Figure S1. Relative replication of WR and Lister strain viruses in human cancer cells compared to
normal human bronchial epithelial cells (NHBE). Viral titres were compared at 96 hours postinfection. Mean viral replication ± SEM was determined by TCID50 assay on CV1 cells. A ratio greater
than 1 indicates superior viral replication in the respective cancer cells compared to non-malignant
cells (NHBE). Statistical analysis was performed using a two-way ANOVA test; * p<0.05, ** p<0.01,
*** p<0.001, ns indicating no statistical significance.
Figure S2. Vaccinia virus titres in harvested tumours from ICRF nu/nu mice bearing CMT93 flank
tumours following IT vaccinia virus treatment. CMT93 cells were seeded by subcutaneous injection
into the right flank of 36 ICRF nu/nu mice. When tumours reached 4-5mm in diameter, 12 mice each
received three IT injections of PBS, WRDD or VVL15 (3 x 1x10 7 pfu). On days 5, 10, 15 and 20 three
mice from each group were sacrificed and tumours were harvested and frozen. Samples were then
homogenised and repeatedly freeze-thawed. Lysates underwent DNA extraction. Mean viral
replication ± SEM were determined by TCID50 assay on CV1 cells. Statistical analysis of viral titres at
stated time-points was performed using a two-way ANOVA test; * p<0.05, ** p<0.01, *** p<0.001,
ns indicating no statistical significance.
Figure S3. Vaccinia virus titres in harvested tumours from ICRF nu/nu mice bearing PT45 flank
tumours following IT vaccinia virus treatment. PT45 cells were seeded by subcutaneous injection
into the right flank of 30 ICRF nu/nu mice. When tumours reached 4-5mm in diameter, 12 mice each
received three IT injections of PBS, WRDD or VVL15 (3 x 1x10 7 pfu). On days 7, 12 and 20 three mice
from each group were sacrificed and tumours were harvested and frozen. Samples were then
homogenised and repeatedly freeze-thawed. Lysates underwent DNA extraction. Mean viral
replication ± SEM were determined by TCID50 assay on CV1 cells. Statistical analysis of viral titres at
stated time-points was performed using a two-way ANOVA test; * p<0.05, ** p<0.01, *** p<0.001,
ns indicating no statistical significance.
Figure S4. Ovarian biodistribution of vaccinia virus mutants in ICRF nu/nu mice bearing flank
CMT93 tumours, as determined by immunohistochemistry. CMT93 cells were seeded by
subcutaneous injection into the right flank of 60 ICRF nude mice. When tumours reached 4-5mm in
diameter, 12 mice each received single IV injections of PBS, WRDD or VVL15 (1 x 1x10 7 PFU). On days
2, 6, 8 and 12 four mice from each group were sacrificed and ovaries were harvested and frozen. IHC
for vaccinia coat protein was performed and representative images at day 12 are shown at 200x
magnification; images were taken with a Zeiss Axioplan microscope fitted with a Zeiss AxioCam MRc
camera.