|Year : 2017 | Volume
| Issue : 1 | Page : 10-13
The cross-contaminated cell lines of adenoid cystic carcinoma: A crucial concern
Nan-Nan Liu1, Sen-Lin Zhang2, Wei Chen2
1 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University; Department of General Dentistry, Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing, Jiangsu, China
2 Department of Stomatology, Affiliated Jinling Hospital, Nanjing Medical University, Nanjing, Jiangsu, China
|Date of Submission||12-Nov-2016|
|Date of Acceptance||29-Dec-2016|
|Date of Web Publication||21-Mar-2017|
Department of Stomatology, Affiliated Jinling Hospital, Nanjing Medical University, 305 East Zhongshan Road, Nanjing, Jiangsu 210002
Source of Support: None, Conflict of Interest: None
Adenoid cystic carcinoma (ACC) is a common type of salivary gland cancer, characterized by slow growth but high incidence of distant metastasis. Distant metastasis occurs in 25%–50% of ACC patients and more commonly in lungs. However, the molecular mechanism underlying the aggressive growth of ACC remains obscure. Human tumor cell lines are extremely useful in studying cancer biology, under both in vitro and in vivo condition. Unfortunately, the tumor cell lines are susceptible to cross-contamination, by other cell lines, during routine culture. Recent studies have proved that most of the ACC cell lines, established for preclinical research, are either cross-contaminated or nonhuman cells. Here, we review the literature assessing the identity of ACC cell lines, dated up to September 1, 2016, using PubMed, EMBASE, and the China National Knowledge Infrastructure databases with the following keywords: “adenoid cystic carcinoma,” “cell line,” and “characterization” or “contamination.” According to the results, only salivary adenoid cystic carcinoma (SACC)-83/SACC-lung metastasis, the HPV-transformed ACC-112 and the two newly established moderately differentiated adenocarcinoma-ACC-01 and LACC-1 cell lines are authentic ACC cell lines. These results suggest that, in the future, it is crucial to authenticate the ACC cell lines before research.
Keywords: Adenoid cystic carcinoma, cell line, cross-contamination
|How to cite this article:|
Liu NN, Zhang SL, Chen W. The cross-contaminated cell lines of adenoid cystic carcinoma: A crucial concern. Transl Surg 2017;2:10-3
| Introduction|| |
Adenoid cystic carcinoma (ACC) is a common subtype of salivary gland malignancy. It represents 25% of malignant tumors in the major salivary glands and 50% in the minor salivary glands. ACC was first recognized and described as a malignant epithelial neoplasm of salivary gland origin, by Robin, Lorain, and Laboulbene in 1853. Further, the term ACC was first used by Spies in 1930. ACC not only originates from salivary glands but also from other glands such as lacrimal, breast, and tongue glands. The neoplasm is characterized by heterogeneous phenotypic features and biologically demonstrates a persistently progressive behavior. It has a protracted clinical course involving local recurrence, perineural invasion, and distant hematogenous metastasis, particularly to the lungs. Despite the various treatment options available, a poor long-term prognosis of ACC patients is often observed, mainly due to the above reasons.
Human tumor cell lines are extremely useful for studying cancer biology and are widely used to model cancer growth and therapy, under both in vitro and in vivo conditions. Unfortunately, the tumor cell lines are susceptible to cross-contamination by other cells during routine culture, as evidenced in the past decades. This phenomenon has been reported for more than 45 years, and it is estimated that 15%–36% of all cell lines are mixed with or mistaken for other cancer cell lines., Recently, many ACC cell lines were reported to be cross-contaminated. Hence, here, we review the literature assessing the identity of ACC cell lines.
| Literature Search Strategy|| |
The PubMed, EMBASE, and the China National Knowledge Infrastructure databases were searched with the following keywords: “adenoid cystic carcinoma,” “cell line,” and “characterization” or “contamination,” dating up until September 1, 2016. The references of the eligible articles and textbooks were also manually searched to find other potential studies. Any disagreement among authors was resolved by discussion.
| Results|| |
Till date, approximately ten ACC cell lines, including ACC-2, ACC-3, ACC-M, ACC-S, ACC-NS, ACC-112, CAC-2, salivary adenoid cystic carcinoma (SACC)-83, and SACC-lung metastasis (LM), have been established.,,,,,,, Among them, the paired cell lines (ACC-M/ACC-2 and SACC-LM/SACC-83) are most widely used in the research on ACC. The ACC-2 cell line was established from a 28-year-old female patient and ACC-3 cell line from a 49-year-old male patient, in 1986. ACC-M cell line was established from an aggressively LM subclone, screened from ACC-2, by combination of in vivo selection and in vitro cloning. The average LM rate of ACC-2 and ACC-M cells was 18% and 96%, respectively. The other partner cell lines, used in ACC research, are SACC-LM and SACC-83. The SACC-83 cell line was derived from a patient pathologically diagnosed as SACC in sublingual gland in 1990. SACC-LM is the aggressively LM clone of SACC-83 established with similar methods, as explained above. The LM rate was 85% for SACC-LM, whereas only 33% for SACC-83. These ACC cell lines are not only housed in Biological Resource Centers such as “China Center for Type Culture Collection” but also have been exchanged between laboratories.
