(A) In vitro collagen tube formation assays using different subsets of CD11b⁺ myeloid cells. Both Gr1⁺CD11b⁺CD11c^– cells and Gr1^–CD11b⁺CD11c^– cells in the presence of 2.5% tumor-conditioned medium successfully induced ECs to form tube-like structures. Gr1^–CD11b⁺CD11c⁺ DCs did not induce efficient EC tube formation. Graph shows mean ± SEM of 2 independent experiments. (B) VEGF and bFGF expression in CD11b⁺Gr1⁺ myeloid cells contribute to angiogenesis. Splenic CD11b⁺Gr1⁺ myeloid cells were exposed to tumor-conditioned medium. Left: Flow cytometric analysis of Stat3^+/+ and Stat3^–/– CD11b⁺Gr1⁺ myeloid cells isolated from spleens of tumor-bearing mice for phospho-Stat3 (p-Stat3) and VEGF levels. Levels of bFGF in the indicated cells were determined by the Luminex system (see Methods). Graph shows mean ± SEM of 2 independent experiments. Right: Neutralization of VEGF and VEGF together with bFGF partially but significantly reduced myeloid cell–mediated angiogenesis. Graph shows mean ± SEM of 2 independent experiments with triplicates. (C) VEGF and bFGF are important for Stat3-dependent, tumor-derived MDSC–mediated angiogenesis. MDSCs purified from B16 tumors were used for tube formation assays in the presence of the indicated antibodies. Data are mean ± SEM of 3 independent experiments, each involving 10–15 pooled mice per group done in triplicates. (D) Stat3 activity in tumor-associated myeloid cells contributes to elevated expression of multiple angiogenic factors. Stat3^+/+ and Stat3^–/– CD11b⁺ myeloid cells were directly isolated from B16 tumors, and real-time RT-PCR was performed to detect RNA levels of the indicated genes. RNA levels of each indicated gene relative to a housekeeping gene (either 18s or GAPDH) in Stat3^+/+ myeloid cells were assigned as 1. Data are mean ± SEM (n = 3). *P < 0.05; **P < 0.01.