AF647-RIS is internalized by TAMs in vivo.

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AF647-RIS is internalized by TAMs in vivo. AF647-RIS is internalized by TAMs in vivo. A and B, time-lapse images from a single optical plane showing uptake of small (A) and large (B) AF647-RIS complexes (red) by F4/80-labeled macrophages (green); images correspond to Supplementary Movies S3 and S4 and are representative of three independent experiments, with at least three single cells analyzed per experiment. C, 60-μm maximal intensity projection from the surface of 4T1 breast tumor 24 hours after injection of AF647-RIS. Blue, collagen; green, F4/80; red, AF647-RIS. images correspond to Supplementary Movie S5. Scale bars, 10 μm in A and B, and 100 μm in C. Time stamp, hh:mm:ss after injection of AF647-RIS. D, FACS staining of mammary epithelial cells for EpCAM and tumor-infiltrating leukocytes (TIL) for CD45. E, histogram, AF647-RIS signal in mammary epithelial cells and TILs in mice treated with PBS (blue) or AF647-RIS (red). F, the pseudocolor plot showing AF647-RIS and F4/80 gated on CD45+ cells in mice treated with PBS (left) or AF647-RIS (right). G, histogram, AF647-RIS signal in CD45+F4/80− TILs and CD45+F4/80+ TAMs in mice treated with PBS (blue) or AF647-RIS (red). H, quantification of AF647-RIS (BP) uptake. MFI, mean fluorescence intensity. I, FACS analysis of AF647-RIS (red) or PBS (blue) uptake by subpopulations of F4/80+ TAMs. Data are the mean ± SEM and are representative of at least two independent experiments with n = 3 mice in each group. Simon Junankar et al. Cancer Discovery 2015;5:35-42 ©2015 by American Association for Cancer Research