Extracellular vesicles deliver sodium iodide symporter protein and promote cancer cell radioiodine therapy

Abstract

Extracellular vesicles (EVs) are a promising carrier for various cargos with antitumor effects, but their capacity to transfer the ability to transport radioiodine for cancer theranostics remains unexplored. Herein, we tested the hypothesis that EVs can be loaded with the sodium iodide symporter (NIS) protein and efficiently deliver the payload to recipient cancer cells to facilitate radioiodine uptake. The results revealed that donor cells either transduced with an adenoviral vector for transient expression or engineered for stable overexpression secreted EVs that contained substantial amounts of NIS protein but not NIS mRNA. Huh7 liver cancer cells treated with EVs secreted from each of the donor cell types showed significantly increased plasma membrane NIS protein, indicating efficient payload delivery. Furthermore, intact function of the delivered NIS protein was confirmed by significantly increased radioiodine transport in recipient cancer cells that peaked at 48 h. Importantly, NIS protein delivered by EVs significantly enhanced the antitumor effects of ¹³¹I radiotherapy. These results reveal that EVs are a promising vehicle to deliver NIS protein to cancer cells in sufficient amounts for radioiodine-based theranostics.

Figure 1

EGFP and NIS proteins in transiently and stably expressing cancer cells. (A) EGFP fluorescence in Huh7 cells transduced with NIS/EGFP adenovirus (Huh7/NIS-Adv cells). (B) Western blots of NIS protein obtained from the cell membrane fraction (top) and whole cell lysates (bottom) of Huh7, Huh7/NIS-Adv, MDAMB231, and MDAMB/NIS-stable cancer cells. (C) ¹²⁵I uptake (right) in Huh7/NIS-Adv and MDAMB/NIS-stable cells compared to respective Huh7 and MDAMB control cells. Bars for uptake are mean ± SD of uptake in fmol/mg protein obtained from triplicate samples per group. WT wild type. ^‡P < 0.0001.

Figure 2

TEM and size distribution of EVs from NIS-expressing cancer cells. (A) Nanosight analysis profiles of EVs derived from Huh/NIS-Adv cells (top) and MDAMB231/NIS-stable donor cells (bottom). (B) Representative TEM images of EVs derived as above. Magnifications are × 10,000 and × 200,000, and scale bars are 1 μm and 20 nm, respectively.

Figure 3

NIS protein and NIS mRNA in EVs from NIS-expressing cancer cells. (A) Western blots and quantified band intensities of NIS (top) and CD63 protein (bottom, control) in EVs derived from Huh7, Huh7/NIS-Adv, MDAMB231, and MDAMB/NIS-stable donor cells. (B) RT-PCR for NIS mRNA (top) and GADPH mRNA (bottom, control) in EVs from respective donor cells. MDAMB231 and MDAMB231/NIS-stable cells were used for NIS mRNA negative control (NC) and positive control (PC), respectively.

Figure 4

NIS protein and ¹²⁵I uptake in cancer cells treated with EVs. (A) Western blots of NIS protein obtained from the plasma membrane fractions of recipient Huh7 liver cancer cells treated with EVs derived from Huh7/NIS-Adv and MDAMB/NIS-stable donor cells (n = 2 per group). (B) Time dependence of ¹²⁵I uptake in recipient Huh7 cells treated with EVs s derived from Huh7/NIS-Adv cells. (C) ¹²⁵I uptake in recipient Huh7 and HepG2 human liver cancer cells, and T47D human breast cancer cells in 24-well plates at 48 h after treatment with 25 µg per well of EVs derived from Huh7/NIS-Adv or MDAMB/NIS-stable donor cells. All cell uptake data are the mean ± SD of values from triplicate samples per group with or without perchlorate inhibition. *P < 0.05; **P < 0.01; ^‡P < 0.0001, compared to controls.

Figure 5

¹³¹I radiotherapy in cancer cells treated with EVs. Huh7 liver cancer cells were incubated for 48 h with vehicle (null), 10 µg of control EVs, or 10 µg of EVs derived from Huh7/NIS-Adv donor cells. The cells then underwent radiotherapy with 20 or 40 µCi of ¹³¹I for 7 h and were assessed for survival. Data are mean ± SD of % survival obtained from triplicate samples per group. **P < 0.01; ^‡P < 0.001 compared to untreated controls for each group.