Publications by authors named "Jarno Drost"

42 Publications

Defects in 8-oxo-guanine repair pathway cause high frequency of C > A substitutions in neuroblastoma.

Proc Natl Acad Sci U S A 2021 Sep;118(36)

Princess Máxima Center for Pediatric Oncology, 3584 CS, Utrecht, The Netherlands;

Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. Our whole-genome sequencing data analysis revealed a high frequency of somatic cytosine > adenine (C > A) substitutions in primary neuroblastoma tumors, which was associated with poor survival. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of or Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. Tumor organoid models with CNL of or show increased 8-oxoG levels compared to wild-type cells. We used CRISPR-Cas9 genome editing to create knockout clones of and in neuroblastoma cells. Whole-genome sequencing of single-cell and knockout clones identified an increased accumulation of C > A substitutions. Mutational signature analysis of these and knockout clones revealed enrichment for C > A signatures 18 and 36, respectively. Clustering analysis showed that the knockout clones group together with tumors containing or CNL. In conclusion, we demonstrate that defects in 8-oxoG repair cause accumulation of C > A substitutions in neuroblastoma, which contributes to mutagenesis and tumor evolution.
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http://dx.doi.org/10.1073/pnas.2007898118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8433536PMC
September 2021

Organoid-based drug screening reveals neddylation as therapeutic target for malignant rhabdoid tumors.

Cell Rep 2021 Aug;36(8):109568

Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS Utrecht, the Netherlands; Oncode Institute, Heidelberglaan 25, 3584 CS Utrecht, the Netherlands. Electronic address:

Malignant rhabdoid tumors (MRTs) represent one of the most aggressive childhood malignancies. No effective treatment options are available, and prognosis is, therefore, dismal. Previous studies have demonstrated that tumor organoids capture the heterogeneity of patient tumors and can be used to predict patient response to therapy. Here, we perform drug screening on patient-derived normal and tumor organoids to identify MRT-specific therapeutic vulnerabilities. We identify neddylation inhibitor MLN4924 as a potential therapeutic agent. Mechanistically, we find increased neddylation in MRT organoids and tissues and show that MLN4924 induces a cytotoxic response via upregulation of the unfolded protein response. Lastly, we demonstrate in vivo efficacy in an MRT PDX mouse model, in which single-agent MLN4924 treatment significantly extends survival. Our study demonstrates that organoids can be used to find drugs selectively targeting tumor cells while leaving healthy cells unharmed and proposes neddylation inhibition as a therapeutic strategy in MRT.
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http://dx.doi.org/10.1016/j.celrep.2021.109568DOI Listing
August 2021

Prognostic Factors for Wilms Tumor Recurrence: A Review of the Literature.

Cancers (Basel) 2021 Jun 23;13(13). Epub 2021 Jun 23.

Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS Utrecht, The Netherlands.

In high-income countries, the overall survival of children with Wilms tumors (WT) is ~90%. However, overall, 15% of patients experience tumor recurrence. The adverse prognostic factors currently used for risk stratification (advanced stage, high risk histology, and combined loss of heterozygosity at 1p and 16q in chemotherapy-naïve WTs) are present in only one third of these cases, and the significance of these factors is prone to change with advancing knowledge and improved treatment regimens. Therefore, we present a comprehensive, updated overview of the published prognostic variables for WT recurrence, ranging from patient-, tumor- and treatment-related characteristics to geographic and socioeconomic factors. Improved first-line treatment regimens based on clinicopathological characteristics and advancing knowledge on copy number variations unveil the importance of further investigating the significance of biological markers for WT recurrence in international collaborations.
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http://dx.doi.org/10.3390/cancers13133142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8268923PMC
June 2021

Single cell derived mRNA signals across human kidney tumors.

Nat Commun 2021 06 23;12(1):3896. Epub 2021 Jun 23.

Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.

