Exploitation of chick embryo environments to reprogram MYCN-amplified neuroblastoma cells to a benign phenotype, lacking detectable MYCN expression

Abstract

Neuroblastoma is a paediatric cancer that arises from the sympathetic ganglia (SG) or adrenal gland. Tumours that occur in patients under 18 months of age have a particularly good prognosis and frequently undergo spontaneous regression. This led to the hypothesis that developmental cues in the youngest patients may prompt belated differentiation and/or apoptosis of the tumour cells. To test our hypothesis, we have injected MYCN-amplified neuroblastoma cells into the extra embryonic veins of chick embryos at embryonic day 3 (E3) and E6 and analysed the response of these Kelly cells at E10 and E14. Amplification of the MYCN gene occurs in up to 30% of tumours and is normally associated with a very poor prognosis. Kelly cells injected at E3 follow neural crest pathways and integrate into neural locations such as SG and the enteric nervous system although never into the adrenal gland. Additionally they migrate to non-neural locations such as the heart, meninges, jaw regions and tail. The cells respond to their respective microenvironments and in SG, some cells differentiate, they show reduced cell division and crucially all cells have undetectable MYCN expression by E10. In non-neural locations, cells form more rapidly dividing clumps and continue to express MYCN. The downregulation of MYCN is dependent on continuous and direct interaction with the sympathetic ganglion environment. We propose that the MYCN-amplicon in the Kelly cells retains the ability to correctly interpret the environmental cues leading to downregulation of MYCN.

Use of the Chick Embryo Model in Uveal Melanoma

“…One possibility is that additional genetic alterations are necessary to increase the metastatic potential of these cells as compared with the parental cell line. The effects of cancer-specific molecular aberrations on tumour mass formation, angiogenesis and dissemination have been examined for a variety of tumour cell types using the CAM assay [23,24]. Laurent et al [25] examined the effects of protein tyrosine phosphatase type IV A member 3 (PTP4A3), a predictor of uveal melanoma metastasis, when introduced into the OCM1 uveal melanoma cell line, on tumour cell dissemination using the CAM assay.…”

Evaluating the effectiveness of transferrin receptor‐1 (TfR1) as a magnetic resonance reporter gene

“…Fast Green is used as a guide for the injection as it allows the monitoring of cell delivery at the site of the injection, but is quickly cleared from the embryo. Fertilized eggs of White Leghorn chicken were windowed at E3 as described previously (38) and the cells were then injected into the midbrain of embryos in ovo using a microcapillary pipette. After receiving the cells, embryos were allowed to grow for a further 48 h. Eggs were maintained at 38°C and 40% humidity and all animal work followed UK regulations (consolidated version of ASPA 1986).…”

CDK inhibitors reduce cell proliferation and reverse hypoxia-induced metastasis of neuroblastoma tumours in a chick embryo model

“…No metastatic cells were seen prior to the formation of a primary tumour on the CAM and they were only seen in embryos that formed a primary tumour. This confirms that the cells within the embryo were derived from the primary tumour rather than from the initial implantation of cells on the CAM at E7 [13,14,[20][21][22][23]. Thus the CAM tumour model is excellent for investigating response mechanisms to new therapeutic agents in a cost-effective and timely manner.…”

“…They were maintained at 37°C with 5% CO 2 were passaged using 0.05% Trypsin/EDTA (Sigma Aldrich). Cell lines were transduced with green fluorescent protein (GFP) lentivirus as described previously [13,22]. For hypoxic studies, cells were maintained at 37 °C with 5% CO 2…”