Publications by authors named "Anne C Rios"

34 Publications

LGR6 marks nephron progenitor cells.

Dev Dyn 2021 Apr 13. Epub 2021 Apr 13.

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 multi-step 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. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/dvdy.346DOI Listing
April 2021

The frontier of live tissue imaging across space and time.

Cell Stem Cell 2021 Apr;28(4):603-622

Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC 27708, USA. Electronic address:

What you see is what you get-imaging techniques have long been essential for visualization and understanding of tissue development, homeostasis, and regeneration, which are driven by stem cell self-renewal and differentiation. Advances in molecular and tissue modeling techniques in the last decade are providing new imaging modalities to explore tissue heterogeneity and plasticity. Here we describe current state-of-the-art imaging modalities for tissue research at multiple scales, with a focus on explaining key tradeoffs such as spatial resolution, penetration depth, capture time/frequency, and moieties. We explore emerging tissue modeling and molecular tools that improve resolution, specificity, and throughput.
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http://dx.doi.org/10.1016/j.stem.2021.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034393PMC
April 2021

Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids.

Nat Protoc 2021 Mar 10. Epub 2021 Mar 10.

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

Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7-21 d; generation of genetically manipulated clonal organoid cultures takes 14-21 d; and organoid expansion for xenotransplantation takes >4 weeks.
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http://dx.doi.org/10.1038/s41596-020-00474-1DOI Listing
March 2021

High-throughput drug screening reveals Pyrvinium pamoate as effective candidate against pediatric MLL-rearranged acute myeloid leukemia.

Transl Oncol 2021 May 2;14(5):101048. Epub 2021 Mar 2.

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

Pediatric MLL-rearranged acute myeloid leukemia (AML) has a generally unfavorable outcome, primarily due to relapse and drug resistance. To overcome these difficulties, new therapeutic agents are urgently needed. Yet, implementing novel drugs for clinical use is a time-consuming, laborious, costly and high-risk process. Therefore, we applied a drug-repositioning strategy by screening drug libraries, comprised of >4000 compounds that are mostly FDA-approved, in a high-throughput format on primary MLL-rearranged AML cells. Here we identified pyrvinium pamoate (pyrvinium) as a novel candidate drug effective against MLL-rearranged AML, eliminating all cell viability at <1000 nM. Additional screening of identified drug hits on non-leukemic bone marrow samples, resulted in a decrease in cell viability of ∼50% at 1000 nM pyrvinium, suggesting a therapeutic window for targeting leukemic cells specifically. Validation of pyrvinium on an extensive panel of AML cell lines and primary AML samples showed comparable viabilities as the drug screen data, with pyrvinium achieving IC values of <80 nM in these samples. Remarkably, pyrvinium also induced cell toxicity in primary MLL-AF10 AML cells, an MLL-rearrangement associated with a poor outcome. While pyrvinium is able to inhibit the Wnt pathway in other diseases, this unlikely explains the efficacy we observed as β-catenin was not expressed in the AML cells tested. Rather, we show that pyrvinium co-localized with the mitochondrial stain in cells, and hence may act by inhibiting mitochondrial respiration. Overall, this study shows that pyrvinium is highly effective against MLL-rearranged AML in vitro, and therefore represents a novel potential candidate for further studies in MLL-rearranged AML.
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http://dx.doi.org/10.1016/j.tranon.2021.101048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933809PMC
May 2021

Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue.

Nat Protoc 2021 Feb 24. Epub 2021 Feb 24.

Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

Multiphoton intravital imaging is essential for understanding cellular behavior and function in vivo. The adipose-rich environment of the mammary gland poses a unique challenge to in vivo microscopy due to light scattering that impedes high-resolution imaging. Here we provide a protocol for high-quality, six-color 3D intravital imaging of regions across the entire mouse mammary gland and associated tissues for several hours while maintaining tissue access for microdissection and labeling. An incision at the ventral midline and along the right hind leg creates a skin flap that is then secured to a raised platform skin side down. This allows for fluorescence-guided microdissection of connective tissue to provide unimpeded imaging of mammary ducts. A sealed imaging chamber over the skin flap creates a stable environment while maintaining access to large tissue regions for imaging with an upright microscope. We provide a strategy for imaging single cells and the tissue microenvironment utilizing multicolor Confetti lineage-tracing and additional dyes using custom-designed filters and sequential excitation with dual multiphoton lasers. Furthermore, we describe a strategy for simultaneous imaging and photomanipulation of single cells using the Olympus SIM scanner and provide steps for 3D video processing, visualization and high-dimensional analysis of single-cell behavior. We then provide steps for multiplexing intravital imaging with fixation, immunostaining, tissue clearing and 3D confocal imaging to associate cell behavior with protein expression. The skin-flap surgery and chamber preparation take 1.5 h, followed by up to 12 h of imaging. Applications range from basic filming in 1 d to 5 d for multiplexing and complex analysis.
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http://dx.doi.org/10.1038/s41596-020-00473-2DOI Listing
February 2021

Anti-GD2-IRDye800CW as a targeted probe for fluorescence-guided surgery in neuroblastoma.

Sci Rep 2020 10 19;10(1):17667. Epub 2020 Oct 19.

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

Neuroblastoma resection represents a major challenge in pediatric surgery, because of the high risk of complications. Fluorescence-guided surgery (FGS) could lower this risk by facilitating discrimination of tumor from normal tissue and is gaining momentum in adult oncology. Here, we provide the first molecular-targeted fluorescent agent for FGS in pediatric oncology, by developing and preclinically evaluating a GD2-specific tracer consisting of the immunotherapeutic antibody dinutuximab-beta, recently approved for neuroblastoma treatment, conjugated to near-infrared (NIR) fluorescent dye IRDye800CW. We demonstrated specific binding of anti-GD2-IRDye800CW to human neuroblastoma cells in vitro and in vivo using xenograft mouse models. Furthermore, we defined an optimal dose of 1 nmol, an imaging time window of 4 days after administration and show that neoadjuvant treatment with anti-GD2 immunotherapy does not interfere with fluorescence imaging. Importantly, as we observed universal, yet heterogeneous expression of GD2 on neuroblastoma tissue of a wide range of patients, we implemented a xenograft model of patient-derived neuroblastoma organoids with differential GD2 expression and show that even low GD2 expressing tumors still provide an adequate real-time fluorescence signal. Hence, the imaging advancement presented in this study offers an opportunity for improving surgery and potentially survival of a broad group of children with neuroblastoma.
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http://dx.doi.org/10.1038/s41598-020-74464-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573590PMC
October 2020

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids.

J Vis Exp 2020 06 5(160). Epub 2020 Jun 5.

Princess Máxima Center for Pediatric Oncology; Department of Cancer Research, Oncode Institute, Hubrecht Institute-KNAW Utrecht; Cancer Genomics Center (CGC);

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.
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http://dx.doi.org/10.3791/60709DOI Listing
June 2020

Tissue-resident ductal macrophages survey the mammary epithelium and facilitate tissue remodelling.

Nat Cell Biol 2020 05 27;22(5):546-558. Epub 2020 Apr 27.

Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.

Macrophages are diverse immune cells that reside in all tissues. Although macrophages have been implicated in mammary-gland function, their diversity has not been fully addressed. By exploiting high-resolution three-dimensional imaging and flow cytometry, we identified a unique population of tissue-resident ductal macrophages that form a contiguous network between the luminal and basal layers of the epithelial tree throughout postnatal development. Ductal macrophages are long lived and constantly survey the epithelium through dendrite movement, revealed via advanced intravital imaging. Although initially originating from embryonic precursors, ductal macrophages derive from circulating monocytes as they expand during puberty. Moreover, they undergo proliferation in pregnancy to maintain complete coverage of the epithelium in lactation, when they are poised to phagocytose milk-producing cells post-lactation and facilitate remodelling. Interestingly, ductal macrophages strongly resemble mammary tumour macrophages and form a network that pervades the tumour. Thus, the mammary epithelium programs specialized resident macrophages in both physiological and tumorigenic contexts.
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http://dx.doi.org/10.1038/s41556-020-0505-0DOI Listing
May 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

Understanding and Targeting Tumor Cell Invasion in Diffuse Intrinsic Pontine Glioma.