Tumor cells may share some patterns of gene expression with their cell of origin, providing clues into the differentiation state and origin of cancer. Here, we study the differentiation state and cellular origin of 1300 childhood and adult kidney tumors. Using single cell mRNA reference maps of normal tissues, we quantify reference "cellular signals" in each tumor. Quantifying global differentiation, we find that childhood tumors exhibit fetal cellular signals, replacing the presumption of "fetalness" with a quantitative measure of immaturity. By contrast, in adult cancers our assessment refutes the suggestion of dedifferentiation towards a fetal state in most cases. We find an intimate connection between developmental mesenchymal populations and childhood renal tumors. We demonstrate the diagnostic potential of our approach with a case study of a cryptic renal tumor. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer.
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http://dx.doi.org/10.1038/s41467-021-23949-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8222373PMC
June 2021

Revealing the spatio-phenotypic patterning of cells in healthy and tumor tissues with mLSR-3D and STAPL-3D.

Nat Biotechnol 2021 10 3;39(10):1239-1245. Epub 2021 Jun 3.

Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.

Despite advances in three-dimensional (3D) imaging, it remains challenging to profile all the cells within a large 3D tissue, including the morphology and organization of the many cell types present. Here, we introduce eight-color, multispectral, large-scale single-cell resolution 3D (mLSR-3D) imaging and image analysis software for the parallelized, deep learning-based segmentation of large numbers of single cells in tissues, called segmentation analysis by parallelization of 3D datasets (STAPL-3D). Applying the method to pediatric Wilms tumor, we extract molecular, spatial and morphological features of millions of cells and reconstruct the tumor's spatio-phenotypic patterning. In situ population profiling and pseudotime ordering reveals a highly disorganized spatial pattern in Wilms tumor compared to healthy fetal kidney, yet cellular profiles closely resembling human fetal kidney cells could be observed. In addition, we identify previously unreported tumor-specific populations, uniquely characterized by their spatial embedding or morphological attributes. Our results demonstrate the use of combining mLSR-3D and STAPL-3D to generate a comprehensive cellular map of human tumors.
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http://dx.doi.org/10.1038/s41587-021-00926-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611791PMC
October 2021

Generation of Human Kidney Tubuloids from Tissue and Urine.

J Vis Exp 2021 04 16(170). Epub 2021 Apr 16.

Princess Máxima Center for Pediatric Oncology; Oncode Institute;

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high efficiency. Normal kidney tubuloids recapitulate many aspects of their tissue of origin. They represent distinct nephron segments - most notably of the proximal tubule, loop of Henle, distal tubules, and collecting duct - and can be used to study normal kidney physiology. Furthermore, tubuloid technology facilitates disease modeling, e.g., for infectious diseases as well as for cancer. Obtaining kidney epithelial cells for tubuloid generation is, however, dependent on leftover surgical material (such as partial) nephrectomies) or needle biopsies. The ability to grow tubuloids from urine would provide an alternative, less invasive source of healthy kidney epithelial cells. It has been previously shown that tubuloid cultures can be successfully generated from only a few milliliters of freshly collected urine. This article describes the protocols to generate and propagate ASC-derived human kidney tubuloid cultures from tissue and urine samples.
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http://dx.doi.org/10.3791/62404DOI Listing
April 2021

LGR6 marks nephron progenitor cells.

Dev Dyn 2021 Nov 6;250(11):1568-1583. Epub 2021 May 6.

Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.

Background: Nephron progenitor cells (NPCs) undergo a stepwise process to generate all mature nephron structures. Mesenchymal to epithelial transition (MET) is considered a multistep process of NPC differentiation to ensure progressive establishment of new nephrons. However, despite this important role, to date, no marker for NPCs undergoing MET in the nephron exists.

Results: Here, we identify LGR6 as a NPC marker, expressed in very early cap mesenchyme, pre-tubular aggregates, renal vesicles, and in segments of S-shaped bodies, following the trajectory of MET. By using a lineage tracing approach in embryonic explants in combination with confocal imaging and single-cell RNA sequencing, we provide evidence for the multiple fates of LGR6+ cells during embryonic nephrogenesis. Moreover, by using long-term in vivo lineage tracing, we show that postnatal LGR6+ cells are capable of generating the multiple lineages of the nephrons.

Conclusions: Given the profound early mesenchymal expression and MET signature of LGR6 cells, together with the lineage tracing of mesenchymal LGR6 cells, we conclude that LGR6+ cells contribute to all nephrogenic segments by undergoing MET. LGR6+ cells can therefore be considered an early committed NPC population during embryonic and postnatal nephrogenesis with potential regenerative capability.
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http://dx.doi.org/10.1002/dvdy.346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8597161PMC
November 2021

Modeling of Embryonal Tumors.

