Article Figures & Data
Figures
- Figure 1.
VCN-01 exerts a potent oncolytic effect in pediatric osteosarcoma cell lines, 531MII and in 143B. A, illustrative scheme of genetic modifications in VCN-01 genome. B, cell viability analyses of osteosarcoma cell lines after viral treatment. 143B and 531MII cell lines were infected with VCN-01 at the indicated MOIs. Cell viability was assessed using MTT assays 5 days after infection. Data are shown as the percentage (mean ± SD) of cells alive after infection with VCN-01. C, viral protein expression in osteosarcoma cell lines infected with VCN-01 assessed by Western blot analysis. D, quantification of VCN-01 replication in the indicated cell lines. Viral counts were determined three days after VCN-01 infection at 1MOI by culture infection antihexon staining–based method in HEK293 cells and expressed as pfu per millilitre. Data are shown as the mean ± SD of three independent experiments. E, correlation of viral PH20 and fiber mRNA expression measured by qRT-PCR in osteosarcoma cell lines previously infected with VCN-01. mRNA levels are presented as 2−ΔΔCt standardized with their constitutive gene and compared with an uninfected control.
- Figure 2.
VCN-01 local or systemic administration showed a safe toxicity profile in osteosarcoma models in vivo. A and B, body weight plotting of animals treated locally (A) or systemically (B) with VCN-01 at the indicated pfu. Mice from the different groups were weighed every week during treatment. Data are shown as the median ± SD within each group at each time point. C, histologic analysis of mice livers bearing an orthotopic osteosarcoma and treated locally with VCN-01 at 108 pfu. H&E (top, magnification ×400) and E1A IHC (bottom, magnification ×400). D, Histologic analysis of mice livers bearing a lung metastatic osteosarcoma and treated systemically with VCN-01 at 108 pfu. Representative micrographs of H&E and E1A IHC of mice livers from indicated groups of the metastatic osteosarcoma model. Images (×400 magnification) showed no viral presence in mice livers, and no signs of hepatotoxicity were found.
- Figure 3.
VCN-01 antisarcoma effect in the orthotopic osteosarcoma model with the 531MII cell line. Tumors were developed by orthotopic injection of 500,000 531MII cells in the tibial tuberosity of female nude mice and were sacrificed 90 days later. A, analyses of tumor burden development. Tumor volume in the mice tibias was measured at different days until the end of experiment. B, PET images of tumor burden. Representative images of PET analyses of control mice or treated with VCN-01 (107 or 108 pfu). C, quantification of tumor burden by PET with the radiotracer 18F-FDG. SUVmax was calculated using the formula SUV = [tissue activity concentration (Bq/cm3)/injected dose (Bq)] × body weight. Represented are the mean ± SD, SUV values of the tumors of all animals in the same group (Wilcoxon test). D, representative macroscopic images of mice tibias from all treated groups. E, tumor volume evaluation. Quantification of tumor volume was calculated by the formula: volume (mm3) = [(long diameter × perpendicular diameter 2)/2]. Represented are the mean ± SD of each tumor in the same group (Student t test). F, H&E staining of tibias from mice of indicated therapy groups. Representative photomicrographs of a control and two animals that received the different dose protocols of VCN-01 (magnification ×100). G, quantification of fiber and PH20 mRNA expression of cDNA from orthotopic osteosarcoma tumors by qRT-PCR and its correlation graph. mRNA levels are presented as 2−ΔΔCt standardized with their constitutive gene and compared with an uninfected control. Data are given as the mean ± SD.
- Figure 4.
VCN-01 administered systemically reduced osteosarcoma lung metastasis in vivo. Lung metastases were induced by endovenous injection of 2 × 106 531-MII cells in the tail of female nude mice. Animals were randomized as positive control (no treatment), VCN-01 group 107 pfu (107 pfu/week × 4 weeks) and VCN-01 group 108 pfu (108 pfu/week × 4 weeks). At day 60 animals were sacrificed. A, Quantification of lung tumor burden (531-MII) in mice at the end of the experiment. Bar representation of tumor area relative to total lung area. The values represent mean percentage tumor area of animals from each group, as obtained from a specifically designed program that measured both tumor area and total lung area from which was calculated the percentage of tumor area. B, Representative H&E photomicrographs at 200× of lung lesions from mice of the three groups described. C, Quantification of number of lung tumor nodules. Bar representation of tumor number represent different quantifications of different observers. D, 200× vimentin (V9antigen) immunochemistry images showing metastatic implants in lung of mice from untreated and VCN-01 treated groups. E, Quantification of fiber and PH20 mRNA expression of cDNA from osteosarcoma tumors by qRT-PCR and its correlation graph. mRNA levels are presented as 2−ΔΔCt standardized with their constitutive gene and compared to an uninfected control.
Additional Files
Supplementary Data
- Supplemental Figure 1 - Supplemental Figure 1
- Supplemental Figure 2 - Supplemental Figure 2
- Supplemental Figure 3 - Supplemental Figure 3
- Supplemental Figure 4 - Supplemental Figure 4
Volume 22, Issue 9
