5D). Collectively, these results indicate that knockdown of SIRPα on Mψ promotes tumor progression in vivo. The above results demonstrated that tumor-exposed SIRPα-KD Mψ produced larger amounts of proinflammatory cytokines than control cells in vitro. Here, we found that adoptive transfer of SIRPα-KD Mψ also increased expression of tumor promoting cytokines in both Hepa1-6 and H22-derived tumor tissues (Fig. 6A,B). Furthermore, immunostaining ITF2357 concentration assays showed that the density of CD31+ endothelial cells, considered the marker of microvessel neogenesis, was higher in Hepa1-6 tumors receiving an intravenous injection of SIRPα-KD Mψ than that of control Mψ (Fig. 6C). Meanwhile,

the KD group also showed an increased expression of vascular endothelial growth factor (VEGF) (Supporting Fig. 5). Interestingly, it FK506 was shown that the stromal cells, including Mψ, were the major source of VEGF production other than tumor cells, indicating an important role of Mψ in tumor neovascularization (Supporting Fig. 5). HIF1α, whose stability is associated with the activation of Akt and NF-κB, is

essential for angiogenesis.[24] Luciferase reporter gene assay showed that the activity of hypoxia transcriptional response element (HRE) was increased in SIRPα-KD Mψ upon exposure to Hepa1-6 cells, and the protein level of HIF1α was also increased in SIRPα-KD Mψ (Fig. 6D,E). In accordance with this, the reporter activity of the downstream molecules, such as NFAT, COX2, and VEGF, was increased by 2-4-fold compared with control Mψ (Fig. 6D). These results

indicate that SIRPα negatively regulates the stability medchemexpress of HIF1α on Mψ in response to tumor, suggesting that SIRPα plays an important role in tumor angiogenesis. SIRPα is a cell surface protein containing the ITIM motif domains which are known to exert an inhibitory function through recruitment of phosphatase enzyme SHP2 to its phosphorylated tyrosine residues. We analyzed whether SIRPα phosphorylation was increased when cocultured with tumor cells. As shown in Fig. 7A, tyrosine phosphorylation of SIRPα was increased in response to Hepa1-6 cells, together with enhanced binding to SHP2. SHP2 was constitutively associated with SIRPα even when SIRPα had the undetectable phosphorylation level at the basal time. Moreover, knockdown of SHP2 by siRNA transfection significantly decreased phosphorylation of IκBα and Akt compared with control Mψ when cocultured with tumor (Fig. 7B). As expected, the amount of IL6 and TNFα production was about 2-fold lower in SHP2-KD Mψ than control (Fig. 7C). To investigate how SHP2 was involved in the regulation of NF-κB and Akt signaling pathways, we performed coimmunoprecipitation experiments by targeting SHP2 on SIRPα-KD and control Mψ upon exposure to Hepa1-6. Tumor cells induced an interaction of SHP2 with IKKβ and PI3K regulatory subunit p85 (PI3Kp85) in Mψ, which was critical in the activation of the NF-κB and Akt pathway, respectively (Fig. 7D).