11R-P53 and GM-CSF Expressing Oncolytic Adenovirus Target Cancer Stem Cells with Enhanced Synergistic Activity

Abstract

Targeting cancer stem cells with oncolytic virus (OV) holds great potential for thorough elimination of cancer cells. Based on our previous studies, we here established 11R-P53 and mGM-CSF carrying oncolytic adenovirus (OAV) SG655-mGMP and investigated its therapeutic effect on hepatocellular carcinoma stem cells Hep3B-C and teratoma stem cells ECCG5. Firstly, the augmenting effect of 11R in our construct was tested and confirmed by examining the expression of EGFP with Fluorescence and FCM assays after transfecting Hep3B-C and ECCG5 cells with OVA SG7605-EGFP and SG7605-11R-EGFP. Secondly, the expressions of 11R-P53 and GM-CSF in Hep3B-C and ECCG5 cells after transfection with OAV SG655-mGMP were detected by Western blot and Elisa assays, respectively. Thirdly, the enhanced growth inhibitory and augmented apoptosis inducing effects of OAV SG655-mGMP on Hep3B-C and ECCG5 cells were tested with FCM assays by comparing with the control, wild type 5 adenovirus, 11R-P53 carrying OVA in vitro. Lastly, the in vivo therapeutic effect of OAV SG655-mGMP toward ECCG5 cell-formed xenografts was studied by measuring tumor volumes post different treatments with PBS, OAV SG655-11R-P53, OAV SG655-mGM-CSF and OAV SG655-mGMP. Treatment with OAV SG655-mGMP induced significant xenograft growth inhibition, inflammation factor AIF1 expression and immune cells infiltration. Therefore, our OAV SG655-mGMP provides a novel platform to arm OVs to target cancer stem cells.

Keywords: 11R.; GM-CSF; OAV; P53; cancer stem cells.

Figure 1

Construction of the OVA SG655-mGMP. A: Linearized depiction of plasmids for SG655-mGM-CSF, SG655-mGMPC and SG655-mGMP, the E1A gene and mGM-CSF expressing cassettes were placed under hTERT promoter and MCMV promoter respectively, the 11R-P53 expressing cassette was inserted in the E3 area in SG655-mGMP; B: The construct was confirmed by restriction enzyme digestions with BglII, EcoRV and HindIII, showing expected DNA fragments of 8465+1613bp, 7575+2114+389bp and 7476+2602bp during agarose gel electrophoresis; C: Agarose gel electrophoresis of PCR products showed expected bands of 1227bp and 426bp for 11R-P53 and mGM-CSF fragments; D: Elisa assay showed effective expression of mGM-CSF and 11R-P53 of SG655-mGMP and SG655-mGMPC clones (SG655-mGMP: 1226.01, 820.36, 1197.63, 1358.33 ng/mL for clone 1-4; SG655-GMPC: 1506.68, 2341.83, 2008.64 ng/mL for clone 1-3, respectively); E: Western blot assay showed effective expression of 11R-P53 and P53 in 293 cell clones after transfection with SG655-mGMP and SG655-mGMPC.

Figure 2

The augmenting effect of 11R within the construct. A: The expression of EGFP of Hep3B-C and ECCG5 cells post transfection was augmented by 11R via showing enhanced green fluorescence intensity; B: The improving effect of 11R on the expression of EGFP in Hep3B-C cells was further verified by FCM assay after transfection with OAV at different MOI through showing higher percent of EGFP positive cells (with V.S. without 11R: 13.8% V.S. 4.2%, 16.4% V.S. 9.8%, 44.6% V.S. 23.1%, MOI=1, 2, 5); C: Elisa assay was applied to detect the secretion of GM-CSF in ECCG5 cell culture supernatants post transfection with OAV SG655-mGMP and SG655-mGMPC at different MOI, and showed effective expression (MOI=20: 81.35 and 43.91 ng/mL, MOI=100: 91.23 and 53.59 ng/mL, MOI=200: 118.79 and 70.90 ng/mL for SG655-mGMP and SG655-mGMPC transfection respectively). D: The expression of 11-P53 was tested by Western blot through using anti-P53 antibody, and showed that 11R-P53 was efficiently expressed in both Hep3B-C and ECCG5 cells with slight heavier weight than P53.

Figure 3

The growth inhibition and apoptosis induction of SG655-mGMP toward cancer stem cells. A: CCK-8 assay was applied to test the effect of OAV SG655-mGMP on the growth of Hep3B-C and ECCG5 cells, and showed that uncoupled P53 expressing OAV SG655-mGMPC inhibited the growth of both Hep3B-C and ECCG5 cells slightly, when compared to the control virus, while the 11R-P53 expressing OAV SG655-mGMP exhibited more remarkable inhibitory effect on the growth of Hep3B-C and ECCG5 cells; B: FCM assay was then used to test the effect of OAV on Hep3B-C cells, and showed that wild type OAV and 11R-P53 expressing OAV SG7605-11R-p53 induced higher percent of apoptotic cells (6.0% and 7.7% respectively) than the control (0.2%), and OAV SG655-mGMP induced the highest percent of apoptotic cells from Hep3B-C cells (12.5%).

Figure 4

SG655-mGMP exerted synergetic effect toward cancer stem cells in vivo. A: The mouse models were established; B: SG655-11R-P53 and SG655-mGM-CSF treatments reduced the volume of treated xenografts in an undistinguishable pattern with the control PBS treatment, while the SG655-mGMP treatment exhibited most powerful inhibition on the growth of xenografts by reducing the volume to lowest level; C: The IHC assay showed that OAV SG655-11R-P53 and SG655-mGM-CSF treatments improved the expression of inflammation factor AIF1 slightly compared to the control PBS treatment, while the OAV SG655-mGMP treatment significantly improved the expression of AIF1. D: The enhancing effect on immune cells infiltration of OAV SG655-11R-P53, SG655-mGM-CSF and SG655-mGMP treatments were examined by FCM through comparing to PBS treatment, and showed that OAV SG655-11R-P53 and SG655-mGM-CSF enhanced the infiltration of immune cells with modest level, while the OVA SG655-mGMP exhibited most powerful function (NK cell: 0.6%, 2.5%, 1.8% and 3.2%; Macrophage: 0.9%, 6.0%, 5.7% and 9.5%; Granulocyte: 2.2%, 6.1%, 6.8% and 9.0%; DC: 1.1%, 5.5%, 5.4% and 6.1% for PBS, SG655-11R-P53, SG655-mGM-CSF and SG655-GMP, respectively).