Breast cancer cell lines carry cell line-specific genomic alterations that are distinct from aberrations in breast cancer tissues: Comparison of the CGH profiles between cancer cell lines and primary cancer tissues

Cancer cell lines are routinely used for various kinds of biomedical research under the assumption that cell lines reflect the genotypic and phenotypic characteristics of primary tumor tissues. However, such cell lines do not always faithfully represent genomic alterations and gene expression observed in tumor tissue specimens [1‐4], and therefore the use of cell lines may lead to erroneous conclusions in some instances. In order to avoid erroneous conclusions in experiments using the cell lines, first of all, it is important to clarify the extent of similarities and differences in genomic aberrations between cancer cell lines and primary cancer tissues.

According to the commonly accepted model for cancer development, somatic mutations accumulate in a cell in the process of tumorigenesis. In clinically overt cancers, not only a large number of genomic aberrations are detected but also genomic instability successively yields genomic alterations in a cancer cell. This theory explains why the number of genomic aberrations is greater in advanced cancers than in early cancers [5‐8]. The established cell lines also undergo genomic changes with multiple passages in culture [9‐11]. Some of the genomic alterations detected in the cell lines are considered as a result of selective pressure to adapt to the culture conditions, while others may be just incidental [12, 13]. This theory raises an additional question in regard to whether there are genomic aberrations specific for cell lines, or in vitro-specific genomic aberrations. In this context, it is crucial to distinguish genomic aberrations in tumor tissues from the secondary changes with cultivation. The differentiation between these aberrations is practically difficult, because available data on difference in the genomic changes between cell lines and tumor tissue specimens are very limited at present [13, 14]. The comparison of genomic profiles obtained from cell lines with those from primary tumor tissues is one of the best ways to determine the difference in genomic aberrations between cell lines and primary tumor tissues and to identify recurrent celll lines-specific genomic aberrations.

This study examined the DNA copy number aberrations (DCNAs) of 24 breast cancer cell lines and 35 primary breast cancer tissues using array-based comparative genomic hybridization (aCGH). The present paper showed that the breast cancer cell lines preserved genomic alterations detected in primary cancer tissue specimens and that the cell lines concurrently carried secondary genomic alterations. Some of the secondary genomic alterations were recurrent and cell line-specific.

CGH profiles were considerably different between the cell lines and tumor tissue specimens (Figure 1), and the array data reported in this manuscript are available on the following websites: http://​cibex.​nig.​ac.​jp/​cibex2/​ExperimentMiame.​do?​queryExperimenta​lDesignAccession​=​CBX105. The number of DCNAs was more in the cell lines than in the tissue specimens as a general trend. The average number of copy number gains and losses were 651.7 ± 148.8 (standard deviation) and 1180.7 ± 433.8, respectively in cell lines, while they were 424.2 ± 215.9 and 548.0 ± 324.7 in the tumor tissue specimens (Figure 2). However, no statistical difference was found between these two groups. The average number of clones with DNA amplification was statistically different between the cell lines and the tissue specimens (P = 0.012), 41.3 ± 29.9 for the cell lines and 15.9 ± 18.6 for the tissue specimens (Figure 2).

DCNAs were frequently detected on the all chromosomes in the cell lines, but inter-regional differences in the frequency were apparent. DCNAs detected In >50% of the cell lines were as follows: clones with copy number gains and losses were detected at 1q, 5p, 8q and 20q and at 1p, 3p, 4p, 6p, 8p, 9p, 10q, 11p, 13q, 15q, 17p, 18q and X, respectively (Figure 1a). DCNAs detected in >50% of the tissue specimens were as follows: gains of 1q and 8q, and losses of 8p 11q, 16q and 17p were detected (Figure 1b). There were DCNAs shared by the cell lines and tumor tissue specimens and recurrent DCNAs in the tissue specimens were generally frequent in the cell lines. The copy number gains of 1q and 8q were frequent in both the cell lines and tissue specimens (Table 1). In particular, the gain of 1q44 was detected in 13 (54.3%) of 24 cell lines and in 19 (54.3%) of 35 tissue specimens. The gain of 8q22.1 was detected in 15 (62.5%) of the cell lines and in 22 (62.9%) of the tissue specimens. The copy number losses of 11q and 17p were frequent in both the cell lines and the tissue specimens. Notable copy number losses of 17p11.2 and 11q23.2 were frequent; 17p11.2 loss was detected in 13 (54.2%) of the cell lines and 19 (54.3%) of the tissue specimens and 11q23.2 loss was detected in 15 (62.5%) of the cell lines and 22 (62.9%) of the tissue specimens (Table 1).

The clone-by-clone comparison of the DCNAs between the cell lines and the tumor tissues provided detailed information concerning the difference in DCNAs between two different sample groups. Gains of 5p14-p15, 20q11 and 20q13 and losses of 4p13-p16, 18q12, 18q21, Xq21.1 and Xq26-q28 were detected almost exclusively in the cell lines (Figure 3). For instance, the copy number gain of clones located on 20q13.33 and 20q13.13 were detected in as many as 75% of the cell lines, but it was a rare event (around 3% of tumors) in the primary tumor tissues (P = 5.68 × 10^-9 and P = 1.23 × 10^-8, respectively)(Table 2). The frequency of the Xq27.1 loss was detected in 16 (66.7%) of the cell lines, but not detected in the tissue specimens (P = 1.53 × 10^-8) (Table 2).

