Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes
2022; Springer Nature; Volume: 14; Issue: 10 Linguagem: Inglês
10.15252/emmm.202216001
ISSN1757-4684
AutoresMichael T. Meister, Marian J.A. Groot Koerkamp, Terezinha de Souza, Willemijn B. Breunis, Ewa Frazer‐Mendelewska, Mariël Brok, Jeff DeMartino, Freek Manders, Camilla Calandrini, Hinri Kerstens, Alex Janse, M. Emmy M. Dolman, Selma Eising, Karin P.S. Langenberg, Marc van Tuil, Rutger R. G. Knops, Sheila Terwisscha van Scheltinga, Laura S. Hiemcke‐Jiwa, Uta Flucke, Johannes H. M. Merks, Max M. van Noesel, Bastiaan B. J. Tops, Jayne Y. Hehir‐Kwa, Patrick Kemmeren, Jan J. Molenaar, Marc van de Wetering, Ruben van Boxtel, Jarno Drost, Frank C. P. Holstege,
Tópico(s)CAR-T cell therapy research
ResumoArticle2 August 2022Open Access Source DataTransparent process Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes Michael T Meister Michael T Meister orcid.org/0000-0001-8311-721X Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Conceptualization, Supervision, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Marian J A Groot Koerkamp Marian J A Groot Koerkamp orcid.org/0000-0002-0867-0821 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Data curation, Investigation, Methodology Search for more papers by this author Terezinha de Souza Terezinha de Souza Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Data curation, Investigation, Visualization Search for more papers by this author Willemijn B Breunis Willemijn B Breunis Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Department of Oncology and Children's Research Center, University Children's Hospital Zürich, Zürich, Switzerland Contribution: Investigation, Methodology Search for more papers by this author Ewa Frazer-Mendelewska Ewa Frazer-Mendelewska orcid.org/0000-0003-0903-869X Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Investigation Search for more papers by this author Mariël Brok Mariël Brok orcid.org/0000-0001-6018-900X Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Investigation Search for more papers by this author Jeff DeMartino Jeff DeMartino orcid.org/0000-0001-7366-4789 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Investigation, Visualization Search for more papers by this author Freek Manders Freek Manders orcid.org/0000-0001-6197-347X Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Investigation, Visualization Search for more papers by this author Camilla Calandrini Camilla Calandrini Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Investigation Search for more papers by this author Hinri H D Kerstens Hinri H D Kerstens Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Data curation Search for more papers by this author Alex Janse Alex Janse Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Data curation Search for more papers by this author M Emmy M Dolman M Emmy M Dolman Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Children's Cancer Institute, Lowy Cancer Centre, UNSW Sydney, Kensington, NSW, Australia School of Women's and Children's Health, Faculty of Medicine, UNSW Sydney, Kensington, NSW, Australia Contribution: Resources Search for more papers by this author Selma Eising Selma Eising Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Karin P S Langenberg Karin P S Langenberg orcid.org/0000-0002-6780-0585 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Marc van Tuil Marc van Tuil Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Investigation Search for more papers by this author Rutger R G Knops Rutger R G Knops Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Sheila Terwisscha van Scheltinga Sheila Terwisscha van Scheltinga Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Laura S Hiemcke-Jiwa Laura S Hiemcke-Jiwa Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Uta Flucke Uta Flucke Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Johannes H M Merks Johannes H M Merks orcid.org/0000-0001-7659-1028 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Max M van Noesel Max M van Noesel orcid.org/0000-0003-0503-5838 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Bastiaan B J Tops Bastiaan B J Tops Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Jayne Y Hehir-Kwa Jayne Y Hehir-Kwa Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Patrick Kemmeren Patrick Kemmeren orcid.org/0000-0003-2237-7354 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Center for Molecular Medicine, UMC Utrecht and Utrecht University, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Jan J Molenaar Jan J Molenaar Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Marc van de Wetering Marc van de Wetering Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Ruben van Boxtel Ruben van Boxtel Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Jarno Drost Corresponding Author Jarno Drost [email protected] orcid.org/0000-0002-2941-6179 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Resources, Writing - review & editing Search for more papers by this author Frank C P Holstege Corresponding Author Frank C P Holstege [email protected] orcid.org/0000-0002-8090-5146 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Center for Molecular Medicine, UMC Utrecht and Utrecht University, Utrecht, The Netherlands Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft Search for more papers by this author Michael T Meister Michael T Meister orcid.org/0000-0001-8311-721X Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Conceptualization, Supervision, Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Marian J A Groot Koerkamp Marian J A Groot Koerkamp orcid.org/0000-0002-0867-0821 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Data curation, Investigation, Methodology Search for more papers by this author Terezinha de Souza Terezinha de Souza Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Data curation, Investigation, Visualization Search for more papers by this author Willemijn B Breunis Willemijn B Breunis Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Department of Oncology and Children's Research Center, University Children's Hospital Zürich, Zürich, Switzerland Contribution: Investigation, Methodology Search for more papers by this author Ewa Frazer-Mendelewska Ewa Frazer-Mendelewska orcid.org/0000-0003-0903-869X Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Investigation Search for more papers by this author Mariël Brok Mariël Brok orcid.org/0000-0001-6018-900X Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Investigation Search for more papers by this author Jeff DeMartino Jeff DeMartino orcid.org/0000-0001-7366-4789 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Investigation, Visualization Search for more papers by this author Freek Manders Freek Manders orcid.org/0000-0001-6197-347X Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Investigation, Visualization Search for more papers by this author Camilla Calandrini Camilla Calandrini Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Investigation Search for more papers by this author Hinri H D Kerstens Hinri H D Kerstens Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Data curation Search for more papers by this author Alex Janse Alex Janse Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Data curation Search for more papers by this author M Emmy M Dolman M Emmy M Dolman Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Children's Cancer Institute, Lowy Cancer Centre, UNSW Sydney, Kensington, NSW, Australia School of Women's and Children's Health, Faculty of Medicine, UNSW Sydney, Kensington, NSW, Australia Contribution: Resources Search for more papers by this author Selma Eising Selma Eising Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Karin P S Langenberg Karin P S Langenberg orcid.org/0000-0002-6780-0585 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Marc van Tuil Marc van Tuil Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Investigation Search for more papers by this author Rutger R G Knops Rutger R G Knops Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Sheila Terwisscha van Scheltinga Sheila Terwisscha van Scheltinga Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Laura S Hiemcke-Jiwa Laura S Hiemcke-Jiwa Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Uta Flucke Uta Flucke Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Johannes H M Merks Johannes H M Merks orcid.org/0000-0001-7659-1028 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Max M van Noesel Max M van Noesel orcid.org/0000-0003-0503-5838 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Bastiaan B J Tops Bastiaan B J Tops Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Jayne Y Hehir-Kwa Jayne Y Hehir-Kwa Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Patrick Kemmeren Patrick Kemmeren orcid.org/0000-0003-2237-7354 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Center for Molecular Medicine, UMC Utrecht and Utrecht University, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Jan J Molenaar Jan J Molenaar Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Marc van de Wetering Marc van de Wetering Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Ruben van Boxtel Ruben van Boxtel Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Resources Search for more papers by this author Jarno Drost Corresponding Author Jarno Drost [email protected] orcid.org/0000-0002-2941-6179 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Oncode Institute, Utrecht, The Netherlands Contribution: Resources, Writing - review & editing Search for more papers by this author Frank C P Holstege Corresponding Author Frank C P Holstege [email protected] orcid.org/0000-0002-8090-5146 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Center for Molecular Medicine, UMC Utrecht and Utrecht University, Utrecht, The