Genetic variability in a frozen batch of MCF-7 cells invisible in routine authentication affecting cell function

Abstract

Common recommendations for cell line authentication, annotation and quality control fall short addressing genetic heterogeneity. Within the Human Toxome Project, we demonstrate that there can be marked cellular and phenotypic heterogeneity in a single batch of the human breast adenocarcinoma cell line MCF-7 obtained directly from a cell bank that are invisible with the usual cell authentication by short tandem repeat (STR) markers. STR profiling just fulfills the purpose of authentication testing, which is to detect significant cross-contamination and cell line misidentification. Heterogeneity needs to be examined using additional methods. This heterogeneity can have serious consequences for reproducibility of experiments as shown by morphology, estrogenic growth dose-response, whole genome gene expression and untargeted mass-spectroscopy metabolomics for MCF-7 cells. Using Comparative Genomic Hybridization (CGH), differences were traced back to genetic heterogeneity already in the cells from the original frozen vials from the same ATCC lot, however, STR markers did not differ from ATCC reference for any sample. These findings underscore the need for additional quality assurance in Good Cell Culture Practice and cell characterization, especially using other methods such as CGH to reveal possible genomic heterogeneity and genetic drifts within cell lines.

Replication Study: The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors

“…So while some aspects, such as cell line, strain and sex of mice, number of cells injected, and the injection site of antibody treatment were maintained, others were unknown or not easily controlled for. These include variables such as cell line genetic drift (Hughes et al, 2007; Kleensang et al, 2016), circadian biological responses to therapy (Fu and Kettner, 2013), mouse strain stocks (Clayton and Collins, 2014), housing temperature in mouse facilities (Kokolus et al, 2013), and the obesity and microbiome of recipient mice (Klevorn and Teague, 2016; Macpherson and McCoy, 2015). Additionally, a differential response to immunotherapy can occur due to heterogeneity in individual tumor microenvironments (Grosso and Jure-Kunkel, 2013), which has also been observed in the clinical setting (Ascierto and Marincola, 2014; Stevenson, 2014).…”

Replication Study: Melanoma genome sequencing reveals frequent PREX2 mutations

“…So while some aspects, such as cell line, site of injection, number of cells injected, and strain and sex of mice were maintained, others were unknown or not easily controlled for. These include variables such as PREX2 copy number in transformed cells, circadian biological responses to therapy (Fu and Kettner, 2013), the microbiome of recipient mice (Macpherson and McCoy, 2015), housing temperature in mouse facilities (Kokolus et al, 2013), and cell line drift (Hughes et al, 2007; Kleensang et al, 2016). Additionally, the accumulation of mutations during cell passage in vitro can drive cell lines towards a malignant phenotype that is observed in vivo (Gregoire et al, 2001; Hurlin et al, 1991).…”

Good Cell Culture Practice for stem cells and stem-cell-derived models

“…Next-generation sequencing can be used to achieve nucleotide level resolution. These higher resolution genomic methods provide a lot of data but are yet to be routinely used as it is not yet defined what differences may constitute a threat to cell safety for cell therapy applications or impact on reproducibility of research (Kleensang et al, 2016). Given the complexity of these methods, current guidelines by ISCBI (2009) recommend the use of G-banding.…”

“…(not detectable with STR) were found in cultures from a major cell bank (Kleensang et al, 2016). Also, these systems do not generally work well in non-human species although STR panels have been developed for non-human species.…”