Front Cell Dev Biol 2021 26;9:640633. Epub 2021 Feb 26.

Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands.

A subset of pediatric tumors affects very young children and are thought to arise during fetal life. A common theme is that these embryonal tumors hijack developmental programs, causing a block in differentiation and, as a consequence, unrestricted proliferation. Embryonal tumors, therefore typically maintain an embryonic gene signature not found in their differentiated progeny. Still, the processes underpinning malignant transformation remain largely unknown, which is hampering therapeutic innovation. To gain more insight into these processes, and research models are indispensable. However, embryonic development is an extremely dynamic process with continuously changing cellular identities, making it challenging to define cells-of-origin. This is crucial for the development of representative models, as targeting the wrong cell or targeting a cell within an incorrect developmental time window can result in completely different phenotypes. Recent innovations in cell models may provide more versatile platforms to study embryonal tumors in a scalable manner. In this review, we outline different models that can be explored to study embryonal tumorigenesis and for therapy development.
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http://dx.doi.org/10.3389/fcell.2021.640633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952537PMC
February 2021

Somatic mutations and single-cell transcriptomes reveal the root of malignant rhabdoid tumours.

Nat Commun 2021 03 3;12(1):1407. Epub 2021 Mar 3.

Princess Máxima Center for Pediatric Oncology, 3584CS, Utrecht, the Netherlands.

Malignant rhabdoid tumour (MRT) is an often lethal childhood cancer that, like many paediatric tumours, is thought to arise from aberrant fetal development. The embryonic root and differentiation pathways underpinning MRT are not firmly established. Here, we study the origin of MRT by combining phylogenetic analyses and single-cell mRNA studies in patient-derived organoids. Comparison of somatic mutations shared between cancer and surrounding normal tissues places MRT in a lineage with neural crest-derived Schwann cells. Single-cell mRNA readouts of MRT differentiation, which we examine by reverting the genetic driver mutation underpinning MRT, SMARCB1 loss, suggest that cells are blocked en route to differentiating into mesenchyme. Quantitative transcriptional predictions indicate that combined HDAC and mTOR inhibition mimic MRT differentiation, which we confirm experimentally. Our study defines the developmental block of MRT and reveals potential differentiation therapies.
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http://dx.doi.org/10.1038/s41467-021-21675-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930245PMC
March 2021

Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells.

Elife 2021 Jan 18;10. Epub 2021 Jan 18.

Amsterdam UMC, location VUmc, Vrije Universiteit Amsterdam, Clinical Genetics, Cancer Center Amsterdam, Amsterdam, Netherlands.

Germline mutations in the Folliculin () tumor suppressor gene cause Birt-Hogg-Dubé (BHD) syndrome, a rare autosomal dominant disorder predisposing carriers to kidney tumors. is a conserved, essential gene linked to diverse cellular processes but the mechanism by which prevents kidney cancer remains unknown. Here, we show that disrupting in human renal tubular epithelial cells (RPTEC/TERT1) activates TFE3, upregulating expression of its E-box targets, including RRAGD and GPNMB, without modifying mTORC1 activity. Surprisingly, the absence of FLCN or its binding partners FNIP1/FNIP2 induces interferon response genes independently of interferon. Mechanistically, FLCN loss promotes STAT2 recruitment to chromatin and slows cellular proliferation. Our integrated analysis identifies STAT1/2 signaling as a novel target of FLCN in renal cells and BHD tumors. STAT1/2 activation appears to counterbalance TFE3-directed hyper-proliferation and may influence immune responses. These findings shed light on unique roles of FLCN in human renal tumorigenesis and pinpoint candidate prognostic biomarkers.
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http://dx.doi.org/10.7554/eLife.61630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899648PMC
January 2021

Unmet needs for relapsed or refractory Wilms tumour: Mapping the molecular features, exploring organoids and designing early phase trials - A collaborative SIOP-RTSG, COG and ITCC session at the first SIOPE meeting.

Eur J Cancer 2021 02 18;144:113-122. Epub 2020 Dec 18.

Department of Medical Oncology and Hematology, Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale Dei Tumori, Milan, Italy.

