Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells

Abstract

Despite the therapeutic potential of nucleic acid drugs, their clinical application has been limited in part by a lack of appropriate delivery systems. Exosomes or microvesicles are small endosomally derived vesicles that are secreted by a variety of cell types and tissues. Here, we show that exosomes can efficiently deliver microRNA (miRNA) to epidermal growth factor receptor (EGFR)-expressing breast cancer cells. Targeting was achieved by engineering the donor cells to express the transmembrane domain of platelet-derived growth factor receptor fused to the GE11 peptide. Intravenously injected exosomes delivered let-7a miRNA to EGFR-expressing xenograft breast cancer tissue in RAG2(-/-) mice. Our results suggest that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs.

Figure 1

Epidermal growth factor receptor (EGFR) ligands on the outer surfaces of the exosomes. (a) Diagrams of the modified epidermal growth factor (EGF) and GE11 proteins. Signal peptide, Igκ-chain leader sequence; HA, hemagglutinin epitope tag (YPYDVPDYA); Linker, (GGGGS) 3; Myc, Myc epitope (EEKLISEEDL); platelet-derived growth factor receptor (PDGFR) transmembrane domain, transmembrane domain from platelet-derived growth factor receptor. (b) Western blots of HA-tagged constructs in exosomes obtained from culture supernatants of human embryonic kidney cell line 293 (HEK293) cells that had been transfected with pDisplay encoding EGF or GE11. The quality of each exosome preparation was confirmed by hybridization with antihuman leukocyte antigen (HLA) antibodies. (c) For flow cytometry, exosomes from transfected HEK293 cells were incubated with latex beads and stained with anti-Myc tag antibodies. Tetraspanin CD81 was used as a positive control for the exosomes. (d) Immunoelectron microscopy showed that HA-tagged constructs were present on exosomes purified from the supernatants of cells transfected with pDisplay encoding EGF or GE11. Bars = 100 nm. The percentages of HA-positive exosomes are indicated in the graph. Data are expressed as means ± SD.

Figure 2

Uptake of epidermal growth factor (EGF)- and GE11-positive exosomes by breast cancer cell lines. (a) Flow cytometric analysis of epidermal growth factor receptor (EGFR) expression on HCC70, HCC1954, and MCF-7 breast cancer cells. (b) Uptake of fluorescently labeled exosomes by the breast cancer cell lines was detected using flow cytometry. PKH67-labeled exosomes were incubated with the breast cancer cell lines at 37 °C or 4 °C for 4 hours. The degree of uptake was relatively low at 4 °C. (c) Intracellular PKH67-labeled exosomes were detected in HCC70 cells (arrows) using confocal fluorescence microscopy. (d) Flow cytometric analysis of EGFR expression on MCF-7 cells, which were stably infected with retrovirus expressing EGFR. (e) Uptake of PKH67-labeled EGF- and GE11-positive exosomes was compared using MCF-7 cells expressing high levels of EGFR and control cells. (f) Uptake of PKH67-labeled EGF- and GE11-positive exosomes was compared among EGFR^Low HCC70, EGFR^High, and control cells.

Figure 3

The activity of encapsulated small-interfering RNA (siRNA) in luciferase assays. Luciferase-specific siRNA (siLuc) was encapsulated in exosomes, which were incubated for 48 hours with HCC70 cells stably expressing firefly luciferase. Data are expressed as means ± SD. n = 3; *P < 0.05.

Figure 4

Migration of GE11-positive exosomes to tumor tissues characterized by high levels of epidermal growth factor receptor (EGFR) expression. (a) Exosomes labeled with XenoLight DiR (near-infrared) were intravenously injected (4 µg of purified exosomes) into mice bearing transplanted HCC70 cells. Brain, heart, spleen, liver, lung, kidney, small intestine, colon, and tumor tissues were harvested 24 hours postinjection for ex vivo imaging. The migration of fluorescently labeled exosomes was detected with an in vivo imaging system (IVIS). (b) The intensity of fluorescent signals from the tumor was measured using an IVIS. Data are expressed as means ± SD. n = 5; *P < 0.05.

Figure 5

Inhibition of breast cancer development in vivo using GE11-positive exosomes containing let-7a. Human embryonic kidney cell line 293 (HEK293) cells expressing GE11 were transfected with synthetic let-7a. Exosomes containing let-7a were purified from culture supernatants and intravenously injected (1 µg of purified exosomes, once per week for 4 weeks) into mice bearing luciferase-expressing HCC70 cells. (a) Let-7a levels in the purified exosomes were measured using quantitative PCRs. Data are expressed as means ± SD. n = 3. (b) Representative images of tumors 4 weeks postinjection are shown. (c) Luciferase signals from the tumors were measured using an in vivo imaging system (IVIS). Data are expressed as means ± SD. n = 5; *P < 0.05.