Abstract
BackgroundNeuroblastoma 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.MethodsAs 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.ResultsTreatment 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.ConclusionDifferentiation 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.
Highlights
Neuroblastoma is a paediatric cancer that despite multimodal therapy still has a poor outcome for many patients with high risk tumours
Coverslips were removed from wells and fixed with 4% paraformaldehyde for 10 min, blocked with 1% Bovine serum albumin (BSA), 0.1% Triton X100 in 0.12 M phosphate pH 7.4 for 30 min and stained overnight at 4 °C with 1:50 dilution of Ki67 (Abcam ab16667) followed by 1:500 Goat anti rabbit Alexa 594 (Life Technologies) for one hour at room temperature both diluted in blocking buffer
Assessment of All-trans retinoic acid (ATRA) effects by measuring cell proliferation and expression of differentiation markers The effect of ATRA on MNA neuroblastoma cells has been well characterised in terms of morphology and immunofluorescence of differentiation markers [18,19,20]
Summary
Neuroblastoma is a paediatric cancer that despite multimodal therapy still has a poor outcome for many patients with high risk tumours. New pre-clinical models are required to test therapies in a rapid cost effective way in order to identify the most useful agents. Whilst many agents tested in vitro look promising, remarkably few are as successful in preclinical models or eventually patients. Mouse models are expensive and time consuming there is a need for additional models. These models should be rapid, cost effective and NC3Rs compliant in order to contribute to the identification of novel therapies which have the potential to progress to successful preclinical/clinical trials and have a significant impact on the disease
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