire.dtl.DTLVardef@9ccaa1org.highwire.dtl.DTLVardef@1bb429b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO The Figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 Unported license. C_FIG

systems biology↗

bioRxiv2024

Plectin-mediated cytoskeletal crosstalk as a target for inhibition of hepatocellular carcinoma growth and metastasis.

The most common primary malignancy of the liver, hepatocellular carcinoma (HCC), is a heterogeneous tumor entity with high metastatic potential and complex pathophysiology. Increasing evidence suggests that tissue mechanics plays a critical role in tumor onset and progression. Here we show that plectin, a major cytoskeletal crosslinker protein, plays a crucial role in mechanical homeostasis and mechanosensitive oncogenic signaling that drives hepatocarcinogenesis. Our expression analyses revealed elevated plectin levels in liver tumors, which correlated with poor prognosis for HCC patients. Using autochthonous and orthotopic mouse models we demonstrated that genetic and pharmacological inactivation of plectin potently suppressed the initiation and growth of HCC. Moreover, plectin targeting potently inhibited the invasion potential of human HCC cells and reduced their metastatic outgrowth in the lung. Proteomic and phosphoproteomic profiling linked plectin-dependent disruption of cytoskeletal networks to attenuation of oncogenic FAK, MAPK/Erk, and PI3K/AKT signatures. Importantly, by combining cell line-based and murine HCC models, we show that plectin inhibitor plecstatin-1 (PST) is well-tolerated and potently inhibits HCC progression. In conclusion, our study demonstrates that plectin-controlled cytoarchitecture is a key determinant of HCC development and suggests that pharmacologically induced disruption of mechanical homeostasis may represent a new therapeutic strategy for HCC treatment.

cancer biology↗

bioRxiv2023

Multilevel Omics-Readouts of in vitro Perturbation Studies are Determined by Memory Effects from Subculture

Mass spectrometry-based omics technologies are increasingly used to map drug effects to biological pathways by identifying significant molecular events. Significance is influenced by the effect size and the variation of each molecular parameter. While the former is largely determined by the biological system, the latter can be tuned by the experimental workflow. Here, we unequivocally show that memory effects originating from subculture of colon carcinoma cells before treating with arsenic trioxide exacerbate the variation of multiple omics levels, including eicosadomics, proteomics and phosphoproteomics, without necessarily impacting on effect size. Real-time monitoring of individual samples enables control over subculture homogeneity and improves the median variation >2-fold across omics levels. This considerably facilitated mode of action deconvolution and resulted in a bilevel perturbation network of 321 causal conjectures. Controlling memory effects from subculture revealed key signaling cascades and transcriptional regulatory events that extend the molecular understanding of arsenic trioxide in solid tumors.

systems biology↗

bioRxiv2022

ROS induction as a strategy to target persister cancer cells with low metabolic activity in NRAS mutated melanoma

Metabolic reprogramming is an emerging hallmark of resistance to cancer therapy but may generate vulnerabilities that can be targeted with small molecules. Multi-omics analysis revealed that NRAS-mutated melanoma cells with a mesenchymal transcriptional profile adopt a quiescent metabolic program to resist cellular stress response induced by MEK-inhibitor resistance. However, as a result of elevated baseline ROS levels, these cells become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intra-tumor heterogeneity requires the combination of a ROS-inducer and a MEK-inhibitor to target both tumor growth and metastasis. By ex vivo pharmacoscopy of 62 human metastatic melanomas, we found that MEK-inhibitor resistant tumors significantly benefitted from the combination therapy. Finally, we profiled 486 cancer cell lines and revealed that oxidative stress responses and translational suppression are biomarkers of ROS-inducer sensitivity, independent of cancer indication. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS-inducers in melanoma and other cancers. Statement of SignificanceTargeted-therapy resistance in cancer arises from genetic selection and both transcriptional and metabolic adaptation. We show that metabolic reprogramming sensitizes resistant cells to ROS-induction in combination with pathway inhibitors. Predictive biomarkers of metabolic sensitivity to ROS-inducing agents were identified in many cancer entities, highlighting the generalizability of this treatment approach. Graphical summary O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cancer biology↗

