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Princess Máxima Center for Pediatric Oncology

8 ARTICLES PUBLISHED IN JoVE

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Cancer Research

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models
Rocco Sciarrillo 1,2,3, Anna Wojtuszkiewicz 1, Irsan E. Kooi 4, Valentina E. Gómez 3, Ugo Boggi 5, Gerrit Jansen 6, Gert-Jan Kaspers 1,7, Jacqueline Cloos *1, Elisa Giovannetti *3,8,9
1Department of Pediatric Oncology/Hematology, VU University Medical Center, 2Department of Hematology, VU University Medical Center, 3Department of Medical Oncology, VU University Medical Center, 4Department of Clinical Genetics, VU University Medical Center, 5Division of General and Transplant Surgery, Azienda Ospedaliera Universitaria Pisana, Universita’ di Pisa, 6Amsterdam Immunology and Rheumatology Center, VU University Medical Center, 7Princess Máxima Center for Pediatric Oncology, 8Cancer Pharmacology Lab, AIRC Start-Up Unit, University of Pisa, 9Institute of Nanoscience and Nanotechnology, CNR-Nano

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

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Medicine

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients
Sylvia F. Boj 1, Annelotte M. Vonk 2, Marvin Statia 1, Jinyi Su 1, Johanna F. Dekkers 3, Robert R. G. Vries 1, Jeffrey M. Beekman 2, Hans Clevers 1,4
1Foundation Hubrecht Organoid Technology, 2Department of Pediatric Pulmonology, Regenerative Medicine Centre Utrecht, Wilhelmina Children's Hospital, University Medical Centre Utrecht, 3Department of Stem Cells and Cancer, Walter and Eliza Hall Institute of Medical Research, 4Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

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Cancer Research

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia
Jacqueline Cloos *1,2, Jeffrey R. Harris *3, Jeroen J.W.M. Janssen 1, Angele Kelder 1, F. Huang 3, Gerrit Sijm 1, Maike Vonk 1, Alexander N. Snel 1, Jennifer R. Scheick 1, Willemijn J. Scholten 1, Jannemieke Carbaat-Ham 1, Dennis Veldhuizen 1, Diana Hanekamp 1, Yvonne J.M. Oussoren-Brockhoff 1, Gertjan J.L. Kaspers 2,4, Gerrit J. Schuurhuis 1, A. Kate Sasser 3, Gert Ossenkoppele 1
1Department of Hematology, VU University Medical Center, 2Pediatric Oncology/Hematology, VU University Medical Center, 3Janssen Research & Development, LLC, 4Princess Máxima Center for Pediatric Oncology

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

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Genetics

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector
Mariliis Tark-Dame 1, Blaise Weber 1, Mara de Sain 1, Damar Tri Anggoro 1, Rechien Bader 1, Aimee Walmsley 1, Rurika Oka 1, Maike Stam 1
1Swammerdam Institute for Life Sciences, University of Amsterdam

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

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Genetics

Characterizing Mutational Load and Clonal Composition of Human Blood
Axel Rosendahl Huber 1, Freek Manders 1, Rurika Oka 1, Ruben van Boxtel 1
1Princess Máxima Center for Pediatric Oncology

Somatic mutation patterns in cells reflect previous mutagenic exposure and can reveal developmental lineage relationships. Presented here is a methodology to catalogue and analyze somatic mutations in individual hematopoietic stem and progenitor cells.

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Biology

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids
Ravian L. van Ineveld *1,2,3, Hendrikus C.R. Ariese *1,2,3, Ellen J. Wehrens 1,2,3, Johanna F. Dekkers *1,2,3,4, Anne C. Rios *1,2,3
1Princess Máxima Center for Pediatric Oncology, 2Department of Cancer Research, Oncode Institute, Hubrecht Institute-KNAW Utrecht, 3Cancer Genomics Center (CGC), 4Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center (UMC) Utrecht

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

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Neuroscience

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents
Rianne Haumann *1,2, Elvin ’t Hart *2, Marc P. P. Derieppe 2, Helena C. Besse 3, Gertjan J. L. Kaspers 1,2, Eelco Hoving 2, Dannis G. van Vuurden 1,2, Esther Hulleman 1,2, Mario Ries 3
1Amsterdam UMC, Vrije Universiteit Amsterdam, Pediatric Oncology, Cancer Center Amsterdam, 2Princess Máxima Center for Pediatric Oncology, 3Imaging Division, Utrecht University

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

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Biology

Generation of Human Kidney Tubuloids from Tissue and Urine
Camilla Calandrini 1,2, Jarno Drost 1,2
1Princess Máxima Center for Pediatric Oncology, 2Oncode Institute

Human kidney tubuloid cultures represent a valuable in vitro model to study kidney physiology and disease. Tubuloids can be established from kidney tissue (healthy and diseased) as well as urine, the latter representing an easily obtainable and less invasive source of research material.

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