Development of a human neuroblastoma organoid model

Neuroblastoma (NB) is the most prevalent solid malignancy in infants, with approximately 20% of cases classified as high-risk due to amplification of the MYCN oncogene - a marker associated with aggressive tumor behavior and poor prognosis. Despite therapeutic advances, relapsed high-risk NB accounts for nearly 15% of pediatric cancer-related mortality. Studies using non-human models may yield misleading results due to species-specific differences in sympathoblast development -the fetal precursors of NB cells - which differ markedly between mice and humans. To address this, we developed and characterized an iPSC-derived human neuroblastoma organoid model that enables the study of early tumorigenesis in a tissue-like context. First, we developed a differentiation protocol to generate human neural crest (NC) organoids from hiPSCs. Key neural crest markers, detected by fluorescent microscopy and Western Blot, were used to characterize the cell types present in the organoids. We then engineered transgenic iPSCs harboring a removable STOP cassette upstream of the MYCN gene (MYCN-GFP hiPSCs) and control iPSCs lacking MYCN (GFP hiPSCs). We verified the success of genomic integration by PCR and differentiated NC organoids from the transgenic hiPSCs using the previously established protocol. Then NC organoids were transduced with a Cre-expressing adeno-associated virus (AAV) that induced MYCN expression in a sympathoblast-specific manner. Changes in organoid size and GFP expression were observed over time with flow cytometry and fluorescent microscopy. The resulting neuroblastoma-like (NB) organoids were characterized by fluorescent microscopy and bulk RNA-sequencing. NB organoids were also treated with chemotherapeutics and were implanted subcutaneously into mice. Successful generation of NC organoids from wild type and transgenic iPSC lines was validated by the expression of neural crest markers. The formation of tumor tissue was observed in organoids generated from MYCN-GFP hiPSC lines after AAV-mediated MYCN overexpression in targeted cells. The resulting NB organoids displayed hallmark features of high-risk NB and were sensitive to clinically relevant chemotherapeutics. NB organoids could also engraft and expand in vivo in mice. We have created a hiPSC-based three-dimensional human neuroblastoma organoid model that includes tumor cells as well as healthy cells of neural crest origin forming the tumor environment. This innovative human organoid model of high-risk neuroblastoma provides a platform to study tumor initiation and investigate resistance mechanisms emerging after repeated chemotherapy cycles. Its application in drug screening may facilitate the discovery of novel, more effective therapies tailored to aggressive NB subtypes.