Category: Publications

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Stromal-derived interleukin 6 drives epithelial-to-mesenchymal transition and therapy resistance in esophageal adenocarcinoma

The tissue dynamics that govern maintenance and regeneration of the pancreas remain largely unknown. In particular, the presence and nature of a cellular hierarchy remains a topic of debate. Previous lineage tracing strategies in the pancreas relied on specific marker genes for clonal labeling, which left other populations untested and failed to account for potential widespread phenotypical plasticity. Here we employed a tracing system that depends on replication-induced clonal marks. We found that, in homeostasis, steady acinar replacement events characterize tissue dynamics, to which all acinar cells have an equal ability to contribute. Similarly, regeneration following pancreatitis was best characterized by an acinar self-replication model because no evidence of a cellular hierarchy was detected. In particular, rapid regeneration in the pancreas was found to be driven by an accelerated rate of acinar fission-like events. These results provide a comprehensive and quantitative model of cell dynamics in the exocrine pancreas.

Authors

Eva A. Ebbing, Amber P. van der Zalm, Anne Steins, Aafke Creemers, Simone Hermsen, Rosa Rentenaar, Michelle Klein, Cynthia Waasdorp, Gerrit K. J. Hooijer, Sybren L. Meijer, Kausilia K. Krishnadath, Cornelis J.A. Punt, Mark I. van Berge Henegouwen, Suzanne S. Gisbertz, Otto M. van Delden, Maarten C. C. M. Hulshof, Jan Paul Medema, Hanneke W. M. van Laarhoven and Maarten F. Bijlsma.

Link

https://doi.org/10.1073/pnas.1820459116

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Microdissected pancreatic cancer proteomes reveal tumor heterogeneity and therapeutic targets

The tissue dynamics that govern maintenance and regeneration of the pancreas remain largely unknown. In particular, the presence and nature of a cellular hierarchy remains a topic of debate. Previous lineage tracing strategies in the pancreas relied on specific marker genes for clonal labeling, which left other populations untested and failed to account for potential widespread phenotypical plasticity. Here we employed a tracing system that depends on replication-induced clonal marks. We found that, in homeostasis, steady acinar replacement events characterize tissue dynamics, to which all acinar cells have an equal ability to contribute. Similarly, regeneration following pancreatitis was best characterized by an acinar self-replication model because no evidence of a cellular hierarchy was detected. In particular, rapid regeneration in the pancreas was found to be driven by an accelerated rate of acinar fission-like events. These results provide a comprehensive and quantitative model of cell dynamics in the exocrine pancreas.

Authors

Tessa Y.S. Le Large, Giulia Mantini, Laura L. Meijer, Thang V. Pham, Niccola Funel, Nicole C.T. van Grieken, Bart Kok, Jaco Knol, Hanneke W.M. van Laarhoven, Sander R. Piersma, Connie R. Jimenez, G. Kazemier, Elisa Giovannetti, and Maarten F. Bijlsma

Link

https://doi.org/10.1172/jci.insight.138290

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Continuous clonal labeling reveals uniform progenitor potential in the adult exocrine pancreas

The tissue dynamics that govern maintenance and regeneration of the pancreas remain largely unknown. In particular, the presence and nature of a cellular hierarchy remains a topic of debate. Previous lineage tracing strategies in the pancreas relied on specific marker genes for clonal labeling, which left other populations untested and failed to account for potential widespread phenotypical plasticity. Here we employed a tracing system that depends on replication-induced clonal marks. We found that, in homeostasis, steady acinar replacement events characterize tissue dynamics, to which all acinar cells have an equal ability to contribute. Similarly, regeneration following pancreatitis was best characterized by an acinar self-replication model because no evidence of a cellular hierarchy was detected. In particular, rapid regeneration in the pancreas was found to be driven by an accelerated rate of acinar fission-like events. These results provide a comprehensive and quantitative model of cell dynamics in the exocrine pancreas.