Netherlands Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft Search for more papers by this author Author Information Michael T Meister1,2, Marian J A Groot Koerkamp1,2, Terezinha Souza1,2, Willemijn B Breunis1,3, Ewa Frazer-Mendelewska1,2, Mariël Brok1,2, Jeff DeMartino1,2, Freek Manders1,2, Camilla Calandrini1,2, Hinri H D Kerstens1, Alex Janse1, M Emmy M Dolman1,4,5, Selma Eising1, Karin P S Langenberg1, Marc Tuil1, Rutger R G Knops1, Sheila Terwisscha Scheltinga1, Laura S Hiemcke-Jiwa1, Uta Flucke1, Johannes H M Merks1, Max M Noesel1, Bastiaan B J Tops1, Jayne Y Hehir-Kwa1, Patrick Kemmeren1,6, Jan J Molenaar1, Marc Wetering1,2, Ruben Boxtel1,2, Jarno Drost *,1,2 and Frank C P Holstege *,1,6 1Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands 2Oncode Institute, Utrecht, The Netherlands 3Department of Oncology and Children's Research Center, University Children's Hospital Zürich, Zürich, Switzerland 4Children's Cancer Institute, Lowy Cancer Centre, UNSW Sydney, Kensington, NSW, Australia 5School of Women's and Children's Health, Faculty of Medicine, UNSW Sydney, Kensington, NSW, Australia 6Center for Molecular Medicine, UMC Utrecht and Utrecht University, Utrecht, The Netherlands *Corresponding author. Tel: +31 88 9727684; E-mail: [email protected] author. Tel: +31 88 972 72 72; E-mail: [email protected] EMBO Mol Med (2022)14:e16001https://doi.org/10.15252/emmm.202216001 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4–8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions. Synopsis The first collection of comprehensively characterized tumor organoid (tumoroid) models of pediatric rhabdomyosarcomas (RMS) as novel preclinical models for this highly aggressive pediatric cancer entity. This is the first collection of purely mesenchymal tumoroid models and only the second tumoroid collection of pediatric cancers. RMS tumoroid models faithfully recapitulate molecular alterations of the parent tumor with retained transcriptional and clonal heterogeneity. They are rapidly established and expanded with the ability to perform drug screening as fast as 27 days after sample acquisition (median 81 days). Lastly, they are amenable to CRISPR/Cas9 editing to recapitulate mutations conferring poor prognosis in RMS (e.g., in TP53). Introduction Rhabdomyosarcoma (RMS) is a type of malignant tumor of mesenchymal origin (Yang et al, 2014) and forms the most common soft tissue sarcoma in children and adolescents (Li et al, 2008). Historically, RMS has been divided into two main subtypes based on histology. Whereas embryonal RMS (eRMS) displays cellular heterogeneity and hallmarks of immature skeletal myoblasts (Patton & Horn, 1962), alveolar RMS (aRMS) cells are distributed around an open central space, thereby resembling pulmonary alveoli (Enterline & Horn, 1958). eRMS is more frequently observed in children under 10, accounting for two-thirds of all RMS cases, and generally has a better prognosis than aRMS, which is more common in adolescents and young adults (Perez et al, 2011). In aRMS, a sole genetic driver alteration is usually observed, caused by a chromosomal translocation resulting in a fusion gene between either PAX3 or PAX7 and FOXO1. In contrast, eRMS is genetically more heterogeneous, harboring mutations in several common oncogenes or tumor suppressor genes (Shern et al, 2014). Other subtypes of RMS have recently been recognized (WHO, 2020). RMS treatment is guided by protocols developed by multinational collaborative groups and includes systemic chemotherapy in addition to local therapy (radiotherapy and/or surgery; Skapek et al, 2019). The prognosis of RMS has improved over the last decades (Bisogno et al, 2019). For patients with high-risk, refractory, or relapsed disease, prognosis remains poor however, despite an immense treatment burden (Pappo et al, 1999; Mascarenhas et al, 2019). Thus, development of new therapeutic options is of critical importance for these patients. Development of such treatment options requires in vitro models and may therefore benefit from application of organoid technology. The basis of this technology is that given a suitable growth environment, tissue stem cells self-renew as well as give rise to natural progeny which organize according to their preferred growth modality without the need for artificial cell immortalization. The technology was first established in healthy epithelial tissue from mouse small intestine (Sato et al, 2009) and soon adapted to various other healthy and diseased epithelial tissues, including cancer (Clevers, 2016). Tumor organoid (tumoroid) systems are proving useful in cancer research as they display genetic stability over extended culture periods, retaining the molecular characteristics of the tumor they are derived from. While dedicated co-culturing tumoroid systems of tumor and nontumor cells are starting to be developed (Yuki et al, 2020), the majority of tumoroid systems consist only of tumor cells. Tumoroid