Figure 7. Anti-tumor effect of the combination treatment of MART1 plasmid with Ad, Ad^M, Ad^GshT, and Ad^MGshT.

A. Diagram of the experimental design. C57BL/6 mice were injected with 7 × 10⁵ B16BL6-CAR/E1B55 cells in 100 μL on day -5 and injected intramuscularly with 50 μg/50 μL of MART1 plasmid into the rear quadriceps on day -2. C57BL/6 tumor-bearing mice were treated with intratumoral injections of 1 × 10⁹ PFU/50 μL of adenovirus on day 1, 3, and 5. B. Tumor volume was monitored and recorded every 2 days until the end of the study. Values represent the mean ± SE (5 animals per group) (Top, Left). Overall survival was determined throughout a 15-day time course (Top, Right). Photographs of C57BL/6 tumor-bearing mice treated with virus were obtained at day 11 after virus infection (Bottom). C. Representative immunohistochemical analysis of recombinant adenovirus-infected tumor sections was done as follows. 3 animals per group of C57BL/6 mice were injected with 7 × 10⁵ B16BL6-CAR/E1B55 cells/100 μL on day -5 and then treated with intramuscular injections of 50 μg/50 μL of MART1 plasmid into the rear quadriceps on day -2. C57BL/6 tumor-bearing mice were treated with intratumoral injections of 1 × 10⁹ PFU/50 μL of various kinds of adenovirus on day 1, 3, and 5. Tumors were collected at day 11 for histological analysis. Paraffin sections of tumor tissue were stained with anti-adenovirus type 5, anti-CD8, anti-CD4, anti-NK1.1, anti-CD11b+c (Top) Results are given as the relative intensity of adenovirus type 5, CD4, CD8, NK/NKT and DC cells to PBS of each 3 mouse. Horizontal black bars indicate mean values (Bottom). D. Flow cytometric analysis of various types of tumorigenic or splenic immune cells after combination treatment of MART1 plasmid with Ad or Ad^MGshT. 3 animals per group of mice bearing B16BL6-CAR/E1B55 cells were sacrificed and tumorigenic or splenic immune cells were isolated after combination treatment of MART1 plasmid with Ad or Ad^MGshT as indicated in detail in (A). To set the gate, T-cell gating of positive CD3 using Jurkat cell was performed. Each cell was then costained with anti-CD3 and anti-CD8 (or anti-CD4) for the detection of T cells. Otherwise, each cell was then costained with anti-NK1.1 and anti-CD122 after gating with anti-CD3 and anti-NK1.1 for the detection of NKT cells or for the detection of regulatory T cells, each cell was then costained with anti-CD25 and anti-FOXP3 after gating with anti-CD4 and anti-CD25. Numbers in inset are percentage of cells in a given quadrant. All flow cytometric results are representative of three experiments (Left, Middle). Right results are the average percentage of corresponding immune cells in each 3 mouse. Horizontal black bars indicate mean values. E. Immunohistochemical analysis was performed as indicated in (C) by staining with anti-IFN-γ antibody (Left). Results are given as the relative intensity of IFN-γ to PBS of each 3 mouse. Horizontal black bars indicate mean values (Right). F. IHC using Ad5 antibody was performed to examine the residual adenovirus at the indicated day after infection of oncolytic control adenovirus was administered intratumorally (1 × 10⁹ PFU per tumor in 50 μl of PBS) on days 1, 3, and 5. Representative confocal immunofluorescence staining of tumor sections was done as described in Materials and methods to confirm the increased mature TIDCs (G., green color) or decreased regulatory T cells (H., orange color) after combination treatment of MART1 plasmid with Ad^MGshT (Left). TIDC or regulatory T cells were counted on images taken from multiple fields per mouse (n = 3/group). Results are given as the average percentage of CD11b^loCD11c^hi+ cells or CD4⁺CD25⁺ cells in each 3 mouse. Horizontal black bars indicate mean values (Right).