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. 2016 Aug 9;7(32):51815-51828.
doi: 10.18632/oncotarget.10122.

E1A-engineered Human Umbilical Cord Mesenchymal Stem Cells as Carriers and Amplifiers for Adenovirus Suppress Hepatocarcinoma in Mice

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Free PMC article

E1A-engineered Human Umbilical Cord Mesenchymal Stem Cells as Carriers and Amplifiers for Adenovirus Suppress Hepatocarcinoma in Mice

Zhenzhen Li et al. Oncotarget. .
Free PMC article

Abstract

Gene therapy is an attractive approach for hepatocellular carcinoma (HCC) patients. Nevertheless, efficient transgene delivery remains a challenge. In this study, we explored a new targeted system based on human umbilical cord-derived mesenchymal stem cells (HUMSCs), which were engineered to deliver adenovirus to tumor sites, and to replicate and assemble into new adenovirus against HCC. Our results showed that HUMSCs infected by Ad-hTERTp-IL24 followed by LentiR.E1A infection could specifically migrate to HepG2 tumor cells and support adenoviral replication in vitro and in vivo 36 h after LentiR.E1A infection. Ad-hTERTp-IL24 specifically inhibited HepG2 cells growth, and this inhibitory effect was enhanced by low doses of 5-fluorouracil (5-Fu), because the expression levels of coxsackie adenovirus receptor (CAR) and integrin ανβ3 on tumor cells were significantly increased, causing higher viral uptake. Compared with the no treatment groups, Ad-hTERTp-IL24 and LentiR.E1A co-loaded HUMSCs exhibited significant anti-tumor activity in vivo, particularly in combination with low doses of 5-Fu. In summary, this study provides a promising targeted gene therapeutic strategy dependent on the tumor tropism of HUMSCs, to improve the outcome of virotherapy for tumor patients especially those with metastatic diseases.

Keywords: HUMSC; adenovirus delivery; gene therapy; hepatocellular carcinoma.

Conflict of interest statement

The authors declare no potential conflict of interest.