models can be expanded, facilitating high-throughput screening approaches such as small molecule or CRISPR/Cas9-knockout screening (Bleijs et al, 2019). To date, tumoroid approaches have been primarily applied to cancers derived from epithelial cells (i.e., carcinomas). Recent studies demonstrate that deriving tumoroid models from nonepithelial cancer is feasible but this has as yet not been achieved for pure mesenchymal cancers (Fusco et al, 2019; Jacob et al, 2020; Saltsman et al, 2020; Abdullah et al, 2021; Yamazaki et al, 2021). Application to tumors of mesenchymal origin such as RMS would be of obvious benefit. Tumoroid models of pediatric nephroblastoma (Wilms' tumor) have been described, which, depending on the subtype, can contain stromal cells (Calandrini et al, 2020). In addition, cells derived from synovial sarcoma and other adult soft tissue sarcomas can grow to a limited extent on fetal calf serum, which, although undefined in terms of the required essential growth factors, also indicates feasibility (Brodin et al, 2019; Boulay et al, 2021). Furthermore, in vitro propagation of RMS tumor cells derived from patient-derived xenograft (PDX) mouse models has recently been shown (Manzella et al, 2020). Although these results are encouraging, no directly patient-derived collection of tumoroid models of malignant tumors of pure mesenchymal origin (i.e., sarcomas) has been generated and studied after growth for extensive periods in well-defined media components. In this study, we therefore set out to develop and apply approaches for generating a collection of tumoroid models that covers the major RMS subtypes, a pediatric cancer of mesenchymal origin with poor outcome for high-risk patients. Besides generating and extensively characterizing the tumoroid collection, we also investigated applicability for drug screening and genetic modification (Fig 1A). Figure 1. A collection of RMS tumoroid models that represent the diverse clinical presentation of RMS A. Tumor organoid (tumoroid) pipeline. WGS, whole-genome sequencing; RNA-seq, mRNA sequencing; liq N2, liquid nitrogen. B. Overview of available RMS tumoroid models in the collection separated by primary versus metastatic site and exact tumor location. The color of the inner circle indicates the histological subtype while the color of the outer circle indicates the presence or absence of a fusion transcript. Letters within the circle indicate disease instance. Asterisks mark tumoroid models derived from the same patient but from distinct tumor samples. C. Brightfield microscopy images of two representative RMS tumoroid models from a fusion-negative embryonal and a PAX3-FOXO1 fusion-positive alveolar tumoroid model grown in a two-dimensional monolayer in two magnifications as indicated by the scale bars. Download figure Download PowerPoint Results A protocol to collect and process RMS tumor samples for tumoroid model establishment and propagation Before starting to generate a collection of RMS tumoroid models, we first optimized sample acquisition and logistics between surgery, pathology, and organoid culture labs (Materials and Methods). In parallel to optimizing sample acquisition, we also optimized sample processing, including testing different formulations of growth media by a combination of systematic and trial and error approaches (Discussion). RMS tumor samples are diverse. Most samples are small needle biopsies (i.e., 16-gauge tru-cut), as large resection specimens are mostly restricted to pretreated RMS or to treatment-naïve paratesticular fusion-negative eRMS (FN-eRMS). In addition, a subset of samples (4% here) are not solid, being acquired as bone marrow aspirates of infiltrating tumor cells (Fig 1B). Samples are plated as minced pieces embedded in a droplet of extracellular matrix (ECM) substitute (Basement-Membrane Extract, BME) and as single-cell suspensions in BME-supplemented medium. Outgrowth of tumor cells to tumoroid models can occur from both modalities. In the case of successful outgrowth of initially plated cells, cells organize as two-dimensional monolayers (Fig 1C). This appears to be the cells' preferred growth modality, as plating them as single-cell suspensions in BME droplets results in cells escaping the surrounding matrix and sinking to the bottom of the culture plate from which they continue to grow in a monolayer. Therefore, cells are further propagated and expanded in this way. We considered an RMS tumoroid model to be successfully established if, over the course of culturing, the expression of specific tumor markers is retained and the culture expansion is at least sufficient for drug screening, all as described below (Table 1). Table 1. Overview of available RMS tumoroid models in the collection. Patients are numbered to allow identification of RMS tumoroid models derived from the same patient. For additional clinical parameters see Dataset EV1. Tumoroid model Patient Patient