New Optical Imaging Reporter-labeled Anaplastic Thyroid Cancer-Derived Extracellular Vesicles as a Platform for In Vivo Tumor Targeting in a Mouse Model

Abstract

Extracellular vesicles (EVs), originating from multivesicular bodies by invagination of the endosomal membrane, are communication channels between distant cells. They are natural carriers of exogeneous cellular materials and have been exploited as drug delivery carriers in various diseases. Here, we found that tumor cell-derived EVs can be used as efficient targets in tumors by monitoring with an optical reporter system. Anaplastic thyroid cancer (CAL62) cell-derived EVs with Renilla luciferase (Rluc) were used to target CAL62 tumors in a mouse model. Optical imaging revealed that cancer cell-derived EVs (EV-CAL62/Rluc) targeted the original tumor (CAL62) in mice within 30 min after systemic injection. Furthermore, fluorescence imaging revealed that EV-CAL62/Rluc were internalized into CAL62 tumors in the mice. Ex vivo Optical imaging further confirmed the in vivo finding. Here, we successfully monitored the tumor targeting ability of tumor cell-derived EVs by optical imaging. Based on these results, tumor cell-derived EVs are highly effective natural carriers for drug delivery for cancer therapies.

Figure 1

Generation of stable reporter gene expression in a cancer cell line. (A) Representative bioluminescent imaging of the in vitro luciferase assay in CAL62 and CAL-62/Rluc cells. (B) In vitro luciferase assay in CAL62 and CAL62/Rluc cells. Data are expressed as the mean ± standard deviation (SD). (C) Representative bioluminescent imaging of the in vitro luciferase assay in CAL62 and CAL62/Effluc cells. (D) In vitro luciferase assay in CAL62 and CAL62/Effluc cells. Data are expressed as the mean ± standard deviation (SD). (E) Western blot analysis of the Rluc and Effluc proteins in CAL62, CAL62/Rluc, and CAL62/Effluc cells; β-actin was used as an internal control. (F) RT-PCR analysis to determine the expression of the Rluc and Effluc genes in CAL62, CAL62/Rluc, and CAL62/Effluc cells; GAPDH served as an internal control.

Figure 2

Characterization of EVs. (A) Examination of EVs from CAL62/Rluc cells using electron microscopy Scale bar: 100, 200 nm. (B) Western blotting analysis of EVs. CD63 and TSG101 (EV marker proteins) GM130, CALNEXIN and PCNA (cell marker proteins) were detected by specific antibodies. (C,D) EV size and concentrations were determined by DLS (Avg. 93.1 ± 21.3 nm). (D) Distribution of EVs population in the isolated EVs.

Figure 3

EV-CAL-62/Rluc showed EV-specific Rluc activity and serum stability in vitro. (A) Representative bioluminescent imaging of an in vitro luciferase assay in EVs from CAL-62 and CAL-62/Rluc cells. (B) Quantitative in vitro luciferase data in EVs are expressed as the mean ± SD. (C) Western blot analysis of the Rluc protein in EVs from CAL-62, CAL-62/Rluc cells, detected by means of Rluc-specific antibodies. TSG served as loading control. (D) Stability of Rluc in EV-CAL62/Rluc and recombinant Rluc protein in serum. Time course of stability of Rluc at 37 °C in 20% FBS/PBS buffer. (E) Representative confocal images of Rluc (green) and DiD (red) in EV-CAL62/Rluc/DiD or EV-CAL62/DiD or EV-CAL62 in CAL62 cells. Scale bars: 10 μm.

Figure 4

Internalization of EVs into cancer cells. (A,B) Representative flowcytometry images of CAL62 or CAL62/Effluc cells incubated with unlabeled EV-CAL62/Rluc or DiD-labeled EV-CAL62/Rluc. (B) Quantification of DiD positive cells from flowcytometry of CAL62 or CAL62/Effluc cells treated with unlabeled EV-CAL62/Rluc or DiD-labeled EV-CAL62/Rluc was represented in bar graph (p = 0.112).

Figure 5

In vivo visualizing and monitoring tumor-derived EVs targeting the same tumor. (A) Image of Effluc activity of CAL62/Effluc nude mice at six weeks after subcutaneous injection of CAL62/Effluc cells. (B) Quantification of tumor BLI signals from derived EV-CAL62/Rluc (n = 3) or PBS (n = 3) mouse groups.(C) In vivo imaging of EV-CAL62/Rluc intravenously administered to naïve or CAL62/Effluc tumor-bearing mice. (D) Quantification of Rluc (EV-CAL62/Rluc) signals in the tumor region of mice mentioned in 3C.

Figure 6

Ex vivo and Subcellular visualization of EV-CAL62/Rluc in tumors. (A) Representative ex vivo bioluminescent imaging of EV-CAL62/Rluc excised tumor from EV-CAL62/Rluc or PBS intravenously administered to CAL62/Effluc tumor-bearing mice (n = 3). (B) Quantification of EV-CAL62/Rluc signal from the tumor above mentioned in (A); the values are expressed as mean ± SD. (C) Representative confocal images of Rluc in harvested tumors (CAL62/Effluc) from mice described in Fig. 3C after 120 min. Scale bars: 50 μm.