bioRxiv2020

Finger Sweat Analysis Enables Short Interval Metabolic Biomonitoring in Humans

Metabolic biomonitoring in humans is typically based on the sampling of blood, plasma or urine. Although established in the clinical routine, these sampling procedures are often associated with a variety of compliance issues and are impractical for performing time-course studies. The analysis of the minute amounts of sweat sampled from the fingertip enables a solution to this challenge. Sweat sampling from the fingertip is non-invasive and robust and can be accomplished repeatedly by untrained personnel. This matrix represents a rich source for metabolomic phenotyping, which is exemplified by the detection of roughly 50000 features per sample. Moreover, the determined limits of detection demonstrate that the ingestion of 200 g of a xenobiotic may be sufficient for its detection in sweat from the fingertip. The feasibility of short interval sampling of sweat from the fingertips was confirmed in three time-course studies after coffee consumption or ingestion of a caffeine capsule, successfully monitoring all known caffeine metabolites. Fluctuations in the rate of sweat production were accounted for by mathematical modelling to reveal individual rates of caffeine uptake, metabolism and clearance. Biomonitoring using sweat from the fingertip has far reaching implications for personalised medical diagnostics and biomarker discovery.

biochemistry↗

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↗

bioRxiv2020

Eicosanoid content in fetal calf serum accounts for reproducibility challenges in cell culture

Reproducibility issues regarding in vitro cell culture experiments are related to genetic fluctuations and batch-wise variations of biological materials such as fetal calf serum (FCS). Genome sequencing may control the former, while the latter may remain unrecognized. Using a U937 macrophage model for cell differentiation and inflammation, we investigated whether the formation of effector molecules was dependent on the FCS batch used for cultivation. High resolution mass spectrometry was used to identify FCS constituents and to explore their effects on cultured cells evaluating secreted cytokines, eicosanoids and other inflammatory mediators. Remarkably, the FCS eicosanoid composition showed more batch-dependent variations than the protein composition. Efficient uptake of fatty acids from medium by U937 macrophages and inflammation-induced release thereof was evidenced using C13-labelled arachidonic acid, highlighting rapid lipid metabolism. For functional testing, FCS batch-dependent nanomolar concentration differences of two selected eicosanoids, 5-HETE and 15-HETE, were balanced out by spiking in. Culturing U937 cells at these defined conditions indeed resulted in significant proteome alterations indicating HETE-induced PPAR{gamma} activation, independently corroborated by HETE-induced formation of peroxisomes observed by high-resolution microscopy. In conclusion, the present data demonstrate that FCS-contained eicosanoids, subject to substantial batch-wise variation, may modulate cellular effector functions in cell culture experiments.

