Single-Cell Sequencing Uncovers Genetic Roots of Cancer Tumor Diversity

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Researchers at The University of Texas MD Anderson Cancer Center have found that a rare resistance mutation first thought to appear in melanoma (pictured) following treatment with a targeted therapy has instead turned out to be hiding in the tumor all along. [Definiens]

Single cell sequencing has revealed the genetic diversity underlying the COLO829 melanoma cell line, which is a benchmark for evaluating somatic genetic alterations. A University of Southern California (USC) team performed a single experiment that determined shallow copy number profiling across many such cells can provide important biological insights, including their possible evolutionary paths.

This study was just published in Nature Communications Biology. The lead author is Enrique I. Velazquez-Villarreal, MD, PhD, MPH,  and assistant professor of Translational Genomics at USC-Keck School of Medicine. The senior author is David W. Craig, PhD, co- director of the Institute of Translational Genomics at Keck.

The researchers aimed to resolve the overall complexity and clonality of the COLO829 cell line, which has already been well studied using multiple other technologies. However, earlier studies found conflicting and/or indeterminate copy numbers in the line. To better understand COLO829’a genetic makeup, the USC scientists performed shallow, single-cell sequencing across 1475 such cells.

Tumors are genetically heterogenous, but they are typically studied by bulk sequencing of millions of tumor cells at once, which delivers a read out reflecting an average of the tumor’s genetic features. While this bulk approach offers a broad view of the tumor’s genetic makeup, it can miss small populations of cancer cells that differ from the majority, but are still important to characterizing the tumor as a whole.

Velazquez-Villarreal’s team used single-cell copy number profiling, an emerging technique, developed by 10X Genomics as well as novel analysis methods that integrated their results with those of historical methods. They found at least four major sub-populations of cells, also known as clones. These sub-groups are believed to have mutated from the first cancer cell at some point during tumor line’s evolution.

“We used this approach to examine a standard cancer cell-line, examined thousands of times by many different labs,” said Craig. “What was really surprising here, was with this technology we uncovered complexity we did not expect. This line actually consistently became a mixture of different types of cells. Reexamining decades of prior work on this line – now with this new information – we have new insights into tumor evolution.”

The ability to identify sub-clones in cancer tissue could provide important biological insights into how cancer progresses, how it spreads and why it can become resistant to treatment. In addition, this study supports the use of shallow single-cell DNA sequencing, a technique that has advanced considerably since it was introduced. Early single-cell RNA-seq assays were labor intensive and could only process small number of cells in a single experiment. Thanks to technological advances researchers can use the technique to analyze hundreds of thousands of cells in parallel, which also reduces cost.

“Instead of analyzing tissue DNA that is the average of thousands of cells, we analyzed the individual DNA of close to 1500 cells within a single experiment,” said Enrique Velazquez-Villarreal, lead author and assistant professor of translational genomics at Keck School of Medicine at USC. “Studying cancer at this higher resolution, we can discover information that lower-resolution bulk sequencing misses.”

The USC researchers say they plan to share their data in the hope that more cancer researchers will focus on single-cell sequencing. The team is also using their technique to study genetic diversity in clinical cancer specimens so as to better understand the early molecular changes that lead to aggressive and tough-to-treat advanced cancers.

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