Figures

Figure 1
Figure 1. Identification of HUMSCs, the specific activity of hTERT promoter and adenoviral transfection efficiency
A. Representative histogram overlays of FACS analysis showed the isolated HUMSCs were positive for CD73, CD90 and CD105. B. Representative histogram overlays of FACS analysis showed the negative antigens of HUMSCs: CD34, CD19 and CD45. C. The representative microscope image showed differentiation of HUMSCs into the adipogenic lineages in vitro. Cells were stained with Oil-red O. D. The representative microscope image showed differentiation of HUMSCs into the osteogenic lineages in vitro. Cells were stained with Alizarin red. (C, D) scale bar = 200 μm. E. The schematic representation displayed the vectors involved in the study. F. The specific transcriptional activity of hTERT promoter in different cell lines was detected by a dual-luciferase reporter assay. G. Adenoviral transfection efficiencies at different MOI after 48 hours in three independent HUMSC samples were evaluated by FACS and expressed as mean ± SD. H. The representative images depicted the transfection efficiency of MSCs with 500 MOI Ad-Track. 48 hours after infection, HUMSCs carrying GFP were observed under fluorescent field (left panel) and bright field (right panel), scale bar = 100 μm.
Figure 2
Figure 2. Replication-deficient adenoviruses amplified in HUMSC.LentiR.E1A and were eventually released from the cells
A. The mRNA expression level of E1A at indicating time points after LentiR.E1A infection. B. The total concentration (intracellularly and in the supernatant) of viral DNA was measured at different time points in in three independent samples of HUMSC sequentially infected by Ad-Track and LentiR.E1A / LentiR. The start of LentiR.E1A infection was set as 0 h. C. The intracellular and supernatant concentration of viral DNA were detected at different time points respectively. D. Electron micrographs show viral particles in the HUMSC sequentially infected by Ad-Track and LentiR.E1A. Adenovirus particles were shown by the black arrow. Left panel: at low magnification; Right panel: at high magnification. E. The infection efficiency of HepG2 was detected by FACS 72 h after co-culture of virus-loaded (Ad-Track and LentiR.E1A) HUMSCs and HepG2 with different ratios in three independent samples.
Figure 3
Figure 3. Migration capacity of virus-loaded HUMSCs to hepatocarcinoma in vitro and in vivo
A. Representative photographs showed the migrated HUMSCs stained with crystal violet in vitro migration assays using Transwell plates. B. The numbers of migrated HUMSCs in three independent samples were expressed as mean ± SD. C. The homing capability of HUMSC.LentiR+Ad-Luc to tumor sites was monitored by bioluminescence imaging using Xenogen imaging system at indicated times after tail vein injection. D. The homing capability of virus-loaded HUMSC.LentiR.E1A+Ad-Luc to tumor sites was monitored in vivo.
Figure 4
Figure 4. Specific tumor suppressing effects of Ad-hTERTp-IL24 and low dose of 5-Fu could enhance growth inhibition of HepG2 cells
A. The cell viability of HepG2 cells was measured every 24 hours after infection with different adenoviruses at MOI 100 by CCK8 assays. The results were expressed as survival rate of untreated control.** P<0.01. B. The cell viability of MRC-5 cells was measured every 24 hours after infection with different adenoviruses at MOI 100 by CCK8 assays. C. The inhibitory rate curve of HepG2 cells treated with 5-Fu gradient concentrations was analyzed. D. The survival rates of HepG2 cells infected by adenoviruses at 100 MOI in the presence or absence of low doses of 5-Fu were tested by CCK8 assay and expressed as mean ± SD. *P<0.05, ** P<0.01 compared with 5-Fu treatment group; #P<0.05, ##P<0.01 compared with Ad-hTERTp-IL24 treatment group. E. Apoptosis ratios were detected when 5-Fu in combination with Ad-hTERTp-IL24 or AdTrack and expressed as mean ± SD. *P<0.05, ** P<0.01 compared with 5-Fu treatment group; #P<0.05, ##P<0.01 compared with Ad-hTERTp-IL24 treatment group. (F) Western blot showed proteins involved in apoptosis pathway when 5-Fu in combination with Ad-hTERTp-IL24 or AdTrack. All the data represent the averages of three independent experiments.
Figure 5
Figure 5. CAR and ανβ3 expression levels essential for virus internalization were increased in response to low doses of 5-Fu causing excessive adenoviral uptake
A. Representative images showed the transfection of HepG2 with 100 MOI Ad-Track in the presence or absence of low-dose of 5-Fu (2μg/ml). 48 hours after infection, HepG2 cells were observed under fluorescent field (above panel) and bright field (below panel). B. Histogram overlays of flow cytometry revealed that the fluorescence intensity of each group in the Figure 5A. The red peak represented group of adenovirus in combined with 5-Fu; the blue peak represented group of adenovirus alone; the green peak represented group of negative control. C. The infection efficiencies of adenovirus for HepG2 at different MOIs were improved by low doses of 5-Fu. D. The expression level of CAR on the surface of HepG2 cells was detected every 24 hours after low doses of 5-Fu treatment. E. The expression level of ανβ3 on the surface of HepG2 cells was detected every 24 hours after low doses of 5-Fu treatment. F. The expression level of ανβ5 on the surface of HepG2 cells was detected every 24 hours after low doses of 5-Fu treatment. *P<0.05, ** P<0.01 compared with group of without 5-Fu treatment.
Figure 6
Figure 6. Tumor suppressing effects of Ad-hTERTp-IL24 loaded MSC.LentiR.E1A in combination with 5-Fu against HepG2 xenograft tumors
A. The tumor volumes of different groups were measured every 3 days after treatment. Points indicate the mean values (n=5); bars indicate SD. *P<0.05, ** P<0.01 compared with PBS group. B-F. The target protein expression was detected in different groups by confocal microscopy. The red fluorescence represented IL-24 proteins; the green fluorescence represented the cells infected by adenovirus. The blue fluorescence showed the nuclei. (B) PBS; (C) MSC.LentiR+Ad-hTERTp-IL24; (D) MSC.LentiR.E1A+Ad-Track; (E) MSC.LentiR.E1A+Ad-hTERTp-IL24; (F) The lung tissue of the group of MSC.LentiR.E1A+Ad-hTERTp-IL24. G-L. TUNEL staining revealed apoptosis in the tumors of different treatment groups. Cy3-labeled TUNEL positive cells on the sections were detected by confocal microscopy. (G) PBS; (H) 5-Fu; (I) MSC.LentiR+Ad-hTERTp-IL24+5-Fu; (J) MSC.LentiR.E1A+Ad-Track+5-Fu; (K) MSC.LentiR.E1A+Ad-hTERTp-IL24; (L) MSC.LentiR.E1A+Ad-hTERTp-IL24+5-Fu.

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