Wilms tumour (WT) accounts for about 6% of all childhood cancers and overall survival of WT is about 90% in international protocols. However, for WT subgroups with much poorer prognoses, i.e. typically high-risk (unfavorable) histology and/or relapse, there is an unmet need to better understand the biology of WT and to translate biological findings into clinics through early phase clinical trials that evaluate innovative therapies. The main challenges are the small numbers of children suitable for early phase trials, the genetic heterogeneity of WT and the low number of somatic mutations that are currently considered 'druggable'. Accordingly, a joint meeting between clinical and biology experts from the international cooperative groups of the Renal Tumour Study Group of the International Society of Paediatric Oncology, the Renal Tumour Committee of the Children's Oncology Group and the European Innovative Therapies for Children with Cancer consortium and parents representatives was organised during the first SIOPE meeting in Prague, 2019. We reviewed WT molecular features, ongoing/planned early phase trials and explored available knowledge on organoid technology. The key messages were: (1) relapsed WT should undergo whenever possible thorough molecular characterization and be enrolled in protocols or trials with systematic data collecting and reporting; (2) WT displays few known 'actionable' targets and currently no novel agent has appeared promising; (3) we need to improve the enrolment rate of WT candidates in early phase trials especially for the relatively small subgroup of relapses with an adverse prognostic signature; (4) despite some agnostic early phase trials existing, development of WT-focused trials are warranted; (5) growing organoids with parallel testing of drug panels seems feasible and may direct individual treatment and encourage clinical researchers to incorporate the most promising agents into early phase trials.
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http://dx.doi.org/10.1016/j.ejca.2020.11.012DOI Listing
February 2021

Next-Generation Surrogate Wnts Support Organoid Growth and Deconvolute Frizzled Pleiotropy In Vivo.

Cell Stem Cell 2020 11 19;27(5):840-851.e6. Epub 2020 Aug 19.

Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.

Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.
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http://dx.doi.org/10.1016/j.stem.2020.07.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655723PMC
November 2020

Characteristics and Outcome of Children with Renal Cell Carcinoma: A Narrative Review.

Cancers (Basel) 2020 Jul 3;12(7). Epub 2020 Jul 3.

Princess Máxima Center for Pediatric Oncology, 3584 CS Utrecht, The Netherlands.

Pediatric renal cell carcinoma (RCC) is a rare type of kidney cancer, most commonly occurring in teenagers and young adolescents. Few relatively large series of pediatric RCC have been reported. Knowledge of clinical characteristics, outcome and treatment strategies are often based on the more frequently occurring adult types of RCC. However, published pediatric data suggest that clinical, molecular and histological characteristics of pediatric RCC differ from adult RCC. This paper summarizes reported series consisting of ≥10 RCC pediatric patients in order to create an up-to-date overview of the clinical and histopathological characteristics, treatment and outcome of pediatric RCC patients.
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http://dx.doi.org/10.3390/cancers12071776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407101PMC
July 2020

Paediatric metanephric tumours: a clinicopathological and molecular characterisation.

Crit Rev Oncol Hematol 2020 Jun 24;150:102970. Epub 2020 Apr 24.

Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.

To characterize metanephric tumours in children, we performed a literature review investigating paediatric metanephric adenomas (MA), metanephric stromal tumours (MST) and metanephric adenofibromas (MAF). Including two patients from our own institution (MA, MAF), 110 individual cases (41 MA, 20 MAF, 49 MST) were identified. Additionally, fifteen composite tumours were identified, with areas of MA/MAF and Wilms tumour (WT) or papillary carcinoma. No distinct clinical or radiological features could be defined. In pure metanephric tumours, histologically proven distant metastases were reported once (MA), relapse was reported once (MST) and one tumour-related death occurred (MST). Somatic BRAF-V600E mutations were tested in 15 cases, and identified in 3/6 MA, 3/3 MAF, and 6/6 MST. In our institution the MA harboured a somatic KRAS-G12R mutation. Overall, paediatric metanephric tumours are difficult to discriminate from other renal tumours at presentation, behave relatively benign, and the occurrence of composite tumours warrants analysis of underlying (genetic) pathways.
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http://dx.doi.org/10.1016/j.critrevonc.2020.102970DOI Listing
June 2020

Organoid models of childhood kidney tumours.

