Biology subjects

Ebert, M. P.

Publications and source records attributed to Ebert, M. P..

11 recordsLinked to original sources

bioRxiv2026

Multi-omics, organoid-based modeling reveals an SRC/mTOR-dependent fetal-like stem cell trajectory in colorectal cancer

BackgroundSingle-cell atlases have described diverse stem cell states in colorectal cancer (CRC), however, the overarching trajectories of those states and the underlying functional mechanisms, including their relevance for drug sensitivity, need better understanding. MethodsWe established 64 patient-derived organoids from microsatellite-stable colorectal cancers, characterized their transcriptomes and genomes, and performed drug screening with 62-140 clinically approved substances. We analyzed additional published transcriptome data from patient-derived organoids (72 patients from three independent datasets), TCGA-CRC data (466 patients), and single-cell transcriptomes of tumor biopsies (123,000 cells from six independent cohorts) to establish a functional and molecular landscape of CRC stem cells. We performed mechanistic follow-up analyses by mass-spectrometry-based proteomics, large-scale kinase inhibition assays and immunofluorescence analyses. ResultsWe find a continuous landscape of CRC stem cells that is characterized by distinct developmental programs: adult stem cell-to fetal-like regenerative states and transition between differentiation programs. By large-scale drug perturbations and multi-omics modeling, we identify a regenerative/fetal-like stem cell trajectory characterized by PI3K/mTOR dependency. We find the identified developmental axes conserved in organoid, clinical, as well as single-cell data, and the fetal-like PI3K/mTOR-dependent state to be associated with poor clinical prognosis. Mechanistically, PI3K/mTOR vulnerability is linked to a lack of adaptive capability due to suppressed mRNA translation and associated with an upregulated SRC signaling network. ConclusionsOur work moves beyond a molecular CRC landscape by combined functional perturbation analyses in organoids. This enables mechanistic modeling of stem cell state regulation and identifies an SRC/mTOR-dependent regenerative state in CRC, which might allow improved therapeutic targeting in the future.

cancer biology↗

bioRxiv2026

Macrophages target PVR/CD155 on colorectal cancer cells via REVERBalpha

Background&AimsPatients with microsatellite-instable (MSI) colorectal cancer (CRC) benefit from immune checkpoint therapy. For patients with microsatellite-stable (MSS) non-MSI tumors targeting alternative immune checkpoints, such as the Poliovirus receptor (PVR/CD155), may extend response to checkpoint inhibitors and thereby improve outcomes. The drugable transcription factor REVERB (NR1D1) is a master repressor of macrophage function. We hypothesized that regulation of PVR allows elimination of tumor cells by macrophage-directed precision therapy. MethodsHuman CRC cell lines (MSS+: HT29, SW480; MSI+: HCT116), patient-derived organoids (MSS+ PDOs) and tissues were assessed by PCR, Western blot, immunohistochemistry and flow cytometry. 3D co-cultures of CRC cells with macrophages derived from THP1 monocytic leukemia cells or peripheral blood of healthy donors were analysed by microscopy and viability assays. Functional perturbation of PVR and REVERB was achieved by CRISPR/Cas9-sgRNA gene modification, synthetic ligands or blocking antibodies (Abs). ResultsREVERB bound a cognate DNA-element in the -1 kb human PVR gene promoter, and its agonist (SR9009) super-repressed, whereas its antagonist (SR8278) de-repressed transcription of PVR mRNA in macrophages. Macrophages with CRISPR-modified REVERB were unresponsive to ligand, devoid of PVR protein, showed more phagocytosis, tumor cell efferocytosis and expression of genes related to host immunity (PDL1, TLR4 e.a.) than clones with the wild-type receptor. Macrophages lowered the viability of tumor cells, potentiated by PVR blocking Ab or PVR knock-out in tumor cells. Consistently, REVERB antagonist augmented tumor cell death in co-cultures with macrophages and PVR blocking Ab. ConclusionCo-addressing the checkpoint axis REVERB-PVR may represent a novel intervention strategy for patients with MSS+ CRC.

cancer biology↗

bioRxiv2025

Immunoglobulin G binding as a quantitative marker of hepatocellular death across acute and chronic liver injury

