Biology subjects

Clevers, H.

Publications and source records attributed to Clevers, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Highly plastic macrophage niches orchestrate acquired quiescence and reactivation in breast-cancer bone metastasis

Recurrence and metastasis remain major causes of cancer mortality, sustained by therapy-resistant micrometastatic cells. Bone is a frequent site of breast-cancer relapse, yet the cues that reawaken disseminated cells remain poorly defined. We identify a previously unrecognized, highly plastic CXCL16 macrophage population that integrates tumor-associated macrophage programs found in distant metastatic sites such as lung and brain with non-tumor disease-associated traits in bone marrow. These CXCL16 macrophages establish a transient niche that restrains disseminated cancer-cell proliferation. Single-cell transcriptomics delineate functional remodeling of myeloid niches within the bone metastatic microenvironment: a CXCL16 macrophage niche that transiently constrains metastatic growth, and G-CSF macrophage and neutrophil niches that reignite tumor outgrowth. In primary tumors, cancer-associated fibroblasts (CAFs) aberrantly secrete G-CSF in response to cancer-cell signals, expanding G-CSF-receptor-positive subset of cancer cells with high metastatic potential. In advanced human bone metastases, CXCL16 macrophages localize to CAF-rich stroma but are excluded from cancer-cell clusters, indicating immune evasion. Together, these findings uncover CAF-bone-marrow cross-talk as a therapeutic target linking stromal inflammation, immune remodeling, and metastatic progression.

cancer biology↗

bioRxiv2026

A Dual-Pathway Wnt-IL-13 Fusion Protein Enhances Human Intestinal Regeneration Through Tuft Cell Activation

Compromised intestinal regenerative responses drive severe inflammatory conditions, such as inflammatory bowel disease and graft-versus-host disease, affecting millions of people worldwide each year. Despite extensive research, effective therapies remain limited, and no curative treatments are currently available. We recently discovered that human intestinal tuft cells promote tissue repair following injury through Wnt and IL-4/IL-13 signaling pathways. Building on this discovery, here we report the engineering and functional validation of a synthetic Wnt-IL-13 fusion protein that simultaneously activates both the Wnt and IL-4/IL-13 signaling pathways to enhance human intestinal tuft cell activity. Employing human organoids technology, we demonstrate that this therapeutic approach promotes mucosal healing.

biochemistry↗

bioRxiv2026

The mood stabilizer lithium alters behaviour and physiology via the gut brain axis.

Lithium, introduced 75 years ago by John Cade1, remains the most effective mood stabilizer for bipolar disorder2. Lithium is proposed to modulate an array of cellular pathways, many ubiquitous to all cells, with pleiotropic roles unlinked to bipolar disorder or lithium responsiveness in genome-wide association studies3,4. These mechanisms cannot explain lithiums specific effects on mood and behaviour. We demonstrate that lithiums primary action is in the periphery, not in the brain itself. Lithium acts in the gut to trigger behavioural and physiological changes, akin to those associated with a torpor-like state, that protect individuals from ingested toxins. Lithium activates gastrointestinal enterochromaffin (EC) cells via their Trpm2 cation channels to modulate afferent vagal and area postrema inputs to the brain. Eliminating these inputs by focal brain lesions eliminates lithiums effects, as does ablation of EC cells or their Trpm2 expression. Lithiums Trpm2-dependent activation of EC cells also occurs in human gut tissue, providing translational relevance for our discovery. These findings challenge the prevailing perception that lithium acts directly on the brain. Via a previously unsuspected gut-brain pathway, lithium engages brain circuitry that reduces arousal and interaction with the external world, therapeutic goals in the manic phase of bipolar disorder.

neuroscience↗

bioRxiv2026

A robust human airway organoid platform enables scalable expansion and trajectory mapping of pulmonary neuroendocrine cells