Nat Rev Urol 2020 Jun;17(6):311-313

Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands.

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http://dx.doi.org/10.1038/s41585-020-0315-yDOI Listing
June 2020

An organoid biobank for childhood kidney cancers that captures disease and tissue heterogeneity.

Nat Commun 2020 03 11;11(1):1310. Epub 2020 Mar 11.

Oncode Institute, Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS, Utrecht, The Netherlands.

Kidney tumours are among the most common solid tumours in children, comprising distinct subtypes differing in many aspects, including cell-of-origin, genetics, and pathology. Pre-clinical cell models capturing the disease heterogeneity are currently lacking. Here, we describe the first paediatric cancer organoid biobank. It contains tumour and matching normal kidney organoids from over 50 children with different subtypes of kidney cancer, including Wilms tumours, malignant rhabdoid tumours, renal cell carcinomas, and congenital mesoblastic nephromas. Paediatric kidney tumour organoids retain key properties of native tumours, useful for revealing patient-specific drug sensitivities. Using single cell RNA-sequencing and high resolution 3D imaging, we further demonstrate that organoid cultures derived from Wilms tumours consist of multiple different cell types, including epithelial, stromal and blastemal-like cells. Our organoid biobank captures the heterogeneity of paediatric kidney tumours, providing a representative collection of well-characterised models for basic cancer research, drug-screening and personalised medicine.
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http://dx.doi.org/10.1038/s41467-020-15155-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066173PMC
March 2020

Embryonal precursors of Wilms tumor.

Science 2019 12;366(6470):1247-1251

Great Ormond Street Hospital for Children NHS Foundation Trust, London WC1N 3JH, UK.

Adult cancers often arise from premalignant clonal expansions. Whether the same is true of childhood tumors has been unclear. To investigate whether Wilms tumor (nephroblastoma; a childhood kidney cancer) develops from a premalignant background, we examined the phylogenetic relationship between tumors and corresponding normal tissues. In 14 of 23 cases studied (61%), we found premalignant clonal expansions in morphologically normal kidney tissues that preceded tumor development. These clonal expansions were defined by somatic mutations shared between tumor and normal tissues but absent from blood cells. We also found hypermethylation of the locus, a known driver of Wilms tumor development, in 58% of the expansions. Phylogenetic analyses of bilateral tumors indicated that clonal expansions can evolve before the divergence of left and right kidney primordia. These findings reveal embryonal precursors from which unilateral and multifocal cancers develop.
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http://dx.doi.org/10.1126/science.aax1323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914378PMC
December 2019

Xenograft and organoid model systems in cancer research.

EMBO J 2019 08 8;38(15):e101654. Epub 2019 Jul 8.

Oncode Institute, Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.

Patient-derived tumour xenografts and tumour organoids have become important preclinical model systems for cancer research. Both models maintain key features from their parental tumours, such as genetic and phenotypic heterogeneity, which allows them to be used for a wide spectrum of applications. In contrast to patient-derived xenografts, organoids can be established and expanded with high efficiency from primary patient material. On the other hand, xenografts retain tumour-stroma interactions, which are known to contribute to tumorigenesis. In this review, we discuss recent advances in patient-derived tumour xenograft and tumour organoid model systems and compare their promises and challenges as preclinical models in cancer research.
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http://dx.doi.org/10.15252/embj.2019101654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6670015PMC
August 2019

Tubuloids derived from human adult kidney and urine for personalized disease modeling.

Nat Biotechnol 2019 03 4;37(3):303-313. Epub 2019 Mar 4.

Hubrecht Institute-Royal Netherlands Academy of Arts and Sciences, Utrecht, the Netherlands.

