Fig. 6: CCR7+ dendritic cell trafficking and MMP9-dependent entry into the sentinel lymph node are critical for immunoradiotherapy efficacy.
A (left) Cartoon schema of the experimental approach. WT animals injected with 4MOSC1 tumors were treated with tdRT→αPD-1 with or without sentinel lymph node lymphatic channel ablation (SLN LCA) or non-sentinel lymph node lymphatic channel ablation (nSLN LCA). SLN LCA and nSLN LCA procedures were performed as described in the methods, involving precise surgical ablation of lymphatic channels. B Flow cytometric analysis of dendritic cell percentages in the sentinel lymph node post-treatment (n = 5 mice per group, p = 0.0379). Data are presented as mean values ± SEM; p values calculated by two-sided unpaired Student’s t test. C Flow cytometric analysis of Ovalbumin-specific T cells (4MOSC1-LucOS model) in SLN and nSLN with and without SLN LCA (left). Quantification of normalized percentages relative to control (right) (n = 4 mice per group, p = 0.0054). Data are presented as mean values ± SEM; p values calculated by two-sided unpaired Student’s t test. D Tumor growth curves for control, tdRT→αPD-1, nSLN LCA + tdRT→αPD-1, and SLN LCA + tdRT→αPD-1 groups (n = 10 mice per group, p < 0.0001). Best-fit lines and p values calculated by simple linear regression (two-sided). E (left) Cartoon schema of the experimental approach. WT animals injected with 4MOSC1 tumors were treated with tdRT→αPD-1 and MMP9 inhibitor. (right) CCR7 expression on dendritic cells and quantification of normalized percentages relative to control (n = 5 mice per group, p = 0.0022). MMP9 inhibition was achieved using a specific inhibitor (Sigma 444293) at 0.4 mg/mouse/dose delivered intratumorally in 8 μL volume on post-transplant day 3, 5 and 7, as previously described38,40. SLN were mapped and harvested 48 hours after completion of treatment. F Flow cytometric analysis of CCR7 + MHCII+ dendritic cells in the SLN post-treatment with and without MMP9 inhibition. Quantification of normalized percentages relative to control (n = 5 mice per group, p < 0.0001). Data are presented as mean values ± SEM; p values calculated by two-sided unpaired Student’s t test. G Tumor growth curves for tdRT→αPD-1 with and without MMP9 inhibition (n = 10 mice per group, p < 0.0001). Data are presented as mean values ± SEM. Best-fit lines and p values calculated by simple linear regression (two-sided). H TCR analysis showing productive frequency in the sentinel lymph node and tumor for tdRT→αPD-1 (Morisita Index: 0.042, Prod Clonality (norm): 14.9) and SLN LCA + tdRT→αPD-1 (Morisita Index: 0.022, Prod Clonality (norm): 0.45) groups. I TCR clonal analysis showing enriched CDR3 sequences in the sentinel lymph node and tumor for tdRT→αPD-1 and SLN LCA + tdRT→αPD-1 groups. Enriched CDR3 sequences were identified through TCR sequencing and analyzed for clonal distribution. Created in BioRender. Saddawi-Konefka, R. (2025) https://BioRender.com/m675tdd. Source data are provided as a Source Data file.