Pulmonary neuroendocrine cells (PNECs) are rare chemosensory epithelial cells, facultative stem cells, and a cell-of-origin for neuroendocrine lung cancers, yet the mechanisms governing their differentiation and heterogeneity are poorly understood. Here we establish NEr-fAOs, a human fetal airway organoid platform that robustly enriches PNECs, and identify a cooperative requirement for dual GSK3 and NOTCH inhibition to drive directed PNEC differentiation. This strategy yields stable cultures with up to 60-fold expansion of PNECs whose transcriptomes closely match fetal and adult PNECs. In addition to PNEC-enrichment, NEr-fAOs retain diverse airway epithelial cell types, preserving epithelial complexity. Time-resolved single-cell transcriptomics maps PNEC trajectories in NEr-fAOs, resolving precursor and mature states. Comparative analyses further reveal a distal airway bias in NEr-fAOs and enrichment for lower-airway progenitors. NEr-fAOs thus provide a scalable, tractable platform to dissect human PNEC biology and distal airway progenitor hierarchies relevant to lung development, cancer, and disease.

developmental biology↗

bioRxiv2025

Human embryo implantation involves Syncytin-2/MFSD2A-mediated heterokaryon formation with maternal endometrium

Human embryo implantation involves attachment of the blastocyst to the endometrial epithelium to subsequently gain access to the underlying stromal compartment. The blastocyst is believed to cross the epithelium either by migration through, or upon apoptosis of, the endometrial epithelial cell layer. Yet, how the blastocyst exactly traverses the endometrial epithelium remains unknown. Here, we describe an in vitro implantation model of human blastoids and hormonally matured endometrial organoids amenable to high-resolution live imaging. We demonstrate that the initial step of implantation is mediated by the direct fusion of blastoid cells with endometrial epithelial cells. Blastoids express the fusion proteins Syncytin-1 and -2, while the endometrial epithelium mainly expresses the fusion co-receptor for Syncytin-2, called MFSD2A. CRISPR-induced loss of MFSD2A in the endometrial epithelium prevents blastoids from attaching and abolishes fusion. Together, these findings support a model in which fetal-maternal cell fusion constitutes the critical initiating mechanism of human embryo implantation, with endometrial MFSD2A playing an indispensable role in this process.

developmental biology↗

bioRxiv2025

BEST4/CA7+ and goblet cells are interdependent regulators of intestinal mucus homeostasis

The intestinal mucus layer is essential for the integrity of the intestinal barrier. It is produced by goblet cells, whose depletion is common in colonic inflammation but remains poorly understood. Here, we show that goblet cell survival relies on a reciprocal dependence with newly discovered BEST4/CA7+ cells. We developed a method to follow BEST4/CA7+ and goblet cells in time from birth to death in human colon organoids. Notably, goblet cells induce BEST4/CA7+ fates in sister cells and other neighbors, using DLL1-mediated lateral activation of Notch-signaling. BEST4/CA7+ cells in turn promote goblet survival, with the latter depleting rapidly after differentiation in absence of BEST4/CA7+ cells. This apoptosis inhibition does not require direct cell-cell contact and instead depends on their shared lumen. Such differentiation and survival interdependencies may be relevant beyond the maintenance of mucosal homeostasis.

cell biology↗

bioRxiv2025

Fetal reversion from diverse lineages sustains the intestinal stem cell pool and confers stress resilience

Plasticity is a central mechanism underlying the robust regenerative capacity of the intestinal epithelium. Two major plasticity have been described: spatial plasticity, in which differentiated cells revert to crypt base columnar cells (CBCs), and fetal reversion into revival stem cells (revSCs). However, the relationship among these two stem cell populations and differentiated cells remains to be clarified. Here, we demonstrated the bidirectional interconversion between CBCs and revSCs. Using lineage tracing, injury models and villus culture, we show that absorptive enterocytes can reprogram into revSCs and regenerate CBCs. These findings position fetal reversion as an entry point to spatial plasticity, establishing a regenerative hierarchy where CBCs, revSCs, and enterocytes collectively orchestrate intestinal repair. Furthermore, we identified revSCs as a highly stress-tolerant stem cell population, whose emergence is crucial for preserving the stem cell pool. Our results establish fetal reversion as a cellular escape mechanism safeguarding epithelial regeneration under inflammatory conditions.

