Figure 2. Deletion of PDPN in TAMs inhibits tumor lymphatic growth and metastasis.

(A-E) 4T1 tumor growth (A), lung metastasis (B), representative images of black ink-injected lungs from 4T1 tumor-bearing mice (C), representative images of lung sections stained with hematoxylin-eosin (H&E) (D) and histologically assessed metastatic area in lungs (E) of WT→WT or Pdpn KO→WT chimeras. Data show representative values of 4 independent experiments.

(F) Quantifications of 4T1 tumor sections stained for a lymphatic endothelial cell specific marker LYVE1.

(G-H) Representative images and quantifications of inguinal lymph nodes from 4T1 tumor-bearing mice, stained for cytokeratin 14 (CK14) as a marker of cancer cells.

(I-J) Quantification of lung metastasis (I) and representative images of black ink-injected lungs (J) from mice in which lymphadenectomy of tumor-draining lymph nodes was performed 7 days prior to 4T1 cell inoculation.

(K-O) Quantification of lung metastasis (K), metastatic index (L), VEGFR3⁺ lymphatics in tumors (M), representative images of tumor sections stained for VEGFR3 (N) and 4T1 tumor growth (O) in WT→WT and Pdpn KO→WT mice treated with a VEGFR3-blocking antibody (Mf431C1) or with an isotype (rat IgG) control by bi-weekly intratumoral injections (40 μg per 1 g of body weight).

(P-Q) Quantification (P) and representative images (Q) of F4/80⁺ TAMs (yellow arrows) directly interacting with VEGFR3⁺ LVs in 4T1 tumors.

(R) qRT-PCR for Pdpn mRNA in CD11b⁺, F4/80⁺ TAMs and CD11b⁺, Ly6G⁺ neutrophils isolated from E0771 tumors bore by Csf1r;Pdpn^wt/wt or Csf1r;Pdpn^lox/lox mice.

(S-T) Quantification (S) and representative images (T) of F4/80⁺ TAMs (yellow arrows) directly interacting with VEGFR3⁺ LVs in E0771 tumors.

(U-V) Quantification (U) and representative images (V) of E0771 tumor sections stained for a lymphatic endothelial cell specific marker LYVE1.

(W) E0771 lung metastasis.

Statistical analysis: *P<0,05; **P<0,005; ***P<0,0005; ****P<0,00005. Graphs show mean ± SEM. Scale bars: 20 μm (N, Q, T); 50 μm (V); 100 μm (G); 2,5 mm (D). DAPI, 49,6-diamidino-2-phenylindole.