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Single-Cell Method Maps Colorectal Cancer Genomic Instability, Epigenetics, Gene Expression

SAN FRANCISCO (GenomeWeb) – In an effort to better understand colorectal cancer progression, researchers from Peking University in China have developed a sequencing method to simultaneously analyze copy number variants, methylation, and gene expression of single cells.

The study, published today in Science, is the latest in a growing effort to harness single-cell sequencing to look at more than just gene expression, combining that data with epigenetic, chromosome conformation, and other features within the cell.

The study builds off previous work the group published in 2016 in Nature Cell Research. In that publication, the researchers described a single-cell triple omics sequencing technique, scTrio-seq, for analyzing gene expression, copy number alterations, and methylation at CpG sites.

For its latest study, the group incorporated single-cell bisulfite sequencing for genome-wide methylation profiling and improved detection efficiency, dubbing the new approach scTrio-seq2. In addition, they upped the number of cells profiled to around 1,900 from 25 in the previous study.

The researchers applied their method to single cells from 12 patients with colorectal cancer, including 10 for whom they had matching primary and metastatic data, which enabled them to identify the genetic lineages that developed from clonal mutations in each patient and to correlate expression, copy number, and methylation data.

For 10 patients, the researchers had samples from primary as well as metastatic tumors, which enabled them to track how the tumor evolved to metastasis. In addition, sampling from multiple regions within the tumor allowed them to identify intra-tumor heterogeneity.

The researchers used both copy number information and methylation data to identify genetic lineages within a patient's primary tumor and were able to track the tumor's evolution through metastasis. For example, in one patient, they identified 12 genetic sublineages in the primary tumor. The single cell data demonstrated that the metastases were less complex than the primary tumor, indicating that the metastatic tumors were likely clonal. It also allowed them to identify that two metastases had a common origin.

Looking specifically at the methylation data, the researchers found that while methylation was consistent among cells within the same genetic sublineage — even through metastasis — methylation levels varied between the lineages and was different than in adjacent normal cells. In the cancer cells, DNA hypomethylation was particularly enriched in long terminal repeats, long interspersed nuclear elements, and heterochromatin regions, while DNA hypermethylation was enriched in CpG islands and open chromatin.

When the researchers looked at the relationship between methylation status and chromosomal alterations, they found that six chromosomes tended to show greater levels of demethylation than others and that three of those demethylated chromosomes also had recurrent chromosomal alterations.

"Single-cell multi-omics sequencing provides insights and resources for understanding the molecular alterations that occur during [colorectal cancer] progression and metastasis," the authors wrote.