Publications

<p>Overview of cells per sample included in analysis.</p>

Small round cell sarcomas (SRCS) are highly aggressive tumors in soft tissues and bone of mostly children and young adults. Despite being different in many aspects, including genetics, possible cell-of-origin, and pathology, patients with any of these entities all receive the same therapeutic regimen. Although several pre-clinical models of Ewing sarcoma have been established, such as cell lines and patient-derived tumor xenografts, few models exist for other SRCS. Here, we describe a pediatric SRCS tumor organoid (tumoroid) biobank containing long-term tumoroid cultures with different translocations, including EWSR1::FLI1, EWSR1::ERG, CIC::DUX4, and BCOR-rearrangements. Using histology, whole genome sequencing and RNA sequencing, we demonstrate that these tumoroids retain histological characteristics, known marker gene expression and chromosomal rearangements of their matching patient tumors. In addition, we compare mutation clusters in the tumoroids across patient-matched longitudinal samples, which shows that cellular heterogeneity is maintained. Drug screening on the tumoroid models unveils entity-specific drug sensitivity to various cytotoxic compounds and targeted compounds, including MCL-1 inhibitors for CIC::DUX4 sarcomas. Taken together, this newly established SRCS patient-derived tumoroid biobank represents a promising source of material for future basic cancer research and drug screening.

Movie S5 from Oral Mucosal Organoids as a Potential Platform for Personalized Cancer Therapy

<div>Abstract<p>Increasing knowledge about signal transduction pathways as drivers of cancer growth has elicited the development of “targeted drugs,” which inhibit aberrant signaling pathways. They require a companion diagnostic test that identifies the tumor-driving pathway; however, currently available tests like estrogen receptor (ER) protein expression for hormonal treatment of breast cancer do not reliably predict therapy response, at least in part because they do not adequately assess functional pathway activity. We describe a novel approach to predict signaling pathway activity based on knowledge-based Bayesian computational models, which interpret quantitative transcriptome data as the functional output of an active signaling pathway, by using expression levels of transcriptional target genes. Following calibration on only a small number of cell lines or cohorts of patient data, they provide a reliable assessment of signaling pathway activity in tumors of different tissue origin. As proof of principle, models for the canonical Wnt and ER pathways are presented, including initial clinical validation on independent datasets from various cancer types. <i>Cancer Res; 74(11); 2936–45. ©2014 AACR</i>.</p></div>

Abstract Background and significance: The identification of leukemia-initiating cells (LIC) in myeloid leukemias allowed for focused therapeutic approaches to prevent drug-resistance and relapse. In B cell leukemia and lymphoma, LIC occur at a high frequency (Rehe 2013), are phenotypically diverse (Aoki 2015) and can arise from any stage of B cell development (Le Viseur 2008). While self-renewal of HSCs and other organ-stem cells is regulated by a developmental hierarchy, self-renewal in the B cell lineage is induced by positive selection events, i.e. induced by antigen-receptor (BCR) signaling when B cells bind to their cognate antigen. Leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) is a Wnt target gene and through binding to its ligand R-spondin, Lgr5 modulates Wnt signaling strength. Lgr5 is widely used as stem cell marker in multiple organs, however its expression levels are very low in hematopoietic cells. Results: Unexpectedly, B cells upregulate Lgr5-expression by >20-fold, (i) when they express the pre-BCR for the first time and (ii) when they encounter antigen for the first time in germinal centers. Engagement of BCR signaling on B cell lymphomas strongly increased LGR5 expression, which was sensitive to inhibition of SYK and BTK kinases in the BCR pathway. In addition, in patients with pediatric or adult ALL, or B cell chronic lymphocytic leukemia (CLL), higher than median mRNA levels of LGR5 at the time of diagnosis were associated with poor clinical outcome and higher likelihood of drug-resistance and relapse. For these reasons, we studied the function of Lgr5 during normal B cell development and malignant transformation. Conditional ablation of Lgr5fl/fl alleles during earliest stages of B cell development (Mb1-Cre) resulted in a >100-fold reduction of absolute B cell numbers. In addition, inducible deletion of Lgr5 in cultured pre-B and mature B cells caused rapid cell death, demonstrating that Lgr5 function is an essential requirement for B cell development. Most studies in epithelial cells suggest a role of Lgr5 as potentiator of WNT-signaling. However, Cre-mediated deletion of Lgr5 in B cells caused cell death in parallel with massive accumulation of nuclear b-catenin. Our genetic experiments in leukemia models demonstrated that LGR5 is critical for leukemia-initiation and survival. Cre-mediated deletion of Lgr5 and b-catenin accumulation abolished colony forming capacity and the ability of leukemia cells to initiate fatal disease in transplant recipients. Likewise, inducible activation of a gain-of-function mutant of b-catenin resulted in rapid cell death in B cells. Conclusion: Lgr5 is widely established as a marker of organ stem cells with self-renewal capacity. In the absence of the developmental stem cell hierarchy in B cells, we conclude that Lgr5 expression is a biomarker of positive selection in B cells. Lgr5-expression and positive selection is induced by BCR-engagement (e.g. by antigen) or oncogenic mimics of BCR signaling in B cell malignancies (e.g. transforming oncogenes that engage the BCR pathway). Unlike in epithelial cells, LGR5 expression in B cells restricts the levels of nuclear b-catenin and enables B cell survival through negative regulation of Wnt-signaling. Disclosures No relevant conflicts of interest to declare.

Abstract Pre-clinical disease models are essential for the development of novel therapies and help better understand the molecular processes. In recent years, a novel groundbreaking technology to generate patient specific in vitro cultures based on adult stem-cell organoids, has gained widespread interest for the development of new therapies and as a predictive diagnostic tool. ASC-organoids can be generated at high efficiency from both healthy and diseased tissue (resections as well as biopsies) including most carcinomas. These patient derived cultures can be expanded for prolonged periods of time while maintaining many properties of the tissue they were derived from. At HUB, we have created comprehensive ‘living biobanks' from tumors of different origin like colon, lung, breast, pancreas and ovary. These biobanks have been characterized at the genetic and transcriptional level. These analyses have shown that our ‘living biobanks' capture disease heterogeneity at the genetic and molecular level. Because organoids can be expanded long term, drug screening is feasible in these models. HUB has developed procedures for (high-throughput) compound screening in organoids including small molecules and biologics. In this setting, organoids have been used to identify novel targets, identify lead compounds and stratify potential responders based on their genetic or molecular profile. Recent advances in the field of immuno-oncology have revolutionized the treatment for cancer patients. Despite of the improvements and due to the complex and highly regulated nature of the immune system, treatment does not have a similar effect on all tumors. We have created a co-culture model of tumor derived organoids and immune cells. In these co-cultures, the cytotoxic effect of the immune cell on the tumor can be measured in a reproducible and quantitative manner. Currently we are expanding existing co-culture models to incorporate different immune cells and organoid tumor types. In addition, organoids can serve as a patient avatar in clinical development or diagnostics. In this setting, organoids could add a functional test for therapy response in a patient centered setting providing a unique tool for personalized medicine. Citation Format: Jasper Mullenders, Annemarie Buijs, Inge Van Maanen, Carol Piani, Soura Mardpour, Hans Clevers, Rob Vries, Sylvia Boj. Patient derived organoids: Bringing the "patient in the lab" for pre-clinical and clinical development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-008.

<p>The single-cell architecture of Ewing sarcoma - part 1</p>