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. 2016 Jul 25:6:30418.
doi: 10.1038/srep30418.

In Vivo therapeutic potential of mesenchymal stem cell-derived extracellular vesicles with optical imaging reporter in tumor mice model

Senthilkumar Kalimuthu  1 , Prakash Gangadaran  1 , Xiu Juan Li  1 , Ji Min Oh  1 , Ho Won Lee  1 , Shin Young Jeong  1 , Sang-Woo Lee  1 , Jaetae Lee  1   2 , Byeong-Cheol Ahn  1
Affiliations

In Vivo therapeutic potential of mesenchymal stem cell-derived extracellular vesicles with optical imaging reporter in tumor mice model

Senthilkumar Kalimuthu et al. Sci Rep. .

Abstract

Mesenchymal stem cells (MSCs) can be used as a therapeutic armor for cancer. Extracellular vesicles (EVs) from MSCs have been evaluated for anticancer effects. In vivo targeting of EVs to the tumor is an essential requirement for successful therapy. Therefore, non-invasive methods of monitoring EVs in animal models are crucial for developing EV-based cancer therapies. The present study to develop bioluminescent EVs using Renilla luciferase (Rluc)-expressing MSCs. The EVs from MSC/Rluc cells (EV-MSC/Rluc) were visualized in a murine lung cancer model. The anticancer effects of EVs on Lewis lung carcinoma (LLC) and other cancer cells were assessed. EV-MSC/Rluc were visualized in vivo in the LLC-efffuc tumor model using optical imaging. The induction of apoptosis was confirmed with Annexin-V and propidium iodide staining. EV-MSC/Rluc and EV-MSCs showed a significant cytotoxic effect against LLC-effluc cells and 4T1; however, no significant effect on CT26, B16F10, TC1 cells. Moreover, EV-MSC/Rluc inhibited LLC tumor growth in vivo. EV-MSC/Rluc-mediated LLC tumor inhibitory mechanism revealed the decreased pERK and increased cleaved caspase 3 and cleaved PARP. We successfully developed luminescent EV-MSC/Rluc that have a therapeutic effect on LLC cells in both in vitro and in vivo. This bioluminescent EV system can be used to optimize EV-based therapy.

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Figures

Figure 1
Figure 1. Characterization of EV-MSC and 1 EV-MSC/Rluc.
(A) Coomassie Brilliant Blue staining. Protein was isolated from naïve MSC and MSC/Rluc cells and their EVs. Equivalent amounts (50 μg) of protein were run on a 10% SDS gel and stained with Coomassie blue. (B) Western blotting analysis of EV marker proteins. ALIX and CD63, positive EV marker proteins, were detected in the MSC/Ruc EV. GM-130 (golgi marker) and calnexin (endoplasmic reticulum marker), negative marker proteins for EV, was not detected in MSC/Rluc EVs. (C) Analysis of EV-MSC and EV-MSC/Rluc by TEM. Image shows EVs with lipid-bilayer (Scale bars, 100 nm), (D) EV-MSC and EV-MSC/Rluc size analyzed by NanoSight. Data are expressed as the mean ± standard deviation (SD) of three independent experiments.
Figure 2
Figure 2. Rluc activity and Rluc protein expression in EV-MSC and EV-MSC/Rluc.
(A) Representative BLI of Renilla luciferase activity of EV-MSC and EV-MSC/Rluc. BLI was performed with different concentration of MSC- and MSC/Rluc-derived EVs in a 96 well plate. (B) Quantitative Rluc activity of EV-MSC and EV-MSC/Rluc. Data are expressed as the mean ± standard deviation (SD) of three independent experiments. (C) Western blot detection of Rluc protein. Rluc protein expression was analyzed in EV-MSC and EV-MSC/Rluc. Alix was immunoprobed as a loading control (D) Dot blot detection of Rluc protein in EV-MSC/Rluc.
Figure 3
Figure 3. Cellular internalization analysis of MSC/Rluc derived EV into LLC cells by confocal microscopy.
LLC cells were incubated with 20 μg of MSC/Rluc-derived EVs that were labelled with DiD for 4 h. Without EV-MSC/Rluc was used as negative control. Scale bar = 10 μm.
Figure 4
Figure 4. Effect of EV-MSC and EV-MSC/Rluc on LLC-1 effluc activity, and apoptosis mechanism.
(A,C) Representative BLI of EV-MSC and MSC/Rluc-EV-treated LLC-effluc activity. The viability of LLC-effluc cells decreased with increasing concentration of EV-MSC and EV-MSC/Rluc. (B,D) Quantitative effluc activity of LLC-effluc cells. (E) Annexin V and PI staining for analyzing LLC cell apoptosis. The percentage of apoptotic cells was increased after treatment with 10 or 20 μg/mL of EV-MSC/Rluc for 24 h. (F) Representative western blot analyzing pERK1/2, cleavage of apoptosis markers caspase 3 and PARP levels in EV MSC/Rluc-treated LLC cells. The fold changes were normalized for pERK with total ERK. Cleaved PARP and cleaved caspase 3 normalized with β-actin. Data are expressed as the mean ± standard deviation (SD) of three independent experiments, *p < 0.05, **p < 0.01., ***p < 0.001 (by Student’s t-test).
Figure 5
Figure 5. In vivo and ex vivo BLI to monitor the effect of EV-MSC/Rluc treatment.
(A) BLI of tumor LLC-effluc activity in mice subcutaneously injected with 1 × 106 LLC-effluc cells and treated with vehicle or EV-MSC/Rluc (50 μg) twice. (B) Quantitative BLI of LLC-effluc activity. (C) Tumor weight. (D) BLI imaging of LLC-effluc activity from ex vivo tumors (E) Ex vivo quantitative BLI of LLC-effluc activity. Values are expressed as the mean ± standard deviation (SD), *p < 0.05, **p < 0.01 (by Student’s t-test).
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