cell biology↗

bioRxiv2025

A collection of patient-derived intestinal organoid lines reveals epithelial phenotypes associated with genetic drivers of pediatric inflammatory bowel disease

Pediatric Inflammatory Bowel Disease (IBD) is a chronic condition characterized by per-sistent intestinal inflammation in children and adolescents. Despite a rising global incidence, the underlying causes and optimal management strategies for pediatric IBD are still not fully understood. Compared to adult IBD, pediatric IBD frequently presents with distinct disease phenotypes, and is more commonly linked to rare monogenic variants that cause intestinal epithelial barrier dysfunction or affect the function of mucosal immune cells. While more than 100 genes have been associated with early-onset IBD, the roles of many of these genes in the intestinal epithelium and the mechanisms by which genetic variants contribute to disease remain poorly defined. Here we aimed to improve our understanding of intestinal epithelium dysfunction in early-onset IBD by conducting extensive molecular and cellular characterization to gain insights into patient-specific epithelial phenotypes and identify therapeutic targets. We generated intestinal epithelial organoids (IEOs) from 94 pediatric IBD patients, representing diverse clinical characteristics and including those with monogenic variants (BTK n=4, TTC7A n=3, IL10RA n=1, LRBA n=1, STXBP2 n=1, TTC37 n=1, TRNT1 n=1, PLCG2 n=1, DKC1 n=1, POLA1 n=1), and 46 non-IBD controls. This effort resulted in the largest RNA-seq dataset of pediatric IBD intestinal epithelial organoids to date, encompassing both baseline conditions and post-immunological stimulation, serving as a valuable resource for future research. We observed that IEOs effectively initiate inflammation upon stimulation with bacterial lysate, regardless of disease status, origin, or mutation status. Inflammatory stimulation triggered single-gene upregulation of IBD-linked SERPINA1 and LIFR across the IBD population compared to controls, suggesting their role in intestinal epithelial innate immune responses. However, co-expression network analysis showed no consistent transcriptional signatures across the entire IBD group at the systems level. Instead, differences emerged between controls and specific genotypes (TTC7A, STXBP2, LRBA), with STXBP2 and LRBA sharing an upregulated transcriptional response of IL-1 and SLC30-mediated zinc trafficking pathways. These findings underscore the potential of IEOs as a valuable model for studying IBD and offer key insights that could guide the development of targeted therapies for both monogenic and non-monogenic forms of IBD. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/659052v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@10a4d9org.highwire.dtl.DTLVardef@b6cdadorg.highwire.dtl.DTLVardef@179216dorg.highwire.dtl.DTLVardef@181a4e4_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2025

Human colon stem cells are the principal epithelial responders to bacterial antigens

Intestinal epithelial cells (IECs) are capable of mounting an adequate antimicrobial inflammatory response to pathogens while tolerating commensals. The underlying regulatory mechanisms of immune sensitivity remain incompletely understood, particularly in the context of human IECs. To enhance our understanding of the immune response of IECs to bacterial epithelial barrier breach, we investigated whether epithelial responsiveness is contingent on cell identity and cell polarization. We exposed human intestinal organoids to bacterial antigens to study their immune responses. Notable discrepancies were observed in the specific reactions exhibited by intestinal stem cells (ISCs) and enterocytes. It was determined that basolateral exposure of IECs to bacterial antigens resulted in a robust response, whereas apical exposure elicited a significantly more modest response. We identified ISCs as the responders, while the reaction of enterocytes was found to be attenuated. The regulation of bacterial responsiveness in enterocytes occurs at multiple levels, including the modulation of NF{kappa}B activation and post-transcriptional control of mRNA stability. Our findings demonstrate that differentiated non-responsive enterocytes can be sensitized to bacterial antigens through the activation of the WNT pathway. These findings extend the crucial role of WNT signaling for intestinal epithelial homeostasis and regulation of stem cell maintenance, proliferation, differentiation, and tissue architecture in the gut. Additionally, they reveal a new function of WNT signaling in regulating microbial responses within the intestinal environment.

immunology↗

bioRxiv2025

A human organoid model of alveolar regeneration reveals distinct epithelial responses to interferon-gamma

