Imaging of STSA-1 tumors (A, C) and canine soft tissue sarcoma cells (B, D).

<p>(<b>A</b>) Histologic section of a canine soft tissue sarcoma from the limb of a golden retriever dog, hematoxylin and eosin stain (H&E, ×200 magnification). (<b>B</b>) Cytology of canine soft tissue sarcoma cells STSA-1 isolated from a subcutaneous mass on a golden retriever dog, Wright-Giemsa stain (×1000 magnification). (<b>C</b>) Canine soft tissue sarcoma STSA-1 xenograft, right flank, athymic nude mouse (H&E, ×200 magnification). (<b>D</b>) Transmitted light microscopy of uninfected STSA-1 cells in MEM-C culture (×100 magnification).</p

Staining characteristics of cells isolated from a spontaneous tumor growth on a golden retriever dog.

<p>Staining characteristics of cells isolated from a spontaneous tumor growth on a golden retriever dog.</p

Comparison of the replication capacity of the vaccinia virus strains GLV-1h68 and LIVP1.1.1 in canine soft tissue sarcoma cells.

<p>For the viral replication assay, STSA-1 cells grown in 24-well plates were infected with either GLV-1h68 or LIVP1.1.1 at an MOI of 0.1. Cells and supernatants were collected for the determination of virus titer at various time points. Viral titers were determined as pfu per well in triplicates by standard plaque assay in CV-1 cell monolayers. Averages plus standard deviation are plotted. The data represent two independent experiments. The statistical significance was analyzed using two-way ANOVA followed Bonferroni post-test on log transformed PFU data. *, **, and *** indicate P<0.05, 0.01, and 0.001, respectively.</p

FACS characterization and comparison of tumor single cell suspensions derived from infected and uninfected STSA-1 tumors at 7 dpvi (n = 3)

A<p>Ratios greater than 1 indicate an increased accumulation of host immune cells in virus-infected tumors.</p><p>Abbreviations: macs: macrophages; DC: dendritic cells; NK: natural killer cells; NKT: natural killer T cells; n.a.: not applicable.</p

Determination of vascular density using CD31 immunohistochemistry in virus- treated and non-treated tumors at 7 dpvi.

<p>(<b>A</b>) Blood vessel density in STSA-1 tumors The vascular density was measured in CD31-labeled tumor cross-sections (n = 3 mice per group) and presented as mean values +/− standard deviations. The study was repeated in an independent experiment. There were no significant differences between groups (P>0.05, Student's t-test). (<b>B</b>) <b>Fluorescence intensity of the CD31 signal of blood vessels</b>. The fluorescence intensity of the CD31-labelling represented the average brightness of all vessel-related pixels and determined as described by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037239#pone.0037239-Weibel2" target="_blank">[32]</a>. The fluorescence signal was measured in 18 images of each tumor (n=3 mice per group). Shown are the mean values / standard deviations. The study was repeated in an independent experiment. ( P0.001, P0.01, Student's t-test)</p

Growth of canine soft tissue sarcoma tumors in virus-and mock-treated mice.

<p>(<b>A</b>) Groups of STSA-1 tumor-bearing nude mice (n = 6) were either treated with a single dose of 1×10⁷ pfu GLV-1h68, LIVP1.1.1 or with PBS (mock control). Tumor size was measured twice a week. Two-way analysis of variance (ANOVA) with Bonferroni post-test was used for comparison of two corresponding data points between groups. *, **, and *** indicate P<0.05, 0.01, and 0.001, respectively. (<b>B</b>) Animals with established STSA-1 flank tumors (>600 mm³) were distributed into two experimental groups (n = 4 per group). Flank tumors were treated with injections of a single dose of LIVP1.1.1 (1×10⁷ pfu) or PBS alone as control. The statistical significance was confirmed by Student's t-test (***p<0.001).</p

Survival curves of LIVP1.1.1-treated and non-treated STSA-1 tumor-bearing mice.

<p>The comparison of the survival between the different treatment groups (n = 5) was statistically evaluated by Kaplan-Meier and log-rank (Mantel-Cox) tests (GraphPad Prism, San Diego, CA). P<0.05 was considered statistically significant. ** P = 0.0039.</p