Differences in Metabolic Cargo in Extracellular Vesicles Released by Metastatic Breast Cancer Cells Sorted by Migratory Phenotype

Abstract

We are interested in the relationship between tumor heterogeneity in migration modes and packaging of metabolic cargo in tumor-derived EVs. To acquire different subpopulations of cancer cells with different migratory properties, we used transwells to phenotypically sort metastatic cancer cells by their migratory phenotype into highly migratory cells and weakly migratory cells. When injected in vivo, the weakly migratory cells showed increased metastatic potential. Previously, we investigated microvesicles released by the migratory subpopulations and found that the microvesicles from weakly migratory cells activate CAFs by transferring Tg2, promoting collective cancer cell migration. RNA-seq data of the migratory subpopulations demonstrated that the migratory subpopulations differentially expressed metabolic proteins, with the upregulation of glycolysis associated transcripts in highly migratory cells and the upregulation of oxidative phosphorylation transcripts in the weakly migratory cells. We investigated the differences in metabolic cargo between the migratory subpopulations including metabolic proteins and ATP. In prior work from our lab, we performed iTRAQ proteomics on our microvesicle populations to show distinctly different protein cargo between the microvesicles released by cells of differing migratory ability. We further analyze this data to investigate the differing metabolic enzymes in the EVs that have a role in glycolysis, oxidative phosphorylation, lipid metabolism, and amino acid metabolism. Since the cells have differentially expressed metabolic pathways, we hypothesized that ATP levels differ between the subpopulations and their released EVs. We investigated the ATP in the EVs: comparing between EV subtypes, quantifying ATP production with the addition of metabolic inhibitors, and determining the location of ATP on the EV membrane. Overall, we investigated two classes of metabolic cargo in the EVs: metabolic enzymes and ATP. This work highlights the heterogeneity of microvesicles within one cell line, and how cellular heterogeneity in migratory phenotype results in the heterogeneity of extracellular vesicle metabolic cargo. By investigating metabolic cargo, this work identifies potential targets that may have a role in changing specific recipient cell behavior, including recipient cell metabolism, collagen remodeling, and purinergic signaling.