Confocal immunofluorescence analysis of mouse tumors identifies a distinguishing TEM signature. (A) N202 mammary tumors (n = 5) grown subcutaneously in Tie2-GFP transgenic mice and analyzed for GFP (green) and Cd68 (red) expression. Confocal planes are shown individually and after merging (merge). The left panels show GFP⁺ blood vessels and abundant Cd68⁺ macrophages; scale bar represents 120 μm. High-magnification photos (right panels) show perivascular Tie2-GFP⁺Cd68⁺ TEMs (arrows); scale bar represents 60 μm. For each tumor, at least 3 sections were analyzed. (B) Mammary tumors spontaneously arising in MMTV-PyMT transgenic mice (n = 4) previously transplanted with Tie2-GFP BM cells and analyzed for GFP (green), Lyve1 or Mrc1 (red), and F4/80 (blue) expression. Abundant F4/80⁺ macrophages are evenly distributed within the tumor mass, whereas GFP⁺Lyve1⁺ or GFP⁺Mrc1⁺ TEMs cells are mainly found in stromal septa surrounding tumor cell nests. Virtually all the Tie2-GFP⁺ cells express Lyve1 and Mrc1. Note that some of the Tie2-GFP⁻ Lyve1⁺ or Mrc1⁺ cells may represent host-derived, nontransgenic TEMs. Scale bar represents 60 μm. For each tumor, at least 10 sections were analyzed for each marker.