Christopher Korch

Deoxyribonucleic Acid Profiling Analysis of 40 Human Thyroid Cancer Cell Lines Reveals Cross-Contamination Resulting in Cell Line Redundancy and Misidentification

CONTEXT Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated. OBJECTIVE We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis. RESULTS Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines. CONCLUSIONS Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.

DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination

OBJECTIVES Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines. METHODS Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed. RESULTS Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively. CONCLUSIONS Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models.

Population-Based Estimate of the Contribution of TP53 Mutations to Subgroups of Early-Onset Breast Cancer: Australian Breast Cancer Family Study

Although germline TP53 mutations have been identified in women with breast cancer from families meeting Li-Fraumeni criteria, their contribution to breast cancer per se is not well known, but is thought to be minimal. We aimed to determine the prevalence of germline TP53 mutations in subgroups of early-onset breast cancer. Germline TP53 mutation status was assessed by DNA sequencing, screening for heterozygous single-nucleotide polymorphisms, and Multiplex Ligation-Dependent Probe Amplification analyses. From an Australian population-based series of invasive breast cancers, we studied (a) 52 women diagnosed before age 30 years unselected for family history [very early-onset (VEO)] and (b) 42 women diagnosed in their 30s with two or more first- or second-degree relatives with breast or ovarian cancer [early-onset family history (EO-FH)]. Of the VEO group, two (4%) had a mutation: G13203A (exon 6 missense) in a 24-year-old and a large 5,338-bp genomic deletion in a 26-year-old. Neither had a family cancer history that met Li-Fraumeni criteria. Of the EO-FH group, three (7%) had a mutation: T13240G (a known intron 5 splicing mutation) in a 36-year-old from a classic Li-Fraumeni family; G12299A (exon 4 missense) in a 33-year-old from a Li-Fraumeni-like family; and 14058delG (exon 7 frame-shift) in a 39-year-old with a family cancer history that did not meet Li-Fraumeni criteria. Germline TP53 mutations play a larger role in early-onset breast cancer than previously thought, and in this context, can be evident outside clinically defined Li-Fraumeni families.

Development of an Integrated Genomic Classifier for a Novel Agent in Colorectal Cancer: Approach to Individualized Therapy in Early Development

Background: A plethora of agents is in early stages of development for colorectal cancer (CRC), including those that target the insulin-like growth factor I receptor (IGFIR) pathway. In the current environment of numerous cancer targets, it is imperative that patient selection strategies be developed with the intent of preliminary testing in the latter stages of phase I trials. The goal of this study was to develop and characterize predictive biomarkers for an IGFIR tyrosine kinase inhibitor, OSI-906, that could be applied in CRC-specific studies of this agent. Methods: Twenty-seven CRC cell lines were exposed to OSI-906 and classified according to IC50 value as sensitive (≤1.5 μmol/L) or resistant (>5 μmol/L). Cell lines were subjected to immunoblotting and immunohistochemistry for effector proteins, IGFIR copy number by fluorescence in situ hybridization, KRAS/BRAF/phosphoinositide 3-kinase mutation status, and baseline gene array analysis. The most sensitive and resistant cell lines were used for gene array and pathway analyses, along with shRNA knockdown of highly ranked genes. The resulting integrated genomic classifier was then tested against eight human CRC explants in vivo. Results: Baseline gene array data from cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which, in combination with IGFIR fluorescence in situ hybridization and KRAS mutational status, was able to predict with 100% accuracy a test set of patient-derived CRC xenografts. Conclusions: These results indicate that an integrated approach to the development of individualized therapy is feasible and should be applied early in the development of novel agents, ideally in conjunction with late-stage phase I trials. Clin Cancer Res; 16(12); 3193–204. ©2010 AACR.

Global Decrease of Histone H3K27 Acetylation in ZEB1-Induced Epithelial to Mesenchymal Transition in Lung Cancer Cells

The epithelial to mesenchymal transition (EMT) enables epithelial cells with a migratory mesenchymal phenotype. It is activated in cancer cells and is involved in invasion, metastasis and stem-like properties. ZEB1, an E-box binding transcription factor, is a major suppressor of epithelial genes in lung cancer. In the present study, we show that in H358 non-small cell lung cancer cells, ZEB1 downregulates EpCAM (coding for an epithelial cell adhesion molecule), ESRP1 (epithelial splicing regulatory protein), ST14 (a membrane associated serine protease involved in HGF processing) and RAB25 (a small G-protein) by direct binding to these genes. Following ZEB1 induction, acetylation of histone H4 and histone H3 on lysine 9 (H3K9) and 27 (H3K27) was decreased on ZEB1 binding sites on these genes as demonstrated by chromatin immunoprecipitation. Of note, decreased H3K27 acetylation could be also detected by western blot and immunocytochemistry in ZEB1 induced cells. In lung cancers, H3K27 acetylation level was higher in the tumor compartment than in the corresponding stroma where ZEB1 was more often expressed. Since HDAC and DNA methylation inhibitors increased expression of ZEB1 target genes, targeting these epigenetic modifications would be expected to reduce metastasis.

