(A and B) Intravascular and extravascular distribution of established B16F10-GFP clusters in mouse lung. (A) Representative confocal images show immunostaining of CD31 (endothelial cell marker, red) and B16-GFP (green) in Nr4a1^–/– mice. B16F10-GFP clusters on the lung surface are indicated by dashed white lines. White and yellow arrows indicate the areas of extravascular and intravascular B16F10-GFP cells, respectively. White and yellow scale bars: 100 and 20 μm, respectively. (B) GFP intensity in 10–22 areas from multiple slides from the deeper regions in lung from WT B6, Nod2^–/–, and Nr4a1^–/– mice (n = 3–4) was determined by use of ImageJ and quantified. Data are presented as mean ± SEM; **P < 0.01; 1-way ANOVA. (C and D) Experimental design (C) and representative 2-photon images (D) showing colocalization of I-NCMs with the established B16F10-GFP metastatic clusters in mouse lung. The I-NCMs were sorted, stained with Hoechst 33342, and adoptively transferred into Nod2^–/– mice bearing B10F10-GFP cells in the lung as described in C. Intravital 2-photon imaging was used for tracing I-NCMs. Blue: Hoechst 33342–stained I-NCMs; green: B16F10-GFP; red: Dx-rhodamine–labeled blood vessel. Colocalization of blue and green signals is indicated in the area circled with a dashed white line in D. Scale bars: 50 μm. (E and F) RFP-labeled I-NCMs (3.5 × 10⁵ cells) were sorted and adoptively transferred into Nr4a1^–/– Nod2^–/– double-mutant mice bearing B10F10-GFP cells in the lung as described for E. Accumulation and distribution of the adoptively transferred RFP–I-NCMs were detected and determined by confocal imaging of lung sections immunostained with CD31 (endothelial cell marker; white), GFP (B16F10-GFP), and RFP (RFP-I-NCMs) in F. Yellow and blue arrows indicate the typical RFP-labeled I-NCMs outside and inside the blood vessel within the B16F10-GFP clusters, respectively. White and yellow scale bars: 50 and 20 μm, respectively. (G and H) Expression of CCL2 in metastatic B16F10 clusters and adjacent areas in mouse lungs. Expression of CCL2 was detected by IHC in the lungs (G) of untreated WT B6 control mice (n = 5, left) and Nr4a1^–/– mice bearing B16F10-GFP melanoma cells (n = 4, right). White and yellow scale bars: 100 and 10 μm, respectively. RFP intensity in 34–96 areas from multiple slides was measured by use of ImageJ and quantified (H). FI, fluorescence intensity. The FI data are presented as mean ± SEM; n = 4–5 mice in each group; *P < 0.05; Forsythe’s and Welch’s ANOVA. (I and J) CCR2 deficiency impaired the accumulation and extravasation of MDP-triggering CCR2⁺ monocytes from blood vessels at the B16F10 metastasis site. Experimental design (I) and representative confocal images (J) showing typical extravasated (J, top row, indicated with white arrows) or intravascular (J, bottom row, indicated with yellow arrows) MDP-triggering CCR2⁺ monocytes in Nr4a1^–/–Nod2^–/– mouse lungs. Scale bars: 20 μm.