Fig 2: H&E staining images and Immunohistochemistry. (A) Representative images of H&E staining of tumor tissues from different groups, with the yellow triangles pointing to areas representing tumor-infiltrating lymphocytes; (B) Comparison of the number of CD4+ cells from immunohistochemistry; (C) Representative images of immunohistochemical staining of tumor tissues from different groups; (D) Comparison of the number of CD8+ cells from immunohistochemistry; *P < 0.05, **P < 0.01, ****P < 0.0001 indicate levels of statistical significance.

Fig 3: Representative flow cytometry staining analysis charts. Comparison of cellular populations of CD3+/CD4+, CD3+/CD8+, and CD4+/Foxp3+ T lymphocytes in glioma animal tumor tissues: control group, FUS only group, PD-1 inhibitor only group, and the combined PD-1 inhibitor + FUS group.

Fig 4: Flow analysis compared the expression of immune cells in tumor tissue, peripheral blood and spleen tissue. Changes of CD3+/CD4+, CD3+/CD8+, CD4+/Foxp3+ T lymphocyte populations in tumor tissues, peripheral blood and spleen tissues in different groups (control group, FUS group, PD-1 inhibitor group and FUS/PD-1 combination group). In tumor tissues, compared with the control group, the number of Th cells in the FUS treatment group and the combination treatment group was relatively increased, the number of CTL cells in the combination treatment group was increased, and the number of Tregs cells in the PD-inhibitor group was higher than that in other treatment groups. In peripheral blood, the number of Th and CTL cells in FUS group was lower than that in other groups. In spleen tissue, Th, CTL and Treg cells in FUS group were lower than those in other groups. *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 5: T-cell infiltration into the dorsal root ganglion contribute to bone cancer pain and is reduced by PP101.Effect of the CCR2-targeting pepducin, PP101 on (A) CD11b+-infiltrating macrophages and (B) CD3+ T-cells at POD 14 (n = 3 per group, 8–10 slices of L1–3 DRG analyzed per animal). (C) Schematic representation of the dynamic of Cx43+ satellite glial cells (SGC), CD11b+, Iba1+ and CD3+ populations in DRG tissue of sham and cancer tumor-bearing rats receiving SCR101 or tumor-bearing rats treated with PP101. Percent of (D) CD4+ and (E) CD8+ infiltrating CD3+ T cells analyzed by flow cytometry. Percent of (F) CD68+ and (G) CD163+ infiltrating F4/80+ monocytes analyzed by flow cytometry (cancer+SCR101 n = 13, cancer+PP101 n = 8). (H) Repeated daily administrations of anti-CD4, anti-CD8, anti-TCRαβ or anti-IgG control antibody (2 μg/rat, i.t.) between days 9 and 14 on mechanical allodynia on (I, J) mechanical allodynia and (K) paw retroflexion score in tumor-bearing animals. (L) Schematic representation of the dynamic weight bearing device and (M) representative weight acquisition for anti-TCRαβ- and anti-IgG antibody-injected tumor-bearing rats. (N-Q) Dynamic weight-bearing measurements performed at POD 13 and 14, (R) tumor score, and histological analysis of (S) CD3+ T-cells and (T) pERK+ neurons in anti-TCRαβ and anti-IgG antibody-injected tumor-implanted animals at POD 14 (cancer+anti-IgG n = 4, cancer+anti-TCRαβ n = 5). Scale bar corresponds to 20 μm in (A-B, and T) and 150 μm in (S). Data represented as mean ± SEM. Kruskal-Wallis followed by Dunn’s multiple comparisons test in (A and B), Mann-Whitney test in (D-G, N-Q, and S-T), Two-Way ANOVA followed by Sidak’s multiple comparison test in (I-K and R). *P < 0.05, **P < 0.01, ***P < 0.001. # compared with sham+SCR101 and * compared with cancer+SCR101 or cancer+IgG1.