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hep26391-sup-0001-suppfig1.tif1672KFigure S1, related to Figure 1. SIRPα expression is reduced on tumor polarized monocytes/Mφ. (A) SIRPα expression on Mφ isolated from HCC tissues of individual patients. (B) Monocytes/Mφ were isolated from peripheral blood and tissues of HCC patients. The representative flow cytometry images are shown. (C) The representative MFI of SIRPα expression on monocytes/Mφ obtained from blood and tissues of HCC patients. (D) Monocytes/Mφ were isolated from peripheral blood and tumor tissues of Hepa1-6-bearing mice. Cells were labeled and the representative flow cytometry images are shown. (E) The representative MFI of SIRPα expression on monocytes/Mφ obtained from blood and tissues of Hepa1-6-bearing mice.
hep26391-sup-0002-suppfig2.tif730KFigure S2. SIRPα expression is reduced in response to certain factors existing in tumor microenvironment. (A) Mφ were cocultured with Hepa1-6 cells for the indicated times, the changes of SIRPα mRNA levels on Mφ were detected by real-time PCR. Data are showed as mean ± SEM of four independent experiments; *p<0.05; **p<0.01. (B) SIRPα protein expression on BMDMs exposed to TNFα (25 ng/ml), hypoxia (0.2% O2) or H2O2 (0.5 mM). Data represent at least three independent experiments.
hep26391-sup-0003-suppfig3.tif435KFigure S3. SIRPα expression on Mφ is reduced by si-RNA transfection or lentivirus infection. (A, B) Mφ were treated with si-RNA transfection or lentivirus infection specifically targeting SIRPα. The efficiency of SIRPα knockdown was measured by immunoblotting (A) and real-time PCR (B).
hep26391-sup-0004-suppfig4.tif1713KFigure S4. MCP-1 and CSF1 are the two of the factors recruitment Mφ to tumors. (A) MCP-1, CSF1 and CCL5 mRNA were analyzed by real-time PCR between Hepa1-6 cells and mice primary hepatocytes. Data represent four individual experiments; **p<0.01. (B) Transwell assay. MCP-1 and CSF1 was transiently knockdown on Hepa1-6 cells and co-cultured with Mφ. 24 hours later, the migrated Mφ were fixed and stained. Data represent four individual experiments.
hep26391-sup-0005-suppfig5.tif2033KFigure S5. Knockdown SIRPα on Mφ promotes VEGF production. VEGF expression was detected in tumor tissues in Fig. 6C by immunohistochemistry assay.
hep26391-sup-0006-suppfig6.tif1882KFigure S6. SIRPα proteins degradation in response to tumor is partially mediated by lysosome. (A) Mφ were treated with TNFα (25ng/ml) or cocultured with Hepa1-6 cells in the presence or absent of anti-TNFα antibody. The cells were then collected and SIRPα expression was determined by immunoblotting (upper). Data are one representative of four experiments. The relative expression of SIRPα in each group was quantified and showed (lower, mean ± SEM); *p<0.05; **p<0.01. (B) Mφ were incubated for 2 h at 37°C in the absence or presence of 100 μM chloroquine, 10 mM NH4Cl, 40 μM MG132, or 5 μM dexamethasone, after which they were cocultured with or without Hepa1-6 cells for 8 h and subjected to immunoblotting analysis using SIRPα-specific antibody. (C) Mφ were transfected with plasmid expression SIRPα-GFP fusion protein (green), the cells were cocultured with Hepa1-6 cells for 6 hours and the lysosomes were labeled by lamp1 (red). The representative pictures are shown.
hep26391-sup-0007-suppfig7.tif4763KFigure S7. Tumor phagocytosis and tumor proliferation. (A) SIRPα-KD and control Mφ (CMFDA-labeled, green) were seeded into TAMRA-labeled Hepa1-6 cells with or without LPS/IFNγ preincubation. The pagocytotic cells were detected as double positive cells and showed as mean ± SEM. (B) Hepa1-6 cells were subcutaneously injected into C57BL/6 mice. Five days later, the medium of SIRPα-KD and control Mφ cocultured with Hepa1-6 cells was collected and subcutaneously injected into the tumor-bearing mice every 2 days. The tumor volume was monitored. Data are showed as mean ± SEM (n=5 mice per group) of two independent experiments; *p<0.05. (C and D) The tumor samples were then subjected to IHC staining with p-Stat3 (C) and Ki67 antibodies (D). (E) Hepa1-6 cells were co-cultured with SIRPα-targeted or control Mφ for the indicated times, cell lysates of Hepa1-6 cells was obtained and immunoblotting was performed by p-Stat3, p-p65, Stat3 and p65 antibodies. Data represent three individual experiments.
hep26391-sup-0008-suppfig8.tif634KFigure S8. (A) Mφ were treated with hypoxia condition (0.2% O2) for the indicated times, SIRPα expression was detected by immunoblotting. Data are one representative of triple independent experiments. (B) C57BL/6 mice were performed with a midline laparotomy, the liver hilum was dissected free of surrounding tissue. All structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes were then occluded with a microvascular clamp (Fine Science Tools) for 60 min. The livers were resected and the SIRPα expression on Mφ was measured by FACS. The data are showed as mean ± SEM of 6 mice.
hep26391-sup-0009-supptab1.doc37KTable S1. Characteristics of the 25 HCC patients
hep26391-sup-0010-supptab2.doc57KTable S2. Relationship between SIRPα expression on Mφ isolated from peritumor tissues and clinicopathologic characteristics in HCC patients (n=25)
hep26391-sup-0011-supptab3.doc60KTable S3. Relationship between SIRPα expression on Mφ isolated from tumor tissues and clinicopathologic characteristics in HCC patients (n=25)
hep26391-sup-0012-supptab4.doc38KTable.S4 Primer sequences for real-time quantitative PCR
hep26391-sup-0013-suppinfo.doc47KSupporting Information.

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