Fig. 4

Attenuated MMP-9 expression in the infiltrating TAMs in tumor-bearing mice infected with Plasmodium parasites led to inhibition of tumor angiogenesis. a-b Tumor-bearing mice were sacrificed on day 17 after infection with Plasmodium parasites. TAMs were sorted, RNA was extracted, and the gene expression in TAMs was then detected through a gene expression (Roche NimbleGen) analysis. Genes with a ≥ 2-fold difference in expression and a q-value ≤0.05 were selected and mapped (a). A molecular annotation system was used to analyze the functions of these differentially expressed genes and the biological processes involved. Genes associated with tumor angiogenesis and that had a ≥ 2-fold difference in expression and a q-value ≤0.05 were selected for further analysis (b). c-f The relative MMP-9 and IGF-1 mRNA levels in the sorted TAMs (c) and tumor tissue (e) were quantified by qRT-PCR. A Western blotting analysis was performed to visualize the expression of MMP-9 and IGF-1 in the sorted TAMs (d, left panel) and tumor tissue (f, left panel). A quantitative analysis was performed using ImageJ software to quantify the endogenous MMP-9 and IGF-1 levels in the sorted TAMs (d, right panel) and tumor tissue (f, right panel). g A FACS-like tissue cytometer analysis system was used to analyze MMP-9 expression in the cells in the tumor margin by immunohistochemical staining with an antibody reactive to MMP-9 on day 17 after infection with Plasmodium parasites. The double-positive scatter plots represent the MMP-9-positive cells in scattergrams (left panel). The percentages represent the ratio of MMP-9-positive cells to total cells in the selected area. The mean intensity (MI) represents the average DAB intensity (right panel). h Representative images of MMP-9-positive cells identified by immunohistochemical staining with an antibody reactive to MMP-9 on day 17 after infection with Plasmodium parasites (original magnification: 400×). The insets show representative cells expressing MMP-9 (left panel) and the number of cells expressing MMP-9 (right panel). i A comprehensive analysis of the correlation among MMP-9 expression, MVD, and macrophage infiltration in the tumors of tumor-bearing mice on day 17 after Plasmodium infection compared with that in the tumors from uninfected tumor-bearing mice. p < 0.05 was considered significant