Single-Cell Lineage Tracing of Metastasis Uncovers an EMT Continuum

Metastasis causes the majority of cancer deaths, but technical limitations have hindered the study of metastatic progression at high resolution. Simeonov and colleagues developed macsGESTALT, an inducible CRISPR–Cas9-based system for high-resolution lineage tracing that enabled both clonal analysis and subclonal phylogenetic reconstruction via static and evolving barcodes. Using a model of metastatic pancreatic ductal adenocarcinoma (PDAC), macsGESTALT constructs were engineered into cells from murine PDAC tumors (LSL-Kras^G12D/+; Trp53^LSL-R172H/+; Pdx1-cre; LSL-Rosa26^YFP/YFP) and transplanted into the pancreata of healthy mice. Following engraftment, CRISPR–Cas9-mediated barcode editing was induced with doxycycline, and, five weeks post-transplantation, tumor cells were harvested from multiple sites across two mice, including the primary tumor, liver, lung, peritoneal metastases, and circulation. Single-cell RNA-sequencing analysis revealed that less than 1% of clones successfully engrafted. Based on size and dissemination of clonal populations, an aggression-scoring system found that aggressive clones were transcriptionally divergent from nonaggressive clones. In one mouse, the most aggressive clones were enriched for expression of mesenchymal markers, whereas nonaggressive clones were enriched for expression of epithelial markers. Trajectory inference revealed that epithelial-to-mesenchymal transition (EMT) gene expression was the main trajectory in this dataset, with genes rising and falling at different rates across the trajectory and supporting the concept of a continuum of EMT states in vivo. Reconstructing subclonal lineages using patterns of edit inheritance revealed that both highly epithelial and highly mesenchymal subclones were small and nonmetastatic, whereas the largest and most disseminated subclones expressed late-hybrid EMT states. Accordingly, patients with PDAC enriched for late-hybrid EMT signatures had significantly increased risk of death. In another mouse, the set of genes most associated with broad dissemination were enriched for S100 genes, previously implicated in PDAC and metastasis, suggesting S100 genes might complement advantages conferred by canonical EMT. Together, this work introduces a novel method to investigate metastasis and sheds light on a continuum of EMT states in metastasis.

Simeonov KP, Byrns CN, Clark ML, Norgard RJ, Martin B, Stanger BZ, et al. Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states. Cancer Cell 2021 Jun 10 [Epub ahead of print].

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