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.

Figure 1. Morphological, phenotypical and gene expression differences of MCF-7 cells from the same ATCC batch (passsage number 154 JHU & passage number 150 BU).

(A) MCF-7 sublines (JHU & BU) display distinct morphological differences. At 0 hours, BU MCF-7 cells and JHU cells have unique morphologies. MCF-7 cells grown and expanded at BU grow in large aggregations while JHU cells grown in the BU laboratory are flat, with cobblestone morphology. (B) Following 72 hours of exposure to estradiol (E2), BU MCF-7 cells displayed significant increases in proliferation (cell count) at concentrations of 0.1, 1 and 10 nM, while JHU cells did not have a significant change in cell count (left). 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 (right). (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. All experiments have been performed in one laboratory (BU) by one individual to exclude any possible inter-laboratory and/or inter-operator effects on reproducibility. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2. Principal Component analysis (PCA) of MCF-7 cells LC-MS metabolomics data from the same ATCC batch.

MCF-7 cells were treated with 0 nM or 1 nM E2 for 4 hours or 24 hours either at Brown University (BU) or Johns Hopkins University (JHU). The data point colors in the graph represent samples from Brown University and at passage number 150 (BU, red) and Johns Hopkins University at passage number 154 (JHU, blue). The shapes represent the treatment time (square: 0 h, circle: 4 h and triangle: 24 h) while the size represents the experimental condition (small: controls and big: 1 nM E2 treatment). QC samples from each were also analyzed. They represent a pool of all samples in an individual experiment (diamond). A total of 1048 features were identified in the two experiments and were used for the multivariate analysis shown here.

Figure 3. Principal Component analysis (PCA) of MCF-7 cells Gene Expression microarray data from the same ATCC batch.

MCF-7 cells were treated with 0 nM or 5 nM propyl pyrazole triol (PPT) for 4 hours or 8 hours (cell culture details see Materials and Methods) either at Brown University (BU) at passage number 150 or Johns Hopkins University (JHU) at passage number 154. The colors in the graph represent samples from Brown University (BU, red) and Johns Hopkins University (JHU, blue). The shapes represent the treatment time (circle: 4 h and triangle: 8 h) while the size represents the experimental condition (small: 0 nM controls and big: 5 nM PPT treatment). A total of 29,787 entities representing detected probes were used for the multivariate analysis shown here.

Figure 4. Unsupervised hierarchical clustering of MCF-7 cells Gene Expression microarray data from the same ATCC batch.

MCF-7 cells were treated with 0 nM or 5 nM propyl pyrazole triol (PPT) for 4 or 8 hours (cell culture details see Materials and Methods) either at Brown University (BU) at passage number 150 or Johns Hopkins University (JHU) at passage number 154. 84 genes covering estrogen receptor signaling, breast and ductal morphogenesis, cellular growth and differentiation, proliferation, tumor progression and epithelial to mesenchymal transition have been selected from the literature. Cluster algorithm used Euclidean distances and Wards linkage criteria on entities and conditions of probes encoding genes. Gene Symbols in bottom labeling columns (often multiple probes represent each gene on microarray). Label plots on the right show conditions corresponding to PPT dose (nM), Time Point (hours) and Institution (Johns Hopkins University, JHU; Brown University, BU), respectively. Color range represents data baselined to the median and log 2 transformed. Comparing two sample from 0 (yellow) to +2 (red) or −2 (blue) would be 4 fold change.

Figure 5. Reproducibility of CGH for two technical replicates of MCF-7 cells.

Comparative Genomic Hybridization of two technical replicates of MCF-7 cells from ATCC lot number 59388743 from Johns Hopkins University at passage number 154 (JHU P154, (A) darkblue and darkcyan), and Brown University and at passage number 150 (BU P150, (B) darkolivegreen and darksalmon) versus human female reference DNA. Within (A,B) respectively, only very minor differences can be seen showing very good reproducibility of CGH. Significant genomic differences detected by the Aberration Detection Method 2 (ADM-2) are indicated by the respective horizontal lines at −2 and +2, respectively. All experiments have been performed in one laboratory (JHU) by one individual to exclude any possible inter-laboratory and/or inter-operator effects on reproducibility.

Figure 6. Direct CGH of MCF-7 genomics DNA from two original ATCC vials of the same batch and after short culture.

Direct Comparative Genomic Hybridization (CGH) of MCF-7 genomic DNA derived from ATCC lot number 59388743, passage 147. Genomic DNA has been directly prepared from two original ATCC vials with the same lot number but shipped at two different time points to two different laboratories (blue) and after few passages in cell culture in the related laboratories (green; JHU passage number 154, BU passage number 150). For the comparative analysis the JHU samples have been defined as reference samples. Both CGH show significant genomic differences on chromosomes 1, 3, 4, 5, 7, 8, 9, 10, 13, 15, 20 and X as detected by the Aberration Detection Method 2 (ADM-2), which are very similar in both comparisons and are indicated by the respective horizontal lines at −1 and +1, respectively. Note, that to show the smaller differences between the different MCF-7 samples in comparison to MCF-7 versus normal female genome (Fig. 5), the Y-axis has been changed. All experiments have been performed in one laboratory (JHU) by one individual to exclude any possible inter-laboratory and/or inter-operator effects on reproducibility.

Figure 7