A constitutional balanced t(3;8)(p14;q24.1) translocation results in disruption of the TRC8 gene and predisposition to clear cell renal cell carcinoma.

Studying the molecular basis of familial renal cell carcinoma (RCC) has allowed identification of novel RCC genes involved in the pathogenesis of both inherited and sporadic RCC. We describe a constitutional balanced t(3;8)(p14;q24.1) translocation found in a brother and sister with bilateral clear cell RCC (CC‐RCC) diagnosed in their forties. Consistent with a prior report, we demonstrated by RT‐PCR of RNA from lymphoblastoid cells fusion mRNAs derived from the fragile histidine triad (FHIT) at 3p14 and TRC8 at 8q24.1 in both affected siblings. Cytogenetic analysis of a CC‐RCC tumor from the affected sister from short‐term tumor cell culture showed both diploid and pseudotetraploid populations containing the translocation and normal appearing chromosomes 3 and 8. Fluorescent in situ hybridization using bacterial artificial chromosomes containing sequences from the FHIT and TRC8 genes demonstrated normal FHIT signals and TRC8 signals on nontranslocated chromosomes in the constitutional blood sample, but the TRC8 signal was absent in a subset of diploid and pseudotetraploid cells from the tumor. The tumor also contained a heterozygous VHL frameshift somatic mutation. These results confirm that balanced translocations disrupting the TRC8 and FHIT genes result in an increased genetic susceptibility for bilateral CC‐RCC. The presence of diploid and tetraploid tumor cells with and without TRC8 deletions on the nontranslocated chromosome suggest that loss of the remaining normal allele of TRC8 may contribute to tumor development at later stages.

TRAIL Receptor Signaling Regulation of Chemosensitivity In Vivo but Not In Vitro

Background Signaling by Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) and Fas ligand (FasL) has been proposed to contribute to the chemosensitivity of tumor cells treated with various other anti-cancer agents. However, the importance of these effects and whether there are differences in vitro and in vivo is unclear. Methodology/Principal Findings To assess the relative contribution of death receptor pathways to this sensitivity and to determine whether these effects are intrinsic to the tumor cells, we compared the chemosensitivity of isogenic BJAB human lymphoma cells where Fas and TRAIL receptors or just TRAIL receptors were inhibited using mutants of the adaptor protein FADD or by altering the expression of the homeobox transcription factor Six1. Inhibition of TRAIL receptors did not affect in vitro tumor cell killing by various anti-cancer agents indicating that chemosensitivity is not significantly affected by the tumor cell-intrinsic activation of death receptor signaling. However, selective inhibition of TRAIL receptor signaling caused reduced tumor regression and clearance in vivo when tested in a NOD/SCID mouse model. Conclusions These data show that TRAIL receptor signaling in tumor cells can determine chemosensitivity in vivo but not in vitro and thus imply that TRAIL resistance makes tumors less susceptible to conventional cytotoxic anti-cancer drugs as well as drugs that directly target the TRAIL receptors.

Gene Expression of Vitamin D Metabolic Enzymes at Baseline and in Response to Vitamin D Treatment in Thyroid Cancer Cell Lines

The association between vitamin D and thyroid cancer is unclear. It is unknown if CYP27A1 or CYP2R1 are present in normal thyroid or cancer cells and there is limited information regarding response to treatment with vitamin D. SV40 immortalized follicular cells (N-thy) and six thyroid cancer cell lines were treated with 10 µ<smlcap>m</smlcap> vitamin D₃, 0.1 µ<smlcap>m</smlcap> 1,25(OH)₂D₃ or vehicle × 24 h. CYP27A1, CYP2R1, CYP27B1 and CYP24A1 mRNA were measured using quantitative real-time-PCR before and after treatment. Cell proliferation was also evaluated in TPC1 and C643 cells after treatment with D₃, 25(OH)D₃ and 1,25(OH)₂D₃. Baseline CYP27A1 and CYP27B1 mRNA were present in all cells, CYP2R1 was higher and CYP24A1 mRNA was lower in cancer cell lines versus N-thy. TPC1 cells had increased CYP24A1 mRNA levels when treated with both D₃ (3.49, p < 0.001) and 1,25(OH)₂D₃ (5.05, p < 0.001). C643 cells showed increased CYP24A1 mRNA expression when treated with 1,25(OH)₂D₃ (5.36, p < 0.001). D₃, 25(OH)D₃ and 1,25(OH)₂D₃ all significantly decreased cell proliferation in TPC1 and C643 cells. Overall, both cancerous and N-thy cell lines express CYP27A1 and CYP2R1 in addition to CYP27B1, establishing the potential to metabolize D₃ to 1,25(OH)₂D₃. Additionally, vitamin D₃, 25(OH)D₃ and 1,25(OH)₂D₃ all had an antiproliferative effect on two thyroid cancer cell lines.

