Neuroblastoma and DIPG Organoid Coculture System for Personalized Assessment of Novel Anticancer Immunotherapies.

Cancer immunotherapy has transformed the landscape of adult cancer treatment and holds a great promise to treat paediatric malignancies. However, in vitro test coculture systems to evaluate the efficacy of immunotherapies on representative paediatric tumour models are lacking. Here, we describe a detailed procedure for the establishment of an ex vivo test coculture system of paediatric tumour organoids and immune cells that enables assessment of different immunotherapy approaches in paediatric tumour organoids. We provide a step-by-step protocol for an efficient generation of patient-derived diffuse intrinsic pontine glioma (DIPG) and neuroblastoma organoids stably expressing eGFP-ffLuc transgenes using defined serum-free medium. In contrast to the chromium-release assay, the new platform allows for visualization, monitoring and robust quantification of tumour organoid cell cytotoxicity using a non-radioactive assay in real-time. To evaluate the utility of this system for drug testing in the paediatric immuno-oncology field, we tested our in vitro assay using a clinically used immunotherapy strategy for children with high-risk neuroblastoma, dinutuximab (anti-GD2 monoclonal antibody), on GD2 proficient and deficient patient-derived neuroblastoma organoids. We demonstrated the feasibility and sensitivity of our ex vivo coculture system using human immune cells and paediatric tumour organoids as ex vivo tumour models. Our study provides a novel platform for personalized testing of potential anticancer immunotherapies for aggressive paediatric cancers such as neuroblastoma and DIPG.

TBX2 is a neuroblastoma core regulatory circuitry component enhancing MYCN/FOXM1 reactivation of DREAM targets.

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.

Anti-GD2 Immunoliposomes for Targeted Delivery of the Survivin Inhibitor Sepantronium Bromide (YM155) to Neuroblastoma Tumor Cells.

PURPOSE: Sepantronium bromide (YM155) is a hydrophilic quaternary compound that cannot be administered orally due to its low oral bioavailability; it is furthermore rapidly eliminated via the kidneys. The current study aims at improving the pharmacokinetic profile of YM155 by its formulation in immunoliposomes that can achieve its enhanced delivery into tumor tissue and facilitate uptake in neuroblastoma cancer cells. METHODS: PEGylated YM155 loaded liposomes composed of DPPC, cholesterol and DSPE-PEG2000 were prepared via passive film-hydration and extrusion method. Targeted (i.e. immuno-)liposomes were prepared by surface functionalization with SATA modified monoclonal anti-disialoganglioside (GD2) antibodies. Liposomes were characterized based on their size, charge, antibody coupling and YM155 encapsulation efficiency, and stability. Flow cytometry analysis and confocal microscopy were performed on IMR32 and KCNR neuroblastoma cell lines. The efficacy of developed formulations were assessed by in-vitro toxicity assays. A pilot pharmacokinetic analysis was performed to assess plasma circulation and tumor accumulation profiles of the developed liposomal formulations. RESULTS: YM155 loaded immunoliposomes had a size of 170 nm and zeta potential of -10 mV, with an antibody coupling efficiency of 60% andYM155 encapsulation efficiency of14%. Targeted and control liposomal formulations were found to have similar YM155 release rates in a release medium containing 50% serum. An in-vitro toxicity study on KCNR cells showed less toxicity for immunoliposomes as compared to free YM155. In-vivo pharmacokinetic evaluation of YM155 liposomes showed prolonged blood circulation and significantly increased half-lives of liposomal YM155 in tumor tissue, as compared to a bolus injection of free YM155. CONCLUSIONS: YM155 loaded immunoliposomes were successfully formulated and characterized, and initial in-vivo results show their potential for improving the circulation time and tumor accumulation of YM155.