“Single-cell resolution covers a much wider spectrum of divergence than what the bulk experiments were showing. Once we clarify the cells’ transcriptional programming, whole exome sequencing will be a must for mutational analysis.”
—Jiannis Ragoussis, PhD, McGill University MUGQIC
One in eight women will be diagnosed with breast cancer during her lifetime, the National Institutes of Health estimates. The statistics are daunting. The breast is the most common female cancer site, and that form of the disease is the world’s leading cause of cancer deaths in women.
Malignant tumors contain many genetically diverse cell subpopulations due to the unchecked cell division that characterizes cancer. Since different cell types respond to treatments differently, making sense of this cell-to-cell variability is key to administering the precise combination of drugs most effective for each individual patient.
Jiannis Ragoussis, PhD, head of Genome Sciences at McGill University and Génome Québec Innovation Centre (MUGQIC) in Montreal, is among the first researchers to implement single-cell whole exome sequencing in breast cancer research. The Greek native has long been a trailblazer; before arriving at MUGQIC in 2013, he spent 11 years at Oxford University’s Wellcome Trust Centre for Human Genetics, where he orchestrated development of genomics platforms and the introduction of next-generation sequencing.
At MUGQIC, Ragoussis is taking next-generation sequencing to individual-cell resolution through pioneering use of single-cell whole exome sequencing on the Fluidigm C1 system. He is revolutionizing the identification and eradication of cancer by cataloging diversity in two different projects, beginning with the most enigmatic breast tumors. His team is focusing on triple-negative breast cancer—one of the most aggressive types, characterized by a high likelihood of spreading and recurring after treatment. Triple-negative breast tumors respond poorly to standard hormonal therapies because they lack receptor expression for estrogen, progesterone and human epidermal growth factor 2 (HER2/neu). Traditional therapies typically target one of these receptors, so triple-negative cases are especially challenging.
Single-cell whole exome sequencing provides a unique view into the biology of cancer cells that is not possible with bulk sequencing methods. It allows Ragoussis to characterize the mutational profiles associated with individual cell populations and track how these change over time, both as cancer progresses and as treatment is administered.
This is especially crucial for improving outcomes in treatment-resistant triple-negative breast cancers, he said. “Mutational profiling can provide information on the variants that are accumulating and allowing the cells to survive after treatment.”
Promising pilot studies
In one of the first uses of the new C1 workflow, Ragoussis is using single-cell whole exome sequencing to examine the heterogeneity in individual cell populations. At this stage of the project there are two overall goals, he explained: “To increase the arsenal of possible compounds by delivering novel meaningful drug targets, and in parallel, to maximize the usage of current compounds.”
First the Ragoussis team is focusing on grouping cells according to their mutation profiles to determine how many distinct profiles exist and to assess whether mutated genes can be classified into distinct networks providing oncogenic clues. “Understanding the heterogeneity and how the tumor cells respond in vitro first is critical to predicting what could work in vivo,” he added.
In a recent pilot study, Ragoussis and colleagues validated this workflow and confirmed it can be integrated into their existing rapid exome-capture protocol. “The Fluidigm workflow is one of the most exciting technical developments we’ve implemented,” said Ragoussis. “The pilot demonstrated the method’s efficiency and feasibility for our research.”
The new workflow produced reliable statistics and covered the whole exome in about 70 percent of cells, by his estimate. His conclusion: “The data generated were comparable in quality to those created from traditional bulk sequencing methods.”
Ragoussis performed the study in collaboration with Morag Park, director of the Goodman Cancer Research Centre at McGill University, whose laboratory creates surgical interspecies tissue graft samples, or xenografts, in mice from primary breast cancer tumors. The transplants foster cancer cell growth while preserving the original tumor’s complexity.
After growing the xenografted cells in culture, the team used C1 to capture and sequence the whole exome of individual cells. To date the researchers have cataloged the exomes of 81 single cells derived from one treatment-resistant tumor.
Now that they’ve identified all the somatic mutations in the tumor, Ragoussis and his team are using the information to define the false discovery rate and determine the number of times a particular mutation must be detected before it’s considered a true statistical finding.
Next the researchers plan to use single-cell exome sequencing to understand the mechanisms underlying treatment resistance. Ragoussis looks forward to combining the single-cell whole exome data with existing protein expression datasets to create functional assays to analyze effects of treatments in vitro. Ideally, that could identify specific treatments that are effective against individual cancer cell types.
Whole exome sequencing 'will be a must'
In a second project, performed in collaboration with McGill’s Richard Kremer, Ragoussis is examining the molecular profile of tumor cells circulating in the blood. Like a tumor, this so-called “liquid biopsy" is filled with diverse cancer cells that can provide a real-time estimate of the current disease state that’s more precise than imaging and less invasive than a traditional biopsy.
Easily obtained blood samples enable monitoring of tumor cell profiles before and after treatment. “This helps us predict metastatic behavior and see whether a treatment has eliminated a particular dangerous cell population,” Ragoussis noted. “The ultimate goal is a personalized medicine approach where patients with treatment-resistant cancers can undergo circulating tumor-cell profiling in order to identify the specific drugs best able to fight their disease.
“Although it’s still early in the project,” said Ragoussis, “it’s clear that within each patient and across patient populations the single-cell resolution covers a much wider spectrum of divergence than what the bulk experiments were showing. Once we clarify the cells’ transcriptional programming, whole exome sequencing will be a must for mutational analysis.”
Ragoussis and his colleagues are thinking big as the 250 MUGQIC researchers fuse new genomic methodologies with bioinformatics to develop technologies spanning a broad spectrum of medical applications.
“Our mission is to establish the genomics methods and make them more widely available,” he said. “Similar single-cell sequencing approaches could provide insight into virtually any disease.”