The genetic information of a tumor is typically obtained by sequencing millions of tumor cells together, rather than individually. While this method offers a broad view of the genetic makeup of the tissue, it can miss small populations of cancer cells within a tumor that are different from the majority of cells. 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. Understanding the genetic diversity of individual cells within a tumor has remained a challenge, due to the current limitations of genomic sequencing.

Using high-throughput single-cell DNA sequencing, a new technique developed by USC researchers and 10x Genomics may offer a higher resolution view into cancer not possible before. Using a microfluidic droplet based single-cell sequencing method, the researchers have simultaneously sequenced the genomes of close to 1,500 single cells, revealing genetic diversity previously hidden in a well-studied melanoma cell line.

“We used this approach to examine a standard cancer cell-line, examined thousands of times by many different labs,” said David Craig, PhD, co-director of the Institute of Translational Genomics at Keck School of Medicine of USC. “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 study, which demonstrates the ability of single-cell sequencing to reveal possible evolutionary trajectories of cancer cells, is published in Nature Communications Biology in a paper titled, “Single-cell sequencing of genomic DNA resolves sub-clonal heterogeneity in a melanoma cell line.

For this study, researchers used an emerging technique called “single-cell copy number profiling” developed by 10X Genomics with novel analysis methods.

The authors performed shallow single-cell sequencing of genomic DNA across 1,475 cells from a cell-line, COLO829, “to resolve overall complexity and clonality.” This melanoma tumor-line has been previously characterized by multiple technologies and is a benchmark for evaluating somatic alterations. The authors wrote that, in some of these past studies, “COLO829 has shown conflicting and/or indeterminate copy number and, thus, single-cell sequencing provides a tool for gaining insight.”

“Instead of analyzing tissue DNA that is the average of thousands of cells, we analyzed the individual DNA of close to 1,500 cells within a single experiment,” said Enrique Velazquez-Villarreal, MD, PhD, 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.”

Their analysis revealed at least four major sub-populations of cells, also known as clones, that are expected to have, at some point during the cancer cell line’s evolution, mutated from the original cancer cell. The authors wrote that, “based on clustering, break-point, and loss of heterozygosity analysis of aggregated data from sub-clones, we identified distinct hallmark events that were validated within bulk sequencing and spectral karyotyping.”

“What if there’s a small population of cells in a tumor that has acquired a change that makes them resistant to therapy?” asked John Carpten, PhD, study author and co-leader of the Translational and Clinical Sciences Program at the USC Norris Comprehensive Cancer Center and co-director of the USC Institute for Translational Genomics. “If you were to take that tumor and just grind it up and sequence it, you may not see that change.”

Carpten asserted that, “If you go to the single-cell level, you not only see it, but you can see the specific population of cells that has actually acquired that change. That could provide earlier access to the molecular information that could help define treatment approaches.”

The researchers hope that more cancer researchers will focus on single-cell sequencing. They are also using their technique to study genetic diversity in clinical cancer specimens as a way to better understand the early molecular changes that lead to aggressive and tough-to-treat advanced cancers.

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