birth year Histology Fusion transcript Patient sex Disease instance of tumoroid establishment Body site of sample used for tumoroid establishment RMS007 1 2011 Alveolar negative Male First relapse Abdomen RMS102 2 2000 Alveolar PAX3-FOXO1 Male Second relapse Lymph node metastasis (clavicle) RMS410 3 2001 Alveolar PAX3-FOXO1 Male Second relapse Bone marrow metastasis RMS000CPU 4 2001 Alveolar PAX3-FOXO1 Male First relapse Kidney RMS127 5 2003 Alveolar PAX3-FOXO1 Male Primary disease Bone marrow metastasis RMS006 6 2004 Alveolar PAX3-FOXO1 Male First relapse Calf RMS013 6 2004 Alveolar PAX3-FOXO1 Male Second relapse Calf RMS108 7 2004 Alveolar PAX3-FOXO1 Female Second relapse Bladder RMS109 7 2004 Alveolar PAX3-FOXO1 Female Second relapse Subcutaneous metastasis RMS110 7 2004 Alveolar PAX3-FOXO1 Female Second relapse Subcutaneous metastasis RMS000FLV 8 2018 Alveolar PAX3-FOXO1 Female Primary disease Arm RMS335 9 2010 Alveolar PAX7-FOXO1 Female Second relapse Lymph node metastasis (groin) RMS000HQC 10 2011 Alveolar PAX7-FOXO1 Male First relapse Cerebral metastasis RMS000HWO 11 2014 Alveolar PAX7-FOXO1 Female Primary disease Leg RMS000HWQ 11 2014 Alveolar PAX7-FOXO1 Female Primary disease Lymph node metastasis (groin) RMS444 12 2003 Embryonal Negative Male Primary disease Paratesticular RMS012 13 2013 Embryonal Negative Male Primary disease Paratesticular RMS000ETY 14 2014 Embryonal Negative Male Primary disease Paratesticular RMS000EEC 15 2005 Embryonal PAX3-WWTR1 Male Primary disease Shoulder Early detection of tumor cells during culturing Tumors consist of a variety of different cell types. These include normal cell types that can grow as well or even better in the provided culture conditions, possibly outcompeting tumor cells (Dijkstra et al, 2020). It would therefore be useful to test for the presence of tumor cells early during culturing to omit the unnecessary propagation of cultures lacking any. At early time points, material is limited, impacting the range of applicable assays. The establishment protocol therefore utilizes an RT-qPCR assay after the first or second passage of cells with probes for standard RMS histopathology markers, that is, DES, MYOG, MYOD1 (WHO, 2020), and the fusion transcript in fusion-positive RMS (FP-RMS; Ponce-Castañeda et al, 2014). We considered a sample positive for tumor cells if at least one of the three genes, plus for FP-RMS the fusion transcript, tests positive. All samples that successfully yield tumoroid models, show positivity for at least one marker gene at this stage, while most models (17 out of 19) are positive for all three marker genes and the fusion transcript if applicable (Fig 2A and B). The RT-qPCR-based approach is therefore a useful tool to determine feasibility at an early stage. Figure 2. Early detection of tumor cells during culturing, retained marker protein expression and heterogeneity in gene expression A. RT-qPCR of early passage RMS tumoroid models shows positivity for at least one gene used in standard-of-care pathology analysis (DES, MYOG, or MYOD1). Conventional RMS cell lines (RD and RH30) were used as positive controls, while two Synovial Sarcoma (SS000DAZ and SS077) tumoroid models were used as negative controls. Gene expression was normalized to the expression of a house-keeping gene and human reference RNA (HREF) via the ΔΔCq method. Each tumoroid line was measured once with four technical replicates with the error bars representing the standard deviation of said technical replicates. B. RT-qPCR of early passage RMS tumoroid models reliably detects the aberrant fusion transcripts. Fusion gene expression was normalized to the expression of a house-keeping gene via the ΔCq method. Each tumoroid line was measured once with four technical replicates with the error bars representing the standard deviation of said technical replicates. C. Morphological (via H&E) and immunohistochemical (IHC) comparison of RMS tumors and derived RMS tumoroid models shows retained marker protein (Desmin, Myogenin and MYOD1) expression and cellular morphology. Scale bars equal 200 μm (RMS012, RMS102) or 100 μm (RMS000HQC). D. t-SNE projection of single-cell transcriptomes from the RMS127 and RMS444 tumoroid models. Plots on the right show the normalized expression values, per single cell, of MYOG, MYOD1, DES, and MKI67, respectively. Source data are available online for this figure. Source Data for Figure 2 [emmm202216001-sup-0005-SDataFig2.xlsx] Download figure Download PowerPoint RMS tumoroid models retain marker protein expression and display heterogeneity in gene expression A hallmark of RMS tumors is the expression of proteins associated with nonterminally differentiated muscle (i.e., Desmin, Myogenin, and MYOD1). Expression of these proteins differs between RMS subtypes (Dias et al, 2000) and can be associated with prognosis (Heerema-Mckenney et al, 2008). To
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