Endogenous DEL-1 restrains melanoma lung metastasis by limiting myeloid cell-associated lung inflammation

Abstract

Distant metastasis represents the primary cause of cancer-associated death. Pulmonary metastasis is most frequently seen in many cancers, largely driven by lung inflammation. Components from primary tumor or recruited leukocytes are known to facilitate metastasis formation. However, contribution of target site-specific host factor to metastasis is poorly understood. Here, we show that developmental endothelial locus-1 (DEL-1), an anti-inflammatory factor abundant in the lung and down-regulated by inflammatory insults, protects from melanoma lung metastasis independently of primary tumor development and systemic immunosurveillance. DEL-1 deficiency is associated with gene profiles that favor metastatic progression with inflammation and defective immunosurveillance. Mechanistically, DEL-1 deficiency primarily influences Ly6G⁺ neutrophil accumulation in lung metastatic niche, leading to IL-17A up-regulation from γδ T cells and reduced antimetastatic NK cells. In support, neutrophil depletion or recombinant DEL-1 treatment profoundly reverses these effects. Thus, our results identify DEL-1 as a previously unrecognized link between tumor-induced inflammation and pulmonary metastasis.

Fig. 1. Melanoma metastasis to the lung but not primary tumor growth is DEL-1 dependent.

(A to E) WT (n = 10) or Del1KO (n = 13) mice were injected intravenously with 5 × 10⁵ DsRed-B16F10 cells and analyzed for lung metastasis after 2 weeks. (A) Representative lung images (top) and corresponding DsRed fluorescence images (bottom). (B) Quantification of lung metastases by FLI depicted in (A). (C) Representative hematoxylin and eosin–stained lungs and (D) quantification of metastatic nodule size and (F) macrometastases from mice in (A). (F to I) DsRed-B16F10 cells were injected subcutaneously into WT and Del1KO mice (n = 6 per group). (F) Tumor sizes and (G) tumor weights were determined at the indicated time points and on day 21, respectively. (H) Representative lung images (top) and corresponding fluorescence images (bottom) showing spontaneous lung metastases. (I) Quantification of lung metastases by FLI depicted in (H). (J and K) Natural killer (NK) cell–mediated lymphoma clearance assay (n = 5 per group) showing representative result (J) and graph (K). (L and M) NK cell degranulation assay (n = 6 per group) showing representative result (L) and graph (M). *P < 0.05; **P < 0.01. Photo credits for (A) and (H): Hyung-Joon Kwon, University of Ulsan.

Fig. 2. Reciprocal regulation of Ly6G⁺ neutrophils and NK cells by DEL-1 in the metastasis-bearing lung.

(A and B) Representative (A) and quantitative (B) flow cytometric analysis of various myeloid cells (CD11b⁺Ly6G⁺ neutrophils, CD11b⁺Ly6C⁺, monocytes, CD11c⁺MHCII⁺ dendritic cells, and F4/80⁺MHCII⁺ M1-like and F4/80⁺CD206⁺ M2-like macrophages) and lymphocytes (CD3⁻NK1.1⁺ NK cells, CD3⁺γδ T cells, CD8⁺ T cells, and CD4⁺ T cells) among CD45⁺ cell populations in the metastasis-bearing lungs of WT and Del1KO mice (n = 10 each group) on day 14 after intravenous injection of B16F10 cells. (C) Representative immunofluorescence staining for Ly6G and NK1.1 in a metastasis-bearing lung from each group of mice depicted in (A). Ly6G⁺ (green) neutrophils and NK1.1⁺ (red) NK cells with 4′,6-diamidino-2-phenylindole (DAPI) counterstain (blue) are shown. Magnified images (right panel for each mouse group) show peritumoral localization of Ly6G⁺ neutrophils. Scale bars, 100 μm. (D and E) Representative (D) and quantitative (E) flow cytometric analyses of CD11b and CD27 expression on NK1.1⁺ NK cells in the metastasis-bearing lungs of WT and Del1KO mice (n = 10 each group) depicted in (A). Horizontal bars indicate the means (B and E). **P < 0.01.

Fig. 3. DEL-1 deficiency promotes melanoma lung metastasis in a neutrophil-dependent manner.