Background & AimsAccurate detection of hepatocellular death is fundamental for understanding liver injury, intoxication, regeneration, and fibrosis. Conventional markers, such as serum transaminases and histopathological scoring, suffer from limited temporal resolution, high variability, and observer dependence. We evaluated immunoglobulin G (IgG) binding as a quantitative and spatially resolved marker of hepatocyte death in acute and chronic liver injury models. MethodsMale C57BL/6 mice were subjected to acute carbon tetrachloride (CCl) intoxication (1600 mg/kg, single dose), dose-escalation (0-800 mg/kg), and chronic injury paradigms including Western diet (WD), WD+CCl, and Mdr2-/- mice with or without a single CCl challenge. The serum ALT and AST levels were measured. Liver sections were stained with IgG, Hematoxylin and Eosin (H&E), bromodeoxyuridine (BrdU), glutamine synthetase (GS), CD26, and alpha-smooth muscle actin (Acta2). Spatial and integrative transcriptomic analyses were performed to characterize the IgG hepatocyte dead regions. ResultsHepatocellular IgG labeling emerged as early as 6h post-CCl, peaked at 72-96h, and declined during regeneration. IgG-positive areas correlated strongly with Ishak necroinflammatory score (r=0.70) and serum transaminase levels (p=0.74-0.85), surpassing both in Receiver Operating Characteristic (ROC) analyses (AUC=0.92-0.95). IgG bound to both apoptotic (TUNEL) and necrotic (TUNEL-) hepatocytes. In chronic liver injury models, IgG deposition was localized to the injury zones and correlated with ALT/AST, irrespective of etiology. Multiplex imaging revealed IgG-positive necrotic cores surrounded by proliferating hepatocytes and Acta2+ myofibroblasts. Spatial transcriptomics identified immune cell enrichment, Fc{gamma}R-mediated signaling, phagocytosis, and vascular remodeling within the IgG-marked regions. ConclusionsIgG immunostaining provides a robust, quantitative, and pathologist-independent readout of hepatocellular death, which scales with injury severity, delineates necrotic zones, and reveals immune-active microenvironments. These findings establish IgG-based detection as a versatile, high-resolution tool for assessing liver injury, regeneration, and fibrosis. Conflict of Interest declarationThe authors declare that they have no affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/690787v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1d9b05eorg.highwire.dtl.DTLVardef@ca2ad8org.highwire.dtl.DTLVardef@c2d384org.highwire.dtl.DTLVardef@cca46d_HPS_FORMAT_FIGEXP M_FIG C_FIG Liver injury induced by toxins, dietary stress, or genetic knockout provokes chemokine release (i.e. CXCL1, CXCL2, and CCL3) and recruitment of immune cells, including T cells, NK cells (NKs), and dendritic cells (DCs). Activated immune cells secrete IgG, which binds to damaged hepatocytes, leading to IgG deposition and opsonization. Opsonized hepatocytes expose "eat me" signals, promoting their phagocytic clearance and contributing to the resolution of liver injury and restoration of hepatic homeostasis.

cell biology↗

bioRxiv2025

TGF-β serves as a critical signaling determinant of liver progenitor cell fate and function

Liver progenitor cells (LPCs) are the smallest cholangiocytes that perform hepatocyte functions to rescue the lives of patients suffering from acute liver failure (ALF) caused by massive hepatic necrosis (MHN). To date, it remains largely unknown how LPCs remain quiescent and become activated following MHN. This study elucidates the essential role of TGF-{beta} in regulating LPC quiescence and activation. Spatial transcriptomics analysis of liver tissues from four MHN-ALF patients revealed that LPCs receive multiple active signals from surrounding macrophages and hepatic stellate cells, including TGF-{beta}, HGF, and EGF. Physiologically, TGF-{beta} inhibits LPC proliferation by impeding G1-S phase transition. Ectopic Smad7 expression remarkably increased LPC proliferation in 3,5-diethoxycarbonyl-1,4-dihydrocollidine-fed mice. Intriguingly, extensive LPC proliferation was observed in ALF patients despite robust TGF-{beta}-p-SMAD2 signaling in activated LPCs. Immunohistochemistry and immunofluorescence revealed significantly elevated expression of p-MET, p-STAT3, p-EGFR, and p-ERK in LPCs, indicating active HGF and EGF signaling. In vitro, either HGF or EGF promoted LPC proliferation despite the presence of TGF-{beta}. Beyond acting as mitogens, HGF and EGF regulate master hepatocyte genes (e.g., HNF4) and cholangiocyte genes (e.g., SOX9) in LPCs. Notably, HGF-dependent HNF4 required TGF-{beta}-activated SMADs. Collectively, TGF-{beta} serves as a critical signaling determinant of LPC fate and function.

