fig5

Figure 5. High NID1 expression promotes mregDC and Treg intratumoral infiltration in vivo. (A) Flowchart for detecting the subcutaneous growth of 1753^p53-/-cmyc NID1 NC and NID1 KD cells in mice (upper). 5 × 10⁴ 1753^p53-/-cmyc NID1 NC/NID1 KD cells were injected into the right dorsal flank of mice (n = 5). Tumors were measured every two days. The mice were sacrificed, and tumors were harvested on day 21. Images of isolated subcutaneous tumors (lower); (B) Body weight (left) and tumor volume (right) changes in mice during tumor progression; (C) Flow cytometry analysis of CD4⁺ T cell and Treg infiltration in subcutaneous tumors from NID1 NC and NID1 KD groups (n = 5); (D) Flow cytometry analysis of PD-L2 and TIM-3 expression on DCs in subcutaneous tumors from NID1 NC and NID1 KD groups (n = 5); (E) Schematic diagram of the DC adoptive transfer experiment. Mice were inoculated with NID1 NC and NID1 KD cells (n = 5). Animals bearing NID1 KD tumors received weekly intravenous injections of PBS, NID1 NC-EV-pulsed DCs, or NID1 KD-EV-pulsed DCs for three consecutive weeks. The mice were sacrificed, and tumors were harvested on day 28; (F) Body weight changes in mice during tumor progression; (G) Images of dissected tumors (left) and tumor growth curves (right); (H) Flow cytometry analysis of the Treg proportion within the tumors (n = 3); (I) Flowchart for assessing orthotopic hepatic growth of 1753^p53-/-cmyc NID1 NC and NID1 KD cells in mice (upper). Images of dissected liver tissues on Day 28; (J) Body weight changes in mice during tumor progression. Flow cytometry analysis of CD4⁺ T cell and Treg proportions in TdLNs (K) and spleen (L) from the NID1 NC and NID1 KD groups (n = 5); (M-O) ELISA determination of NID1, IL-10, and CCL17 levels in mouse serum from the NID1 NC and NID1 KD groups (n = 5). Data are presented as the mean ± SD, with statistical significance determined by one-way ANOVA (multiple groups) (Tukey’s test) or Student’s t-test (two groups). Biological replicates are represented as individual data points. Data are presented as the mean ± SD. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001. NID1: Nidogen 1; NC: negative control; KD: knockdown; EV: extracellular vesicle; DC: dendritic cell; mregDC: mature regulatory dendritic cell; PBS: phosphate-buffered saline; CD4: cluster of differentiation 4; FOXP3: forkhead box protein P3; Treg: regulatory T cell; PD-L2: programmed death-ligand 2; TIM-3: T-cell immunoglobulin and mucin-domain containing-3; TdLN: tumor-draining lymph node; IL-10: interleukin-10; CCL17: C-C motif chemokine ligand 17; ELISA: enzyme-linked immunosorbent assay; SD: standard deviation; ANOVA: analysis of variance; ns: not significant.