(A) Representative lung images (top) and corresponding DsRed fluorescence images (bottom) showing the diminished metastases to the lungs of Del1KO mice (n = 5 per group) treated with anti-Ly6G antibody. Photo credit: Hyung-Joon Kwon, University of Ulsan. (B) Quantification of lung metastases by FLI depicted in (A). (C and D) Representative (C) and quantitative (D) flow cytometric analysis of myeloid cells and lymphocytes, graphed on Ly6C by Ly6G and CD3 by NK1.1 dot plots, respectively, among CD45⁺ cell populations in the metastasis-bearing lungs depicted in (A). (E and F) Representative (E) and quantitative (F) flow cytometric analysis of CD11b and CD27 expression on NK1.1⁺ NK cells depicted in (A). (G and H) Representative (G) and quantitative (H) flow cytometric analysis of intracellular IL-17A expression in different lymphocytes in the metastasis-bearing lung of WT and Del1KO mice (n = 6 per group). (I and J) Representative (I) and quantitative (J) flow cytometric analysis of intracellular IL-17A expression in different lymphocytes depicted in (A). Horizontal bars indicate the means (D, F, H, and J). *P < 0.05; **P < 0.01.

Fig. 4. Melanoma lung metastasis and neutrophil accumulation are ameliorated by Del-1-Fc administration.

(A and B) C57BL/6 mice were treated with intravenous injection of control-Fc or Del-1-Fc 6 hours before an intravenous injection of 2 × 10⁵ B16F10 cells expressing DsRed and twice a week treatment thereafter. (A) Representative lung images (top) and the corresponding DsRed fluorescence images (bottom) showing lung metastases (n = 8 each group) after 14 days of tumor implantation. Photo credit: Hyung-Joon Kwon, University of Ulsan. (B) Quantification of lung metastases by FLI depicted in (A). (C and D) Representative (C) and quantitative (D) flow cytometric analysis of different myeloid cells and lymphocytes, graphed on Ly6C by Ly6G and CD3 by NK1.1 dot plots, respectively, in the metastasis-bearing lungs depicted in (A). Horizontal bars indicate the means (D). *P < 0.05.

Fig. 5. CM from B16F10 cells induces neutrophil accumulation in the lung microenvironment.

(A) Schematic diagram showing the schedule for B16F10-CM treatment. WT and Del1KO mice (n = 8 each group) were intravenously administered with B16F10-CM for two consecutive days. Mice were euthanized the next day after the last B16F10-CM treatment. (B and C) Representative (B) and quantitative (C) flow cytometric analysis of myeloid cells and lymphocytes, graphed on Ly6C by Ly6G and CD3 by NK1.1 dot plots, respectively, among CD45⁺ cell populations in the lungs depicted in (A). (D and E) Representative (D) and quantitative (E) flow cytometric analysis of myeloid cells, graphed on Ly6C by Ly6G dot plots, among CD45⁺ cell populations in the spleens depicted in (A). (F and G) Representative images (F) and quantification (G) of lung metastasis in WT or Del1KO mice (n = 7 each group) after administration of media or B16F10-CM as depicted in (A) and then injection of B16F10-Luc2 cells by luciferase-based bioluminescence imaging (BLI). One day after the last B16F10-CM treatment, mice received an intravenous injection of B16F10-Luc2 cells. Horizontal bars indicate the means (C and E). *P < 0.05; **P < 0.01.

Fig. 6. DEL-1 deficiency promotes neutrophil recruitment to perimetastatic foci in the lung.

(A) Scheme of two-photon intravital imaging. DsRed-B16F10 cells were intravenously injected (day 0) and imaged at day 5 for GFP⁺ neutrophils and DsRed⁺ B16F10 cells. (B and C) Representative images (B) and quantification (C) of neutrophils around metastatic foci in the lungs of LysM-GFP/WT (n = 10) or LysM-GFP/Del1 KO mice (n = 7). Scale bar, 50 μm. (D) Migration trajectories of neutrophils observed for 15 min in a field of view. The starting point for each neutrophil was set to zero. Axis unit = μm. (E to G) Quantification of neutrophil velocity (E), displacement (F), and meandering index (G) from intravital imaging. (H) Broad view of cleared lung tissue 5 days after DsRed-B16F10 cell injection. Scale bar, 300 μm. (I) Counts of neutrophils within 50 μm from the boundaries of metastatic foci. (J) The number of neutrophils inside the metastatic foci. *P < 0.05; ****P < 0.0001.