cell biology↗

bioRxiv2025

CD271 sorting for improved liver cell isolation: Semiautomated and simultaneous preparation of parenchymal and non-parenchymal cells from mouse and human livers

BackgroundA detailed understanding of the dynamic fate changes of hepatocytes, hepatic stellate cells (HSC), Kupffer cells (KC), and liver sinusoidal endothelial cells (LSEC) is critical for studying liver (patho)physiology during disease progression. Current isolation methods often focus on single cell types, limiting utility in comprehensive research. AimTo develop a novel, semi-automated protocol for the simultaneous isolation of hepatocytes and non-parenchymal cells (NPCs), including HSC, KC, and LSEC, from mouse and human, with high yield, purity, and viability from healthy and diseased livers. MethodThe protocol employs a two-step EGTA and collagenase II perfusion for tissue digestion. Hepatocytes were isolated by low-speed centrifugation and a Percoll gradient. Subsequently, magnetic-activated cell separation, using CD271 as a selective surface marker for HSC, CD11b for KC and CD146 for LSEC) was performed. Validation was achieved with immunofluorescence staining, flow cytometry, RT-PCR, and UV fluorescence, whereby yield, purity, and viability were assessed. ResultsWith our method, yield of hepatocytes, HSC, KC, and LSEC, is 33.4{+/-}5.5x10, 5.2{+/-}6.3x10, 12.4{+/-}4.8x10 and 18.2{+/-}8.9x10 cells per healthy mouse liver, respectively, with cell viabilities exceeding 89%, and purity surpassing 90%. CD271 was validated as an effective marker for purifying HSC in healthy and diseased human (n=4-6) and mouse livers. Compared to microfluidic and organ-on-a-chip approaches, with our protocol, we achieve higher yield and purity values while enabling the simultaneous isolation of multiple cell types from a single sample. ConclusionOur semi-automated protocol offers a scalable, reliable, and versatile solution for isolating main liver cell types with high yield, purity, and viability from both healthy and diseased tissues, advancing liver research and facilitating downstream investigations. Impact and implicationsO_LIBroad applicability: The CD271-based method efficiently isolates key liver cell types (Hepatocyte, HSC, KC, LSEC) simultaneously. C_LIO_LIVersatility in disease models: Effective for studying healthy, fibrotic, and damaged liver tissues. C_LIO_LIRobust across variability: Works reliably across different mouse strains, age groups, and conditions. C_LIO_LIHuman research potential: Scalable for high-purity isolation from human liver tissue, enabling translational studies. C_LIO_LIHigh-quality results: Ensures >85% viability and >90% purity, supporting reproducible liver research applications. C_LI

cell biology↗

bioRxiv2024

An organoid platform reveals MEK-PARP co-targeting to enhance radiation response in rectal cancer

Locally advanced rectal cancer is usually treated by neoadjuvant chemoradiotherapy. However, tumor response rates to this treatment vary greatly. Thus, most patients do not reach a complete remission and have to undergo tumor resection. In the present study, we introduce a patient-derived rectal cancer organoid platform that reflects clinical radiosensitivity and use this to screen 1596 drug-radiation combinations. We identify inhibitors of RAS-MAPK signaling, especially MEK inhibitors, strongly synergizing with radiation response. Mechanistically, MEK inhibitors suppressed radiation-induced activation of RAS-MAPK signaling, and selectively downregulated the homologous recombination DNA repair pathway component RAD51, thereby achieving radio-enhancement. Through testing drug-drug-radiation combinations in organoids and cell lines, we identified synergism between PARP and MEK inhibitors to further enhance the effect of radiation. Our data support clinical testing of combined MEK and PARP inhibition with radiotherapy in locally advanced rectal cancers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/597640v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1d77d62org.highwire.dtl.DTLVardef@68b928org.highwire.dtl.DTLVardef@154142eorg.highwire.dtl.DTLVardef@f8f462_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