cell biology↗

bioRxiv2020

Schwann cell plasticity regulates neuroblastic tumor cell differentiation

The remarkable plasticity of Schwann cells (SCs) enables the acquisition of repair-specific functions essential for peripheral nerve regeneration. We hypothesized that this plastic potential is manifested in stromal SCs found within mostly benign-behaving peripheral neuroblastic tumors. To shed light on the cellular state and impact of stromal SCs, we combined transcriptome and proteome profiling of human ganglioneuromas and neuroblastomas, rich and poor in SC-stroma, respectively, as well as human injured nerve explants, rich in repair SCs. The results revealed a nerve repair-characteristic gene expression signature of stromal SCs. In turn, primary repair SCs had a pro-differentiating and anti-proliferative effect on aggressive neuroblastoma cell lines after direct and trans-well co-culture. Within the pool of secreted stromal/repair SC factors, we identified EGFL8, a matricellular protein with so far undescribed function, to induce neuronal differentiation of neuroblastoma cell lines. This study indicates that human SCs undergo a similar adaptive response in two patho-physiologically distinct situations, peripheral nerve injury and tumor development. This response is mediated by EGFL8 and other SC derived factors, which might be of therapeutic value for neuroblastic tumors and nerve regeneration. SYNOPSIS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/019422v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@14cb89forg.highwire.dtl.DTLVardef@1ecd06aorg.highwire.dtl.DTLVardef@66f08corg.highwire.dtl.DTLVardef@3a9962_HPS_FORMAT_FIGEXP M_FIG C_FIG In order to investigate the nature of stromal Schwann cells in benign peripheral neuroblastic tumors (ganglioneuromas), we compared the cellular state of stromal Schwann cells with repair-associated Schwann cells emerging in peripheral nerves after injury. O_LIStromal Schwann cells in ganglioneuromas and repair Schwann cells in injured nerves share the expression of nerve repair-associated genes. C_LIO_LINeuroblastoma cell lines, derived from high-risk metastatic peripheral neuroblastic tumors (neuroblastomas), respond to primary repair Schwann cells and their secretome with increased neuronal differentiation and reduced proliferation. C_LIO_LIStromal and repair Schwann cells express the matricellular protein EGFL8, which is capable to induce neuronal differentiation of neuroblastoma cell lines in recombinant form. C_LI THE PAPER EXPLAINEDO_ST_ABSProblemC_ST_ABSIn response to peripheral nerve damage, Schwann cells (SCs) are able to transform into specialized repair cells essential for nerve cell regeneration. Our previous studies indicated that this reactive/adaptive potential of human SCs is not restricted to injured nerve cells but also emerges in response to peripheral neuroblastic tumor cells. The usually benign subtypes of peripheral neuroblastic tumors, i.e. ganglioneuroblastomas and ganglioneuromas, contain neuronal differentiating tumor cells and are pervaded by various portions of stromal SCs. Of note, the amount of stromal SCs correlates with a favorable tumor behavior and increased patient survival, whereas aggressive subtypes of peripheral neuroblastic tumors, i.e. neuroblastomas, usually lack stromal SCs and have bad prognosis. This enigma prompted us to investigate the molecular wiring and functional state of stromal SCs versus injury-associated repair SCs and how SC signals could be leveraged as therapeutics. ResultOur study revealed that the cellular state of stromal SCs in ganglioneuromas is in many aspects very similar to human repair SCs in injured nerves as both, stromal SCs and repair SCs, are equipped with distinct nerve repair-associated functions. Hence, we exposed different cell lines, derived from high-risk metastatic neuroblastomas, to primary repair SCs or their secretome. The results demonstrated that repair SCs had a pro-differentiating and anti-proliferative effect of on neuroblastoma cell lines upon direct and/or indirect contact. Searching for secreted anti-tumor factors by transcriptome and proteome analyses identified that the matricellular protein EGFL8 was highly expressed in injured nerves and ganglioneuromas. EGFL8 gene expression in peripheral neuroblastic tumors further correlated with increased patient survival. Indeed, treatment of neuroblastoma cell lines with recombinant EGFL8 promoted neuronal differentiation and present EGFL8 as a novel neuritogen. ImpactThese findings demonstrate that stromal SCs are equipped with the tools to exert nerve repair-associated functions on peripheral neuroblastic tumor cells and the tumor microenvironment. We further show that the pool of secreted stromal/repair SC molecules contains yet uncharacterized factors with a therapeutic potential for aggressive neuroblastomas. We conclude that the inherent plasticity (reactive/adaptive potential) of SCs is responsible for the development of usually benign ganglioneuroblastomas and ganglioneuromas and, thus, is of utmost interest to be exploited in future treatment approaches for aggressive neuroblastoma subtypes.

cancer biology↗

bioRxiv2018

iTAP, a novel iRhom interactor, controls TNF secretion by policing the stability of iRhom/TACE

The apical inflammatory cytokine TNF regulates numerous important biological processes including inflammation and cell death, and drives inflammatory diseases. TNF secretion requires ADAM17/TACE, which cleaves TNF from its transmembrane tether, releasing it for signalling. The trafficking of ADAM17/TACE to the cell surface, and stimulation of its proteolytic activity, depends on membrane proteins, called iRhoms. To delineate how the TNF/TACE/iRhom axis is regulated, we performed an immunoprecipitation/mass spectrometry screen to identify iRhom-binding proteins. Here we report a novel protein, that we name iTAP (iRhom tail-associated protein) that binds to iRhoms, enhancing the stability of iRhoms and TACE, preventing their degradation in lysosomes. iTAP-null primary human macrophages, or tissues from iTAP KO mice, are dramatically depleted in the levels of iRhom2 and active TACE, and are, consequently, profoundly impaired in TNF production. Our work illustrates iTAP as a physiological rheostat controlling TNF signalling and a novel target for the control of inflammation.

biochemistry↗