Functional single-cell profiling identifies that exosomes are associated with increased immune cell infiltration in non-metastatic breast cancer

Exosomes mediate intercellular communication in health and disease. Conventional assays are limited in profiling exosomes secreted from large populations of cells and are unsuitable for studying the functional consequences of individual cells exhibiting varying propensity for exosome secretion. In cancer, since exosomes can support the development of the pre-metastatic niche, cells with varying abilities to secrete exosomes can directly impact tumorigenesis.

University of Houston researchers have developed a high throughput single-cell technique that enabled the mapping of exosome secretion dynamics. By utilizing clinically relevant models of breast cancer, they established that non-metastatic cancer cells secrete more exosomes than metastatic cancer cells. Single-cell RNA-sequencing confirmed that pathways related to exosome secretion were enriched in the non-metastatic cells compared to the metastatic cells. The researchers established isogenic clonal cell lines from non-metastatic cells with differing propensities for exosome secretion and showed that exosome secretion is an inheritable property preserved during cell division. Combined in vitro and in vivo studies with these cell lines suggested that exosome secretion can impede tumor formation. In human non-metastatic breast tumors, tumors with higher secretion of exosomes have a better prognosis, higher immune cytolytic activity, and enrichment of pro-inflammatory macrophages compared to tumors with lower secretion of exosomes. This single-cell methodology can become an essential tool that enables the direct integration of exosome secretion with multiple cellular functions.

High-throughput single-cell assay for monitoring exosome secretion

A The overall workflow of the single-cell assay. Cells and anti-CD81 conjugated beads are loaded on the nanowell and incubated for 2-6 hours. The entire array is incubated with fluorescently-labeled antibody against CD63 and imaged using microscopy. B Representative images of individual nanowells containing 67NR cells with different exosome secretion capacity. The insets show single cells and the contour map of CD63 (exosome) intensity. C Comparison of the frequency of exosome secreting cells between 67NR (non-metastatic) and 4T1 (metastatic) breast cancer cells. D The rate of secretion of exosomes by 67NR cells is higher than that of 4T1 cells at two, four, and six hours. Each dot represents a single cell with the median and quantiles of CD63 (exosome) intensity shown across all cells. T-tests were used for comparison. A representative example from three independent repeats is shown.