SINGLE-CELL TRANSCRIPTIONAL LINEAGE TRACING REVEALS COMPLEXITY AND PLASTICITY OF BREAST CANCER PRO-METASTATIC PHENOTYPES

Breast cancer (BC) represents a major clinical hurdle, with metastatic outcome accounting for poor patient prognosis. Phenotypic intra-tumor heterogeneity inversely correlates with prognosis in BC and is considered at the basis of metastatization. Traditionally, BC poly-clonal structure was investigated through the genetic barcoding of cells followed by the tracing of the offspring. However, this analysis did not allow a solid investigation of the transcriptional profile associated to clones throughout tumor progression. In this work, we exploited a recently published library of expressed barcodes to perform concomitant clonal and transcriptional tracing in breast cancer by single-cell RNA sequencing. We applied this technology to the triple-negative BC cell line MDA-MB-231 and the luminal B patient-derived xenograft MBC26 to study the transcriptional profile of clones during BC metastatization. We found that clonal populations in vivo display proliferative heterogeneity, with some clones expanding more than others. Furthermore, we showed that single clones mainly activate one of two distinct transcriptional programs, either highly-proliferative or migratory/stressed/stem-like. Remarkably, we found that most times the same clones in distinct tumors display a high similarity in terms of transcriptional profile. In this scenario, however, we found that the metastatic progression of a clone is not determined by the clonal lineage, as a given clone can spread metastases in one tumor without being metastatic in another one. We found that the metastases to lungs and liver are composed of an overlapping setting of clones, with the vast majority of metastatic cells deriving from a single pro-metastatic clone. Notably, we showed that the pro-metastatic clones upregulate a set of genes that significantly distinguish them from the non-metastatic ones. These genes display a strong and significant clinical value, as they can be condensed in gene signatures predicting worse BC patient prognosis. Mechanistically, the pro-metastatic clones were shown to hyper-activate pathways involved in extracellular matrix (ECM) deposition, hypoxia response, unfolded protein response (UPR), type-I interferon (IFN) response, and migration, thereby reinforcing the role of these pathways in metastasis spreading. In line with this, we demonstrated that the knock-down of five that were upregulated by the pro-metastatic clones and involved in the above-mentioned processes (i.e., ANGPTL4, KCNQ1OT1, ITGB4, LY6E, and IFI6) significantly reduced the metastatic burden in vivo. Therefore, our work provided an unprecedented insight in the clonal phenotype that fosters metastatic progression in BC.