Authors

Sophie C Lodestijn, Tom van den Bosch, Lisanne E Nijman, Leandro F Moreno, Sophie Schlingemann, Vivek M Sheraton, Sanne M van Neerven, Jasper J Koning, Felipe A Vieira Braga, Nanne J Paauw, Maria C Lecca, Kristiaan J Lenos, Edward Morrissey, Daniël M Miedema, Douglas J Winton, Maarten F Bijlsma, Louis Vermeulen

Link

https://doi.org/10.1016/j.stem.2021.07.004

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Marker-free lineage tracing reveals an environment-instructed clonogenic hierarchy in pancreatic cancer

Effective treatments for pancreatic ductal adenocarcinoma (PDAC) are lacking, and targeted agents have demonstrated limited efficacy. It has been speculated that a rare population of cancer stem cells (CSCs) drives growth, therapy resistance, and rapid metastatic progression in PDAC. These CSCs demonstrate high clonogenicity in vitro and tumorigenic potential in vivo. However, their relevance in established PDAC tissue has not been determined. Here, we use marker-independent stochastic clonal labeling, combined with quantitative modeling of tumor expansion, to uncover PDAC tissue growth dynamics. We find that in contrast to the CSC model, all PDAC cells display clonogenic potential in situ. Furthermore, the proximity to activated cancer-associated fibroblasts determines tumor cell clonogenicity. This means that the microenvironment is dominant in defining the clonogenic activity of PDAC cells. Indeed, manipulating the stroma by Hedgehog pathway inhibition alters the tumor growth mode, revealing that tumor-stroma crosstalk shapes tumor growth dynamics and clonal architecture.

Authors

Sophie C Lodestijn , Daniël M Miedema, Kristiaan J Lenos, Lisanne E Nijman, Saskia C Belt, Khalid El Makrini, Maria C Lecca, Cynthia Waasdorp, Tom van den Bosch, Maarten F Bijlsma, Louis Vermeulen.

Link

https://doi.org/10.1016/j.celrep.2021.109852

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Estrogen-related receptor alpha drives mitochondrial biogenesis and resistance to neoadjuvant chemoradiation in esophageal cancer

Neoadjuvant chemoradiotherapy (nCRT) improves outcomes in resectable esophageal adenocarcinoma (EAC), but acquired resistance precludes long-term efficacy. Here, we delineate these resistance mechanisms. RNA sequencing on matched patient samples obtained pre-and post-neoadjuvant treatment reveal that oxidative phosphorylation was the most upregulated of all biological programs following nCRT. Analysis of patient-derived models confirms that mitochondrial content and oxygen consumption strongly increase in response to nCRT and that ionizing radiation is the causative agent. Bioinformatics identifies estrogen-related receptor alpha (ESRRA) as the transcription factor responsible for reprogramming, and overexpression and silencing of ESRRA functionally confirm that its downstream metabolic rewiring contributes to resistance. Pharmacological inhibition of ESRRA successfully sensitizes EAC organoids and patient-derived xenografts to radiation. In conclusion, we report a profound metabolic rewiring following chemoradiation and demonstrate that its inhibition resensitizes EAC cells to radiation. These findings hold broader relevance for other cancer types treated with radiation as well.

Authors

Mark P.G. Dings, Amber P. van der Zalm, Sanne Bootsma, Tatum F.J. van Maanen, Cynthia Waasrop, Tom van den Ende, Daija Liu, Peter Bailey, Jan Koster, Danny A. Zwijnenburg, C. Arnold Spek, Jan P.G. Klomp, Arthur Oubrie, Gerrit K.J. Hooijer, Sybren L. Meijer, Mark I. van Berge Henegouwen, Maarten C. Hulshof, Jacques Bergman, Cesar Oyarce, Jan Paul Medema, Hanneke W.M. van Laarhoven, Maarten F. Bijlsma.