Front Oncol 2020 7;10:92. Epub 2020 Feb 7.

Princess Máxima Center for Pediatric Oncology, Utrecht, Netherlands.

Diffuse Intrinsic Pontine Glioma (DIPG) is a rare, highly aggressive pediatric brain tumor that originates in the pons. DIPG is untreatable and universally fatal, with a median life expectancy of less than a year. Resection is not an option, due to the anatomical location of the tumor, radiotherapy has limited effect and no chemotherapeutic or targeted treatment approach has proven to be successful. This poor prognosis is partly attributed to the tumor's highly infiltrative diffuse and invasive spread. Thus, targeting the invasive behavior of DIPG has the potential to be of therapeutic value. In order to target DIPG invasion successfully, detailed mechanistic knowledge on the underlying drivers is required. Here, we review both DIPG tumor cell's intrinsic molecular processes and extrinsic environmental factors contributing to DIPG invasion. Importantly, DIPG represents a heterogenous disease and through advances in whole-genome sequencing, different subtypes of disease based on underlying driver mutations are now being recognized. Recent evidence also demonstrates intra-tumor heterogeneity in terms of invasiveness and implies that highly infiltrative tumor subclones can enhance the migratory behavior of neighboring cells. This might partially be mediated by "tumor microtubes," long membranous extensions through which tumor cells connect and communicate, as well as through the secretion of extracellular vesicles. Some of the described processes involved in invasion are already being targeted in clinical trials. However, more research into the mechanisms of DIPG invasion is urgently needed and might result in the development of an effective therapy for children suffering from this devastating disease. We discuss the implications of newly discovered invasive mechanisms for therapeutic targeting and the challenges therapy development face in light of disease in the developing brain.
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http://dx.doi.org/10.3389/fonc.2020.00092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020612PMC
February 2020

Snake Venom Gland Organoids.

Cell 2020 01;180(2):233-247.e21

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, 3584 CT Utrecht, the Netherlands; Oncode Institute, Hubrecht Institute, 3584 CT Utrecht, the Netherlands; The Princess Maxima Center for Pediatric Oncology, 3584 CS Utrecht, the Netherlands. Electronic address:

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.
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http://dx.doi.org/10.1016/j.cell.2019.11.038DOI Listing
January 2020

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy.

J Vis Exp 2019 05 3(147). Epub 2019 May 3.

Cancer Genomics Netherlands, Prinses Máxima Center for Pediatric Oncology.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.
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http://dx.doi.org/10.3791/59278DOI Listing
May 2019

High-resolution 3D imaging of fixed and cleared organoids.

Nat Protoc 2019 06 3;14(6):1756-1771. Epub 2019 May 3.

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

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.
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http://dx.doi.org/10.1038/s41596-019-0160-8DOI Listing
June 2019

Comparison of 3-Dimensional and Augmented Reality Kidney Models With Conventional Imaging Data in the Preoperative Assessment of Children With Wilms Tumors.

JAMA Netw Open 2019 04 5;2(4):e192633. Epub 2019 Apr 5.

Department of Pediatric Surgery, Princess Máxima Center for Pediatric Oncology, Utrecht, the Netherlands.

Importance: Nephron-sparing surgery can be considered in well-defined cases of unilateral and bilateral Wilms tumors, but the surgical procedure can be very challenging for the pediatric surgeon to perform.

Objective: To assess the added value of personalized 3-dimensional (3-D) kidney models derived from conventional imaging data to enhance preoperative surgical planning.

Design, Setting, And Participants: In a survey study, the conventional imaging data of 10 Dutch children with Wilms tumors were converted to 3-D prints and augmented reality (AR) holograms and a panel of pediatric oncology surgeons (n = 7) assessed the quality of the different imaging methods during preoperative evaluation. Kidney models were created with 3-D printing and AR using a mixed reality headset for visualization.

