Single-cell DNA sequencing reveals a latedissemination model in metastatic colorectal cancer

Metastasis is a complex biological process that has been difficult to delineate in human colorectal cancer (CRC) patients. Amajor obstacle in understanding metastatic lineages is the extensive intra-tumor heterogeneity at the primary and metastatic tumor sites. To address this problem, we developed a highly multiplexed single-cellDNAsequencing approach to trace the metastatic lineages of two CRC patients with matched liver metastases. Single-cell copy number or mutational profiling was performed, in addition to bulk exome and targeted deep-sequencing. In the first patient, we observed monoclonal seeding, in which a single clone evolved a large numberof mutations prior to migrating to the liver to establish the metastatic tumor. In the second patient, we observed polyclonal seeding, in which two independent clones seeded the metastatic liver tumor after having diverged at different time points from the primary tumor lineage. The single-cell data also revealed an unexpected independent tumor lineage that did not metastasize, and early progenitor clones with the "first hit" mutation in APC that subsequently gave rise to both the primary and metastatic tumors. Collectively, these data reveal a late-dissemination model of metastasis in two CRC patients and provide an unprecedented view of metastasis at single-cell genomic resolution.