Link

https://doi.org/10.1016/j.xcrm.2022.100802

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Cerebral Organoids: A Human Model for AAV Capsid Selection and Therapeutic Transgene Efficacy in the Brain

The development of gene therapies for central nervous system disorders is challenging because it is difficult to translate preclinical data from current in vitro and in vivo models to the clinic. Therefore, we developed induced pluripotent stem cell (iPSC)-derived cerebral organoids as a model for recombinant adeno-associated virus (rAAV) capsid selection and for testing efficacy of AAV-based gene therapy in a human context. Cerebral organoids are physiological 3D structures that better recapitulate the human brain compared with 2D cell lines. To validate the model, we compared the transduction efficiency and distribution of two commonly used AAV serotypes (rAAV5 and rAAV9). In cerebral organoids, transduction with rAAV5 led to higher levels of vector DNA, transgenic mRNA, and protein expression as compared with rAAV9. The superior transduction of rAAV5 was replicated in iPSC-derived neuronal cells. Furthermore, rAAV5-mediated delivery of a human sequence-specific engineered microRNA to cerebral organoids led to a lower expression of its target ataxin-3. Our studies provide a new tool for selecting and deselecting AAV serotypes, and for demonstrating therapeutic efficacy of transgenes in a human context. Implementing cerebral organoids during gene therapy development could reduce the usage of animal models and improve translation to the clinic.

Authors

Josse Depla, Marina Sogorb-Gonzalez, Lance A. Mulder, Vivi M. Heine, Pavlina Konstantinova, Sander J. van Deventer, Katja C. Wolthers, Dasja Pajkrt, Adithya Sridhar, and Melvin M. Evers

Link

https://doi.org/10.1016/j.omtm.2020.05.028

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A Human 2D Primary Organoid-Derived Epithelial Monolayer Model to Study Host-Pathogen Interaction in the Small Intestine

Gut organoids are stem cell derived 3D models of the intestinal epithelium that are useful for studying interactions between enteric pathogens and their host. While the organoid model has been used for both bacterial and viral infections, this is a closed system with the luminal side being inaccessible without microinjection or disruption of the organoid polarization. In order to overcome this and simplify their applicability for transepithelial studies, permeable membrane based monolayer approaches are needed. In this paper, we demonstrate a method for generating a monolayer model of the human fetal intestinal polarized epithelium that is fully characterized and validated. Proximal and distal small intestinal organoids were used to generate 2D monolayer cultures, which were characterized with respect to epithelial cell types, polarization, barrier function, and gene expression. In addition, viral replication and bacterial translocation after apical infection with enteric pathogens Enterovirus A71 and Listeria monocytogenes were evaluated, with subsequent monitoring of the pro-inflammatory host response. This human 2D fetal intestinal monolayer model will be a valuable tool to study host-pathogen interactions and potentially reduce the use of animals in research.

Authors

Thomas Roodsant*, Marit Navis*, Ikrame Aknouch, Ingrid B. Renes, Ruurd M. van Elburg, Dasja Pajkrt, Katja C. Wolthers, Constance Schultsz, Kees C. H. van der Ark, Adithya Sridhar and Vanesa Muncan

* equal contributors

Link

https://doi.org/10.3389/fcimb.2020.00272

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A Novel Organoid Model of Damage and Repair Identifies HNF4α as a Critical Regulator of Intestinal Epithelial Regeneration

Background & aims: Recent evidence has suggested that the intact intestinal epithelial barrier protects our body from a range of immune-mediated diseases. The epithelial layer has an impressive ability to reconstitute and repair upon damage and this process of repair increasingly is seen as a therapeutic target. In vitro models to study this process in primary intestinal cells are lacking.

Methods: We established and characterized an in vitro model of intestinal damage and repair by applying γ-radiation on small-intestinal organoids. We then used this model to identify novel regulators of intestinal regeneration.

Results: We identified hepatocyte nuclear factor 4α (HNF4α) as a pivotal upstream regulator of the intestinal regenerative response. Organoids lacking Hnf4a were not able to propagate in vitro. Importantly, intestinal Hnf4a knock-out mice showed impaired regeneration after whole-body irradiation, confirming intestinal organoids as a valuable alternative to in vivo studies.

Conclusions: In conclusion, we established and validated an in vitro damage-repair model and identified HNF4α as a crucial regulator of intestinal regeneration. Transcript profiling: GSE141515 and GSE141518.

