Circulating Tumor Cells Clustering Triggers DNA Methylation Changes to Enable Metastasis Seeding

The ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Our DNA methylation analysis led us to hypothesize that CTC clusters are characterized by active TF networks that support both stemness and proliferation. To identify whether the stemness- and proliferation-related TF networks are also transcriptionally active in CTC clusters compared to single CTCs, we performed single-cell resolution RNA sequencing analysis of single CTCs and CTC clusters, matched within individual liquid biopsies and isolated from six breast cancer patients with progressive metastatic disease, and of single CTCs and CTC clusters isolated from three xenograft models. In addition, among 2,486 FDA-approved compounds, we identify Na+/K+ ATPase inhibitors that enable the dissociation of CTC clusters into single cells, leading to DNA methylation remodeling at critical sites and metastasis suppression. We performed RNA sequencing analysis of BR16 and Brx50 cells upon treatment to assess the molecular consequences of clusters dissociation. Overall design: The ability of circulating tumor cells (CTCs) to form clusters is key to accelerate the metastatic process. Yet, biological features and vulnerabilities of CTC-clusters remain largely unknown. Here, we profile DNA methylation and RNA expression landscape of single CTCs and CTC-clusters, matched within individual breast cancer patients and mouse models. We find that clustering of CTC leads to a remarkable hypomethylation of binding sites for stemness-related transcription factors. Through a screen with 2'486 FDA-approved compounds, we identify drugs that are able to dissociate CTC-clusters into single cells, leading to DNA re-methylation at critical sites and to a reduction in metastasis-seeding ability. Thus, clustering of CTCs favors accessibility to binding sites of stemness-related transcription factors, and cluster-dissociating compounds enable to suppress the spread of cancer.