*3.4. Tumor-Infiltrating Immune Cells*

The number of CD8 (+) lymphocytes tended to be higher in the CS (+) group than in the CS (−) group, but the difference is not statistically significant (10.81 vs. 2.9, *p* = 0.052; Figure 3). The number of CD4 (+) lymphocytes showed no significant difference between the two groups (6.94 vs. 4.89, *p* = 0.28; Figure 3). The number of immunosuppressive CD163 (+) cells was significantly higher in the CS (+) group than in the CS (−) group (7.7 vs. 2.6, *p* = 0.046; Figure 4). Although the number of immunosuppressive Foxp3 (+) cells showed no significant difference between the two groups (0.5 vs. 0.4, *p* = 0.39; Figure 4), Foxp3/CD8 ratio was significantly higher in the CS (+) group than in the CS (−) group (25.87 vs. 7.25, *p* = 0.0059; Figure 4).

**Figure 3.** Analysis of tumor-infiltrating lymphocytes. Immunohistochemical analysis of CD8 and CD4 (Original magnification, ×20). Typical examples of each staining are shown in both groups. Statistical analysis of each staining is shown. The number of CD8 (+) lymphocytes tends to be higher in the CS (+) group than in the CS (−) group, but the difference is not statistically significant (*p* = 0.052). The number of CD4 (+) lymphocytes shows no significant difference between the two groups (*p* = 0.28). Data represent the mean ± standard error of mean (CD4 and CD8, student's *t*-test).

**Figure 4.** Analysis of immunosuppressive cells and immune checkpoint molecules. Immunohistochemical analysis of CD163, Foxp3, PD-1, and PD-L1 (Original magnification, ×20). Typical examples of each staining are shown in both groups. Black arrow: tumor cell showing positive Foxp3 expression. The number of CD163 (+) tumor-associated macrophages (TAMs) and Foxp3/CD8 ratio are significantly higher in the CS (+) group than in the CS (−) group (CD163: *p* = 0.046, Foxp3/CD8: *p* = 0.0059). The score of PD-L1 tends to be higher in the CS (+) group than in the CS (−) group (*p* = 0.050). Expressions of Foxp3 and PD-1 do not reach statistical significance (Foxp3: *p* = 0.39, PD-1: *p* = 0.39). Data represent the mean ± standard error of mean (CD163, Foxp3, PD-1 and Foxp3/CD8 ratio, student's *t*-test; PD-L1, Mann-Whitney *U* test).
