Abstract
It is well known that in vitro subculture represents a selection pressure on cell lines, and over time this may result in a genetic drift in the cancer cells. In addition, long-term cultures harbor the risk of cross-contamination with other cell lines. The consequences may have major impact on experimental results obtained in various laboratories, where the cell lines no longer reflect the original tumors that they are supposed to represent. Much neglected in the scientific community is a close monitoring of cell cultures by regular phenotypic and genetic characterization. In this report, we present a thorough characterization of the commonly used glioblastoma (GBM) model U-251, which in numerous publications has been wrongly identified as U-373, due to an earlier cross-contamination. In this work, the original U-251 and three subclones of U-251, commonly referred to as U-251 or U-373, were analyzed with regard to their DNA profile, morphology, phenotypic expression, and growth pattern. By array comparative genomic hybridization (aCGH), we show that only the original low-passaged U-251 cells, established in the 1960s, maintain a DNA copy number resembling a typical GBM profile, whereas all long-term subclones lost the typical GBM profile. Also the long-term passaged subclones displayed variations in phenotypic marker expression and showed an increased growth rate in vitro and a more aggressive growth in vivo. Taken together, the variations in genotype and phenotype as well as differences in growth characteristics may explain different results reported in various laboratories related to the U-251 cell line.
Highlights
Since the isolation of the first human immortal cancer cell line (HeLa; from a cervical cancer) in 1952, cell lines represent important biological model systems that are both accessible and easy to handle
We analyzed various batches of U-251 by short tandem repeat (STR) fingerprinting, and detected that we had three subclones of the U-251 cell line: U-251N which is quite similar to the STR profile published for U-251 [7, 14]; one clone which was quite similar to U-251N but with a gain at FGA 20, resulting in three alleles at this locus; and U-251-4q12 which is more heterogeneous at each locus than the other two (Table 1)
Loss of heterozygosity (LOH) at one or more STR alleles is frequently found in cancer tissues and cancer cell lines [16, 17], the STR profile of the original low-passage U-251MG show complete allelic heterogeneity, indicating that it has undergone less selection pressure and genetic drift in culture
Summary
Since the isolation of the first human immortal cancer cell line (HeLa; from a cervical cancer) in 1952, cell lines represent important biological model systems that are both accessible and easy to handle. Cell lines have facilitated cancer research in many ways, such as the study of functional aspects of tumor transformation and gene expression, the elucidation of mechanisms of resistance to therapy, drug screening, and the development of a 2014 The Authors. Cancer cell lines represent important tools to study genetic aberrations and molecular pathways in cancer. Cell lines are known to provide more consistent and reproducible research data than the more heterogeneous and more limited biopsy material, subculture may select for a specific genetic clone within the patient biopsy. Cell lines represent only a small subpopulation of the original tumor. The consistency and reproducibility of cell line studies between different laboratories represents a major challenge based on genetic drift upon long-term subculture and potential cross-contamination
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