Beware imposters: MA-1, a novel MALT lymphoma cell line, is misidentified and corresponds to Pfeiffer, a diffuse large B-cell lymphoma cell line

We noted with some anticipation a recent publication in this journal, describing the establishment of a novel mucosa-associated lymphoid tissue (MALT) lymphoma cell line, MA-1 (Kuo et al., 2011). To our knowledge, this would be the first cell line to be successfully established from gastric MALT lymphoma, an unusual extranodal form of B-cell lymphoma. MALT lymphoma has been extensively characterized over the last several decades, but further discoveries are hampered by lack of access to appropriate in vitro models for the disorder (Du, 2007). Our anticipation was tempered, however, by the knowledge that novel cell lines often do not match up to expectations because of crosscontamination and misidentification. Cultures being handled for prolonged periods—for example, during cell line establishment—can easily be cross-contaminated if cells from another culture are introduced by accident (Capes-Davis et al., 2013). The original culture is almost always overgrown by the imposter cell line, often without the scientist at the microscope being aware of the change. The end result is a misidentified cell line, and a series of publications that relate to the imposter rather than the original tissue, cell type or disease state. Cell line cross-contamination and misidentification are not new. Publications dating from the 1960s to the present day (Masters et al., 2012) have urged the scientific community to thoroughly characterize novel cell lines, and authenticate cell line stocks used in experimental work. Recently, however, a sustained effort has been made to standardize the methods used to authenticate human cell lines. Short tandem repeat (STR) profiling is now accepted as an effective way to authenticate human cell lines. A Standard has been published by the American National Standards Institute (ANSI), giving protocols and guidelines for STR profiling as applied to cell lines (ANSI/ATCC ASN-0002–2011, 2012). The International Cell Line Authentication Committee (ICLAC) was established following publication of the Standard to increase awareness and provide resources to help address these ongoing problems (Masters et al., 2012). The MA-1 cell line was analyzed using STR profiling, and its STR profile was published as part of its characterization (Kuo et al., 2011). However, for a cell line to be authenticated, there is a requirement to compare that profile to another sample—preferably from the same donor. Online databases have been developed to allow comparison to other commonly used cell lines, with results made available for this purpose by cell line repositories worldwide (Dirks et al., 2010). An STR profile for the donor of MA-1 was not made available (Kuo et al., 2011), so we compared the cell line result to several STR profile databases (Dirks et al., 2010). A match was found to the Pfeiffer cell line, held by the American Type Culture Collection (ATCC) and established in 1992 from a patient with nonHodgkin lymphoma (Gabay et al., 1999). Comparison of the two STR profiles shows 100% match across eight core STR loci and the gender marker amelogenin (Table 1). These loci have been shown to unequivocally authenticate 98% of cell line samples when assessed using a dataset of 2,279 samples from four cell banks (Capes-Davis et al., 2013). The MA-1 cell line was subsequently obtained from the authors and retested at the Leibniz Institute German Collection of Microorganisms and Cell Cultures (DSMZ) with identical results. Unless further stock can be found that corresponds to the original donor, we must conclude that the MA-1 cell line is misidentified and cannot be used as a model for MALT lymphoma. Laboratories and cell line repositories worldwide are working to characterize existing cell lines, focusing on authentication and diseasespecific markers and mutations (Ottaviano et al., 2010). This task is made more difficult by lack of

Widespread use of misidentified cell line KB (HeLa): Incorrect attribution and its impact revealed through mining the scientific literature

Continuous cell lines are widely used, but can result in invalid, irreproducible research data. Cell line misidentification is a common problem that can be detected by authentication testing; however, misidentified cell lines continue to be used in publications. Here we explore the impact of one misidentified cell line, KB (HeLa), on the scientific literature. We identified 574 articles between 2000 and 2014 that provided an incorrect attribution for KB, in accordance with its false identity as oral epidermoid carcinoma, but only 57 articles that provided a correct attribution for KB, as HeLa or cervical adenocarcinoma. Statistical analysis of 57 correct and 171 incorrect articles showed that the number of citations to these articles increased over time. Content analysis of 200 citing articles showed there was a tendency to describe the cell line in accordance with the description in the cited paper. Analysis of journal impact factor showed no significant difference between correct and incorrect groups. Articles using KB or citing that usage were most frequently published in the subject areas of pharmacology, pharmacy, oncology, and medicinal chemistry. These findings are important for science policy and support the need for journals to require authentication testing as a condition of publication. Cancer Res; 77(11); 2784-8. ©2017 AACR.