Chronic obstructive pulmonary disease is characterized by inflammation and emphysema, leading to progressive alveolar destruction. Currently, no therapies effectively regenerate the alveolar epithelium. Here, we describe a feeder- and serum-free primary adult human organoid model to investigate how inflammation influences alveolar regeneration. We achieve long-term expansion of multipotent progenitor-like cells, while an alveolar type 2 (AT2) maturation protocol enhances surfactant production and supports tubular myelin formation. Introducing a LATS inhibitor to the expansion condition induces alveolar type 1 (AT1) differentiation while maintaining AT2 cells. Using this platform, we find that interferon-gamma exerts cytotoxic effects on AT1 cells while promoting the growth of regenerating AT2 cells, illustrating how a single inflammatory stimulus can have divergent effects on alveolar epithelial cell types. These findings underscore the nuanced influence of pro-inflammatory cytokines on alveolar regeneration. Our organoid model provides a reductionist platform for mechanistic studies, aimed to identify strategies to enhance alveolar regeneration.

cell biology↗

bioRxiv2025

Mapping the complete influenza A virus infection cycle through single vRNP imaging

Cell-to-cell heterogeneity is a common feature of viral infection that can generate enormous complexity, complicating understanding of infection progression and interpretation of differences between viral variants. To overcome these challenges, we developed a technology to visualize the infection cycle of human influenza A virus (IAV) from start-to-finish in individual living cells with unprecedented spatial and temporal resolution, which identified numerous distinct pathways through which infection can progress. We show that heterogeneous viral gene expression drives infection cycle heterogeneity, and identified genome packaging, vRNP transcriptional activation and host cell division as major determinants of gene expression variability. Our work maps out the complete IAV infection cycle, identifies the origin of infection heterogeneity and provides a broadly-applicable technology for studying IAV and other viruses.

cell biology↗

bioRxiv2024

Mature tuft cell phenotypes are sequentially expressed along the intestinal crypt-villus axis following cytokine-induced tuft cell hyperplasia

Intestinal tuft cells are epithelial sentinels that trigger host defense upon detection of parasite-derived compounds. While representing interesting targets for immunomodulatory therapies in inflammation-driven intestinal diseases, their detailed functioning is poorly understood. Although two distinct intestinal tuft cell types have been described, we reveal common intermediary transcriptomes among tuft cells in mouse and human. Tuft cell-specific reporter knock-ins in organoids show that the two tuft types are sequentially expressed transcriptomic states that represent different maturation stages. Moreover, cytokines interleukin-4 and interleukin-13 only induce lineage specification to Nrep+ tuft-1 cells, while BMP and cholinergic signalling advance differentiation towards immune-related ChAT+ tuft-2 phenotypes. Functionally, both tuft cell states have chemosensory capacity and respond to stimuli like succinate, but reaction probability increases during tuft cell maturation. Our tuft type-specific reporters and optimized differentiation strategy in organoids provide an experimental platform to study the functioning of tuft cells and their unique chemosensory properties.

cell biology↗

bioRxiv2024

Sensitivity to Vγ9Vδ2TCR T cells is imprinted after single mutations during early oncogenesis

V{gamma}9V{delta}2T cells have the unique ability to recognize a broad range of malignant transformed cells. The tumor targeting event involving BTN2A1 and BTN3A1 dimers on the tumor cell surface is critical, leading to full activation of the TCR. Although the molecular mechanisms governing TCR engagement and T cell activation are well-characterized, the role of V{gamma}9V{delta}2 T cells in cancer immune surveillance remains to be fully elucidated, particularly the mechanisms that enable these cells to discriminate between healthy and malignant cells at an early stage of malignant transformation. We employed two independent, genetically engineered step-wise mutagenesis models of human colorectal and breast cancer that mimic the transformation steps leading to tumor formation. We demonstrate that various single oncogenic mutations introduced into healthy organoids or cells, are sufficient to upregulate surface expressed BTN2A1 and enable V{gamma}9V{delta}2 TCR binding to tumor cells. However, full activation of T cells through a V{gamma}9V{delta}2TCR required additional subsequent phosphorylation of juxtamembrane (JTM) amino acids of BTN3A1, leading to the activating heterodimerization of BTN2A1 and 3A1. Using a protein interactome mapping pipeline, we identified PHLDB2, SYNJ2 and CARMIL1 as key players in controlling these delicate dual surface dynamics of BTN2A1 and 3A1 during early transformation. This mode of action allowed V{gamma}9V{delta}2TCR T cells to control tumors in vitro and in vivo, emphasizing the crucial role of these molecules from early mutagenesis, to advanced cancer stages, and highlighting the therapeutic potential of a V{gamma}9V{delta}2TCR.

