Identification of Determinants of Metastatic Behaviour in Breast Cancer Clonal Populations isolated through a CRISPRa-based Barcoding Strategy

Breast cancer is the most commonly diagnosed cancer in women, and one of the most lethal malignancies worldwide. Its high mortality is attributed to the development of metastases in secondary sites of the body that impair the function of vital organs. One of the reasons why the effective eradication of breast cancer remains an unmet clinical challenge is its inherent heterogeneity. In cancer, heterogeneity can be found at different levels: among tumors of the same type, with different cytological and molecular characteristics, or among cell populations within the same tumor. Within a tumor mass, several clonal populations with different phenotypes and characteristics, such as the ability to metastasize and survive in distant organs, co-exist. To deepen our knowledge about what differentiates highly aggressive and less metastatic populations, D2.0R cells were genetically barcoded following the CaTCH (CRISPRa tracing of clones in heterogeneous cell populations) protocol and injected into mice, that developed metastases. Following an assessment of barcode representation, we isolated clonal populations with different metastatic fitness. We isolated the clonal population for each selected barcode, both from the initial polyclonal population (PRE) and from the lung metastasis (POST). RNA sequencing of the different populations revealed clear distinct gene expression profiles among clones with different metastatic fitness, as well as among PRE and POST clonal populations. In line with RNA sequencing results, differential behaviours were observed among clonal populations cultured on a soft substrate, displaying either a mesenchymal- or epithelial-like phenotype. These observations prompted us to investigate the role of E-cadherin, an epithelial marker shown to be differentially expressed, in the metastatic process. Our results showed that E-cadherin strongly impacts the self-organization of the cells in different experimental setups in vitro, suggesting that it may influence metastatic recurrence.