Figure 5.
GRP78 facilitates tumor trafficking of MSCs
(A) MSC-TERT was transfected with 2 μg of pcDNA3.1-Hygro or pcDNA3.1-Hygro-GRP78 plasmid. After 48 h, protein expression was analyzed by western blot (upper), and GRP78 mRNA expression was measured by qRT-PCR (lower). Error bars are mean ± SD from three independent experiments: ∗∗∗∗p < 0.0001. (B) Expression levels of GRP78 and MMP2 in G418-selected MSC-TERT-GRP78 clones. Clones stably overexpressing GRP78 and MMP2 were used for further experiments. (C) Bioluminescence imaging of MSC-TERT or MSC-TERT-GRP78 (clone 2 and clone 4) loaded with Ad-Luc in mice bearing SNU-398 tumors. MSC-TERT and MSC-TERT-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. Each scale bar shows the intensity of bioluminescence; minimum and maximum radiances are indicated. (D) Quantification of adenovirus copy numbers in tissues by qRT-PCR analysis. BALB/c athymic nude mice were transplanted with SNU-398 cells by subcutaneous injection. After 7 days, mice were intravenously injected with 1 × 106 MSC-TERT-GRP78 (clone 4) loaded with Ad-3484-shHSP27-shTGF-β1. On the indicated days after intravenous injection, samples of tumor, liver, lung, spleen, kidney, and heart were collected, and total genomic DNA of each tissue was extracted. A standard curve was created using adenoviral DNA of known concentrations, and the standard curve was used to determine the viral load of each tissue sample. Black points represent individual mice (n = 4–5). (E) Measurement of luciferase activity in mouse tissues. Tumors, livers, and lungs were collected from mice in each group (n = 2–4) at 6 and 24 h after intravenous injection with Ad-Luc/MSC complex. After lysis of thawing frozen tissue powders and centrifugation, the supernatant was used for the detection of luciferase activity. Luciferase activities of tissues were calculated as ratios of firefly luciferase to Renilla luciferase activities, and the activities were reported as the ratios divided by the weight of each tissue.