cancer biology↗

bioRxiv2024

Integrin-activating Yersinia protein Invasin sustains long-term expansion of primary epithelial cells as 2D organoid sheets

Matrigel/BME, a basement membrane-like preparation, supports long-term growth of epithelial 3D organoids from adult stem cells (ASC)1,2. Here, we show that interaction between Matrigels major component Laminin111 with epithelial 6{beta}1-integrin is crucial for this process. The outer membrane protein Invasin of Yersinia is known to activate multiple integrin-{beta}1 complexes, including integrin-6{beta}1. A C-terminal integrin-binding fragment of Invasin, coated on culture plates, mediated gut epithelial cell adhesion. Addition of organoid growth factors allowed multi-passage expansion in 2D. Polarization, junction formation and generation of enterocytes, goblet cells, Paneth cells, and enteroendocrine cells was stable over time. Sustained expansion of other human-, mouse-, and even snake epithelia was accomplished under comparable conditions. The 2D organoid sheet format holds advantages over the 3D in gel format in terms of imaging, accessibility of basal and apical domains and automation for high throughput screening. Invasin represents a fully defined, affordable, versatile, and animal-free complement to Matrigel/BME.

cell biology↗

bioRxiv2024

Divergent lineage trajectories and genetic landscapes in human gastric intestinal metaplasia organoids associated with early neoplastic progression

ObjectiveGastric intestinal metaplasia (IM) is a pre-cancerous stage spanning a morphological spectrum that is poorly represented by human cell line models. We aim to establish and characterize human IM cell models to better understand IM progression along the cancer spectrum. DesignA large human gastric IM organoid (IMO) cohort (n=28) was established, along with normal gastric organoids (n=42) for comparison, and comprehensive multi-omics profiling and functional characterization were performed. ResultsSingle-cell transcriptomes revealed IMO cells spanning a spectrum from hybrid gastric/intestinal to advanced intestinal differentiation, and unveiled lineage trajectories that connected different cycling and quiescent stem and progenitors, highlighting their differences in gastric to IM transition. The hybrid IMO cells showed impaired differentiation potential, high lineage plasticity beyond gastric or intestinal fates, and reactivation of a fetal gene program. Cell populations in gastric IM and cancer tissues were found to be highly similar to those derived from IMOs and exhibited fetal signature. Genomically, IMOs showed an elevated mutation burden, frequent chromosome 20 gain, and epigenetic de-regulation of many intestinal and gastric genes. Functionally, IMOs downregulated FGFR2 and became independent of FGF10 for survival. Several IMOs exhibited a cell-matrix adhesion independent (CMi) subpopulation that displayed chromosome 20 gain but lacked key cancer driver mutations, which could represent the earliest neoplastic precursor of IM-induced gastric cancer. ConclusionsOverall, our IM organoid biobank captured the heterogeneous nature of IM, revealing mechanistic insights on IM pathogenesis and its neoplastic progression, offering an ideal platform for studying early gastric neoplastic transformation and chemoprevention.

