Mentor Name: Adam Wolpaw

Neuroblastoma (NB) accounts ~12% of childhood cancer mortality in addition to significant morbidity among survivors. The most important recent advance in NB therapy has been the use of monoclonal antibodies targeting GD2, which are now FDA approved for use in NB. These antibodies improve survival in patients in first remission and, when combined with chemotherapy, are the most active therapy for relapsed disease. To improve this therapy and optimize combination therapies, we need facile and accurate model systems. Cell lines grown in 2D are easy to use and inexpensive, but do not recapitulate key features of human NB. Mouse models are much more accurate, but expensive and do not allow substantial throughput. To address this issue, we have grown murine NB cells in 3D culture. The advantage of murine cells is that we can produce new source material from transgenic mice, but we can also put our 3D cultures back in an immunocompetent mouse. This allows us to study immunotherapy both in vitro and in vivo. We decided to focus on anti-GD2 antibody therapy. It has been challenging to study anti-GD2 therapy in mouse models because murine cell lines lose GD2 while transitioning from an adrenergic lineage state to a mesenchymal lineage state. Our preliminary results show that GD2 expression is maintained in 3D cultures. The goal of this project is to understand how enzymes controlling GD2 expression and the transcriptome differ between 2D and 3D cultures. Our hypothesis is that culture in 3D maintains key aspects of the adrenergic state important for GD2 expression. In the current proposal, Megan will isolate murine NB cells and grow the cells in both 2D and 3D. She will first measure GD2 surface expression on freshly isolated cells and how it changes over time in each culture condition. This will be done with flow cytometry. She will then compare how expression of the enzymes required for GD2 synthesis – GM3 synthase, GD3 synthase, and GD2 synthase – differ between 2D and 3D culture using western blots. Once she identifies the optimal time point where these cultures differ, she will perform RNA sequencing. She will learn how to analyze this data to identify genes that are differentially expressed, how defined signatures of genes representing the adrenergic and mesenchymal states change, and what other pathways differ. This will help us understand how 2D and 3D cultures differ, identify the best system to study anti-GD2 therapy, and reveal what aspects of the adrenergic transcriptome are required for GD2 expression. Furthermore, this will teach Megan essential wet lab and computational tools to further her contributions to pediatric oncology research.