Single-Cell Cloning of Breast Cancer Cells Secreting Specific Subsets of Extracellular Vesicles

SIMPLE SUMMARY: Extracellular vesicles (EVs) are a pivotal mechanism for long-distance intercellular communication and facilitate the stable transport of biological information. Conventional methods for profiling EVs are focused on the biological cargo obtained from large populations of cells and cannot map the secretion of specific subsets of EVs onto their cell of origin. We developed a high-throughput single-cell cloning method that can identify the kinetics of secretion of specific subsets of EVs. With the aid of this methodology, we illustrate that secretion of specific subsets of EVs can be an inheritable property of cancer cells. Our single-cell methodology enables the direct integration of EV secretion with multiple cellular functions and can enable new insights into cell and disease biology. ABSTRACT: Extracellular vesicles (EVs) mediate communication in health and disease. Conventional assays are limited in profiling EVs secreted from large populations of cells and cannot map EV secretion onto individual cells and their functional profiles. We developed a high-throughput single-cell technique that enabled the mapping of dynamics of EV secretion. By utilizing breast cancer cell lines, we established that EV secretion is heterogeneous at the single-cell level and that non-metastatic cancer cells can secrete specific subsets of EVs. Single-cell RNA sequencing confirmed that pathways related to EV secretion were enriched in the non-metastatic cells compared with metastatic cells. We established isogenic clonal cell lines from non-metastatic cells with differing propensities for CD81(+)CD63(+)EV secretion and showed for the first time that specificity in EV secretion is an inheritable property preserved during cell division. Combined in vitro and animal studies with these cell lines suggested that CD81(+)CD63(+)EV secretion can impede tumor formation. In human non-metastatic breast tumors, tumors enriched in signatures of CD81(+)CD63(+)EV have a better prognosis, higher immune cytolytic activity, and enrichment of pro-inflammatory macrophages compared with tumors with low CD81(+)CD63(+)EVs signatures. Our single-cell methodology enables the direct integration of EV secretion with multiple cellular functions and enables new insights into cell/disease biology.