Fig. 10

Chitin and anti-CHI3L1 treatment reduces CHI3L1-stimulated macrophage integration in lymphatic vessels in the 4T1-based intraductal model. A Representative images and quantification of bioluminescent signals (total flux density in p/s/cm²) in axillary lymph nodes from the IgG control-, anti-CHI3L1- and chitin-treated 4T1-based model at 5 w p.i. (n = 6 for all groups). B Flow cytometric analysis of PDPN positivity on the cell surface of RAW264.7 macrophages following a 24 h, 48 h and 72 h incubation with either PBS, 0.1, 0.5 or 1 µg/ml rmCHI3L1 (n = 6 for all groups). The dot plots highlight the PDPN positivity after a 72 h incubation with PBS versus 1 µg/ml rmCHI3L1. C Adhesion of PBS- and 5 µg/ml rmCHI3L1-treated RAW264.7 macrophages to a 2D LEC monolayer with representative images shown (n = 9 for all groups). D Ability of anti-PDPN, anti-IL13Rα2 and their respective IgG controls to inhibit adhesion of 5 µg/ml rmCHI3L1-treated RAW264.7 macrophages to a 2D LEC monolayer (n = 6 for all groups). E Integration of PBS- and 5 µg/ml rmCHI3L1-treated RAW264.7 macrophages into 3D LEC vessel-like structures with representative images shown (n = 9 for all groups). F Ability of anti-PDPN, anti-IL13Rα2 and their respective IgG controls to inhibit integration of 5 µg/ml rmCHI3L1-treated RAW264.7 macrophages into 3D LEC vessel-like structures (n = 7 for the rat IgG control and rat anti-PDPN group, n = 10 for the goat IgG control and goat anti-IL13Rα2 group). G Dual staining for the TAM marker F4/80 and LEC marker LYVE-1 on primary tumor sections from IgG control-, anti-CHI3L1- and chitin-treated 4T1 tumor-bearing mice at 5 w p.i. (n = 9 for all groups; 3 slides with 3 images per slide). Dashed inserts highlight stained tissue at a larger magnification. Black scale bars = 200 µm, white scale bars = 100 µm, red scale bars = 50 μm, green scale bars = 20 µm. Data are presented as the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001