cancer biology↗

bioRxiv2024

Tuft cells act as regenerative stem cells in the human intestine

In mice, intestinal tuft cells have been described as a long-lived, post-mitotic cell type of which two distinct subsets have been identified, named tuft-1 and tuft-21. By combining analysis of primary human intestinal resection material and intestinal organoids, we identify four distinct human tuft cell states, two of which overlap with their murine counterparts. We show that tuft cell development depends on the presence of Wnt ligands, and that tuft cell numbers rapidly increase upon interleukin (IL)-4 and IL-13 exposure, as reported previously in mouse2-4. This occurs through proliferation of pre-existing tuft cells, rather than through increased de novo generation from stem cells. Indeed, proliferative tuft cells occur in vivo both in fetal and in adult human intestine. Single mature proliferating tuft cells can form organoids that contain all intestinal epithelial cell types. Unlike stem- and progenitor cells, human tuft cells survive irradiation damage and retain the ability to generate all other epithelial cell types. Accordingly, organoids engineered to lack tuft cells fail to recover from radiation-induced damage. Thus, tuft cells represent a damage-induced reserve intestinal stem cell pool in humans.

cell biology↗

bioRxiv2023

Multi-omic dataset of patient-derived tumor organoids of neuroendocrine neoplasms

BackgroundOrganoids are three-dimensional experimental models that summarize the anatomical and functional structure of an organ. Although a promising experimental model for precision medicine, patient-derived tumor organoids (PDTOs) have currently been developed only for a fraction of tumor types. ResultsWe have generated the first multi-omic dataset (whole-genome sequencing, WGS, and RNA-sequencing, RNA-seq) of PDTOs from the rare and understudied pulmonary neuroendocrine tumors (n = 12; 6 grade 1, 6 grade 2), and provide data from other rare neuroendocrine neoplasms: small intestine (ileal) neuroendocrine tumors (n = 6; 2 grade 1 and 4 grade 2) and large-cell neuroendocrine carcinoma (n = 5; 1 pancreatic and 4 pulmonary). This dataset includes a matched sample from the parental sample (primary tumor or metastasis) for a majority of samples (21/23) and longitudinal sampling of the PDTOs (1 to 2 time-points), for a total of n = 47 RNA-seq and n = 33 WGS. We here provide quality control for each technique, and provide the raw and processed data as well as all scripts for genomic analyses to ensure an optimal re-use of the data. In addition, we report somatic small variant calls and describe how they were generated, in particular how we used WGS somatic calls to train a random-forest classifier to detect variants in tumor-only RNA-seq. ConclusionsThis dataset will be critical to future studies relying on this PDTO biobank, such as drug screens for novel therapies and experiments investigating the mechanisms of carcinogenesis in these understudied diseases.

cancer biology↗

bioRxiv2023

Multi-dimensional profiling of hepatoblastomas and patient-derived tumor organoids uncovers tumor subpopulations with divergent WNT activation profiles and identifies pan-hepatoblastoma drug sensitivities

Hepatoblastoma, the most prevalent pediatric liver cancer, almost always carries a WNT-activating CTNNB1 mutation, yet exhibits notable molecular heterogeneity. To characterize this heterogeneity and identify novel targeted therapies, we performed comprehensive analysis of hepatoblastomas and tumor-derived organoids using single-cell RNA-seq, spatial transcriptomics, single-cell ATAC-seq and high throughput drug profiling. We identified two distinct tumor epithelial signatures: hepatic fetal-like and WNT-high embryonal-like signatures, displaying divergent WNT signaling patterns. The liver-specific WNT targets were enriched in the fetal-like group, while the embryonal-like group was enriched in canonical WNT target genes. Gene regulatory network analysis revealed enrichment of regulons related to hepatic function such as bile acid, lipid and xenobiotic metabolism in the fetal-like subgroup but not in the embryonal-like subgroup. In addition, the dichotomous expression pattern of the transcription factors HNF4A and LEF1 allowed for a clear distinction between the fetal- and embryonal-like tumors. We also performed high-throughput drug screening using patient-derived tumor organoids and identified sensitivity to multiple inhibitor classes, most notably HDAC inhibitors. Intriguingly, embryonal-like tumor organoids, but not fetal-like tumor organoids, were sensitive to FGFR inhibitor treatments, suggesting a dependency on FGFR signaling. In summary, our data uncover the molecular and drug sensitivity landscapes of hepatoblastoma and pave the way for the development of targeted therapies.

cancer biology↗