Keywords: Irradiation; Organoids; Regeneration.

Authors

Paula S Montenegro-Miranda, Jonathan H M van der Meer, Christine Jones, Sander Meisner, Jacqueline L M Vermeulen, Jan Koster, Manon E Wildenberg, Jarom Heijmans, Francois Boudreau, Agnes Ribeiro, Gijs R van den Brink, Vanesa Muncan

Link

https://doi.org/10.1016/j.jcmgh.2020.02.007

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ATF2 and ATF7 Are Critical Mediators of Intestinal Epithelial Repair

Background & Aims

Activation factor-1 transcription factor family members activating transcription factors 2 and 7 (ATF2 and ATF7) have highly redundant functions owing to highly homologous DNA binding sites. Their role in intestinal epithelial homeostasis and repair is unknown. Here, we assessed the role of these proteins in these conditions in an intestine-specific mouse model.

Methods

We performed in vivo and ex vivo experiments using Villin-CreERT2Atf2fl/flAtf7ko/ko mice. We investigated the effects of intestinal epithelium-specific deletion of the Atf2 DNA binding region in Atf7-/- mice on cellular proliferation, differentiation, apoptosis, and epithelial barrier function under homeostatic conditions. Subsequently, we exposed mice to 2% dextran sulfate sodium (DSS) for 7 days and 12 Gy whole-body irradiation and assessed the response to epithelial damage.

Results

Activating phosphorylation of ATF2 and ATF7 was detected mainly in the crypts of the small intestine and the lower crypt region of the colonic epithelium. Under homeostatic conditions, no major phenotypic changes were detectable in the intestine of ATF mutant mice. However, on DSS exposure or whole-body irradiation, the intestinal epithelium showed a clearly impaired regenerative response. Mutant mice developed severe ulceration and inflammation associated with increased epithelial apoptosis on DSS exposure and were less able to regenerate colonic crypts on irradiation. In vitro, organoids derived from double-mutant epithelium had a growth disadvantage compared with wild-type organoids, impaired wound healing capacity in scratch assay, and increased sensitivity to tumor necrosis factor-α–induced damage.

Conclusions

ATF2 and ATF7 are dispensable for epithelial homeostasis, but are required to maintain epithelial regenerative capacity and protect against cell death during intestinal epithelial damage and repair.

Authors

Bartolomeus J. Meijer, Francesca P. Giugliano, Bart Baan, Jonathan H.M. van der Meer, Sander Meisner, Manon van Roest, Pim K. Koelink, Ruben J. de Boer, Nic Jones, Wolfgang Breitwieser, Nicole N. van der Wel, Manon E. Wildenberg, Gijs R. van den Brink, Jarom Heijmans, and Vanesa Muncan

Link

https://doi.org/10.1016/j.jcmgh.2020.01.005

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Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning

During the suckling-to-weaning transition, the intestinal epithelium matures, allowing digestion of solid food. Transplantation experiments with rodent fetal epithelium into subcutaneous tissue of adult animals suggest that this transition is intrinsically programmed and occurs in the absence of dietary or hormonal signals. Here, we show that organoids derived from mouse primary fetal intestinal epithelial cells express markers of late fetal and neonatal development. In a stable culture medium, these fetal epithelium-derived organoids lose all markers of neonatal epithelium and start expressing hallmarks of adult epithelium in a time frame that mirrors epithelial maturation in vivo In vitro postnatal development of the fetal-derived organoids accelerates by dexamethasone, a drug used to accelerate intestinal maturation in vivo Together, our data show that organoids derived from fetal epithelium undergo suckling-to-weaning transition, that the speed of maturation can be modulated, and that fetal organoids can be used to model the molecular mechanisms of postnatal epithelial maturation.

Authors

Marit Navis, Tania Martins Garcia, Ingrid B Renes, Jacqueline Lm Vermeulen, Sander Meisner, Manon E Wildenberg, GIjs R van den Brink, Ruurd M van Elburg, Vanesa Muncan

Link

https://doi.org/10.15252/embr.201846221