In this study, we assembled a panel of 85 head and neck cell lines which were authenticated by STR profiling of 122 cell lines from multiple tissues of the head and neck region, including oral cavity, pharynx, larynx, nose, skin, thyroid, and parotid gland tissues; samples of leukoplakia lesions; and primary keratinocytes and normal oral epithelium. The verified cell lines included 61 HNSCC cell lines, 11 thyroid cancer cell lines, 3 cutaneous SCC cell lines, 2 leukoplakia lesion cell lines, 5 immortalized primary keratinocyte cell lines, and 3 immortalized primary epithelial cell lines.
We also identified 37 cell lines, which were either misidentified or cross-contaminated. The misidentification and cross-contamination of mammalian cell cultures continues to be a widespread problem in research, even though awareness of this problem dates back more than 45 years. It has been estimated that the incidence of research papers that report the use of cell cultures that were misidentified or cross-contaminated is 15–36% (7
), and a number of recent articles and editorials have highlighted the significance of this problem for the global research community (16
). This study identified authentication problems with 30% of the cell lines that were analyzed. If these problems had remained undetected, these invalid cell lines could have resulted in false conclusions about head and neck cancers that could delay progress in understanding and treating these deadly diseases. We hope that the data presented here can serve as a standard reference for head and neck cancer researchers and facilitate the validation of the cell lines in their own laboratories. Proof of cell line authentication is now required by many journals and is a relatively simple and inexpensive technique.
STR profiling has been reported to be an efficient and reproducible method to verify the true origin of human cell lines (26
). In this study, we performed STR profiling using 9 different genetic markers also utilized by the ATCC (http://www.atcc.org
) in order to facilitate comparison of our data to data in the ATCC’s STR database. STR profiling without appropriate controls can occasionally lead to slightly ambiguous results. For example, the STR profile of JHU028 did not exactly match the profile of the A549 lung cancer cell line. Two alleles were found to be slightly different, amelogenin and THO1 (). The amelogenin marker is known be to somewhat unstable, and samples from males do not always display the Y amelogenin marker, which could be the case for the A549 cell line, which was derived from the tumor of man (27
). Loss of the Y chromosome is common even in peripheral blood lymphocytes and frequently seen in HNSCC cells from older males (28
). The discrepancy in the THO1 marker could have a number of possible explanations, including cross-contamination and genomic instability. Additionally, it is mathematically possible that 2 unique cell lines will have identical STR profiles when only 9 loci are analyzed.
While STR analysis provides an effective way for cell-line comparison and identification of cross contamination, it is almost impossible to determine whether or not a cell line originates from a specific source unless it matches the signature of the donor tissue. The most accurate way to authenticate a cell line is to perform STR profiling of control tissue from the tumor from which the cell line is derived or early-passage stocks of the cells. As these resources often do not exist for many older cell lines, it is often necessary to make judgments about the validity of a cell line based on comparisons to published data and those on the website of cell line collections such as the ATCC (15
). Brenner et al. (13
) reported STR profiles for 73 UM-SCC cell lines and, in some cases, either early-passage tumor cells or normal fibroblasts were available to confirm the genotyping. Of significance to the present study, there was concordance between the results in that study and the results with the same lines in this study. Many of the factors that influenced our decisions about cell line validity are described in the Results section. As we found that contamination can occur even during the stage of primary culture, a new cell line ideally should have a corresponding tumor signature recorded when it is established, and that data should be stored with de-identified clinical information including whether the cell line was derived from previously untreated tissue, or tumor tissue that resulted from treatment failures and the treatment rendered. Much of these data were unavailable during our analysis of these cell lines. In general, we erred on the side of caution and eliminated questionable cell lines from our collection. The 85 unique head and neck cell lines that we report here represent one of the largest panels of cell lines assembled for the head and neck cancer research community.
HPV infection plays an important role in the pathogenesis of oropharyngeal cancers, and HPV DNA is identified in approximately 40–80% of oropharyngeal cancers (29
). However, we found that only 1 of the 56 HNSCC lines evaluated for HPV status was HPV-16 positive. This finding may be due to the fact that only 5 cell lines were derived from oropharyngeal tissue and the fact that the majority of these cell lines were established before the observation of the HPV-associated oropharyngeal cancer epidemic (29
). This finding could also reflect an inherent difference in the biology of HPV-positive oropharyngeal cancers. Patients with oropharyngeal squamous cell carcinoma associated with HPV has been shown in several reports to have improved survival, suggesting HPV-driven disease responds well to treatment and is perhaps less aggressive (31
). The lack of HPV-positive cell lines in our panel suggests that future efforts should be made to identify or derive HPV-positive cell lines in order to study this subset of HNSCC further given the growing importance of this subset of HNSCC (30
). Another important driver of HNSCC is TP53
). We have concurrently examined this gene along with the in vivo
growth characteristics of the HNSCC cell lines among our panel in a separate report (35
), and this study exemplifies how the present report can serve as a foundation for large-scale examination of the biology of HNSCC.
Molecular and genetic heterogeneity have been identified in many tumor types, including breast, colon, and lung tumors. This heterogeneity influences the clinical course of the disease and can predict response to therapy. Cell line panels often reflect the heterogeneity present in primary tumors, and these panels can be used as tools to understand the clinical disease. We believe that the panel of unique head and neck cell lines that we have assembled will shed light on the clinical heterogeneity present in HNSCC. An indication of this heterogeneity can be seen in the range of in vitro
morphologies that we observed. Mesenchymal tumors or so-called epithelial-mesenchymal transition indicates a poor prognosis in HNSCC and other tumor types (36
). We identified at least 18 cell lines with some indication of mesenchymal morphology. This panel should be useful for future studies on the role of mesenchymal characteristics in HNSCC. This panel may also improve our understanding of other aspects of HNSCC, and we and others are currently undertaking a number of studies of the molecular and genetic heterogeneity of this cell line panel and its in vitro
and in vivo
In conclusion, we have identified a new cell line resource for the head and neck cancer research community. This panel of 85 unique cell lines represents many tissues of the head and neck region. Nearly 30% of the cell lines that were assayed during the validation process had been previously misidentificated or cross-contaminated. This finding should serve as a strong rationale to researchers that cell line authentication is critical to efficient and productive research on head and neck cancer. We hope that this panel of 85 genomically verified cell lines will improve our understanding of HNSCC and facilitate the identification of novel therapeutic interventions.
Statement of Translational Relevance
Human cell lines are vital reagents for studying cancer biology, and pre-clinically modeling novel cancer therapeutics, but recent reports have revealed that many cell lines are either misidentified or cross-contaminated with other cell lines. This fact remains true in the case of head and neck cancer cells lines. To clarify the derivation, and lineage of head and neck cancer cell lines for investigators working in this field, we assembled and performed STR analysis on a large panel of such cell lines collected from multiple investigators. Using this method, we found that 85 of the 122 head and neck cell lines had unique genetic profiles. This panel of validated and characterized cell lines serves as a resource for the head and neck cancer research community, to help further our understanding of neoplastic diseases of the head and neck region and to improve our ability to prognosticate and treat them.