A novel strategy of transferring NIS protein to cells using extracellular vesicles leads to increase in iodine uptake and cytotoxicity

Abstract

Background: This study was designed to explore a novel approach for transferring NIS protein to cells using extracellular vesicle (EV) and enhancing iodine avidity in hepatocellular carcinoma (HCC) cells.

Methods: We transfected the HCC cells (Huh7) with NIS gene, designated as Huh7/NIS, and isolated the EVs from them. Presence of NIS protein in EVs and EV-mediated transport of NIS protein to recipient Huh7 cells were tested using Western blotting. We also examined radioiodine uptake in Huh7 cells treated with EV-Huh7/NIS.

Results: Successful transfer of NIS protein into Huh7 cells was confirmed by WB and microscopy. EVs showed high levels of NIS protein in them. Treatment of Huh7 cells with EV-Huh7/NIS increased the NIS protein level and enhanced ¹²⁵I uptake in recipient Huh7 cells. In addition, EV-huh7/NIS pre-treatment enhanced the cytotoxicity of ¹³¹I therapy against Huh7 cells by inducing increased DNA damage/increased γH2A.X foci formation.

Conclusion: This is the first-of-its-kind demonstration of successful transportation of the NIS protein to cells via EVs, which increased radioiodine uptake. This approach can revert radioiodine-resistant cancers into radioiodine-sensitive cancers.

Keywords: extracellular vesicle; hepato-cellular carcinoma; iodine uptake; sodium iodide symporter (NIS).

Figure 1

Establishment of double-gene expression in Huh7 cells. Notes: (A) CMV promoter driven hNIS and EGFP was expressed in cells. (B) Western blotting analysis for the finding of NIS proteins from Huh7/NIS cells, not from Huh7 cells. (C) Confocal microscopy analysis to detect NIS and GFP protein in Huh7/NIS cells, not from Huh7 cells. Abbreviations: CMV, cytomegalovirus; IRES, internal ribosomal entry site.

Figure 2

Characterization of EVs isolated from Huh7/NIS. Notes: (A) Morphology of EV-Huh7/NIS confirmed by transmission electron microscopy, arrow indicates the lipid bilayer (scale bar: 100 nm). (B) Size and Zeta potential of EV-Huh7/NIS determined by ELS (n=3; average diameter: 169.2±71.5 nm). (C) Western blot analysis of EV-Huh7/NIS and Huh7/NIS. Abbreviations: EVs, extracellular vesicles; ELS, electrophoretic light scattering.

Figure 3

EV-mediated transfer of NIS protein to cells enhanced ¹²⁵I uptake. Notes: (A) Western blot analysis of Huh7 cells after EV-Huh7/NIS (0–40 μg/mL) treatment for 24 hours. (B) Quantification of band intensity by GelQuant software represented in bar graph. (C) ¹²⁵I uptake assay for Huh7 cells after EV-Huh7/NIS (0–40 μg/mL) treatment for 24 hours (n=5). Mean ± SD of experiments is shown. Student’s t-test was used. Abbreviation: EV, extracellular vesicle.

Figure 4

Effect of EV-Huh7/NIS, ¹³¹I, ¹³¹I-mediated effects by the EV-Huh7/NIS pretreatment of Huh7 cells. Notes: γH2A.X and DAPI were visualized by blue and green, respectively (scale bar: 20 μm). The yellow dotted square indicates the cropping region of overlay images. Abbreviation: EV, extracellular vesicle.