Expression of Gas6 in the tumor stroma. (A) RT-PCR analysis revealing detectable Gas6 expression in CT26 tumors in WT but not in Gas6^−/− mice (n = 7-8; # indicates not detectable). (B) Staining for Gas6 in CT26 tumors grown in WT mice showing Gas6 expression (red) in tumor-infiltrating leukocytes, identified on the basis of their characteristic morphology by an expert pathologist. (C) Double immunostaining of CT26 tumors for Gas6 (red) and Mac3 (blue-gray) in WT mice. Most Mac3⁺ macrophages express Gas6 (brownish color: merger of blue and red; red arrowheads), whereas a few macrophages do not express Gas6 (blue arrowheads). (D) Gas6 was undetectable in macrophages in Gas6^−/− mice (blue-gray; blue arrows). (E) RT-PCR analysis revealing greater Gas6 expression in FACS sorted CD45⁺ leukocytes than CD31⁺ endothelial cells in CT26 tumors in WT mice (n = 4; P = .002). (F) RT-PCR analysis, showing Gas6 up-regulation in CD45⁺ leukocytes upon infiltration in CT26 tumors. The source of the CD45⁺ leukocytes is indicated: TM (tumor infiltrated), BM (bone marrow), PB (peripheral blood; n = 4; P < .001). (G) RT-PCR analysis showing increased Gas6 expression in CT26 tumor-infiltrating macrophages (n = 4) in comparison with tissue resident macrophages isolated from spleen, lung, and peritoneum (n = 3 each; P < .05). (H) RT-PCR analysis of different subfractions of WBCs infiltrating CT26 tumors (n = 3; P < .05). Scale bars represent 50 μm in panels B-D. *Denotes statistical significance (P < .05). Error bars in panels A, E, F, G, and H show SEM; all subsequent error bars are defined similarly.