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. Preclinical 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 RNAsequencing 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.

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.

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.

Breast cancer is the most prevalent type of malignancy in women with ∼1.7 million new cases diagnosed annually, of which the majority express ERα (ESR1), a ligand-dependent transcription factor. Genome-wide chromatin binding maps suggest that ERα may control the expression of thousands of genes, posing a great challenge in identifying functional targets. Recently, we developed a CRISPR-Cas9 functional genetic screening approach to identify enhancers required for ERα-positive breast cancer cell proliferation. We validated several candidates, including CUTE, a putative ERα-responsive enhancer located in the first intron of CUEDC1 (CUE-domain containing protein). Here, we show that CUTE controls CUEDC1 expression, and that this interaction is essential for ERα-mediated cell proliferation. Moreover, ectopic expression of CUEDC1, but not a CUE-domain mutant, rescues the defects in CUTE activity. Finally, CUEDC1 expression correlates positively with ERα in breast cancer. Thus, CUEDC1 is a functional target gene of ERα and is required for breast cancer cell proliferation.

Tumor cells may produce many of the same messenger RNAs (mRNA) as the cell they derive from. The relative abundance of these mRNAs, the transcriptiomic profile, may provide clues into the origin and development of tumors. Here we investigated the cellular origins of 1,300 childhood and adult renal tumors, spanning 7 different subtypes. We decomposed tumor bulk transcriptomes into single cell components, measuring the abundance of single cell derived reference "cellular signals" in each tumor. We quantified the extent to which each tumor utilized fetal cellular signals, finding that all childhood renal tumors are definitively fetal. This replaces the long-held presumption of "fetalness" with a precise, quantitative readout of immaturity. Analyzing cellular signals in each tumor type, we recapitulated previous findings for some, whilst providing novel insights into other, less well understood tumor types. For example, our analyses predicted fetal interstitial cells as the cell of origin of the infant kidney tumor, congenital mesoblastic nephroma, and demonstrate that another childhood kidney cancer, malignant rhabdoid tumor, arises from mesodermally derived cells in early development. We found remarkable uniformity in the cell signal of each tumor type, indicating the possible therapeutic and diagnostic utility of cellular signal decomposition. We demonstrated this utility with an example of a child with a cryptic renal tumor, which had not been identifiable by conventional diagnostic work-up but was clearly classified with our approach. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer.

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, in vitro and in vivo 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 in vitro cell models may provide more versatile platforms to study embryonal tumors in a scalable manner. In this review, we outline different in vitro models that can be explored to study embryonal tumorigenesis and for therapy development.

Recent advances in in vitro culture technologies, such as adult stem cell-derived organoids, have opened up new avenues for the development of novel, more physiologic human cancer models. Such preclinical models are essential for efficient translation of basic cancer research into novel treatment regimens. We succeeded in growing organoids from a range of pediatric solid tumors, including Wilms’ tumors, renal cell carcinomas, and different types of rhabdoid tumors (i.e., AT/RT, MRT). Tumor organoids retain many characteristics of parental tumor tissue. For instance, Wilms’ tumor organoids retain the cellular heterogeneity of tumors, as they are composed of an intricate network of different cell types. Moreover, we demonstrate that tumor organoids are amenable to gene editing and high-throughput drug screens. In conclusion, our pediatric cancer organoids capture disease and tissue heterogeneity and provide a platform for basic cancer research, drug screening, and personalized medicine. Citation Format: Camilla Calandrini, Frans Schutgens, Rurika Oka, Thanasis Margaritis, Tito Candelli, Luka Mathijsen, Carola Ammerlaan, Ravian van Ineveld, Sepideh Derakhshan, Lars Custers, Philip Lijnzaad, Harry Begthel, Hinri Kerstens, Maarten Rookmaker, Marianne Verhaar, Patrick Kemmeren, Ronald de Krijger, Kathy Pritchard-Jones, Anne Rios, Marry van den Heuvel-Eibrink, Frank Holstege, Ruben van Boxtel, Hans Clevers, Jarno Drost. Patient-derived organoids in pediatric cancer research [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr IA27.

Malignant rhabdoid tumor (MRT) is a highly malignant and often lethal childhood cancer. MRTs are genetically defined by bi-allelic inactivating mutations in SMARCB1, a member of the BRG1/BRM-associated factors (BAF) chromatin remodeling complex. Mutations in BAF complex members are common in human cancer, yet their contribution to tumorigenesis remains in many cases poorly understood. Here, we studied derailed regulatory landscapes as a consequence of SMARCB1 loss in the context of MRT. Our multi-omics approach on patient-derived MRT organoids revealed a dramatic reshaping of the regulatory landscape upon SMARCB1 reconstitution. Chromosome conformation capture experiments subsequently revealed patient-specific looping of distal enhancer regions with the promoter of the MYC oncogene. This intertumoral heterogeneity in MYC enhancer utilization is also present in patient MRT tissues as shown by combined single-cell RNA-seq and ATAC-seq. We show that loss of SMARCB1 drives patient-specific epigenetic reprogramming underlying MRT tumorigenesis.