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

Neuroblastoma is a paediatric cancer with a poor prognosis. This is in part due to widespread metastasis at time of presentation, which is refractory to current treatment modalities. New therapeutic agents that can control not only tumour growth but also metastasis are urgently needed. The differentiation therapy, retinoic acid, is currently used in clinic, leading to terminal differentiation of neuroblastoma cells thus reducing tumour growth in the primary tumour as well as at metastatic sites. However, retinoic acid only works in a subset of patients. We investigated the potential of CDK inhibitors, Palbociclib and RO-3306, on neuroblastoma cell differentiation, tumour progression and metastasis by utilising a 3R compliant cost effective preclinical chick embryo model. In both SK-N-AS and BE(2)C cell lines, when engrafted on the chorioallantoic membrane of chick embryos, we observed a reduction of tumour cell proliferation as well as a reduction in hypoxia preconditioning-driven metastasis by 60%. In addition, the expression of a panel of genes with known roles in metastasis, which increased upon hypoxia-preconditioning, was largely reduced by a CDK1 inhibitor. These results provide a promising alternative to currently existing therapies and might aid the development of new treatment protocols for retinoic acid-resistant patients.

Optimising the chick chorioallantoic membrane xenograft model of neuroblastoma for drug delivery.

BACKGROUND: Neuroblastoma is a paediatric cancer that despite multimodal therapy still has a poor outcome for many patients with high risk tumours. Retinoic acid (RA) promotes differentiation of some neuroblastoma tumours and cell lines, and is successfully used clinically, supporting the view that differentiation therapy is a promising strategy for treatment of neuroblastoma. To improve treatment of a wider range of tumour types, development and testing of novel differentiation agents is essential. New pre-clinical models are therefore required to test therapies in a rapid cost effective way in order to identify the most useful agents. METHODS: As a proof of principle, differentiation upon ATRA treatment of two MYCN-amplified neuroblastoma cell lines, IMR32 and BE2C, was measured both in cell cultures and in tumours formed on the chick chorioallantoic membrane (CAM). Differentiation was assessed by 1) change in cell morphology, 2) reduction in cell proliferation using Ki67 staining and 3) changes in differentiation markers (STMN4 and ROBO2) and stem cell marker (KLF4). Results were compared to MLN8237, a classical Aurora Kinase A inhibitor. For the in vivo experiments, cells were implanted on the CAM at embryonic day 7 (E7), ATRA treatment was between E11 and E13 and tumours were analysed at E14. RESULTS: Treatment of IMR32 and BE2C cells in vitro with 10 µM ATRA resulted in a change in cell morphology, a 65% decrease in cell proliferation, upregulation of STMN4 and ROBO2 and downregulation of KLF4. ATRA proved more effective than MLN8237 in these assays. In vivo, 100 µM ATRA repetitive treatment at E11, E12 and E13 promoted a change in expression of differentiation markers and reduced proliferation by 43% (p < 0.05). 40 µM ATRA treatment at E11 and E13 reduced proliferation by 37% (p < 0.05) and also changed cell morphology within the tumour. CONCLUSION: Differentiation of neuroblastoma tumours formed on the chick CAM can be analysed by changes in cell morphology, proliferation and gene expression. The well-described effects of ATRA on neuroblastoma differentiation were recapitulated within 3 days in the chick embryo model, which therefore offers a rapid, cost effective model compliant with the 3Rs to select promising drugs for further preclinical analysis.