The frequency of amplification was higher in the cell lines than in the tissue specimens (Table 3). DNA amplification was frequent at a wide chromosomal region 17q12-q21 in both the cell lines and tissue specimens, though other BAC clones with frequent DNA amplification were considerably different between these two sample groups (Table 3). The amplification of the chromosomal region 17q12 including ERBB2/HER2 was detected in 10 (41.7%) of 24 cell lines and 6 (17.1%) of 35 tissue specimens (P = 0.037).

Making a comparison of the CGH profiles between the established cell lines and their parental tumor tissue specimens is practically impossible, because the source tissue specimens are no longer obtainable. Therefore, the comparison of the genomic profiles obtained from cell lines with those from primary tumor tissues is one of the best ways to determine the difference in genomic aberrations between cell lines and primary tumor tissues and to identify recurrent cell lines-specific genomic aberrations.

The array-based CGH revealed a large number of DCNAs including recurrent ones in both breast cancer cell lines and primary breast cancer tissues. There was a tendency that the average number of DCNAs was greater in cell lines than in primary breast cancer tissue specimens, 1832.4 (45.5% of spots) and 972.2 clones (24.1%) for a cell line and tumor tissue, respectively. This result is consistent with the data reported by Naylor and colleagues [24]. The comparison of CGH profiles between cell lines and tumor tissues revealed gains of 1q and 8q and losses of 8p, 11q, 16q and 17p as recurrent DCNAs shared by two groups. Although there are some variations in the CGH patterns of breast cancers between studies, copy number gains of 1q, 8q, 11q, 17q and 20q and losses of 6q, 8p, 9p, 13q, 16q and 17p were previously reported as recurrent aberrations in breast cancers [14, 15, 24‐27]. Gains of 1q44, 1q21 and 8q21-q23 and a loss of 17p11-p13 were detected in both of the cell lines and the tumor tissues at high rates (>50% of both samples). The present observations support the hypothesis that the cell lines basically preserve the genomic alterations that have occurred in primary tumor tissues [13, 24]. These recurrent DCNAs detected in both cell lines and tumor tissues are though to be closely relevant to the development and progression of breast cancer. The clone-by-clone comparison of DNA copy numbers between cell lines and tumor tissues allowed detection of recurrent DCNAs exclusively in breast cancer cell lines as well as recurrent DCNAs shared by two groups. Gains of 5p14-15, 20q11 and 20q13 and losses of 4p13-p16, 18q12, 18q21, Xq21.1 and Xq26-q28 were detected almost exclusively in the cell lines. Although the resolution of the BAC array used in this study is low in comparison to the tiling arrays, this study revealed a distinct difference in the patterns of the copy number aberrations between primary tumor tissues and cell lines. When data of cell lines are compared between the present CGH platform and others including tiling arrays, the chromosomal regions identified as copy number aberrations in this study are compatible with those provided by tiling arrays [27, 28]. Indeed, gains of 8q and 20q were frequently detected by the 4K array slides as well as by tiling arrays. The present data provided by the 4K array platform are thus considered to be reliable.

It is particularly worth noting that some of recurrent DCNAs that are frequently detected in cell lines are hardly detected in primary cancer tissues. These DCNAs correspond to cell line-specific or in vitro-specific alterations [13, 24, 29]. Interestingly, these recurrent DCNAs identified in breast cancer cell lines were also detected in cell lines of other types, such as gastric cancer [30], lung cancer [31], colon cancer [32] and pancreatic cancer cell lines [33]. Established cell lines possibly carry the cell line-specific DCNAs regardless of the parental tumor types. DCNA profiles primarily depend on organs and tissues from which cancer develops [13, 34, 35]. Breast epithelial cells transformed in vitro show genomic alterations similar to those of cell lines [36]. Therefore, taking these observations into consideration, it is legitimate to consider that in vitro environments endow cells with genomic aberrations of which cell line-specific DCNAs are critical for cells to survive and proliferate in vitro. Indeed, it is known that the pattern of CGH profiles change in varying degree with the cell passage numbers [9].

The difference in the CGH profiles found between the cell lines and tumor tissues is not attributed to just variations in genomic alterations between parental histological types, because DCNAs detected exclusively in cell lines are not explained by the difference in histologic types [37‐39]. Accordingly, this also strengthens the view that recurrent DCNAs detected exclusively in cell lines can be called cell line-specific aberrations.

The average number of amplified clones was more than double in the cell lines than the tumor tissues (41.3 vs. 15.9). In particular, the frequency of amplification for clones on 17q12-q21, encompassing many genes including ERBB2/HER2 that is the most frequent amplified gene in breast cancers [40, 41], was higher in the cell lines than in tissue specimens. The amplification frequency of the BAC clone harboring ERBB2/HER2 was in 41.7% of the cell lines and 17.1% of the tumor tissues. The amplification of ERBB2/HER2 is usually detected in no more than 20% of breast cancer patient [42]. Therefore, it can be said that additional DNA amplifications occur not only in the chromosomal region but also in other regions in the cell lines.