Long thought to be genetically stable and identical, cancer cell lines harbor significant levels of genetic variation, which may help explain why it can be hard to reproduce findings in cell line-based research.
Cancer cell lines evolve in ways that affect how they respond to drugs
Cell lines form the backbone of cancer research. These individual groups of cells, typically collected from patients' tumor samples and cultured to grow indefinitely in the laboratory, enable everything from basic genetic research to drug discovery.
But while scientists have thought that individual cell lines remain genetically uniform even as they continue to grow and divide, they can in fact evolve in ways that dramatically change their responses to drugs, say researchers from the Broad Institute and collaborating institutions. This continuing evolution of cells within cell lines — potentially driven by the laboratory conditions in which they are grown — may help explain why different studies that use the same cell lines often have conflicting results.
These findings, reported in Nature, suggest that scientists need to take extra care to ensure that cell line-based models of cancer accurately reflect the tumor they are studying. The research team has released an online tool, Cell STRAINER, to help scientists benchmark their models.
"The main message here is not that cell lines and culture-based models are bad. Rather, you should know your model, and understand its properties and limitations," said Broad core institute member and Cancer Program director Todd Golub, who was the study's co-senior author, along with Broad associate member and Dana-Farber Cancer Institute (DFCI) adult neuro-oncologist Rameen Beroukhim. "Knowing requires a level of genetic and genomic characterization beyond what we usually think about. Skipping this sort of careful characterization is not an option."
Evolution in a lab dish
Researchers have long recognized that cell lines do not perfectly mimic their tumor of origin. Still, they are considered representative and stable enough to provide accurate insights into cancer biology and drug response. In fact, every cancer treatment available today was at some point tested in these laboratory models.
However, scientists often struggle to reproduce study results from cell lines. In addition, no one has yet undertaken a systematic effort to measure how, and the extent to which, the cells in cancer cell lines change genetically over time, and whether those changes affect drug responses.
"You can find many examples in the literature pointing out inconsistencies in drug sensitivity data from cell line experiments," said Uri Ben-David, the study's first author and a postdoctoral fellow in the Golub lab. "We wanted to look for signs of evolution and connect the dots between changes within lines and those inconsistencies."
The researcher’s first began to suspect that these cell lines evolve when they reanalyzed sequencing data from 106 lines housed in two large collections: the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE) and the Sanger Institute's Genomics of Drug Sensitivity in Cancer. These lines should be genetically identical in both collections, but the team found high levels of variability. For instance, 19 percent of the genetic mutations the team found were present in only one collection or the other.
The team then ran deep molecular analyses on 27 and 23 strains, respectively, of two widely used cell lines: the estrogen receptor-positive breast cancer line MCF-7, and the lung cancer line A549. The analysis included whole-genome DNA sequencing, targeted DNA sequencing of nearly 450 genes commonly mutated in cancer, and bulk and single-cell RNA-sequencing. Each of the strains represented a batch of cells from the line with a distinct history (e.g., different kinds of lab manipulation, different length of time in culture, different original source).
Their data revealed striking genomic differences between strains, ranging from single base pair mutations to large-scale changes in genome structure (e.g., losses of entire chromosome arms) to major changes in gene expression — all indicating that the cells lines were neither as stable nor as identical as researchers thought. These genetic and expression differences also affected the strains' growth rates, cell size and shape, and other traits.
Microscopy images of three strains of the MCF-7 breast cancer cell line. (Courtesy Uri Ben-David)
Among the 27 MCF-7 strains, for example, the team found widespread duplications and deletions of whole genes throughout the genome. Only 13 percent of the duplications and 21 percent of the deletions were seen in all strains; 7 and 13 percent, respectively, were seen in only one. Additionally, as many as 1,574 genes — including ones that are both biologically and therapeutically important — showed at least a two-fold difference in expression between different strains. The team even noted significant expression differences between individual cells within the same strains.
The team extended their findings by carrying out targeted sequencing on an additional 11 cancer cells lines representing a variety of tumor types (e.g., prostate, colon, liver, skin). Across the board, the team found extensive genetic variability within lines.
What drives these changes? By conducting experiments with different laboratory conditions, the team found that even changing the kind of nutrient media in which a cell line is cultivated can give some cells within a strain a growth advantage over others, allowing genetically distinct populations to evolve. And by extracting single cells and growing them in culture, the team also found that the progeny of isolated single cells could spontaneously acquire new mutations, showing that new, genetically diverse populations of cells could arise within a cell line strain from individually isolated ones.
Strain variation affects drug sensitivity
To understand how these genetic changes might affect drug response, the team tested the 27 MCF-7 strains' against 321 drugs. Indeed, any given drug's ability to slow the growth of or kill a strain's cells correlated with the strain's genetic features and its expression of genes targeted by the drug.
For example, strains lacking expression of the estrogen receptor (ER) — and ER expression is a hallmark of the MCF-7 line — were less sensitive than usual to tamoxifen (which interferes with the ER on breast cancer cells).
The findings complement those of another led by Ben-David, Beroukhim, and Golub that examined patient-derived xenograft (PDX) models (which are generated by implanting tumor cells from a cancer patient into a mouse). In 2017, they reported in Nature Genetics that over time human cancer cells in PDX models lose characteristic genetic features seen in patients and gain new features not encountered in humans, changes that correlated with shifts in the models' drug sensitivities.
A change in perspective, and an opportunity
Taken together, the findings highlight a need for scientists to understand how the cells they are using may have diverged genetically from their parent line, so as to make accurate comparisons across experiments.
To that end, the study team has developed and released an online tool, Cell STRAINER, to help researchers estimate how much a cell line strain they use has diverged genetically from reference samples in the Cancer Cell Line Encyclopedia collection.
The team also note that the cell lines' capacity for evolution could actually provide an opportunity to study a variety of aspects of cancer biology and tumor evolution.
"Because the strains have the same general genetic background, you could test the same compound on two strains and use their features to study the compound's mechanism of action, or the influences of gene expression differences on sensitivity," said Golub, who is also the Charles A. Dana Investigator in Human Cancer Genetics at DFCI. "Or you could investigate tumor heterogeneity and the selective pressures that cause strains to evolve."
"Technology has given us new perspectives on how cancer cells evolve in patients, and we need to apply that same perspective to the models we use to study cancer in the laboratory," said Broad institute scientist and Cancer Program associate director Jesse Boehm, who leads efforts to generate cell models from patient tumors and was not involved in the study. "We’ve learned that tumors aren’t static in patients. Now, we’ve learned that the laboratory models we use to study cancer are also subject to evolutionary pressures.”
"These findings tell us as a field that we need to adopt more nuanced view about cell models," Boehm continued, "and adjust the practices we use to generate, propagate, and employ such models accordingly."
Support for this study came from the National Cancer Institute, the Human Frontier Science Program, the Gray Matters Brain Cancer Foundation, the Koch Institute-Dana-Farber/Harvard Cancer Center Bridge Project, BroadNext10, and other sources. Todd Golub is an investigator with the Howard Hughes Medical Institute.
Ben-David U, et al. Genetic and transcriptional evolution alters cancer cell line drug response. Nature. Online August 8, 2018. DOI: 10.1038/s41586-018-0409-3.
Ben-David U, et al. Patient-derived xenografts undergo mouse-specific tumor evolution. Nature Genetics. Online October 9, 2017. DOI: 10.1038/ng.3967.