Elsevier

European Journal of Cancer

Volume 50, Issue 3, February 2014, Pages 628-637
European Journal of Cancer

Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours

https://doi.org/10.1016/j.ejca.2013.11.015Get rights and content

Abstract

Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour models.

Introduction

Neuroblastoma is a neuroendocrine tumour that arises from the peripheral sympathetic nervous system [1]. International Neuroblastoma Staging System (INSS) stage 1 and 2 tumours display excellent prognosis while stage 3 and 4 tumours have poor clinical outcome with a survival rate of 30% [2], [3], [4], [5]. Neuroblastoma can genomically be characterised by aberrations such as gain of chromosome 17q, partial loss of chromosome 1p or 11q and MYCN amplification [6]. MYCN amplification occurs in about 20% of tumours and strongly correlates with poor prognosis [7], [8]. Gain of 17q is the most frequent genomic abnormality which is present in over 90% of high grade neuroblastomas [9].

Cancer cell lines have been regarded as valid systems to study cancer biology. Nevertheless, these cell systems are cultured in non-physiological conditions and maintained for many years allowing considerable artificial adaptation. The genetic makeup and phenotypic characteristics of these cells can thus differ substantially from their original tumours [10], [11]. In recent years, protocols have been devised to culture cells from fresh tumours in serum-free conditions in neural stem cell medium [12], [13]. Such cells retained much of the characteristics of the original tumours and were able to initiate tumours in immune-compromised mice. They were therefore frequently called tumour initiating cells (TICs), a terminology that we use in this paper to refer to cells cultured by these protocols. TICs isolated from neuroblastoma have recently been reported, but the origin of these cells has been under debate [14], [15], [16], [17].

Here we report the unambiguous establishment of TIC lines from neuroblastoma. These TICs reflect the primary tumours based on the genotype and phenotype and are not contaminated with cells from the haematopoietic lineage. We also show xenografts of neuroblastoma TICs recapitulate the genotype of primary neuroblastoma and showed similar mRNA expression compared to primary tumours as opposed to xenografts from classical neuroblastoma cell lines.

Section snippets

Patients, isolation and culture of neuroblastoma TICs

Freshly resected human neuroblastoma tissue was obtained directly at surgery. Primary neuroblastoma cells were derived by mechanical disaggregation followed by enzymatic digestion of sheared tumour tissues with Liberase DH (500 μl/25 ml) (Roche) for 45 mins. Digests were passed through a 40 μM cell strainer (BD Biosciences) and cells were cultured in TIC total medium containing Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) (GIBCO) supplemented with 40 ng/ml basic fibroblast

Clinical and genomic characteristics of patients’ tumours

Tumour material was obtained from six stages 4 neuroblastoma patients aged 15–115 months. Primary tumours were typed by haematoxylin–eosin (H&E) and by immunohistochemistry staining for NCAM, CHGA and SYP (Fig. 1A and B and Supplementary Fig. 1). All primary tumours were positive for nuclear imaging using MIBG I127 and all patients were positive for urine catecholamines. aCGH analysis showed MYCN amplification in four out of six tumours and multiple other aberrations at chromosomes 1p36, 11q and

Discussion

In this study, we isolated and cultured eight neuroblastoma TICs from primary neuroblastoma tumours and bone marrow metastases in serum-free medium supplemented with bFGF and EGF. We use the terminology of tumour initiating cells (TICs) to refer to our cell lines although we have not studied stem cell properties of these cell lines in depth. Nevertheless, we will continue to call these cell lines TICs as they were established with the protocols and techniques previously used to establish TIC

Conflict of interest statement

None declared.

Acknowledgements

This research was supported by grants from Stichting Villa Joep, the Tom Voute Fonds and KWF Foundation.

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