Fig 2: Agpat4 silencing in CRC suppresses T cell-dependent tumor progression. a Eight-week-old male NCG mice were intraperitoneally inoculated with sh-Agpat4- or sh-NC-transfected MC-38 cells (4 × 106 cells in 100 μl of PBS per mouse). Twelve days later, the mice were sacrificed, and the tumor nodules were collected and weighed. (n = 6). b Agpat4-silenced MC-38 cells increased the percentage of CD8+ T cells in epididymal fats. Six-week-old mice were intraperitoneally engrafted with sh-Agpat4- or sh-NC-transfected MC-38 cells (4 × 106 cells in 100 μl of PBS per mouse) on day 0 and were sacrificed for the analysis of total T cells, CD4+ T cells and CD8+ T cells in the stromal vascular fraction (SVF) of epididymal fats on days 0, 3, and 5. T cells were marked by the phenotype CD45+CD3+CD11b−. CD4+ T cells were defined by the phenotype CD45+CD3+CD11b−CD8−CD4+. CD8+ T cells were defined by the phenotype CD45+CD3+CD11b−CD4−CD8+. Representative results are shown. c Percentage of IFN-γ+ cells in the CD8+ T cells described above in b. (n = 5). d Six-week-old male C57BL/6 mice were intraperitoneally inoculated with MC-38 cells (4 × 106 cells in 100 μl of PBS per mouse) on day 0 and then treated with anti-CD8 antibody or IgG as a control 3 times (days 2, 4, and 6) at a dose of 100 μg/day. Mice were sacrificed for tumor observation on day 14. (n = 5). The data in a–d represent the means ± s.e.ms. (***P < 0.005; Student’s t test)

Fig 3: CD8+ T cells depletion experiments and ex vivo analysis of immune cells.a Primary (*p = 0.0345, ***p = 0.0001) and b distant (***p = 0.0001) tumor growth curves of CT26 colorectal bilateral tumor-bearing mice treated with Saline, Saline+RT, H@Gd-NCPs+RT and H@Gd-NCPs+RT + αCD8a, (n = 8 biologically independent animals). [Gd3+] = 30 mg kg−1, [Hemin] =12.5 mg kg−1 and [αCD8a] = 10 mg kg−1. Treatments were performed on days 0 and 6. X-ray radiation therapy was performed 6 h after nanomedicines intravenous injection (black arrow). RT 6 Gy × 2 with fractions delivered 6 days apart and only primary tumors received radiation therapy. Anti-CD8a antibody was treated via intraperitoneal injection 6 h after radiation therapy (red arrow). Data (a, b) were presented as mean ± SEM. c Primary (***p = 0.0001, ***p = 0.0001) and d distant (***p = 0.0001, ***p = 0.0001) CT26 tumor weight (n = 8 biologically independent animals). e, f Growth curves of e primary and f distant individual tumors in the H@Gd-NCPs + RT and H@Gd-NCPs + RT + αCD8a groups. g, h The percentages of CD8+ T cells in the g primary (***p = 0.0001, ***p = 0.0001) and h distant (***p = 0.0002, ***p = 0.0003) tumors analyzed by flow cytometry (n = 6 biologically independent animals). i The percentages of macrophages (F4/80+ and CD11b+) in the primary tumors analyzed by flow cytometry (n = 6 biologically independent animals). Tumor tissues (g–i) in different groups were harvested on day 18 for flow cytometry analysis. Data (c, d, g–i) were presented as mean ± SD. Two-sided Student’s t-test was used to calculate the statistical difference between two groups. N.S. represented non-significance, and *p < 0.05, ***p < 0.001. Source data are provided as a Source data file.

Fig 4: Colec11–/– mice exhibit less immunosuppressive TME.Tumors excised from Colec11+/+ (WT) or Colec11–/– (KO) mice (d14) were used for analyzing TME. (A–C) Tumor infiltrates analyzed by flow cytometry. (A) CD45+ cells. (B) Subsets of tumor-infiltrating leukocytes analyzed by flow cytometry. Data were analyzed by unpaired t test (n = 18 mice per group, pooled from 4 experiments). Each dot represents an individual mouse. (C) A bar chat representing proportion of subsets in CD45+ cells shown in B. (D) Representative microscopy images of immunochemical staining for CD11b (green)/CD3 (red)/DAPI (blue) and F4/80 (green)/CD3 (red)/DAPI (blue). Scale bar: 50 μm. (E) Representative microscopy images of immunochemical staining for CD8 (red)/DAPI (blue) in tumor edge and core areas. Scale bar: 50 μm. (F). qPCR analysis in tumor tissues. Data were analyzed by unpaired t test (n = 8 mice per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 5: Systemic antitumor immunity induced by aAGd-NW-sensitized radiation.a Schematic illustration of aAGd-NWs sensitized radiation-mediated in situ vaccine. Created with BioRender.com. b–d Tumor growth curves (b), isolated tumor photographs (c), and tumor weights (d) of BALB/c mice immunized with RT and aAGd-NWs+RT treated tumor cells, respectively (n = 8 mice). e Flow cytometry analysis of CD11c+ and H2Kb-SIINFEKL+ DCs in TDLNs after Vehicle, GGd-NCPs, aAGd-NWs, Vehicle+RT, GGd-NCPs+RT, aAGd-NWs+RT treatments (n = 3 mice). f Flow cytometry analysis of DCs maturation (CD80+ and CD86+ gated within CD11c+) in TDLNs (n = 8 mice). g Tumor growth curves of CT26 colorectal tumor-bearing mice (n = 8 mice). Black and red arrows indicated drug/radiation and anti-CD8a/anti-PD-L1 treatments, respectively. RT (5 Gy × 2) was performed on day 0 and 6, respectively. Anti-CD8a/anti-PD-L1 antibodies (10.0 mg kg−1 × 4 with fractions delivered 3 days apart) were administered via intraperitoneal injection 6 h after X-ray irradiation. h, i Percentages of CD4+ (h) and CD8+ (i) T cells infiltrating within tumor tissues as detected by flow cytometry (n = 4 mice for the combinational group and n = 6 mice for other groups). j Relative multiples of IFN-γ secretion in the tumor tissues as detected by ELISA kit (n = 4 mice for the combinational group and n = 6 mice for other groups). All data were shown as mean ± SD. Two-tailed Student’s t-test was used to calculate statistical differences between two groups, and one-way ANOVA analysis of variance was used for multiple groups. p values > 0.05 represented nonsignificance (N.S.) and p values < 0.05 represented statistically significant. Source data are provided as a Source Data file.