Adult stem cell-derived organoids are three-dimensional epithelial structures that recapitulate fundamental aspects of their organ of origin. We describe conditions for the long-term growth of primary kidney tubular epithelial organoids, or 'tubuloids'. The cultures are established from human and mouse kidney tissue and can be expanded for at least 20 passages (>6 months) while retaining a normal number of chromosomes. In addition, cultures can be established from human urine. Human tubuloids represent proximal as well as distal nephron segments, as evidenced by gene expression, immunofluorescence and tubular functional analyses. We apply tubuloids to model infectious, malignant and hereditary kidney diseases in a personalized fashion. BK virus infection of tubuloids recapitulates in vivo phenomena. Tubuloids are established from Wilms tumors. Kidney tubuloids derived from the urine of a subject with cystic fibrosis allow ex vivo assessment of treatment efficacy. Finally, tubuloids cultured on microfluidic organ-on-a-chip plates adopt a tubular conformation and display active (trans-)epithelial transport function.
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http://dx.doi.org/10.1038/s41587-019-0048-8DOI Listing
March 2019

Profiling proliferative cells and their progeny in damaged murine hearts.

Proc Natl Acad Sci U S A 2018 12 7;115(52):E12245-E12254. Epub 2018 Dec 7.

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Centre Utrecht, 3584 CT Utrecht, The Netherlands;

The significance of cardiac stem cell (CSC) populations for cardiac regeneration remains disputed. Here, we apply the most direct definition of stem cell function (the ability to replace lost tissue through cell division) to interrogate the existence of CSCs. By single-cell mRNA sequencing and genetic lineage tracing using two Ki67 knockin mouse models, we map all proliferating cells and their progeny in homoeostatic and regenerating murine hearts. Cycling cardiomyocytes were only robustly observed in the early postnatal growth phase, while cycling cells in homoeostatic and damaged adult myocardium represented various noncardiomyocyte cell types. Proliferative postdamage fibroblasts expressing follistatin-like protein 1 (FSTL1) closely resemble neonatal cardiac fibroblasts and form the fibrotic scar. Genetic deletion of in cardiac fibroblasts results in postdamage cardiac rupture. We find no evidence for the existence of a quiescent CSC population, for transdifferentiation of other cell types toward cardiomyocytes, or for proliferation of significant numbers of cardiomyocytes in response to cardiac injury.
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http://dx.doi.org/10.1073/pnas.1805829115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310797PMC
December 2018

Organoids in cancer research.

Nat Rev Cancer 2018 07;18(7):407-418

Princess Máxima Centre for Paediatric Oncology, Utrecht, Netherlands.

The recent advances in in vitro 3D culture technologies, such as organoids, have opened new avenues for the development of novel, more physiological human cancer models. Such preclinical models are essential for more efficient translation of basic cancer research into novel treatment regimens for patients with cancer. Wild-type organoids can be grown from embryonic and adult stem cells and display self-organizing capacities, phenocopying essential aspects of the organs they are derived from. Genetic modification of organoids allows disease modelling in a setting that approaches the physiological environment. Additionally, organoids can be grown with high efficiency from patient-derived healthy and tumour tissues, potentially enabling patient-specific drug testing and the development of individualized treatment regimens. In this Review, we evaluate tumour organoid protocols and how they can be utilized as an alternative model for cancer research.
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http://dx.doi.org/10.1038/s41568-018-0007-6DOI Listing
July 2018

LncRNA-OIS1 regulates DPP4 activation to modulate senescence induced by RAS.

Nucleic Acids Res 2018 05;46(8):4213-4227

Division of Oncogenomics, Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.

Oncogene-induced senescence (OIS), provoked in response to oncogenic activation, is considered an important tumor suppressor mechanism. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nt without a protein-coding capacity. Functional studies showed that deregulated lncRNA expression promote tumorigenesis and metastasis and that lncRNAs may exhibit tumor-suppressive and oncogenic function. Here, we first identified lncRNAs that were differentially expressed between senescent and non-senescent human fibroblast cells. Using RNA interference, we performed a loss-function screen targeting the differentially expressed lncRNAs, and identified lncRNA-OIS1 (lncRNA#32, AC008063.3 or ENSG00000233397) as a lncRNA required for OIS. Knockdown of lncRNA-OIS1 triggered bypass of senescence, higher proliferation rate, lower abundance of the cell-cycle inhibitor CDKN1A and high expression of cell-cycle-associated genes. Subcellular inspection of lncRNA-OIS1 indicated nuclear and cytosolic localization in both normal culture conditions as well as following oncogene induction. Interestingly, silencing lncRNA-OIS1 diminished the senescent-associated induction of a nearby gene (Dipeptidyl Peptidase 4, DPP4) with established role in tumor suppression. Intriguingly, similar to lncRNA-OIS1, silencing DPP4 caused senescence bypass, and ectopic expression of DPP4 in lncRNA-OIS1 knockdown cells restored the senescent phenotype. Thus, our data indicate that lncRNA-OIS1 links oncogenic induction and senescence with the activation of the tumor suppressor DPP4.
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http://dx.doi.org/10.1093/nar/gky087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934637PMC
May 2018

A surgical orthotopic organoid transplantation approach in mice to visualize and study colorectal cancer progression.

