Neuroblastoma is a challenging childhood malignancy, with a very high percentage of patients relapsing following acquisition of drug resistance, thereby necessitating the identification of mechanisms of drug resistance as well as new biological targets contributing to the aggressive pathogenicity of the disease. In order to investigate the molecular pathways that are involved with drug resistance in neuroblastoma, we have developed and characterised cisplatin resistant sublines SK-N-ASCis24, KellyCis83 and CHP-212Cis100, integrating data of cell behaviour, cytotoxicity, genomic alterations and modulation of protein expression. All three cisplatin resistant cell lines demonstrated cross resistance to temozolomide, etoposide and irinotecan, all of which are drugs in re-initiation therapy. Array CGH analysis indicated that resistant lines have acquired additional genomic imbalances. Differentially expressed proteins were identified by mass spectrometry and classified by bioinformatics tools according to their molecular and cellular functions and their involvement into biological pathways. Significant changes in the expression of proteins involved with pathways such as actin cytoskeletal signalling (p = 9.28E-10), integrin linked kinase (ILK) signalling (p = 4.01E-8), epithelial adherens junctions signalling (p = 5.49E-8) and remodelling of epithelial adherens junctions (p = 5.87E-8) pointed towards a mesenchymal phenotype developed by cisplatin resistant SK-N-ASCis24. Western blotting and confocal microscopy of MYH9, ACTN4 and ROCK1 coupled with invasion assays provide evidence that elevated levels of MYH9 and ACTN4 and reduced levels of ROCK1 contribute to the increased ROCK1-independent migratory potential of SK-N-ASCis24. Therefore, our results suggest that epithelial-to-mesenchymal transition is a feature during the development of drug resistance in neuroblastoma.

Traditional 2D cell cultures do not completely capture the 3D architecture of cells and extracellular matrix contributing to a gap in our understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results. Here, we demonstrated the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. The ability to test drugs in this reproducible and controllable tissue-engineered model system will help reduce the attrition rate of the drug development process and lead to more effective and tailored therapies. Importantly, such 3D cell models help to reduce and replace animals for pre-clinical research addressing the principles of the 3Rs.

OBJECTIVES. The purpose of this study was to determine how the method of assessment affects patient report of human immunodeficiency virus (HIV) risks. METHODS. Patients at a sexually transmitted disease clinic randomly received either a written self-administered questionnaire or an audio self-administered questionnaire delivered by cassette player and headset. These questionnaires were followed by face-to-face interviews. RESULTS. Audio questionnaires had fewer missing responses than written questionnaires. Audio questionnaires also identified more unprotected vaginal intercourse and sexual partners suspected or known to have HIV infection or acquired immunodeficiency syndrome than did written questionnaires. Although both the audio and written questionnaires identified more risks than the face-to-face interviews, the difference in the mean number of reported risks between the audio questionnaires and the face-to-face interviews was greater than that between the written questionnaires and the face-to-face interviews. CONCLUSIONS. Audio questionnaires may obtain more complete data and identify more HIV risk than written questionnaires. Research is warranted about whether audio questionnaires overcome barriers to the completion and accuracy of HIV risk surveys. This study emphasizes the need to elucidate the relative strengths and weaknesses of written questionnaires, audio questionnaires, and face-to-face interviews for HIV risk assessment.

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