Single-nucleus sequencing approach developed at CSHL finds cancer development is more staccato than legato.
Scientists claim the genetic changes that underlie tumor development and metastasis may involve populations of cancer cells undergoing a process termed punctuated clonal expansion rather than the more gradual evolution that has traditionally been postulated. The Cold Spring Harbor Laboratory (CSHL) researchers used a new technique known as single-nucleus sequencing (SNS) to quantify genomic copy number within the nuclei of single cancer cells.
The research is published in Nature in a paper titled “Tumour evolution inferred by single-cell sequencing.”
Genomic analysis can help evaluate the role of copy number variation in disease, but most methods are not designed to resolve mixed populations of cells, point out CSHL’s Michael Wigler, Ph.D., and colleagues. “In tumors, where genetic heterogeneity is common, very important information may be lost that would be useful for reconstructing evolutionary history.”
In order to evaluate the genomic relationships between mixed populations of cancer cells, Dr. Wigler’s team developed the SNS method, which involves the flow-sorting of nuclei, combined whole-genome amplification, and next-generation sequencing. They used the technique to analyze the nuclei of 100 cells taken from a polygenomic breast cancer tumor and 100 cells from a monogenomic breast cancer tumor and its liver metastasis.
They found three distinct populations of cells in the polygenomic tumor, each of which comprised cells with similar copy number profiles. Interestingly, chromosomal breakpoint analysis suggested these three subpopulations all emerged when the tumor was much smaller. This is in contrast to previous theories that genetically heterogeneous tumor cell populations become mixed via gradual evolution.
“We could infer that these most probably represent three distinct clonal expansions of the tumor,” remarks lead author Nicholas Navin, Ph.D., who is now assistant professor at the University of Texas MD Anderson Cancer Center.
Analysis of cells from the monoclonal tumor and its liver metastasis, meanwhile, indicated that the primary tumor mass formed by a single clonal expansion of an aneuploid cell and that one of the cells from this expansion subsequently seeded the metastatic tumor with little further evolution. The analysis further suggested that although the primary and metastatic tumor cells were closely related, they were cleanly separated, indicated that the two populations had not mixed since the metastasis developed.
“Differences in the profiles that distinguish primary and metastatic tumor populations were in the degree of copy number changes,” Dr. Navin notes. The authors add, “Our findings are consistent with previous findings using bulk DNA, which indicate that copy number profiles in primary tumors are highly similar to the metastases.”
“Thus, the metastatic cells emerge from a main advanced expansion and not from an earlier intermediate or a completely different subpopulation. In contrast to gradual models, this pattern reflects the sudden emergence of a tumor cell whose rate of effective population growth markedly exceeds its rate of genomic evolution.”
Interestingly, the CSHL researchers also found that up to 30% of the tumor cells they evaluated in both primary tumors were of a genetically diverse type of cell they termed pseudodiploid. Traditional methods of tumor cell characterization have not previously been able to distinguish these cells, they note. The pseudodiploid cells appear not to travel to the site of metastasis, and their profiles are characterized by nonrecurring copy number changes (including whole chromosome arms) that are not shared between any two pseudodiploid cells nor with the corresponding tumor profiles.
“These data indicate that unlike the aneuploid cells, pseudodiploids do not undergo clonal expansions in the tumor,” the authors suggest. “The relative abundance of pseudodiploid cells in primary tumors does, however, indicate that they may emerge from an ongoing aberrant process that generates genomic diversity in the tumor.”
The researchers admit that their inferences are drawn from the study of only two cancers. Nevertheless, in contrast with the hypothesis that tumors evolve by a process of gradual evolution, the punctuated clonal evolution pattern of tumor growth indicated by the SNS analyses suggests that “major clonal expansion events are relatively infrequent and may provide accessible targets for cancer therapy,” concludes CSHL co-author Professor James Hicks.