Oncolytic Vaccinia Virus Therapy of Salivary Gland Carcinoma

Figure 1. Effective early infection by GLV-1h68 of human salivary carcinoma cell lines. Five cell lines were infected by GLV-1h68 at multiplicity of infection 1.0 and visualized under a green fluorescent protein (GFP) filter. All 5 cell lines showed progressively increasing GFP marker gene expression within 24 hours (hematoxylin-eosin, original magnification ×10).

Figure 2. In vitro quantification of gene expression and viral replication by GLV-1h68 in human salivary carcinoma cell lines. A, Cell lines were infected by GLV-1h68 at multiplicity of infection (MOI) 1.0. β-Galactosidase (β-gal) assay was performed at 12 hours. B, Cell lines were infected by GLV-1h68 at MOI 1.0. Luciferase activity was recorded at 24 hours. C, Cell lines were infected by GLV-1h68 at MOI 0.1 and incubated for 72 hours. Viral titers were quantified by plaque assays. Limit lines represent standard error. PFU indicates plaque-forming units.

Figure 3. GLV-1h68 exhibited significant oncolytic effects in human salivary carcinomas in vitro. Five cell lines were infected by GLV-1h68 at varying multiplicity of infection (MOI) (0.01, 0.1, 1.0, 5.0), and lactate dehydrogenase assay was performed for 7 days to assess cell viability. All 5 cell lines were susceptible to significant cytotoxic effects at MOI 5.0. Three cell lines demonstrated exquisite sensitivity to GLV-1h68, even at MOI 0.01. Limit lines represent standard error.

Figure 4. Therapy of human salivary carcinoma flank tumor in vivo. A, Established undifferentiated mucoepidermoid carcinoma flank tumors were injected with GLV-1h68 (5 × 10⁶ plaque-forming units [PFU]), and luciferase activity was measured following retro-orbital coelenterazine injections during a 16-day period. GLV-1h68 bioluminescence imaging in vivo showed selective prolonged virus-mediated luciferase activity in flank tumors. B, Quantification of luciferase activity during a 16-day period using software assessment of emitted photons was performed (n = 3). The late reduction of luciferase activity correlated with tumor size regression. C, A single injection of GLV-1h68 resulted in complete regression of established undifferentiated mucoepidermoid flank tumor in vivo. D, Established flank tumors (n = 5 per group) were treated with a single intratumoral injection of GLV-1h68 (5 × 10⁶ PFU). Measured tumor volumes for the GLV-1h68 group demonstrated nearly complete regression. In contrast, measured tumor volumes for the control phosphate-buffered saline–treated group demonstrated significant tumor progression. E, There was no observed change in animal weight related to viral administration. B and D, Limit lines represent standard error.

Figure 5. Therapy of human salivary carcinoma orthotopic tumor in vivo. A, Established orthotopic parotid undifferentiated mucoepidermoid tumors were injected with GLV-1h68 (5 × 10⁶ plaque-forming units [PFU]), and luciferase activity was measured following retro-orbital coelenterazine injections during a 20-day period. GLV-1h68 bioluminescence imaging in vivo showed selective prolonged virus-mediated luciferase activity in orthotopic parotid tumors. B, Quantification of luciferase activity during a 20-day period using software assessment of emitted photons was performed (n = 3). The late reduction of luciferase activity correlated with tumor size regression. C, A single injection of GLV-1h68 resulted in significant regression of established undifferentiated mucoepidermoid orthotopic parotid tumor in vivo. D, Established orthotopic parotid tumors (n = 5 per group) were treated with a single intratumoral injection of GLV-1h68 (5 × 10⁶ PFU). Measured tumor volumes for the GLV-1h68 group demonstrated significant tumor regression. In contrast, measured tumor volumes for the control phosphate-buffered saline–treated group demonstrated extensive tumor progression. E, No change was observed in animal weight related to viral administration. B and D, Limit lines represent standard error.

Figure 6. In contrast to subcutaneous flank xenografts, orthotopic parotid tumors demonstrated more aggressive behavior, with extracapsular extension as well as bone and muscle invasion. A, Stain of subcutaneous flank undifferentiated mucoepidermoid carcinoma demonstrates well-encapsulated tumor. B, Stain of orthotopic parotid undifferentiated carcinoma demonstrates tumor growth within the parotid gland. C, Stain of orthotopic parotid undifferentiated carcinoma demonstrates extensive bone invasion. D, Stain of orthotopic parotid undifferentiated carcinoma demonstrates extensive muscle invasion (hematoxylin-eosin, original magnification ×10).