Figure 9.

Most macrophages in orthotopic mouse breast cancer models are myeloid-derived lymphatic endothelial cell progenitors. A–C: EMT6 (A) and MDA-MB-231 (B and C) tumors were double stained for CD11b and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve-1; A and B) or podoplanin (Pdpn; C). White arrows indicate myeloid-lymphatic hybrid cells expressing CD11b and lymphatic endothelial cell (LEC) markers, whereas white arrowheads indicate cells positive only for CD11b. D: CD11b⁺ tumor-associated macrophages (TAMs), isolated by fluorescence-activated cell sorting from MDA-MB-231 tumors, were analyzed by flow cytometry for myeloid progenitor markers Ly6C and Ly6G as well as LEC markers vascular endothelial growth factor receptor 3 (Vegfr-3), Lyve-1, and Pdpn. E: Representative histograms of CD11b⁺/Lyve-1⁺ and CD11b⁺/Pdpn⁺ cells demonstrating shifts in tumor-derived versus peritoneal macrophages from non–tumor-bearing mice. F: TAMs were sorted for CD11b⁺/Pdpn⁺ and Pdpn⁻ cells. F: Pdpn⁺, Pdpn⁻, and whole CD11b⁺ populations were analyzed by real-time PCR for lymphatic marker expression. Results are the mean values from two independent experiments. All analyzed targets, except CD34, were significantly up-regulated in the Pdpn⁺ group compared with other groups. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (determined by t-test). Scale bar = 100 μm (A–C). Original magnification, ×400 (A–C). CoupTF2, chicken ovalbumin upstreampromoter transcription factor 2; Itga9, integrin subunit alpha 9; Nrp2, neuropilin; Prox1, prospero homeobox protein 1; Tlr, toll-like receptor; Vegfc, vascular endothelial growth factor C.