Main Outcomes And Measures: Differences in the assessment of 4 anatomical structures (tumor, arteries, veins, and urinary collecting structures) using questionnaires. A Likert scale measured differences between the imaging methods, with scores ranging from 1 (completely disagree) to 5 (completely agree).

Results: Of the 10 patients, 7 were girls, and the mean (SD) age was 3.7 (1.7) years. Compared with conventional imaging, the 3-D print and the AR hologram models were evaluated by the surgeons to be superior for all anatomical structures: tumor (median scores for conventional imaging, 4.07; interquartile range [IQR], 3.62-4.15 vs 3-D print, 4.67; IQR, 4.14-4.71; P = .008 and AR hologram, 4.71; IQR, 4.26-4.75; P = .002); arteries (conventional imaging, 3.62; IQR, 3.43-3.93 vs 3-D print, 4.54; IQR, 4.32-4.71; P = .002 and AR hologram, 4.83; IQR, 4.64-4.86; P < .001), veins (conventional imaging, 3.46; IQR 3.39-3.62 vs 3-D print, 4.50; IQR, 4.39-4.68; P < .001 and AR hologram, 4.83; IQR, 4.71-4.86; P < .001), and urinary collecting structures (conventional imaging, 2.76; IQR, 2.42-3.00 vs 3-D print, 3.86; IQR, 3.64-4.39; P < .001 and AR hologram, 4.00; IQR, 3.93-4.58; P < .001). There were no differences in anatomical assessment between the two 3-D techniques (the 3-D print and AR hologram).

Conclusions And Relevance: In this study, the 3-D kidney models were associated with improved anatomical understanding among the surgeons and can be helpful in future preoperative planning of nephron-sparing surgery for Wilms tumors. These models may be considered as a supplementary visualization in clinical care.
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http://dx.doi.org/10.1001/jamanetworkopen.2019.2633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481457PMC
April 2019

Intraclonal Plasticity in Mammary Tumors Revealed through Large-Scale Single-Cell Resolution 3D Imaging.

Cancer Cell 2019 04 28;35(4):618-632.e6. Epub 2019 Mar 28.

Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC 3010, Australia. Electronic address:

Breast tumors are inherently heterogeneous, but the evolving cellular organization through neoplastic progression is poorly understood. Here we report a rapid, large-scale single-cell resolution 3D imaging protocol based on a one-step clearing agent that allows visualization of normal tissue architecture and entire tumors at cellular resolution. Imaging of multicolor lineage-tracing models of breast cancer targeted to either basal or luminal progenitor cells revealed profound clonal restriction during progression. Expression profiling of clones arising in Pten/Trp53-deficient tumors identified distinct molecular signatures. Strikingly, most clones harbored cells that had undergone an epithelial-to-mesenchymal transition, indicating widespread, inherent plasticity. Hence, an integrative pipeline that combines lineage tracing, 3D imaging, and clonal RNA sequencing technologies offers a comprehensive path for studying mechanisms underlying heterogeneity in whole tumors.
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http://dx.doi.org/10.1016/j.ccell.2019.02.010DOI Listing
April 2019

Long-term expanding human airway organoids for disease modeling.

EMBO J 2019 02 14;38(4). Epub 2019 Jan 14.

Oncode Institute, Hubrecht Institute-KNAW and UMC Utrecht, Utrecht, The Netherlands

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the study of hereditary, malignant, and infectious pulmonary disease.
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http://dx.doi.org/10.15252/embj.2018100300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6376275PMC
February 2019

Foxp1 Is Indispensable for Ductal Morphogenesis and Controls the Exit of Mammary Stem Cells from Quiescence.

Dev Cell 2018 12 25;47(5):629-644.e8. Epub 2018 Oct 25.

Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, VIC 3010, Australia. Electronic address:

Long-lived quiescent mammary stem cells (MaSCs) are presumed to coordinate the dramatic expansion of ductal epithelium that occurs through the different phases of postnatal development, but little is known about the molecular regulators that underpin their activation. We show that ablation of the transcription factor Foxp1 in the mammary gland profoundly impairs ductal morphogenesis, resulting in a rudimentary tree throughout life. Foxp1-deficient glands were highly enriched for quiescent Tspan8 MaSCs, which failed to become activated even in competitive transplantation assays, thus highlighting a cell-intrinsic defect. Foxp1 deletion also resulted in aberrant expression of basal genes in luminal cells, inferring a role in cell-fate decisions. Notably, Foxp1 was uncovered as a direct repressor of Tspan8 in basal cells, and deletion of Tspan8 rescued the defects in ductal morphogenesis elicited by Foxp1 loss. Thus, a single transcriptional regulator Foxp1 can control the exit of MaSCs from dormancy to orchestrate differentiation and development.
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http://dx.doi.org/10.1016/j.devcel.2018.10.001DOI Listing
December 2018

Imaging organoids: a bright future ahead.

Nat Methods 2018 01;15(1):24-26

Princess Maxima Center and the Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Utrecht, the Netherlands.

Organogenesis, tissue homeostasis and organ function involve complex spatial cellular organization and tissue dynamics. The underlying mechanisms of these processes, and how they are disrupted in disease, are challenging to address in vivo and ethically impossible to study in human. Organoids, three-dimensional (3D) stem cell cultures that self-organize into ex vivo 'mini-organs', now open a new window onto cellular processes within tissue. Light microscopy is a powerful approach to probe the cellular complexity that can be modeled with organoids. This combination of tools is already leading to exciting synergies in stem cell and cancer research.
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http://dx.doi.org/10.1038/nmeth.4537DOI Listing
January 2018

Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling.

Nat Commun 2017 11 20;8(1):1627. Epub 2017 Nov 20.

ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

The mammary epithelium comprises two primary cellular lineages, but the degree of heterogeneity within these compartments and their lineage relationships during development remain an open question. Here we report single-cell RNA profiling of mouse mammary epithelial cells spanning four developmental stages in the post-natal gland. Notably, the epithelium undergoes a large-scale shift in gene expression from a relatively homogeneous basal-like program in pre-puberty to distinct lineage-restricted programs in puberty. Interrogation of single-cell transcriptomes reveals different levels of diversity within the luminal and basal compartments, and identifies an early progenitor subset marked by CD55. Moreover, we uncover a luminal transit population and a rare mixed-lineage cluster amongst basal cells in the adult mammary gland. Together these findings point to a developmental hierarchy in which a basal-like gene expression program prevails in the early post-natal gland prior to the specification of distinct lineage signatures, and the presence of cellular intermediates that may serve as transit or lineage-primed cells.
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http://dx.doi.org/10.1038/s41467-017-01560-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696379PMC
November 2017

Identification of quiescent and spatially restricted mammary stem cells that are hormone responsive.

Nat Cell Biol 2017 03 13;19(3):164-176. Epub 2017 Feb 13.

ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.

Despite accumulating evidence for a mammary differentiation hierarchy, the basal compartment comprising stem cells remains poorly characterized. Through gene expression profiling of Lgr5 basal epithelial cells, we identify a new marker, Tetraspanin8 (Tspan8). Fractionation based on Tspan8 and Lgr5 expression uncovered three distinct mammary stem cell (MaSC) subsets in the adult mammary gland. These exist in a largely quiescent state but differ in their reconstituting ability, spatial localization, and their molecular and epigenetic signatures. Interestingly, the deeply quiescent MaSC subset (Lgr5Tspan8) resides within the proximal region throughout life, and has a transcriptome strikingly similar to that of claudin-low tumours. Lgr5Tspan8 cells appear to originate from the embryonic mammary primordia before switching to a quiescent state postnatally but can be activated by ovarian hormones. Our findings reveal an unexpected degree of complexity within the adult MaSC compartment and identify a dormant subset poised for activation in response to physiological stimuli.
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http://dx.doi.org/10.1038/ncb3471DOI Listing
March 2017

Derivation of a robust mouse mammary organoid system for studying tissue dynamics.

Development 2017 03 19;144(6):1065-1071. Epub 2016 Dec 19.

Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia

Advances in stem cell research have enabled the generation of 'mini organs' or organoids that recapitulate phenotypic traits of the original biological specimen. Although organoids have been demonstrated for multiple organ systems, there are more limited options for studying mouse mammary gland formation Here, we have built upon previously described culture assays to define culture conditions that enable the efficient generation of clonal organoid structures from single sorted basal mammary epithelial cells (MECs). Analysis of Confetti-reporter mice revealed the formation of uni-colored structures and thus the clonal nature of these organoids. High-resolution 3D imaging demonstrated that basal cell-derived complex organoids comprised an inner compartment of polarized luminal cells with milk-producing capacity and an outer network of elongated myoepithelial cells. Conversely, structures generated from luminal MECs rarely contained basal/myoepithelial cells. Moreover, flow cytometry and 3D microscopy of organoids generated from lineage-specific reporter mice established the bipotent capacity of basal cells and the restricted potential of luminal cells. In summary, we describe optimized conditions for the efficient generation of mouse mammary organoids that recapitulate features of mammary tissue architecture and function, and can be applied to understand tissue dynamics and cell-fate decisions.
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http://dx.doi.org/10.1242/dev.145045DOI Listing
March 2017

The complexities and caveats of lineage tracing in the mammary gland.

Breast Cancer Res 2016 11 25;18(1):116. Epub 2016 Nov 25.

Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia.

Lineage tracing is increasingly being utilised to probe different cell types that exist within the mammary gland. Whilst this technique is powerful for tracking cells in vivo and dissecting the roles of different cellular subsets in development, homeostasis and oncogenesis, there are important caveats associated with lineage tracing strategies. Here we highlight key parameters of particular relevance for the mammary gland. These include tissue preparation for whole-mount imaging, whereby the inclusion of enzymatic digestion can drastically alter tissue architecture and cell morphology, and therefore should be avoided. Other factors include the scoring of clones in three dimensions versus two dimensions, the timing of induction, and the marked variability in labelling efficiency that is evident not only between different mouse models harbouring a similar gene promoter but also within a given strain and even within a single mammary gland. Thus, it becomes crucial to visualise extensive areas of ductal tissue and to consider the intricacies of the methodology for lineage tracing studies on normal mammary development and on potential 'cells of origin' of cancer.
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http://dx.doi.org/10.1186/s13058-016-0774-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5124259PMC
November 2016

Lineage Tracing of Mammary Stem and Progenitor Cells.

Methods Mol Biol 2017 ;1501:291-308

Section of Molecular Cytology and Van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, Universityof Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands.

Lineage tracing analysis allows mammary epithelial cells to be tracked in their natural environment, thereby revealing cell fate and proliferation choices in the intact tissue. This technique is particularly informative for studying how stem cells build and maintain the mammary epithelium during development and pregnancy. Here we describe two experimental systems based on Cre/loxP technology (Cre/loxP and rtTA/tetO-Cre/loxP), which allow the inducible, permanent labeling of mammary epithelial cells following the administration of either tamoxifen or doxycycline.
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http://dx.doi.org/10.1007/978-1-4939-6475-8_15DOI Listing
January 2018

Cytoplasmic NOTCH and membrane-derived β-catenin link cell fate choice to epithelial-mesenchymal transition during myogenesis.

Elife 2016 05 24;5. Epub 2016 May 24.

Australian Regenerative Medicine Institute, Monash University, Clayton, Australia.

How cells in the embryo coordinate epithelial plasticity with cell fate decision in a fast changing cellular environment is largely unknown. In chick embryos, skeletal muscle formation is initiated by migrating Delta1-expressing neural crest cells that trigger NOTCH signaling and myogenesis in selected epithelial somite progenitor cells, which rapidly translocate into the nascent muscle to differentiate. Here, we uncovered at the heart of this response a signaling module encompassing NOTCH, GSK-3β, SNAI1 and β-catenin. Independent of its transcriptional function, NOTCH profoundly inhibits GSK-3β activity. As a result SNAI1 is stabilized, triggering an epithelial to mesenchymal transition. This allows the recruitment of β-catenin from the membrane, which acts as a transcriptional co-factor to activate myogenesis, independently of WNT ligand. Our results intimately associate the initiation of myogenesis to a change in cell adhesion and may reveal a general principle for coupling cell fate changes to EMT in many developmental and pathological processes.
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http://dx.doi.org/10.7554/eLife.14847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917337PMC
May 2016

Essential role for a novel population of binucleated mammary epithelial cells in lactation.

