Macrophages have distinct origins within lung tumors. (A) Lung cryo-sections of tumor free and TC-1^tdTomato tumor bearing MacBlue × Cx3cr1^EGFP/+ mice show the distribution of ECFP⁺ and EGFP⁺ cells within tumor nodules over time. (B) Dot plots show the relative proportion of macrophage subsets in tumor-free, tumor-bearing MacBlue × Cx3cr1^EGFP/+ (WT) and MacBlue × Cx3cr1^EGFP/+ × Ccr2^−/− (Ccr2^−/−) mice at indicated time points, numbers indicate the mean percentage ± SD of ECFP⁺EGFP⁺ TAMs (left panel). Box and whisker graph shows the absolute number per milligram of tissue of indicated myeloid subsets in WT and Ccr2^−/− mice (right panel; n = 10 mice out of three independent experiments, two-way ANOVA with Bonferroni multiple comparisons test was performed). (C) Lung cryo-sections show the distribution of ECFP⁺ and EGFP⁺ cells in TC-1 tumor-bearing MacBlue × Cx3cr1^EGFP/+ (WT), MacBlue × Cx3cr1^EGFP/+ × Ccr2^−/− (Ccr2^−/−) mice and C57Bl6 host parabiont with MacBlue × Cx3cr1^EGFP/+ mice (left panels). Ratio of ECFP⁺/EGFP⁺ cell numbers in lung tumors. Each dot represents mean of ECFP⁺/EGFP⁺ cell ratio from different tumor nodules per mouse, red bars indicate mean, mice are pooled from at least two independent experiments (right panel). (D) Lung cryo-sections show the distribution of ECFP⁺ and EGFP⁺ cells in LLC tumor bearing MacBlue × Cx3cr1^EGFP/+ mouse (day 15; left panel), and ratio of ECFP⁺/EGFP⁺ cells in LLC tumors (middle panel). Each dot represents mean of ECFP⁺/EGFP⁺ cell ratio from different tumor nodules per mouse, red bar indicates mean, mice are pooled from two independent experiments. Dot plot show the relative proportion of macrophages subsets in LLC tumor bearing MacBlue × Cx3cr1^EGFP/+ mice at the indicated time point; numbers indicated the mean percentage ± SD of ECFP⁺EGFP⁺ TAMs (right panel; n = 4 mice out of two independent experiments). (E) Lung cryo-section shows the distribution of ECFP⁺ and EGFP⁺ cells in spontaneous pulmonary metastases from MacBlue × Cx3cr1^EGFP/+ × PyMT-ChOVA mouse (left panel). Middle panel shows the lack of EGFP⁺ cells in pulmonary metastases from C57Bl6 PyMT-ChOVA host parabiont. Right panel shows the quantification based on histological analyses. Each dot represents mean of ECFP⁺/EGFP⁺ cell ratio from different tumor nodules per mouse, red bar indicates mean, all mice are independents. ANOVA with Bonferroni multiple comparisons test were performed for panel B and C and unpaired Student's t test was performed for panel E. For all panels: *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. See also Fig. S2.