Cytogenomic characteristics of murine breast cancer cell line JC

Breast cancer (BC), one of the most frequent human tumors, is genetically and histologically heterogeneous. Treatment options can be adapted according to BC subtype. Still, research is necessary to characterize BC biology better and to study potential new treatment options. Murine BC-cell lines can be used as model systems in this respect. Here for the first time murine BC-cell line JC was cytogenomically characterized as being complex rearranged and near-tetraploid. Multicolor banding and array comparative genomic hybridization were applied and the result was in silico translated to the human genome. Even though being commercially available, cell line JC was yet not much included in BC-research, most likely due to a lack of cytogenomic data. Thus, here comprehensive data is provided on chromosomal aberrations, genomic imbalances and involved breakpoints of JC cell line. Also JC could be characterized as a model for BC of luminal B type, basal-like tumor rather than for luminal A type.


Introduction
Breast cancer (BC) is considered to be one of the most aggressive human cancer forms and is leading among cancer-related deaths, especially in females. Survival rates vary across countries depending on diagnostic regimens, awareness of and possibilities to treat this disease [1,2]. Factors like advanced age, low estrogen-levels, family history of cancer, and certain adverse gene mutations, as well as lifestyle influence incidence of this cancer type [2,3]. BCs are a heterogenic group of tumors, and thus divided into subtypes, according to their molecular profiles, morphology, and expression of specific biomarkers [4]. Therefore, each subtype has a different prognosis, a specific response to treatment and a defined clinical outcome [4,5]. All these go together with repeatedly observed aggressive clinical courses and limitations in breast cancer treatment [6]. BC biomarkers include oncogenes and tumor suppressor genes (TCGs), as well as their and related gene-products. Immunohistochemical markers for tumor cell surface or plasma, as well as for growth in this context are estrogen receptor, progesterone receptor, human epidermal growth factor receptor-2 (ERBB2/ HER-2) and epidermal growth factor receptor, cytokeratin 5 and/or nuclear protein Ki67 expression [1,7]. Based on such expression profiles BC can be classified in (1) luminal A-like, (2) luminal B-like (HER2-positive or HER2-negative), (3) HER2-overexpressing, and (4) triplenegative subtypes [4,7].
According to subtype, treatment regimens for BC are adapted [7,8]. After initial surgery, being commonly applied for most BC subtypes chemo-and radio-therapeutic care follow, most of which have severe side effects.
Thus, new types of medication being targeted only towards BC cells are still a pressing necessity [9]. The latter can be e.g. approached by including animal, especially murine models, in research to study biological pathways of BC and BC progression [10][11][12]. Even though murine cell lines are applied in ample research, still most of them are not characterized in detail genetically [1]. This is also valid for the here studied BC cell line JC, which was established in 1978 from a murine BC, developing spontaneously in a 1.5 years old female BALB/c mouse [13]. JC-cells show papillary adenocarcinoma morphology, and cytogenetics done in 1989 revealed only 26-100 chromosomes per cell, and 40 per cell being found in 28 of the 99 evaluated metaphases [14].
Molecular cytogenetics is considered the most practicable technique to characterize genetic alteration in cancer [15]. Thus, here for the first time, the murine BC cell line JC was studied in detail by multicolor-fluorescence in situ hybridization (FISH) using all murine whole chromosome painting (wcp) probes and FISH-based murine multicolor banding (mcb) approach together with arraycomparative genomic hybridization (aCGH). The in silico translation performed on the data to determine the corresponding homologous genetic alterations in human BC was done as previously described [16].

Cell lines
The cell line JC was obtained from American Type Culture Collection (ATCCR CRL-2116 ™ , Wesel, Germany). After being adherently grown in RPMI-1640 medium containing 10% fetal calf serum with recommended antibiotics, cells were cytogenetically prepared [17], and in parallel whole genomic DNA was extracted from the same passage of cells [16]. Molecular cytogenetic / FISH analyses (see below) on the cell line-derived chromosomes, and aCGH analyses on the extracted DNA (see below) were done. According to the ethical committee (medical faculty) and the Animal Experimentation Commission of the Friedrich Schiller University, there are no ethical agreements necessary for studies involving murine tumor cell lines like JC.

Results
After wcp-based mFISH analyses, JC presented as relatively stable, hypo-tetraploid cell line with 64-66 chromosomes (result not shown). Overall there were 3 clones, which differed only in few alterations.
Clone 2 (53.3%) was present in ~53% had the same karyotype shown in Fig. 1 as 64-66<4n> Overall, FISH-data was in agreement with the aCGH results and copy number alterations and breakpoints are summarized in Fig. 2a. An in-silico-translation of those results to the human genome (only imbalances larger than 3.5 megabase pairs were included) identified the corresponding homologous region in the human genome (Fig. 2b). Genomic details are given in Additional file 1: Table S1.
The corresponding homologous regions for the cell line were compared with common imbalances in human BC, and this revealed copy number variations in regions known to harbor oncogenes and tumor suppressor genes (TSG) with an overlap of 40% (Table 1). The breakpoints of the cell line compared with the chromosomal breaks of human BC overlap to 56% (Table 2). Genetic alterations in cell line JC correlated best with the subtype for human BC acc. to Horlings et al. [32] with basal-like (50%) and luminal B type tumors (67%); no correlation was found with human luminal A type BCs (Table 3).

Discussion
BC is one of most frequent tumors in females, genetically heterogeneous and with an often very aggressive course. Therefore, still basic research is necessary to understand better its biology and to find more efficient treatment strategies. Accordingly, especially murine tumor cell lines are favorite model systems to perform basic research studies, as well as such towards testing of new medications [8,33]. Our group characterized already several murine tumor cell lines by the same test strategy as applied here, and thus delivered previously not available but urgently necessary genetic basic data, including chromosome numbers, observable breakpoints and copy number changes [1,16,17,19,34,35]. Here a commercially available murine BC-cell line was characterized for the first time, cell line JC, which was yet rarely used in research. It may be speculated that this was in major parts due to the fact that there was no genetic information available, yet. This gap was closed by the present study now.
JC cell line presents a near-tetraploidy karyotype, which is typically observable, especially in human [36], however, only present in about 50% of murine tumor cell lines [1,16,17,19,34,35]. Polyploidy promotes malignant transformation of mammary cells and increases cell resistance to drugs [36]; still, it is also a known adaptation of cells to cell culture conditions. In case of JC cell line the only available previous cytogenetic characterization dates back to 1989; at that stage the cell line was extremely chromosomally instable with chromosome numbers reported between 28 and 99. As ~ 30% of the cells had modal chromosome numbers of 40 [14] it must be suggested that since that time a stabilization of the karyotype by tetraploidization took place. However, structurally rearranged chromosomes were not duplicated, as observed in other tetraploid BC cell lines [1]. For observed copy number alterations gain of copy numbers was observed for MYC and loss of heterozygosity for tumor suppressor genes MSH2, PTEN and BRCA2 were determined here.
Overall, murine cell line JC can has here been genetically characterized and it could be shown to be suited

Additional file 1: Table S1
The regions of gain and loss of copy numbers, as well of breakpoints of balanced rearrangements, observed in JC and the corresponding homologue regions in humans are listed as cytoband and position (GRCh37/hg19).