FOXA2 is essential for maintaining the urea cycle in acute liver failure

Hepatic encephalopathy is a lethal complication of acute liver failure (ALF), and is caused by hyperammonemia. Ammonia clearance by the liver requires an intact and complete urea cycle comprising six enzymes, including the rate-limiting enzyme carbamoyl phosphate synthetase I (CPS1). To date, the detailed regulation of CPS1 transcription in order to maintain urea cycle in physiological condition and ALF remains largely unknown. This study scrutinizes the role of pioneer factor forkhead box A 2 (FOXA2) in the regulation of CPS1 transcription, urea cycle performance and hyperammonemia. Physiologically, CPS1 transcription requires FOXA2 to maintain chromatin accessibility on its enhancers, which is essential for CCAAT enhancer-binding protein-alpha (C/EBP) binding to activate gene transcription. In ALF, hepatic C/EBP expression is inhibited by inflammatory mediators such as TGF-{beta} and TNF-. In this setting, retinoic acid receptor synergizes with FOXA2 to maintain CPS1 transcriptions. Once ALF patients suffer from massive hepatic necrosis, liver progenitor cells initiate a transcription network comprising FOXA2 and C/EBP to perform the urea cycle and prevent hyperammonemia. In ALF, hepatic encephalopathy occurs in patients lacking hepatic FOXA2 expression. In mice with acetaminophen-induced ALF, injection of Foxa2-AAV8 maintains urea cycle and prevents hyperammonemia. Taken together, FOXA2 is essential for maintaining the urea cycle. Pharmaceutical induction of hepatic FOXA2 expression might represent a novel approach to treat hepatic encephalopathy in ALF. One Sentence SummaryPioneer factor FOXA2 synergizes with C/EBP or RAR to maintain urea cycle in acute liver failure

molecular biology↗

bioRxiv2023

Rapid dexamethasone treatment inhibits LPS-induced cytokine storm in mice

Severe infection-induced cytokine storm is an urgent medical syndrome with high mortality. To date, no therapy is available. This study shows that high concentrations of lipopolysaccharide (LPS) induce cytokine storm within 48h and thus kill most experimental mice. Rapid, but not late dexamethasone administration remarkably inhibits cytokine storm and rescues LPS-treated mice. Monocytes and macrophages are the major source of cytokine storm. In these cells, pro-inflammatory genes (i.e., Tnf, Il6 and Il1{beta}) have preassembled RNA polymerase II (RNA Pol II), but stay at the pause stage of transcriptional elongation in the absence of stimulation. LPS rapidly activates transcription of these "pre-loaded" genes within 2h. Administration of dexamethasone within this time window inhibits RNA Pol II ser2 binding to the core promoters of pro-inflammatory genes and thus reduces LPS-induced cytokine transcription. Therefore, rapid utilization of dexamethasone might be efficacious to prevent severe bacterium-induced cytokine storm in clinical practice.

molecular biology↗

bioRxiv2023

Insulin determines the effects of TGF-beta on HNF4alpha transcription and epithelial-to-mesenchymal transition in hepatocytes

To date, epithelial-to-mesenchymal transition (EMT) has been observed in cultured hepatocytes, but not in vivo. TGF-{beta} is supposed to initiate EMT in hepatocytes by inhibiting HNF4 through the SMAD2/3 complex. We report that TGF-{beta} does not directly inhibit HNF4, but contributes to its transcriptional regulation by SMAD2/3 recruiting acetyltransferase CBP/p300 to the HNF4 promoter. The recruitment of CBP/p300 is indispensable for C/EBPa binding, another essential requirement for constitutive HNF4 expression in hepatocytes. In contrast to the observed induction of HNF4, SMAD2/3 inhibits C/EBP transcription. Therefore, long-term TGF-{beta} incubation results in C/EBP depletion, which abrogates HNF4 expression. Intriguingly, SMAD2/3 inhibitory binding to the C/EBP promoter is abolished by insulin. Thus, maintaining a high insulin concentration in culture medium ensures constitutive HNF4 and thereby prevents TGF-{beta}-induced hepatocyte EMT. Furthermore, insulin inhibits TGF-{beta}-induced SMAD2/3 binding to the promoters of core EMT transcription factors e.g., SNAI1. SNAI1 transcription requires both SMAD2/3 and FOXO1 in nuclei. Insulin inhibits SNAI1 transcription through impeding SMAD2/3 binding to its promoter and inducing FOXO1 phosphorylation. Hence, insulin is the key factor that prevents TGF-{beta}-induced EMT in hepatocytes.

