Fig 2: Sirpα expression in tumor-associated macrophages (TAMs) increases with tumor progression. a TAMs increased with tumor progression in MC-38 and CT-26 xenograft models. MC-38 or CT-26 cells (1.0 × 106/100 μl of PBS per mouse) were subcutaneously inoculated into 6-week-old WT C57BL/6 or BALB/c mice. TAMs (CD90.2-CD45R-Gr1-CD45+F4/80+) were assessed dynamically by flow cytometry. b Sirpα mRNA levels in TAMs increased with tumor progression. The TAMs described above in (a) were isolated and subjected to real-time PCR assays to detect Sirpα mRNA levels (n = 3). c Real-time PCR assays of Sirpα mRNA levels in TAMs from APCmin+/− mice were performed at the indicated time points (n = 5). d Correlation analysis of tumor weight and TAM Sirpα mRNA levels in APCmin+/− mice was performed (Pearson R = 0.814, P < 0.005, n = 15). e Sirpα protein levels in TAMs from MC-38-based subcutaneous xenograft models were assessed by flow cytometry. f The gating strategy for the analysis of in vivo phagocytosis rates with flow cytometry is shown. g Phagocytosis rates decreased with tumor growth. GFP-tagged MC-38 cells (1.0 × 106/100 μl PBS) were subcutaneously injected into 6-week-old WT C57BL/6 mice. Phagocytizing macrophages were defined as CD45 + F4/80+GFP+ cells. Phagocytosis rates were measured dynamically with flow cytometry. In (a, b, e, g), each sample was pooled from 3 individuals. All data are presented as the mean ± s.e.m. The data in (a–c, e, g) were analyzed using one-way ANOVA

Fig 3: Elk-1-Sirpα regulates phagocytosis and CRC progression. a The FACS gating strategy for in vitro tumor phagocytosis is shown. Macrophages were defined as CD45+F4/80+ cells. MC-38 or CT-26 colorectal cancer cells were GFP+. b Macrophage Elk-1 suppressed MC-38 phagocytosis in a Sirpα-dependent manner (n = 4). c Macrophage Elk-1 suppressed Sirpα-dependent CT-26 phagocytosis (n = 4). d Macrophage Elk-1 suppressed Sirpα-dependent MC-38 phagocytosis in vivo. GFP-tagged MC-38 cells (1.0 × 106/100 μl of PBS) were subcutaneously inoculated into 6-week-old WT or TgElk-1 mice. Each mouse was treated (i.v.) with anti-Sirpα antibodies or IgG as a control on days 1, 3, and 5 at a dosage of 100 μg/day. Two weeks later, tumors were collected, and phagocytosis rates were measured (n = 3). e Mice were treated as described in (d), and tumor volume was recorded dynamically (n = 10). f The survival of mice treated as in (d) is shown (n = 10, ***P < 0.005; ns, not significant; Gehan–Breslow–Wilcoxon test). The data in b–e are shown as the mean ± s.e.m (**P < 0.01; ***P < 0.005; ns, not significant; Student’s t-test)

Fig 4: MGLL deficiency in tumor-associated macrophages results in lipid accumulation. a FACS gating strategy for tissue macrophages and lipid measurement. Debris and doublets were removed, and tissue macrophages were then assessed as CD3-CD45R-Gr1-CD45+F4/80+. The average fluorescence degree of macrophages stained by Bodipy (GFP) was measured. b The percentage of TAMs in inoculated tumors. Six-week-old WT mice were subcutaneously inoculated with CT-26, MC-38 or 4T1 cells and the TAMs were quantified at 1st and 3rd week. Each tested sample was pooled from five individual ones. c Lipid staining of macrophages from spleens (TSMs) or tumors (TAMs). Six-week-old mice were subcutaneously inoculated with MC-38 tumors and sacrificed two weeks later. Tissue macrophages were isolated and stained with Bodipy (Green). The nucleus was visualized by DAPI staining (Blue). This experiment was repeated four times. Representative images are displayed. Scale bars, 10 µm. d The lipid levels in TSMs and TAMs. Six-week-old mice were subcutaneously injected with indicated cells. Two weeks later, TSMs and TAMs were isolated for lipid staining with bodipy. The Geometric mean fluorescence intensity (MFI) of Bodipy in each group was measured. Each tested sample was pooled from five individual ones. e A diagram of glycerolipid metabolism. The blue arrow indicates up-regulation, and the orange arrow indicates down-regulation. A gene-microarray analysis was performed on the peritoneal macrophages, which were treated with regular or conditioned medium from CT-26 cells for 24 h. TG triglyceride, DG diglyceride, MG monoglyceride. f Relative mRNA expression of MGLL in TSMs and TAMs. Six-week-old mice were subcutaneously inoculated with indicated tumors and sacrificed two weeks later. TSMs and TAMs were isolated for real-time PCR assays. g Cellular lipid levels were measured in TSMs and TAMs from the MC-38 tumor-bearing mice as described in c. h Relative mRNA expression of MGLL in TSMs and TAMs. Six-week-old WT or myeloid MGLL transgenic (TgMGLL) mice were subcutaneously inoculated with MC-38 tumors for 2 weeks. TSMs and TAMs were then isolated for mRNA assays of MGLL. i Cellular lipid levels in TSMs and TAMs from the MC-38 tumor-bearing mice as described in h. Data in f–i represent the means ± s.e.ms (n = 5, *P < 0.05, **P < 0.01, ***P < 0.005; student’s t-test; ns not significant)

Fig 5: MGLL potentiates proinflammatory cytokine expression in tumor-associated macrophages. a Quantification of M1-like and M2-like TAMs in MC-38 tumors which were subcutaneously inoculated for 3 weeks in WT or TgMGLL mice. Each tested sample was pooled from five individual ones. The M1-like TAMs were assed as CD3-CD45R-Gr1-GFP-CD45+F4/80+CD11c+CD206-; The M2-like TAMs were assed as CD3-CD45R-Gr1-GFP-CD45+F4/80+CD206+CD11c-. The inoculated MC-38 cells were stably marked with GFP. This test was repeated three times. Representative results were displayed. b mRNA expression of cytokines, as indicated, in the TAMs from WT or TgMGLL mice were measured dynamically (n = 5, ***P < 0.005). c MGLL potentiates M1 and blocks M2 activation of macrophages. The BMDMs from WT or TgMGLL mice were treated with IFN? (10 ng ml-1) + LPS (100 ng ml-1) or IL-4 (10 ng ml-1) for 6 h and then subjected to real-time PCR assays (n = 4, **P < 0.01, ***P < 0.005). d mRNA levels of cytokines in the fl/fl or Mac-mgll-KO mice-derived BMDMs treated with IFN? (10 ng ml-1) + LPS (100 ng ml-1) or IL-4 (10 ng ml-1) for 6 h (n = 4, **P < 0.01, ***P < 0.005). Data in b–d represent the means ± s.e.ms. (Student’s t-test)