Cytogenomic characterization of three murine malignant mesothelioma tumor cell lines

Malignant mesothelioma (MM) is a rare aggressive cancer primary located in pleura and lung. MMs can be divided into biphasic, epithelioid and sarcomatoid subtypes. In majority of cases MMs are induced by asbestos fiber exposure. As latency period after asbestos exposure ranges between ~ 10 and 60 years MMs are mainly observed in elder people. Human MM, being a rare tumor type, lacks detailed cytogenetic data, while molecular genetic studies have been undertaken more frequently. However, murine MM cell lines are also regularly applied to get more insight into MM biology and to test new therapy strategies. Here the murine MM cell lines AB1, AB22 and AC29 were studied by molecular cytogenetics and molecular karyotyping. Interestingly, yet there were no genetic or genomic studies undertaken for these already in 1992 established cell lines. The obtained data on genomic imbalances in these murine cell lines was translated into the human genome as previously reported based on human and murine genomic browsers. It turned out that all three cell lines showed high similarities in copy number variants as observed typically in human MM. Also, all three cell lines were most similar to human epithelioid MMs, and should be used as models therefore.


Background
Malignant mesothelioma (MM) is a rare aggressive tumor-family of pleura and lung, with an incidence of about 0.002% [1,2]. In most of the cases, MMs are located in pleural mesothelium, and only rarely in peritoneal cavities, tunica vaginalis or pericardium. MM can be specifically promoted by exposure to asbestos fibers [3,4]. Besides working with asbestos, accordingly contaminated buildings provide an additional, often unrecognized problem, where affected person can undergo asbestos inhalation, ingestion, or less often, severe exposures via the skin [3,5]. The latency periods for MM after asbestos exposure can range from 1 to 6 decades, and the median age of onset is 72 years [6].
Histomorphologically and according to their growth parameters, MM can be divided into the following, most frequently observed subtypes: (1) biphasic, (2) epithelioid and (3) sarcomatoid. Different median survival times were attributed to each subtype; the best prognosis has the epithelioid, while the worst one has the sarcomatoid subtype [13,17].
As MM is an aggressive tumor with poor prognosis, there is ongoing research to better understand the biology of this cancer type [18,19]. Therefore, also animal models, including murine tumor cell lines are regularly applied, also because human and mouse genomes show homologies within coding sequences of up to 97% [20]. In 1992 Davis and coworkers inoculated asbestos fibers into female BALB/c and CBA mice and established successfully 12 MM cell lines from tumor ascites cells [21]. Here two of these cell lines derived from BALB/c mice, i.e. AB1 and AB12, and one of them from CBA mice (AC29) were studied. Strikingly, in none of these cell lines (cyto) genetic research was undertaken yet to characterize their cytogenomic content. However, the latter data are important to use such cell lines in the best suited way to answer questions about MM-biology or to apply them in tests for new treatment options, i.e. for drug tests meant for the corresponding MM subtype.
Two marker chromosomes could be resolved here, neither by multicolor fluorescence in situ hybridization using all 21 murine whole chromosome paints as probes (mFISH- Fig. 3a) nor by mcb. Thus, most likely they are derivatives of the centromere-near region of any of the murine chromosomes-subband A1, which do not specifically stain by any euchromatic DNA-probe. Accordingly, the marker chromosomes could be left overs of the dic(9;15)(A1;A1) and a del(?)(A1).

aCGH
Array comparative genomic hybridization (aCGH) data (Additional file 1: Table 1) together with which FISH results could be summarized in Figs. 4a, 5a and 6a. These results were translated to the corresponding homologous regions in the human genome as depicted in Figs. 4b, 5b and 6b. All in the evaluation included imbalances were larger than 3.5 mega base pairs.

Data-analyses
The common aberrations and cytogenetic changes that frequently occur in MM [22] revealed for all three cell lines to be less similar to human biphasic MM subtype (

Conclusions
The murine MM cell lines AB1, AB22 and AC29 were studied in this paper for the first time by molecular cytogenetics combined with aCGH. This enabled to determine their genetic alterations and imbalances and align these with human MMs. mFISH using whole chromosome painting probes revealed the general characteristics of the cell lines, like the ploidy, clonal and nonclonal changes as well as numerical and intrachromosomal structural aberrations. By mcb interchromosomal alterations as duplications, deletions or inversions, and chromosomal breakpoints involved could be uncovered, as previously reported [23][24][25][26][27][28]. The aCGH data was aligned with the FISH results and also used to determine breakpoints of unbalanced rearrangements (Additional file 1: Table 1).
The cell lines A1 and AC29 were tetraploid; as in both cell lines derivative chromosomes were present twice, it is possible that polyploidization was a result of cell culture, and tetraploidy was absent in original tumor. Such so-called telomere-driven tetraploidization in the context of cell culture-related factors as trypsin treatment, increasing number of cell-culture passages, and oxygen exposure [29,30] was discussed before. However, as no karyotype of tumor or early Fig. 4 aCGH results for cell line AB1. In a copy number variations detected are summarized with respect to a tetraploid karyotype. Gains are depicted as green bars (one more copy = light green; two more copies = dark green), loss of one copy is depicted as a red bar and loss of two copies is depicted as a dark-red bar. Breaks are registered here as arrows. In b results of in silico translation for AB1 to human genome are shown the same way as in a cell passages of A1 and AC29 are available, this is just speculation and cannot be tested by any means.
Interestingly, a deletion of CDKN2A gene is considered as one of the most typical alterations in human MMs [7][8][9]. In the AB1 and AC29 there was indeed a deletion in the murine homologous region; however in cell line AB22 this region was duplicated (Additional file 1). For other tumor suppressor genes BAP1, NF2 and TP53 thought to play important roles in human MMs [10][11][12][13][14][15][16], there is even less or no concordance in the copy number variant regions of the three cell lines (Additional file 1).
Nonetheless, the overall similarities of copy number variants found in the three murine MM cell lines compared to human MM are striking. A shown in Table 1 all three cell lines can serve as models for human epithelioid MM. As similarities are also high for sarcomatoid MM, also here they may be used as models  Fig. 4 for. However, AB1, AB22 and AC29 are definitely not models for human biphasic MM.

Murine MM cell lines
The murine cell lines AB1 and AC29 were obtained from Cell Bank Australia (Westmead, Australia, order #s CBA-0144 and CBA-0152) and AB22 European Collection of Authenticated Cell Cultures (Salisbury, UK-order# ECACC 10092307). For this study, the cells were cultivated and divided into two portions, worked up cytogenetically (portion 1), and used to extract wholegenomic DNA (portion 2) as previously described [24].

Data analysis and translation
The regions of imbalances and breakpoints in AB1, AB22 and AC29 were characterized after analyses of aCGH and mcb data, and aligned with their human homologous regions using Ensembl Genome Browser, as previously described [24]. The data we obtained was compared with the literature [22] (Tables 1 and 2).