Unfortunately, most of the above ACC cell lines are proven cross-contaminated. Early in 2008, Choi et al. reported that the ACC-2, ACC-3, and ACC-M cell lines had identical genotypes. This result was then authenticated by independent laboratories. In 2009, Phuchareon et al. performed short tandem repeat (STR) analysis on six ACC cell lines: ACC-2, ACC-3, ACC-M, ACC-S, ACC-NS, and CAC-2. Of these, ACC-2, ACC-3, and ACC-M were determined to be HeLa cells, a kind of cervical adenocarcinoma cell line used in the laboratories worldwide. Further, the ACC-S cell line was found composed of T24 urinary bladder cancer cells, whereas the ACC-NS and CAC-2 cells were found contaminated with cells derived from nonhuman mammalian species; the ACC-NS cell line from mouse cells and the CAC-2 cell line from rat. In 2011, Zhao et al. again proved that the ACC-2, ACC-3, and ACC-M cells were HeLa cells, by STR profiling.
However, there are some encouraging results, as paired cell lines, the SACC-83 and SACC-LM, are confirmed as authentic ACC cell lines by Dong et al. in 2011, through STR analysis and immunostaining. Thus, the authenticated pair of SACC-83/SACC-LM cell lines is an extremely useful tool in ACC research. Further, in a recent study by Drier et al., published in Nature Genetics in 2016, ACC-112, the HPV-transformed ACC cell line, was also authenticated.
To avoid the potential cross-contamination threat, some researchers have tried to establish new kinds of ACC cell lines. In the United States, scientists have successfully established a new immortalized ACC cell line named moderately differentiated adenocarcinoma (MDA)-ACC-01, from a 48-year-old male patient with a primary ACC of the base of tongue. The MDA-ACC-01 was authenticated as a true ACC cell line, with hybrid epithelial-myoepithelial feature, through functional experimentation. In China, the researchers have also successfully established and authenticated a new ACC cell line named LACC-1, from a 35-year-old female patient with a solid pattern of ACC on the right lacrimal gland.
| Conclusion|| |
Cell line contamination is a significant generalized global problem, not only of concern to ACC cell lines and not only in China. For example, according to Lin's  study, nearly 30% (37) of a panel of 122 head and neck squamous cell cancer cell lines were either misidentified or cross-contaminated. Another study analyzed 550 leukemia-lymphoma cell lines and reported that 14.9% (82) were cross-contaminated with different cells. These results suggest that any cell lines, including ACC cell lines, should be authenticated before research. We anticipate that these efforts will notably reduce cell contamination and finally improve the quality of ACC study.
Indeed, the NIH has issued a notice that grants applications for studies involving cell lines must include cell line authentication, and journals are beginning to require authentication before publication of research articles. To solve the difficult problem, STR profiling has been reported to be an efficient and reproducible method to verify the true origin of human cell lines.
STR profiling techniques were originally developed for forensic applications. The technology allows easy determination of a cell line's authenticity at minimal cost. It has been reported to be an efficient and reproducible method to verify the true origin of human cell lines. Using nine different genetic markers (Amelogenin, CSF1PO, D13S317, D16S539, D5S818, D7S820, TH01, TPOX, and vWA) also utilized by the American Type Culture Collection (ATCC) (http://www.atcc.org), STR profiling provide an easy method to authenticate cell lines by comparing data in the ATCCs STR database. As early as 2009, at ATCC, approximately 700 out of 1700 tumor cell lines were already STR profiled (http://www.atcc.org/CulturesandProducts/CellBiology/STRProfileDatabase/tabid/174/Default.aspx).
Various studies have defined the cross-contaminated cells as those that match at 80% or more of alleles in STR analysis. In the previous study, the percent matches of ACC-2, ACC-3, and ACC-M cell lines to HeLa cells were 100%, 97%, and 94%, respectively. These data suggest that the original ACC-2, ACC-3, and ACC-M cells had been replaced with HeLa cells. The authors inferred that these three ACC cell lines may have acquired the genetic changes, particularly in vWA, due to passaging effects in HeLa cells during long-time cell culture. As detailed in Phuchareon's  article, these three ACC cell lines were kindly donated by two independent laboratories from China and Japan. As the above three ACC cell lines were established in a single laboratory, laboratory in Fudan University (China), one may guess that perhaps all ACC-2, ACC-3, and ACC-M cell lines across the worldwide biomedical laboratories are actually HeLa cells.
Taken together, it is concluded that the ACC-2, ACC-3, ACC-M, ACC-S, ACC-NS, and CAC-2 cell lines are cross-contaminated [Table 1], whereas the SACC-83, SACC-LM, ACC-112, and the two newly established MDA-ACC-01 and LACC-1 are authentic ACC cell lines [Table 2]. These results suggest that the cell line authentication is necessary and critical before ACC research, and one must avoid the cross-contamination of the cell lines during the process of cell culture and storage.
|Table 1: Misidentified and cross-contaminated adenoid cystic carcinoma cell lines|
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Financial support and sponsorship
This study was supported by grants from the National Natural Science Foundation of China (Grant No. 81102051), and the Natural Science Foundation of Jiangsu Province (Grant No. BK2011659).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]