Nat Protoc 2018 02 4;13(2):235-247. Epub 2018 Jan 4.

Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands.

Most currently available colorectal cancer (CRC) mouse models are not suitable for studying progression toward the metastatic stage. Recently, establishment of tumor organoid lines, either from murine CRC models or patients, and the possibility of engineering them with genome-editing technologies, have provided a large collection of tumor material faithfully recapitulating phenotypic and genetic heterogeneity of native tumors. To study tumor progression in the natural in vivo environment, we developed an orthotopic approach based on transplantation of CRC organoids into the cecal epithelium. The 20-min procedure is described in detail here and enables growth of transplanted organoids into a single tumor mass within the intestinal tract. Due to long latency, tumor cells are capable of spreading through the blood circulation and forming metastases at distant sites. This method is designed to generate tumors suitable for studying CRC progression, thereby providing the opportunity to visualize tumor cell dynamics in vivo in real time by intravital microscopy.
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http://dx.doi.org/10.1038/nprot.2017.137DOI Listing
February 2018

Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer.

Science 2017 10 14;358(6360):234-238. Epub 2017 Sep 14.

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center (UMC) Utrecht, 3584CT Utrecht, Netherlands.

Mutational processes underlie cancer initiation and progression. Signatures of these processes in cancer genomes may explain cancer etiology and could hold diagnostic and prognostic value. We developed a strategy that can be used to explore the origin of cancer-associated mutational signatures. We used CRISPR-Cas9 technology to delete key DNA repair genes in human colon organoids, followed by delayed subcloning and whole-genome sequencing. We found that mutation accumulation in organoids deficient in the mismatch repair gene is driven by replication errors and accurately models the mutation profiles observed in mismatch repair-deficient colorectal cancers. Application of this strategy to the cancer predisposition gene , which encodes a base excision repair protein, revealed a mutational footprint (signature 30) previously observed in a breast cancer cohort. We show that signature 30 can arise from germline mutations.
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http://dx.doi.org/10.1126/science.aao3130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038908PMC
October 2017

Translational applications of adult stem cell-derived organoids.

Development 2017 03;144(6):968-975

Cancer Genomics Netherlands, UMC Utrecht, Utrecht 3584CG, The Netherlands.

Adult stem cells from a variety of organs can be expanded long-term as three-dimensional organotypic structures termed organoids. These adult stem cell-derived organoids retain their organ identity and remain genetically stable over long periods of time. The ability to grow organoids from patient-derived healthy and diseased tissue allows for the study of organ development, tissue homeostasis and disease. In this Review, we discuss the generation of adult stem cell-derived organoid cultures and their applications in disease modeling, personalized cancer therapy and regenerative medicine.
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http://dx.doi.org/10.1242/dev.140566DOI Listing
March 2017

Genetic dissection of colorectal cancer progression by orthotopic transplantation of engineered cancer organoids.

Proc Natl Acad Sci U S A 2017 03 7;114(12):E2357-E2364. Epub 2017 Mar 7.

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center (UMC) Utrecht, 3584 CT Utrecht, The Netherlands;

In the adenoma-carcinoma sequence, it is proposed that intestinal polyps evolve through a set of defined mutations toward metastatic colorectal cancer (CRC). Here, we dissect this adenoma-carcinoma sequence in vivo by using an orthotopic organoid transplantation model of human colon organoids engineered to harbor different CRC mutation combinations. We demonstrate that sequential accumulation of oncogenic mutations in Wnt, EGFR, P53, and TGF-β signaling pathways facilitates efficient tumor growth, migration, and metastatic colonization. We show that reconstitution of specific niche signals can restore metastatic growth potential of tumor cells lacking one of the oncogenic mutations. Our findings imply that the ability to metastasize-i.e., to colonize distant sites-is the direct consequence of the loss of dependency on specific niche signals.
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http://dx.doi.org/10.1073/pnas.1701219114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373343PMC
March 2017

Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening.

