Human lung malignancies are one of the leading causes of cancer-associated death and account for ~17% of annual new tumor diagnoses worldwide. Lung cancers are categorized into the following subtypes: Lung squamous cell lung carcinoma (L-SCC; accounting for ~30%) and non-SCCs, which include e.g., lung adenocarcinomas (L-AC; ~40%) and large cell carcinomas (~10%) (1,2). Approximately 90% of L-SCCs are induced by mutagens, particularly toxins inhaled during smoking (1,3).

Accordingly, clinicians and oncologists are confronted with high numbers of lung cancer patients seeking treatment and cure. There are numerous therapeutic options available, including cytotoxic chemotherapy with carboplatin or cisplatin, or erlotinib or cetuximab to target epidermal growth factor, ipilimumab or nivolumab (inhibitors of the immune system) and ramucirumab (antiangiogenetic mechanism of action) (1). Ongoing research aims to target and interfere with additional metabolic pathways in cancer cells to increase sensitivity and response to therapies in lung cancer patients; numerous studies were performed using murine lung cancer cell lines (4-6).

The majority of commonly used murine cancer cell lines were established decades ago. More uniform in shape than human chromosomes, murine chromosomes are difficult to characterize by banding cytogenetics, which resulted in a lack of detailed genetic characterization despite the widespread use of these cell lines (7,8). The urethane-induced murine lung cancer cell line LA-4 (also known as LA4 or LA 4), utilized in ~70 studies in the literature, exemplifies the cytogenomic under-characterization of murine tumor cell lines. LA-4 was established in 1975(9) from a lung adenoma induced in a (most likely female) A/He mouse. Cells from a primary tumor were cultured and 50 cells were cytogenetically analyzed; on average, 114 (range, 54-133) chromosomes per metaphase were counted. Stoner et al (9) reported that the subclones LA-1 to LA-6 were established from the primary tumor; however, further details were only reported for LA-4 and at present, the other 5 cell lines are no longer available. Reports of LA-4 documented epithelial morphology with slower growth compared to the primary tumor. In addition, LA-4 cells did not have any tumorigenic potential in nude mice. Solid-stain cytogenetics revealed 116 (range, 87-132; 80% of cells within the range of 106-122) chromosomes (9). According to the American Type Culture Collection (ATCC) webpage (accessed from Germany, but cell stocks are the same worldwide if purchased from ATCC, https://www.lgcstandards-atcc.org/Products/All/CCL-196.aspx?geo_country=de#characteristics), LA-4 cells have 38-256 chromosomes per cell, with rearranged chromosomes in ~12% of the metaphases.

As comprehensive cytogenomic characterization of murine tumor cell lines may be performed using murine multicolor banding (mcb) combined with molecular karyotyping (8), the present study provided the first karyotype (including ploidy level) for the LA-4 cell line, with an overview of chromosomal imbalances and in silico translation to regions of homology within the human genome; thus, it was possible to determine for which human tumor type this cell line may be used as model.

The LA-4 cell line is derived from a mutagen (urethane)-induced primary tumor. In 1975, LA-4 cells were demonstrated to have a karyotype with 87-132 chromosomes and 80% of the cells had chromosome numbers ranging from 106-122; no obvious structural aberrations were observed at that time (9). The only other cytogenetic analyses performed revealed structural aberrations in 12% of the cells with chromosome numbers ranging from 38 to 256 (https://www.lgcstandards-atcc.org/Products/All/CCL-196.aspx?geo_country=de#characteristics).

In the present study, the majority of LA-4 cells had 85 to 93 chromosomes, i.e., a hyper-tetraploid karyotype. There were three distinct (pseudo-)dicentric derivatives, two neocentrics, four unbalanced translocations, two chromosomes with terminal deletions and one chromosome with a balanced inversion. Thus, it was not possible to confirm the original observation that LA-4 harbored no structural rearrangements (9). Also, in contrast to the information from ATCC, these derivative chromosomes were present in 100% and not limited to only 12% of the cells, a result confirmed by the aCGH results (https://www.lgcstandards-atcc.org/Products/All/CCL-196.aspx?geo_country=de#characteristics). However, as observed by Stoner et al (9), there was a small subpopulation with only 67-72 chromosomes per cell. The observed dicentric and neocentric chromosomes are unusual and only rarely reported as stable derivatives in tumor cell lines; of note, they have also been observed in human cancer cell lines (13). In previous studies of 23 murine tumor cell lines, these phenomena were only observed in the colorectal cancer cell line CMT-93 [for an overview see (14)].

These results indicate that LA-4 underwent a karyotype evolution during the past 46 years of cell culture. However, the LA-4 cells examined in the present study had an overall stable karyotype, with a tendency towards chromosomal loss in ~12% of cells. Of note, tetraploidization was an early event that was present in the original tumor (9). Previously documented cell line evolution, e.g., for HeLa (15), suggests that variant clones of LA-4 may be present in different laboratories. Thus, it is highly recommended that an aCGH or cytogenetic study of the locally available and used LA-4 cells is performed to compare their chromosomal/genetic constitution with that reported in the present study prior to further experimentation.

LA-4 was established from a benign lung cancer, an adenoma. As reported in the present study, these genetic data establish that LA-4 cells have evolved in vitro, through the acquisition of compounding genetic changes, which were not initially present in 1975(9). However, the comparison in Table I, which aligned LA-4 with specific human lung tumor types, was rather inconclusive at first observation. Aberrations typical for human L-ACs were ~44% concordant and human L-SCCs were ~50% concordant. Of note, LA-4 cells exhibited ~58% concordance with imbalances observed in human HNSCCs. Thus, it may be cautiously proposed that LA-4 cells demonstrate characteristic (genetic) features most consistent with human SCC cells. As their lung origin is indubitable and they are mutagen-induced, similar to the majority of human L-SCCs, LA-4 may be considered a well-suited model for non-metastatic human L-SCC.