Figure 1. Visualization of tumor antigens within discrete migratory and resident myeloid compartments.

(A) B16ZsGreen tumors injected into membrane-tdTomato (mT) expressing mice imaged by confocal microscopy. Representative image. Scale bar = 100 μm. Inlay image is higher magnification of intracellular ZsGreen puncta within mT host cells. Scale bar = 5 μm. (B) Tumor-derived ZsGreen within myeloid cells of the tumor. Plot is mean frequency (top) and mean fluorescence intensity (MFI, bottom) +/− SEM. n = 6. Representative of 3 independent experiments. (C) ZsGreen⁺ myeloid cells from B16ZsGreen tumors stained for tumor-associated antigens, tyrosinase (Tyr, red) and gp100 (yellow). Frequency of colocalization for tumor-derived ZsGreen with tyrosinase and gp100 is quantified (right). Representative images. Arrows indicate puncta. Scale bars = 3 μm. (D) Tumor draining lymph node (tdLN) confocal imaging to detect ZsGreen. Representative image. White box inset (top) shows position of zoomed image (bottom). Top scale bar = 50 μm. Bottom scale bar = 5 μm. (E) Lattice light sheet imaging of sorted DC from B16ZsGreen tdLN. Left image is single z-slice of cell. Representative image. Scale bar = 2 μm. Right is surface rendering to show single z-slice of membrane (red) with remaining depth of z plane in grey. Total 3D volume of ZsGreen is surfaced (green). (F) Tumor-derived ZsGreen within myeloid cells and microparticles (MP) of the tdLN. migratory CD11b (m11b), resident CD11b (r11b). Plots is mean frequency (left), count (top, right) and MFI (bottom, right) +/− SEM. n = 4. Representative of 3 independent experiments. (G) ZsGreen⁺ myeloid cells sorted from B16ZsGreen tdLN and stained for tumor-associated antigens, tyrosinase (Tyr, red) and gp100 (yellow). Colocalization for tumor-derived ZsGreen with tyrosinase and gp100 is quantified (right). Arrows indicate puncta. Representative images. Scale bar = 4 μm. See also Figure S1 and Video S1.