Thyroid Cancer Cell Line Misidentification: An Update

Abstract

Human cancer cell lines remain valuable tools to study the biology of cancer and to test new therapies. Thus, the integrity of cell lines derived from tumors is critical to accurately study tumor-specific mechanisms and response to therapy. Despite the importance of cell lines, approximately 18–36% of cell lines are cross-contaminated or misidentified (1). It is well known that the cell line misidentification problem started after the establishment of the HeLa cell line, which is a common cross-contaminant due to its aggressive properties in culture. Despite this long history, cell line cross-contamination and misidentification remains a significant problem and continues to affect the integrity of biomedical research. In 2008, our group reported that approximately 20 out of 40 thyroid cancer cell lines were either redundant or misidentified with other tumor types (2). These results were quite shocking, given that many of these cell lines had been widely used for the past 20 years. Of note, our studies did not include cell lines derived from medullary thyroid cancer (MTC). In this issue of the JCEM, Nelkin and colleagues (3) report the misidentification of two presumed MTC cell lines, RO-H85-1 and RO-D81-1. Specifically, Nelkin's group used short tandem repeat (STR) profiling, which is an internationally recognized method for the genetic profiling of cell lines (4), and showed that the presumed MTC cell line, RO-H85-1, is genetically identical to the bladder cancer cell line, 647-V, and the presumed MTC cell line, RO-D81-1, is genetically identical to the HT-29 colon cancer cell line. The only observed differences in the genetic profiling between identical cell lines were likely due to genetic instability or loss of heterozygosity, which commonly occurs in culture (2, 5). Based on the timeline of establishment, the RO-H85-1 and RO-D81-1 cell lines are likely derivatives of these more aggressive bladder and colon cancer cell lines, respectively. Consistent with the aggressive nature of the HT-29 cell lines, we previously found that the HT-29 colon cancer cell line was a cross-contaminant of what were thought to be 5 different thyroid cancer cell lines (2). Although STR profiling represents a powerful approach for the genetic authentication of cell lines, it does not provide information on tissue of origin. Therefore, the analysis of phenotypic markers can be useful to further characterize the tissue of origin. For the RO-H85-1 and RO-D81-1 cell lines, little has been reported regarding the expression of MTC-specific genes, except, as noted by Nelkin and colleagues, that the RO-H85-1 cell line was originally reported to produce calcitonin (6). Thus, it is possible that the original RO-H85-1 cell line was indeed of MTC origin and later became cross-contaminated, or that the detection of calcitonin production in this cell line was a false-positive result. Nonetheless, the STR results show that the RO-H85-1 and RO-D81-1 “MTC” cell lines are genetically identical to the HT-29 and 647-V cell lines, respectively, and are likely misidentified with these nonthyroid cell lines. Although the RO-H85-1 and RO-D81-1 cell lines were not widely used MTC models, the loss of these two cell lines nonetheless leaves the MTC field with a limited number of valid cell culture models. Nelkin's group (3) went on to validate two of the most widely used MTC-derived cell lines, the TT and MZ-CRC-1, and found that they are genetically unique. Thus, the TT and MZ-CRC-1 cell lines, along with the future development and validation of additional MTC cell line models, which genetically match their tissue of origin, will be critical models to study MTC biology and test new therapeutic strategies. In conclusion, the misidentification of two additional thyroid cancer cell lines once again emphasizes the importance of authenticating cell lines. STR profiling is a straightforward and economically feasible approach for the genetic profiling of cells. The key advantage to STR profiling is that the number of allelic repeats is converted into a genetic “barcode,” which can be used to search databases of cell line STR profiles. All major cell line repositories, including the American Type Culture Collection (ATCC), German Collection of Microorganisms and Cell Cultures (DSMZ), European Collection of Cell Cultures (ECACC), and Japanese Collection of Research Bioresources Cell Bank (JCRB), maintain STR databases for their cell lines. To maintain the genetic identity of cell lines, it is recommended that laboratories routinely perform STR profiling on cell line stocks. Although many journals and funding agencies recommend cell line authentication, an increasing number of journals now require cell line authentication due to the importance of cell line models in translational research and ultimately future therapies for patients.