Elife 2016 11 15;5. Epub 2016 Nov 15.

Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht, Netherlands.

Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic mutation. Using this panel, we evaluated RAS pathway inhibitors and drug combinations that are currently in clinical trial for RAS mutant cancers. Presence of mutant RAS correlated strongly with resistance to these targeted therapies. This was observed in tumorigenic as well as in normal organoids. Moreover, dual inhibition of the EGFR-MEK-ERK pathway in RAS mutant organoids induced a transient cell-cycle arrest rather than cell death. In vivo drug response of xenotransplanted RAS mutant organoids confirmed this growth arrest upon pan-HER/MEK combination therapy. Altogether, our studies demonstrate the potential of patient-derived CRC organoid libraries in evaluating inhibitors and drug combinations in a preclinical setting.
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http://dx.doi.org/10.7554/eLife.18489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127645PMC
November 2016

The Generation of Organoids for Studying Wnt Signaling.

Methods Mol Biol 2016 ;1481:141-59

Hubrecht Institute-KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.

We established an in vitro culture model in which intestinal epithelial stem cells can grow into three-dimensional, ever-expanding epithelial organoids that retain their original organ identity and genetic stability. Moreover, organoids can easily be genetically modified using different genome modification strategies, including viral delivery of transgenes and CRISPR/Cas9 technology. These combined characteristics make them a useful in vitro model system to study many biological processes including the contribution of cellular signaling pathways to tissue homeostasis and disease. Here we describe our current laboratory protocols to establish human intestinal organoids and how to genetically modify both mouse and human intestinal organoids to study cellular signaling pathways, specifically Wnt signaling. Moreover, we provide a detailed protocol for lentiviral transduction and CRISPR/Cas9-mediated genome modification of organoid cultures.
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http://dx.doi.org/10.1007/978-1-4939-6393-5_15DOI Listing
January 2018

Who Is in the Driver's Seat: Tracing Cancer Genes Using CRISPR-Barcoding.

Mol Cell 2016 08;63(3):352-4

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584CT Utrecht, the Netherlands; Cancer Genomics Netherlands, UMC Utrecht, 3584CG Utrecht, the Netherlands. Electronic address:

Intratumor heterogeneity is thought to be the driving force of tumor evolution and therapy resistance. Yet tools to study these processes are limited. In this issue, Guernet et al. (2016) devised clustered regularly interspaced short palindromic repeats (CRISPR)-barcoding to functionally annotate specific mutations and study clonal evolution in heterogeneous cell populations.
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http://dx.doi.org/10.1016/j.molcel.2016.07.013DOI Listing
August 2016

TGFβ signaling directs serrated adenomas to the mesenchymal colorectal cancer subtype.

EMBO Mol Med 2016 07 1;8(7):745-60. Epub 2016 Jul 1.

Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands Cancer Genomics Center, Amsterdam, The Netherlands

The heterogeneous nature of colorectal cancer (CRC) complicates prognosis and is suggested to be a determining factor in the efficacy of adjuvant therapy for individual patients. Based on gene expression profiling, CRC is currently classified into four consensus molecular subtypes (CMSs), characterized by specific biological programs, thus suggesting the existence of unifying developmental drivers for each CMS Using human organoid cultures, we investigated the role of such developmental drivers at the premalignant stage of distinct CRC subtypes and found that TGFβ plays an important role in the development of the mesenchymal CMS4, which is of special interest due to its association with dismal prognosis. We show that in tubular adenomas (TAs), which progress to classical CRCs, the dominating response to TGFβ is death by apoptosis. By contrast, induction of a mesenchymal phenotype upon TGFβ treatment prevails in a genetically engineered organoid culture carrying a BRAF(V) (600E) mutation, constituting a model system for sessile serrated adenomas (SSAs). Our data indicate that TGFβ signaling is already active in SSA precursor lesions and that TGFβ is a critical cue for directing SSAs to the mesenchymal, poor-prognosis CMS4 of CRC.
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http://dx.doi.org/10.15252/emmm.201606184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931289PMC
July 2016
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