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.
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.
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.
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.
Matthay KK, Reynolds CP, Seeger RC, Shimada H, Adkins ES, Haas-Kogan D, Gerbing RB, London WB, Villablanca JG. Long-term results for children with high-risk neuroblastoma treated on a randomized trial of myeloablative therapy followed by 13-cis-retinoic acid: a children's oncology group study. J Clin Oncol. 2009;27(7):1007–13. CrossRefPubMedPubMedCentral
Ribatti D. The chick embryo chorioallantoic membrane in the study of tumor angiogenesis. Romanian J Morphol Embryol. 2008;49(2):131–5.
Herrmann A, Rice M, Levy R, Pizer BL, Losty PD, Moss D, See V. Cellular memory of hypoxia elicits neuroblastoma metastasis and enables invasion by non-aggressive neighbouring cells. Oncogene. 2015;4:e138. CrossRef
Rytelewski M, Buensuceso A, Leong HS, Deroo BJ, Chambers AF, Koropatnick J. Evaluating the effectiveness of cancer drug sensitization in vitro and in vivo. J Vis Exp. 2015;96: e52388. https://doi.org/10.3791/52388
Carter R, Mullassery D, See V, Theocharatos S, Pizer B, Losty PD, Jesudason E, Moss DJ. Exploitation of chick embryo environments to reprogram MYCN-amplified neuroblastoma cells to a benign phenotype, lacking detectable MYCN expression. Oncogene. 2012;1:e24. CrossRef
Armstrong PB, Quigley JP, Sidebottom E. Transepithelial invasion and intramesenchymal infiltration of the chick embryo chorioallantois by tumor cell lines. Cancer Res. 1982;42(5):1826–37. PubMed
Herrmann A, Moss D, See V. The Chorioallantoic membrane of the Chick embryo to assess tumor formation and metastasis. Tumor Angiogenesis Assays: Methods and Protocols. 2016;1464:97–105. CrossRef
Sung PJ, Boulos N, Tilby MJ, Andrews WD, Newbold RF, Tweddle DA, Lunec J. Identification and characterisation of STMN4 and ROBO2 gene involvement in neuroblastoma cell differentiation. Cancer Lett. 2013;328:168–75.
Shalinsky DR, Bischoff ED, Gregory ML, Gottardis MM, Hayes JS, Lamph WW, Heyman RA, Shirley MA, Cooke TA, Davies PJ, et al. Retinoid-induced suppression of squamous cell differentiation in human oral squamous cell carcinoma xenografts (line 1483) in athymic nude mice. Cancer Res. 1995;55(14):3183–91. PubMed
Russell WMS, Burch RL: The principles of humane experimental technique: Wheathampstead: universities federation for Anim Welf,; 1992.
Sugiura Y, Shimada H, Seeger RC, Laug WE, DeClerck YA. Matrix metalloproteinases-2 and -9 are expressed in human neuroblastoma: contribution of stromal cells to their production and correlation with metastasis. Cancer Res. 1998;58(10):2209–16. PubMed
Borrill Mather G: The Chick embryo; A new drug delivery model for Neuroblastoma. MPhil. University of Liverpool; 2014.
Zage PE, Zeng L, Palla S, Fang W, Nilsson MB, Heymach JV, Zweidler-McKay PA. A novel therapeutic combination for neuroblastoma: the vascular endothelial growth factor receptor/epidermal growth factor receptor/rearranged during transfection inhibitor vandetanib with 13-cis-retinoic acid. Cancer. 2010;116(10):2465–75. PubMed
- Optimising the chick chorioallantoic membrane xenograft model of neuroblastoma for drug delivery
Barry L. Pizer
Paul D. Losty
- BioMed Central
Neu im Fachgebiet Onkologie
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