Recombinant oncolytic poliovirus, PVSRIPO, has potent cytotoxic and innate inflammatory effects, mediating therapy in human breast and prostate cancer xenograft models

Figures

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  Figure 1. PVSRIPO effectively lyses SUM149 breast cancer cells and DU145 prostate cancer cells SUM149 (A) and DU145 (B) cells were seeded at a density of 5 × 104 cells per well. Cells were exposed to various concentrations of PVSRIPO (MOI of 0, 0.1, 1 and 10 MOIs) for 0, 24, 48 and 72 hours. Cell lysis was assessed using crystal violet staining. (C) PVSRIPO propagation following infection (MOI of 10) of DU145 and SUM149 cells was assessed by plaque assay at the designated time points. Data are representative of at least 3 independent experiments; note that titers in (C) are from an assay distinct from (A) and (B).

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  Figure 2. Single intratumoral administration of PVSRIPO results in tumor regression in SUM149 and DU145 xenografts SUM149 and DU145 cells were implanted in athymic nu/nu mice. Tumors were injected with 108 pfu of PVSRIPO when they reached 150-200 mm3. Tumors were collected at 7 days post injection and size and weight were assessed. (A) SUM149 tumor data are representative of five different experiments and a total of 25 mice per group. Graph represents tumor weights (grams) from one representative experiment. (B) DU145 tumor data are representative of five different experiments and a total of 25 mice per group. Graph represents tumor weights (grams) from one representative experiment. ***p<0.001.

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  Figure 3. PVSRIPO upregulates transcripts encoding for innate immune-related gene products in SUM149 and DU145 xenografts SUM149 (A) and DU145 (B) cells were implanted in athymic nu/nu mice. Tumors were injected with 108 pfu of PVSRIPO when they reached 150-200 mm3. Tumors were collected at 24 hours post injection and innate immune-related transcript abundance was assessed. Data are representative of two different experiments and a total of 6 mice per group. Only changes of ≥8-fold are reported. (C) Cytokines/chemokines represented in (A, B) and their function in inflammation; cytokines shown in bold were induced in both tumors. Information about cytokine significance was obtained from Uniprot [43] and NCBI databases.

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  Figure 4. PVSRIPO recruits innate immune cells into SUM149 and DU145 xenografts (A) SUM149 and DU145 cells were implanted in athymic nu/nu mice. Tumors were injected with 108 pfu of PVSRIPO when they reached 150-200 mm3. Tumors were collected at 48 hours post injection and innate immune cell infiltration was assessed by flow cytometry (CD45.2, CD11b, F480, Ly6C, Ly6G, B220, CD335). The numbers reflect percentage of cells in the gated population (boxed area) in the preceding flow quadrant as indicated. Data are representative of three different experiments and a total of 15 mice per group. (B) Tumor infiltrating immune cell percentages from one representative experiment (n=5) in the SUM149 breast tumor model are shown. ***p<0.001. (C) Tumor infiltrating immune cell percentages from one representative experiment (n=5) in the DU145 prostate tumor model are shown. *p<0.05, **p<0.01, ***p<0.001.

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  Figure 5. Innate immune cell infiltration after PVSRIPO is associated with signs of cell-mediated tumor cytotoxicity SUM149 and DU145 cells were implanted in athymic nu/nu mice. Tumors were injected with 108 pfu of PVSRIPO when they reached 150-200 mm3. Tumors were collected at 7 days post injection and immune cell recruitment was assessed by histology (H&E) and immunohistochemistry (CD11b). Data are representative of two different experiments and a total of 10 mice per group. SUM149 (A) and DU145 (B) tumors demonstrate significantly increased infiltration of CD11b+ immune cells. (C, D) SUM149 tumor homogenates from mock (PBS) or PVSRIPO-treated mice were tested (C, top) for markers of neutrophil and innate immune cell inflammation by immunoblot; (C, bottom) for the presence of H2O2; (D) for the presence of TNF-α and IFN-β by ELISA.

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  Figure 6. Single intratumoral administration of PVSRIPO in SUM149 and DU145 xenografts results in tumor regression and survival benefit SUM149 and DU145 cells were implanted in athymic nu/nu mice. Tumors were injected with 108 pfu of PVSRIPO when they reached 150-200 mm3. Tumor growth was monitored daily and mice were sacrificed when tumors reached 2000 mm3. Data are representative of 3 different experiments (n=30). SUM149 (A, B) and DU145 (C, D) tumor growth 7 days post PVRIPO injection and overall survival up to 100 days.

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  Figure 7. PVSRIPO persistence in SUM149 and DU145 xenografts following single intratumoral injection SUM149 (A) and DU145 (B) cells were implanted in athymic nu/nu mice. Tumors were injected with 108 pfu of PVSRIPO when they reached 150-200 mm3. Tumors were isolated at 1, 2, 3, 4 and 7 days post virus inoculation. Pfu per mg of tumor were determined by plaque assay and plotted; data are representative of two independent experiments (n=30).