Fig 2: Anti-tumor activity of the recombinant VV on colon cancer models. (A) Treatment scheme of MC38 tumor model. The subcutaneous tumor model was established by inoculation of 2 × 106 MC38 cells on the right flank of C57BL/6 mice. Mice were administered intratumorally at the indicated day for 3 times with PBS, 1 × 107 PFU of VV-Control or VV-a-TIGIT. (B) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 8 mice for all groups. (C) Kaplan-Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-a-TIGIT. (D) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for all groups. (E) Treatment scheme of CT26 tumor model. The subcutaneous tumor model was established by inoculation of 5 × 105 CT26 cells on the right flank of BALB/c mice. Mice treatment were similar to (A). (F) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 8 mice for all groups. (G) Kaplan–Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-a-TIGIT. (H) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for all groups. (I, J) Immunohistochemistry (IHC) detection of the infiltration of CD8+ T cells (I) and microvascular density (MVD) (J) of the tumor in the CT26 subcutaneous tumor model. The tumor model was established as previously described. When the tumor reached approximately 50 mm3, mice were treated i.t. with PBS, 1 × 107 PFU of VV-Control or VV-a-TIGIT. Seven days after VV injection, tumors were collected from mice, and CD8 and CD31 expression were detected by IHC. (K, L) The a-TIGIT levels in tumor and blood. Samples of the tumor lysates and sera were prepared as previously described in materials and methods and a luciferase assay was used to detect the levels of secreted a-TIGIT. (M) Viral titers in tumor and blood. Samples were collected similar to (K, L), and viral titers were quantified by a TCID50 method. Statistical differences in tumor volume, body weight, CD8+ T cells, MVD, luciferase activity, and viral titer among the groups were evaluated using ANOVA. Statistical differences in survival among the groups were evaluated using the Log-Rank test. ns, no significant differences; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: Treatment of mice with VV-a-TIGIT established long-term tumor-specific immunological memory. (A) Schematic diagram of tumor rechallenge. Ascites tumor model was established and treated as previously described. Mice cured of H22 by VV-a-TIGIT were twice intraperitoneally and once subcutaneously inoculated with the H22 cells, and once subcutaneously inoculated with MC38 cells at the indicated time. (B) Kaplan-Meier survival curves of tumor-naïve or cured mice twice intraperitoneally rechallenged with H22 cells (n=10). (C) Tumor volumes and Kaplan–Meier survival curves of tumor-naïve or cured mice subcutaneously rechallenged with H22 cells (n=10). (D) Tumor volumes and Kaplan–Meier survival curves of tumor-naïve or cured mice subcutaneously rechallenged with MC38 cells (n=10). Statistical differences in tumor volume were evaluated using ANOVA. Statistical differences in survival were evaluated using the Log-Rank test. ns, no significant differences; ****P < 0.0001.

Fig 4: VV-a-TIGIT enhanced immune-cell infiltration and activation in the tumor microenvironment. (A) The treatment scheme. The tumor model was established by intraperitoneally (I.P.) inoculation of 5×106 H22 cells in C57BL/6 mice. After ascites were formed, the tumor-bearing mice were treated with PBS, 1 × 107 PFU of VV-Control or VV-a-TIGIT at a 2-day interval 3 times via I.P. injection. (B) Dynamic detection of the proportion of the tumor cells and lymphocytes in the ascites by flow cytometry. (C, D) Flow cytometric analysis of the proportion of the tumor cells, lymphocytes, and their subsets in the ascites at day 4 after the first VV-a-TIGIT treatment. (E) Kaplan–Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, or VV-a-TIGIT (n=14). (F) The a-TIGIT levels in ascites. Ascites were harvested on day 4 after the first VV-a-TIGIT treatment and a luciferase assay was used to detect the levels of secreted a-TIGIT in ascites. (G) The concentration of murine IFN-? was detected by an ELISA assay in the ascites of mice on day 4 after the first VV-a-TIGIT treatment. Statistical differences in cell proportion, luciferase activity, and IFN-? concentration in the ascites were evaluated using ANOVA. Statistical differences in survival of mice among the groups were evaluated using the Log-Rank test. ns, no significant differences; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 5: The in vivo anti-tumor activity of VV-a-TIGIT depends on CD8+ T cells. (A) Schematic diagram of the experiment for CD8+ T or NK cell depletion. Ascites tumor model was established as previously described. Three days post tumor inoculation, mice were injected intraperitoneally (I.P.) with 500 ug of anti-mouse CD8a or anti-mouse NK1.1 mAB per mouse. (B) The depletion of CD8+ T and NK cells was confirmed by detecting the corresponding cells in ascites by flow cytometry 5 days post the mAB injection. (C) Kaplan–Meier survival curves of tumor-bearing mice treated with PBS, VV-a-TIGIT, VV-a-TIGIT plus anti-CD8a, or VV-a-TIGIT plus anti-NK1.1. (D) Flow cytometric analysis of the proportion of the tumor cells, lymphocytes, and their subsets in the ascites. (E) The level of a-TIGIT in ascites was detected by a luciferase assay on day 4 after the first VV-a-TIGIT treatment. (F) The concentration of murine IFN-? was detected by an ELISA assay in the ascites of mice on day 4 after the first VV-a-TIGIT treatment. Statistical differences in cell proportion, luciferase activity, and IFN-? concentration in the ascites were evaluated using ANOVA. Statistical differences in survival of mice among the groups were evaluated using the Log-Rank test. ns, no significant differences; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.