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. 2022 Sep 17;27:26–47. doi: 10.1016/j.omto.2022.09.004

Figure 6.

Figure 6

Each function of MSC-TERT-tetoneE1B55K-GRP78 works well

(A) Expression levels of E1B55K, GRP78, and MMP2 in G418-selected clones in the presence of 0.5 μg/mL doxycycline. The final clone 21 stably overexpressed GRP78 and MMP2 and was selected as MSC-TERT-tetoneE1B55K-GRP78. (B) Induction of E1B55K expression in MSC-TERT-tetoneE1B55K-GRP78 in the presence of various concentrations of doxycycline. (C) Measuring virus production levels in MSC-TERT-tetoneE1B55K-GRP78. Cells were infected with indicated MOIs of Ad-3484-shHSP27-shTGF-β1 for 4 h and treated with or without doxycycline. After 48 h, all virus particles released from MSC-TERT-tetoneE1B55K-GRP78 were collected and the amount of infectious virus particles was calculated. Error bars are mean ± SD from three independent experiments: ∗∗p < 0.01, ∗∗∗∗p < 0.0001. (D) Quantification of viral production kinetics in vitro after infection of oncolytic virus to MSC-TERT-tetoneE1B55K-GRP78. Cells were infected with 100 MOIs of Ad-3484-shHSP27-shTGF-β1 for 4 h and treated with or without doxycycline. At the indicated time points, all virus particles released from MSC-TERT-tetoneE1B55K-GRP78 were collected and the amount of infectious virus particles was calculated. Data from three independent experiments are shown. (E) Cell viabilities of MSC-TERT-tetoneE1B55K-GRP78 loaded with oncolytic virus at the indicated time points were examined after infection with 100 MOIs of Ad-3484-shHSP27-shTGF-β1 for 4 h and treated with or without doxycycline. (F) Bioluminescence imaging of MSC-TERT or MSC-TERT-tetoneE1B55K-GRP78 loaded with Ad-Luc in mice bearing SNU-398 (upper), A549 (middle), or MIA PaCa-2 (lower) tumors. MSC-TERT and MSC-TERT-tetoneE1B55K-GRP78 distribution was monitored in vivo using the IVIS Spectrum System at 6, 24, 48, and 72 h after tail vein injection of Ad-Luc-loaded MSCs. The photo on the far left of each row shows the transplantation location of the tumor. Each scale bar shows intensity of bioluminescence; minimum and maximum radiances are indicated. (G) In vivo distribution of fluorescent probe-labeled MSCs. MSC-TERT or MSC-TERT-tetoneE1B55K-GRP78 was stained by cell tracker red fluorescent dye and intravenously injected into A549 tumor-bearing mice. At 24 h (upper), 48 h (middle), and 72 h (lower) after intravenous injection, tumor tissues were prepared by necropsy and frozen sectioned. All slides were observed under a fluorescence microscope with excitation at 540–585 nm and emission at 600 nm. Scale bars, 100 μm.