Resident and MoD-TAMs harbor distinct phenotypes and anatomical distribution. (A) Lung cryo-section shows the distribution of ECFP⁺ and EGFP⁺ cells in TC-1 pulmonary tumor nodule located in the vicinity of the pleura. Bar, 50 µm. Dashed lines represent each 50-µm interval, starting from the pleura. Graph represents the relative distribution of EGFP⁺ cells and ECFP⁺ MoD cells as a function of the distance from the surface (pleura; bars represent means ± SD of four mice out of two independent experiments; two-way ANOVA was performed). (B) Lung cryo-sections show the distribution of ECFP⁺ and EGFP⁺ cells in pulmonary tumor nodules located in the alveolar space or near large airways, 15 d after TC-1 inoculation. Graph represents the percentage of EGFP⁺ cells among fluorescent cells in each sub-anatomical compartment (dots represent the mean ratio per mouse, mice are pooled from two independent experiments, and red bars indicate mean. Unpaired Student’s t test was performed). (C) Lung cryo-sections of Tnfrsf11a^Cre; Rosa26^LSL-YFP mice shows YFP⁺ cells in pulmonary tumor nodules located in the pleura or in the alveolar space 15 d after tdTomato⁺ TC-1 inoculation. Graph represents the number of YFP⁺ cells in each sub-anatomical compartment (dots represent the mean ratio per mouse, mice are pooled from two independent experiments, and bars indicate means; one-way ANOVA was performed). (D) Heat map shows a selection of transcripts involved in extracellular matrix interaction and remodeling, differentially expressed between EGFP⁺ and ECFP⁺-TAMs. (E) Expression of indicated genes, relative to GAPDH (2-^ΔCt), as determined by qPCR of FACS-sorted YFP⁺ and YFP⁻ TAMs in Tnfrsf11a^Cre; Rosa26^LSL-YFP mice. Each dot represents one mouse, pooled from two independent experiments. (F) Representative histogram shows VCAM expression gated on TAM subsets from MacBlue × Cx3cr1^EGFP/+ mice (left panel). Middle panel shows the quantification of VCAM1 expression by EGFP⁺- and ECFP⁺-TAMs in MacBlue × Cx3cr1^EGFP/+. Right panel represents the quantification of VCAM1 expression by YFP⁺ and YFP⁻ macrophages in Tnfrsf11a^Cre; Rosa26^LSL-YFP mice. Mice are pooled from three independent experiments, bars indicate means; unpaired Student’s t test was performed. (G) TPLSM 3D reconstruction shows the perivascular location of the EGFP⁺ cell network within a tumor nodule (left panel). Lung cryo-section from TC-1 tumor-bearing MacBlue × Cx3cr1^EGFP/+ at day 15 shows fluorescent subset distribution regarding tumor vasculature using CD31 staining (middle panel). Close interactions of EGFP⁺ cells with the vasculature are indicated (white arrows). Scatter plot represents the relative proportion of perivascular EGFP⁺ cells and ECFP⁺ cells in tumor nodules (right panel; dots represent the mean proportion per mouse pooled from at least two independent experiments; bars indicate means; unpaired Student’s t test was performed). For all panels: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Fig. S3 and Videos 1 and 2.