Figure 4. IFNγ-signaling in tumor-associated lymphatic vessels restrains a tip-like state that promotes lymph node metastasis but inhibits dendritic cell migration.

A. Schematic of lymphatic drainage of the intradermal tumors on the top right side of the back. B. B16-OVA-VEGFC tumor growth (intradermal, 5×10⁵ cells) in Ifnγr1^WT and Ifnγr1^iProx1 mice (n=5). C. Representative gross images of B16-OVA-VEGFC tumor-draining LNs from Ifnγr1^WT and Ifnγr1^iProx1 mice. D. Quantification of black intensity over total LN area, each point represents a mouse, (n=7–8). E. Quantification of Evans blue intensity in B16-OVA-VEGFC tumor-draining LNs from Ifnγr1^WT and Ifnγr1^iProx1 mice, each point represents a mouse, (n=8–9). F. Quantification of CD11C⁺MHCII^highFITC⁺ migratory dendritic cells (mDC) as percent of live CD45⁺ cells in B16-OVA-VEGFC tumors and G. in tumor-draining LNs from Ifnγr1^WT and Ifnγr1^iProx1 mice, each point represents a mouse, (n=8–9). H. Rate of migration as a ratio of FITC⁺CD11C⁺MHCII^high mDC in the LN to the tumor. I. Representative images of B16-OVA-VEGFC tumors from Ifnγr1^WT and Ifnγr1^iProx1 mice. Lymphatic vessels (LYVE-1; red), DCs (CD11c; yellow, and nuclei (DAPI; cyan). Scale bars=100mm. J. Dot plot of select genes expressed in cluster 0, normalized from −1 to 1. Size indicates the percentage of cells in the cluster expressing the transcript. K. Stalk, tip, and Petkova et al module scores projected on combined UMAP. L. Dll4 expression as a function of pseudotime. M. Representative immunofluorescence images of B16-OVA-VEGFC tumors from Ifnγr1^iProx1 mice. Lymphatic vessels (LYVE-1, red); DLL4 (yellow); nuclei (DAPI, cyan). HF=Hair follicle, LV=Lymphatic vessel, scale bar = 100μm. N. Heatmap of tip-like genes expressed by HDLECs treated with PBS or IFNγ (n=3 technical replicates). O. Quantification of the percentage A375 melanoma, each point represents a technical replicate across two independent experiments, or P. bone marrow-derived dendritic cells (DC) that migrate across a transwell in the presence (DLL4-Fc) or absence (IgG-Fc) of adsorbed DLL4, each point represents a BDMCs from a single mouse, across two independent experiments. ****p<0.0001. Student’s t-test. *p<0.05; **p<0.01; ****p<0.0001.