New scDNA sequencing method provides insight into breast cancer progression

Overcoming previous technical challenges with single-cell DNA (cDNA) sequencing, a group led by researchers at the University of Texas MD Anderson Cancer Center developed a new method for sequencing single-molecule scDNA. . This technique revealed for the first time that triple negative breast cancers undergo continuous changes in genetic copy number after an initial explosion of chromosomal instability.

The results, published today in Nature, offer a new, precise and efficient approach for sequencing hundreds of individual cancer cells while providing new information on cancer progression. This information may explain why treatments are not always effective and why researchers are unable to generate homogeneous cell cultures in the laboratory.

This represents a significant advance over our first single-cell DNA sequencing methods, with significantly increased throughput, precision and ease of use. We are now able to resolve very small differences in copy number within the tumor cell population in ways that were not previously possible. “

Nicholas Navin, Ph.D., senior author, associate professor of genetics and bioinformatics and computational biology

The new technique, called Acoustic Cell Tagmentation (ACT), begins with fluorescence-activated cell sorting to isolate single nuclei from thousands of cells. A three-step chemistry process then cuts each cell’s DNA into precise fragments, adds universal adapters, and incorporates barcodes for next-generation sequencing.

Chemistry is performed with Acoustic Liquid Transfer Technology, which uses sound waves to transfer minute volumes of liquid quickly and efficiently. While early approaches to scDNA sequencing took three days from start to finish, the new approach can be completed in just three hours, Navin explained.

With this improved method of sequencing DNA from a limited number of cells, the researchers sought to answer a continuing question about the course of cancer. Navin’s team had previously established that triple negative breast cancers undergo a punctuated course, acquiring copy number changes in an initial burst of chromosomal instability, but it was not known whether cancer cells continued to accumulate changes. after this event.

Led by graduate student Darlan Conterno Minussi, Navin’s team worked with the lab of Franziska Michor, Ph.D., at the Dana-Farber Cancer Institute, to perform copy number analysis on 16,178 single cells of eight triple negative breast cancers and four cell lines. using the ACT technique.

“We found that the punctuated evolution of these cells is followed by transient instability,” Navin said. “After the initial event, there is a period in which the copy number changes accumulate at high rates that eventually slow down to a basal rate.

Understanding that triple negative breast cancers continue to evolve over time may explain why treatments aren’t always effective – a small portion of cancer cells may have acquired a mutation that reflects resistance to a given therapy. In the future, researchers would like to determine whether the number of genetic changes a tumor undergoes is predictive of clinical outcomes.

The results also have implications for preclinical research, as the researchers confirmed that triple-negative breast cancer cell lines also continue to accumulate changes when grown in the lab. Importantly, the researchers have shown that commonly used laboratory cell culture procedures are unable to generate homogeneous populations of tumor cells because they rapidly re-diversify their genomes.

The research team continues to build on this work by investigating other types of cancer, seeking to understand whether this model of cancer progression may be widely applicable beyond triple negative breast cancers.