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

Andre Kleensang,Heng-Hong Li,Melvin E Andersen,Marguerite M Vantangoli,Michael Rosenberg,Albert J Fornace,Carolina B Livi,Kim Boekelheide,Alexandra Maertens,Shelly Odwin-Dacosta,Samantha Madnick,Mounir Bouhifd,Liang Zhao,Thomas Hartung,James D Yager

doi:10.1038/srep28994

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.

Highlights

The results of the morphological, phenotypical and gene expression differences that have been performed in one laboratory (BU) by one individual are given in Fig. 1: Morphologic assessment of the MCF-7 cells showed that Brown University (BU) cells grow in large aggregations while Johns Hopkins University (JHU) cells grow flat, with cobblestone morphology (Fig. 1A)
Gene expression analysis using quantitative PCR of the estrogen receptor target progesterone receptor (PgR) following 6 hours of exposure to E2 indicated that BU MCF-7 cells are responsive to low levels of estrogen (Fig. 1C)
Exposure to the estrogen receptor alpha agonist propyl pyrazole triol (PPT) for 72 hours resulted in a significant increase in proliferation at concentrations of 0.1, 1.0 and 10 nM in the BU subline, while JHU cells did not exhibit significant changes. (C) Gene expression analysis of the estrogen receptor target progesterone receptor (PgR) following 6 hours of exposure to E2 indicated that BU MCF-7 cells are responsive to low levels of estrogen

Summary

Introduction

Later, these findings were confirmed by others[8,9,10] and it is recognized that MCF-7 is heterogeneous with respect to both the expression of hormone receptors and to the utilization of the signaling pathways linked to these receptors, differences that result in phenotypic heterogeneity[11]. Based on data from our Human Toxome Project[14,15], we demonstrate by various techniques that there can be marked cellular and phenotypic heterogeneity in a single batch of cells from a cell bank that are invisible with the usual STR cell authentication protocols, and that this heterogeneity has serious consequences for reproducibility and primary outcomes of experiments

Methods

Results

Conclusion