Figure 3.

CA4-NPs + DC101 reduced the tumor burden while simultaneously increasing the number of intratumoral CD8⁺ T cells. H22 cells were subcutaneously inoculated into Balb/c mice. When the tumor volume reached ~170 mm³, CA4-NPs (45 mg/kg, on a CA4 basis) were administered intravenously on day 0. DC101 (10 mg/kg) was administered intraperitoneally on days 2, 5, and 8. (A) Tumor IHC staining of Ki67 and H&E staining in the PBS and CA4-NP + DC101 groups on day 11. V, viable region of the tumors; N, necrotic region of the tumors. (B) Quantitative analysis of the Ki67⁺ area (Ki67⁺ area/field) for tumor IHC staining using Image J software (n = 3). (C) Quantitative analysis of the necrotic area (necrotic area/field) for tumor H&E staining using Image J software (n = 3). (D) Tumor burden (long axis of tumors) in the PBS and CA4-NP + DC101 groups on day 8 (n = 5). (E1) Immunofluorescent images of the tumors showing blood vessel pericyte coverage in the PBS and CA4-NP + DC101 groups on day 10. CD31⁺ endothelial cells, red; α-SMA⁺ pericytes, green; nuclei, blue. (E2) Immunofluorescent images of the tumors showing Hoechst 33342 tumor perfusion in the PBS and CA4-NP + DC101 groups on day 10. Hoechst 33342⁺ cells, blue; nuclei, green. (E3) Immunofluorescent images of tumors showing FITC-lectin tumor blood vessel perfusion in the PBS and CA4-NP + DC101 groups on day 10. CD31⁺ endothelial cells, red; FITC-lectin-perfused vessels, green; nuclei, blue. (E4) Immunofluorescent images of the tumors showing hypoxia in the PBS and CA4-NP + DC101 groups on day 10. Pimonidazole⁺ cells, red; nuclei, blue. (F1) Quantification of pericyte coverage determined by α-SMA⁺CD31⁺/CD31⁺ staining using ZEN software (Carl Zeiss, Germany) (n = 3). (F2) Quantification of tumor perfusion determined by Hoechst 33342⁺ area/field using Image J software (n = 3). (F3) Quantification of perfused functional tumor blood vessels determined by lectin⁺CD31⁺/CD31⁺ staining using ZEN software (Carl Zeiss, Germany) (n = 3). (F4) Quantification of tumor hypoxia determined by the pimonidazole⁺ area/tumor area using ZEN software (Carl Zeiss, Germany) (n = 3). (G) Representative flow cytometry plots for intratumoral CD4⁺ T (CD3⁺CD4⁺) cells and CD8⁺ T (CD3⁺CD8⁺) cells in the PBS and CA4-NP + DC101 groups on day 10. (H, I) Quantification of intratumoral CD4⁺ T cells (H, n = 3) and CD8⁺ T cells (I, n = 3) detected by flow cytometry. Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001).