A Systematic Evaluation of Factors Affecting Extracellular Vesicle Uptake by Breast Cancer Cells

Abstract

Extracellular vesicles (EVs) are nanometer-scale particles that are secreted by cells and mediate intercellular communication by transferring biomolecules between cells. Harnessing this mechanism for therapeutic biomolecule delivery represents a promising frontier for regenerative medicine and other clinical applications. One challenge to realizing this goal is that to date, our understanding of which factors affect EV uptake by recipient cells remains incomplete. In this study, we systematically investigated such delivery questions in the context of breast cancer cells, which are one of the most well-studied cell types with respect to EV delivery and therefore comprise a facile model system for this investigation. By displaying various targeting peptides on the EV surface, we observed that although displaying GE11 on EVs modestly increased uptake by MCF-7 cells, neuropeptide Y (NPY) display had no effect on uptake by the same cells. In contrast, neurotensin (NTS) and urokinase plasminogen activator (uPA) display reduced EV uptake by MDA-MB-231 cells. Interestingly, EV uptake rate did not depend on the source of the EVs; breast cancer cells demonstrated no increase in uptake on administration of breast cancer-derived EVs in comparison to HEK293FT-derived EVs. Moreover, EV uptake was greatly enhanced by delivery in the presence of polybrene and spinoculation, suggesting that maximal EV uptake rates are much greater than those observed under basal conditions in cell culture. By investigating how the cell's environment might provide cues that impact EV uptake, we also observed that culturing cells on soft matrices significantly enhanced EV uptake, compared to culturing on stiff tissue culture polystyrene. Each of these observations provides insights into the factors impacting EV uptake by breast cancer cells, while also providing a basis of comparison for systematically evaluating and perhaps enhancing EV uptake by various cell types.

Keywords: EVs; breast cancer; exosomes; extracellular vesicles.

FIG. 1.

Evaluation of targeting peptide display on EVs on recipient cell uptake. (A) Schematic summary of EV biogenesis and mechanisms of uptake by recipient cells. (B) Uptake of targeted, CD63-GFP-labeled EVs by MCF-7 cells or MDA-MB-231 cells following 2 h of incubation. “FLAG” refers to EVs displaying a nontargeting FLAG tag as a negative control. Data for both MDA-MB-231 targeting peptides are compared to the same negative control. Experiments were performed in biological triplicate, and error bars indicate one standard deviation. Statistical tests comprise two-tailed Student's t-tests (*p < 0.05). Results are representative of two independent experiments (Supplementary Fig. S3). EVs, extracellular vesicles. Color images available online at www.liebertpub.com/tea

FIG. 2.

Effect of producer–recipient cell type pairing on EV uptake. (A) Illustration of experimental design. (B) Uptake of blank or CD63-GFP-labeled EVs harvested from HEK293FT or MDA-MB-231 cells and delivered to MDA-MB-231, MCF-7, or HEK293FT cells following 2 h of incubation. Experiments were performed in biological triplicate, and error bars indicate one standard deviation. Results are representative of two independent experiments (Supplementary Fig. S6). Color images available online at www.liebertpub.com/tea

FIG. 3.

Enhancement of EV uptake by treatment with polybrene and spinoculation. (A) Illustration of experimental design. (B) Uptake of CD63-GFP-labeled EVs by MCF-7 cells or MDA-MB-231 cells was quantified following incubation for 2 h under the delivery conditions indicated (for conditions including spinoculation, spinoculation was performed for 1 h and then cells were returned to the incubator for an additional 1 h before analysis). Experiments were performed in biological triplicate, and error bars indicate one standard deviation. Statistical tests comprise two-tailed Student's t-tests (*p < 0.05). Results are representative of two independent experiments (Supplementary Fig. S7). Color images available online at www.liebertpub.com/tea

FIG. 4.

Dependence of EV uptake on cell growth matrix stiffness. (A) Illustration of experimental design. (B) Gelatin matrix stiffness quantified by rheology (no cells added). (C) CD63-GFP-labeled EV uptake by MCF-7 cells or MDA-MB-231 cells during culture on various substrates following 2 h of incubation. Experiments were performed in biological triplicate, and error bars indicate one standard deviation. Statistical tests comprise paired two-tailed Student's t-tests, individually comparing each gelatin concentration condition to TCPS (*p < 0.05). Results are representative of two independent experiments (Supplementary Fig. S8). TCPS, tissue culture polystyrene. Color images available online at www.liebertpub.com/tea