cell biology↗

bioRxiv2022

Repeated toxic injuries of murine liver are tolerated through microsteatosis and mild inflammation

The liver has a remarkable capacity to regenerate and thus compensates for repeated injuries through toxic chemicals, drugs, alcohol or malnutrition for decades. However, largely unknown is how and when alterations in the liver occur due to tolerable damaging insults. To that end, we induced repeated liver injuries over ten weeks in a mouse model injecting carbon tetrachloride (CCl4) twice a week. We lost 10% of the study animals within the first six weeks, which was accompanied by a steady deposition of extracellular matrix (ECM) regardless of metabolic activity of the liver. From week six onwards, all mice survived, and in these mice ECM deposition was rather reduced, suggesting ECM remodeling as a liver response contributing to better coping with repeated injuries. The data of time-resolved paired transcriptome and proteome profiling of 18 mice was subjected to multi-level network inference, using Knowledge guided Multi-Omics Network inference (KiMONo), identified multi-level key markers exclusively associated with the injury-tolerant liver response. Interestingly, pathways of cancer and inflammation were lighting up and were validated using independent data sets compiled of 1034 samples from publicly available human cohorts. A yet undescribed link to lipid metabolism in this damage-tolerant phase was identified. Immunostaining revealed an unexpected accumulation of small lipid droplets (microvesicular steatosis) in parallel to a recovery of catabolic processes of the liver to pre-injury levels. Further, mild inflammation was experimentally validated. Taken together, we identified week six as a critical time point to switch the liver response program from an acute response that fosters ECM accumulation to a tolerant "survival" phase with pronounced deposition of small lipid droplets in hepatocytes potentially protecting against the repetitive injury with toxic chemicals. Our data suggest that microsteatosis formation plus a mild inflammatory state represent biomarkers and probably functional liver requirements to resist chronic damage. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=163 HEIGHT=200 SRC="FIGDIR/small/476054v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@11ecc7aorg.highwire.dtl.DTLVardef@1027df5org.highwire.dtl.DTLVardef@9ba0f7org.highwire.dtl.DTLVardef@164f80a_HPS_FORMAT_FIGEXP M_FIG C_FIG The datasets generated via transcriptomics, proteomics as well as blood, histopathological and biochemical analysis were analyzed in an independent and integrative manner. The independent analysis was performed via state-of-the-art statistical approaches i.e. differential and consistently regulated genes and proteins. Combining the results identified three fibrosis phases. Using the KiMONo algorithm, a fibrosis specific multi-omic network was inferred. Within this network we identified several nodes connecting phase III specific features forming 13 distinct multi-omic modules suggesting a tolerance scheme. Some of these modules were experimentally validated and compared to 11 independent human studies of various liver diseases.

systems biology↗

bioRxiv2019

Multiparametric phenotyping of compound effects on patient derived organoids

Patient derived organoids resemble the biology of tissues and tumors, enabling ex vivo modeling of human diseases from primary patient samples. Organoids can be used as models for drug discovery and are being explored to guide clinical decision making. Patient derived organoids can have heterogeneous morphologies with unclear biological causes and relationship to treatment response. Here, we used high-throughput, image-based profiling to quantify phenotypes of over 5 million individual colorectal cancer organoids after treatment with more than 500 small molecules. Integration of data using a joint multi-omics modelling framework identified organoid size and cystic vs. solid organoid architecture as axes of morphological variation across organoids. Mechanistically, we found that organoid size was linked to IGF1 receptor signaling, while a cystic organoid architecture was associated with an LGR5+ stemness program. Treatment-induced organoid morphology reflected organoid viability, drug mechanism of action, and was biologically interpretable using joint modelling. Inhibition of MEK led to cystic reorganization of organoids and increased expression of LGR5, while inhibition of mTOR induced IGF1 receptor signaling. In conclusion, we identified shared axes of variation for colorectal cancer organoid morphology, their underlying biological mechanisms, and pharmacological interventions with the ability to move organoids along them. Image-based profiling of patient derived organoids coupled with multi-omics integration facilitates drug discovery by linking drug responses with underlying biological mechanisms.

genomics↗