Abstract
Tumors often show intra-tumor heterogeneity because of genotypic differences between all the cells that compose it and that derive from it. Recent studies have shown significant aspects of neuroblastoma heterogeneity that may affect the diagnostic-therapeutic strategy. Therefore, we developed a laboratory protocol, based on the combination of the advanced dielectrophoresis-based array technology and next-generation sequencing to identify and sort single cells individually and carry out their copy number variants analysis. The aim was to evaluate the cellular heterogeneity, avoiding overestimation or underestimation errors, due to a bulk analysis of the sample. We tested the above-mentioned protocol on two neuroblastoma cell lines, SK-N-BE(2)-C and IMR-32. The presence of several gain or loss chromosomal regions, in both cell lines, shows a high heterogeneity of the copy number variants status of the single tumor cells, even if they belong to an immortalized cell line. This finding confirms that each cell can potentially accumulate different alterations that can modulate its behavior. The laboratory protocol proposed herein provides a tool able to identify prevalent behaviors, and at the same time highlights the presence of particular clusters that deviate from them. Finally, it could be applicable to many other types of cancer.
Highlights
It is widely accepted that cancer is a highly heterogeneous disease and that subpopulations of cells, within a single tumor, can exhibit distinct genomic profiles
Single cells isolated from two neuroblastoma cell namely
Of the cells isolated from the IMR-32 plate, were considered suitable for analysisof ofathe chromosomal pattern, which allowed highlighting in all 19 IMR-32 single cells thethe presence total chromosomal pattern, which allowed highlighting in all 19 IMR-32 single cells the presence of a total gain of chromosome 6, 2 partial gains, 1 in the chromosomal region between 1p32.3 and 1q44 (194 Mb) gain of chromosome 6, 2 partial gains, 1 in the chromosomal region between 1p32.3 and 1q44 (194 and the other in the chromosomal region between 17q21.31 and 17q25.3 (39 Mb), and a partial loss
Summary
It is widely accepted that cancer is a highly heterogeneous disease and that subpopulations of cells, within a single tumor, can exhibit distinct genomic profiles. Recent technological advances made it possible to analyze nucleic acids and proteins from different areas of a single tumor, as well as within a heterogeneous tumor sample, reaching single-cell resolution. In this way, it is possible to avoid the averaging of bulk analysis and to capture the heterogeneity of cells [2]. NB heterogeneity is related to tumor differentiation and histology: it derives from multipotent Neural Crest Cells (NCCs), forms during embryonic development, and mainly involves the sympathetic nervous system (abdomen, especially the adrenal gland) [4,5]
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