Nat Commun 2016 Apr 22;7:11400. Epub 2016 Apr 22.

Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.

The mammary gland represents a unique tissue to study organogenesis as it predominantly develops in the post-natal animal and undergoes dramatic morphogenetic changes during puberty and the reproductive cycle. The physiological function of the mammary gland is to produce milk to sustain the newborn. Here we view the lactating gland through three-dimensional confocal imaging of intact tissue. We observed that the majority of secretory alveolar cells are binucleated. These cells first arise in very late pregnancy due to failure of cytokinesis and are larger than mononucleated cells. Augmented expression of Aurora kinase-A and Polo-like kinase-1 at the lactogenic switch likely mediates the formation of binucleated cells. Our findings demonstrate an important physiological role for polyploid mammary epithelial cells in lactation, and based on their presence in five different species, suggest that binucleated cells evolved to maximize milk production and promote the survival of offspring across all mammalian species.
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http://dx.doi.org/10.1038/ncomms11400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4844753PMC
April 2016

EGF-mediated induction of Mcl-1 at the switch to lactation is essential for alveolar cell survival.

Nat Cell Biol 2015 Apr 2;17(4):365-75. Epub 2015 Mar 2.

1] ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.

Expansion and remodelling of the mammary epithelium requires a tight balance between cellular proliferation, differentiation and death. To explore cell survival versus cell death decisions in this organ, we deleted the pro-survival gene Mcl-1 in the mammary epithelium. Mcl-1 was found to be essential at multiple developmental stages including morphogenesis in puberty and alveologenesis in pregnancy. Moreover, Mcl-1-deficient basal cells were virtually devoid of repopulating activity, suggesting that this gene is required for stem cell function. Profound upregulation of the Mcl-1 protein was evident in alveolar cells at the switch to lactation, and Mcl-1 deficiency impaired lactation. Interestingly, EGF was identified as one of the most highly upregulated genes on lactogenesis and inhibition of EGF or mTOR signalling markedly impaired lactation, with concomitant decreases in Mcl-1 and phosphorylated ribosomal protein S6. These data demonstrate that Mcl-1 is essential for mammopoiesis and identify EGF as a critical trigger of Mcl-1 translation to ensure survival of milk-producing alveolar cells.
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http://dx.doi.org/10.1038/ncb3117DOI Listing
April 2015

In situ identification of bipotent stem cells in the mammary gland.

Nature 2014 Feb 26;506(7488):322-7. Epub 2014 Jan 26.

1] Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia [2] Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.

The mammary epithelium undergoes profound morphogenetic changes during development. Architecturally, it comprises two primary lineages, the inner luminal and outer myoepithelial cell layers. Two opposing concepts on the nature of mammary stem cells (MaSCs) in the postnatal gland have emerged. One model, based on classical transplantation assays, postulates that bipotent MaSCs have a key role in coordinating ductal epithelial expansion and maintenance in the adult gland, whereas the second model proposes that only unipotent MaSCs identified by lineage tracing contribute to these processes. Through clonal cell-fate mapping studies using a stochastic multicolour cre reporter combined with a new three-dimensional imaging strategy, we provide evidence for the existence of bipotent MaSCs as well as distinct long-lived progenitor cells. The cellular dynamics at different developmental stages support a model in which both stem and progenitor cells drive morphogenesis during puberty, whereas bipotent MaSCs coordinate ductal homeostasis and remodelling of the mouse adult gland.
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http://dx.doi.org/10.1038/nature12948DOI Listing
February 2014

[A tale of stolen kiss in muscle].

Med Sci (Paris) 2012 Mar 6;28(3):264-6. Epub 2012 Apr 6.

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http://dx.doi.org/10.1051/medsci/2012283012DOI Listing
March 2012