Oxidized low-density lipoprotein stimulates CD206 positive macrophages upregulating CD44 and CD133 expression in colorectal cancer with high-fat diet

Figure 1 Increased expression of oxidized low-density lipoprotein and CD206 in the stroma of colorectal cancer tissue. A: Immunofluorescence (IF) images of oxidized low-density lipoprotein (ox-LDL) expression in colorectal tissues (scale bars represent 50 μm); B: Double IF images of CD206-ox-LDL expression in colorectal tissues (scale bars represent 50 μm); C: IF images of LOX-1 expression in colorectal tissues (scale bars represent 50 μm); D: Double IF images of CD206-LOX-1 expression in colorectal tissues (scale bars represent 50 μm); E: Histogram of ox-LDL expression in colorectal tissues based on IF results in Figure 1A; F: Quantification of CD206 and ox-LDL positive cells in colorectal tissues based on IF results in Figure 1B; G: Histogram of LOX-1 expression in colorectal tissues based on IF results in Figure 1C; H: Quantification of CD206 and LOX-1 positive cells in colorectal tissues based on IF results in Figure 1D; I: Double IF images of inducible nitric oxide synthase (iNOS)-F4/80 expression in colorectal tissues (scale bars represent 50 μm); J: Double IF images of CD206-F4/80 expression in colorectal tissues (scale bars represent 50 μm); K: Quantification of iNOS positive macrophages in colorectal tissues based on IF results in Figure 1I; L: Quantification of CD206 positive macrophages in colorectal tissues based on IF results in Figure 1J. Data are shown as the mean ± SD. Statistical analyses were conducted using an unpaired t-test. The boxed area is enlarged in the bottom right corner. ^bP < 0.01, ^cP < 0.001. ox-LDL: Oxidized low-density lipoprotein; iNOS: Inducible nitric oxide synthase.

Figure 2 Establishment of a high-fat diet mouse model. A: Length of colorectal segment of controls and high-fat diet (HFD) mice; B: Quantification of the length of colorectal segment of controls and HFD mice; C: Histopathological colorectal tissue sections of controls and HFD mice (scale bars represent 50 μm). Data are shown as the mean ± SD. Statistical analyses were conducted using an unpaired t-test. ^bP < 0.01. HFD: High-fat diet; H-E stain: Hematoxylin-eosin staining; iNOS: Inducible nitric oxide synthase.

Figure 3 High-fat diet leads to increased expression of oxidized low-density lipoprotein and an increase of CD206 positive macrophages in mouse colorectal stroma. A: Double immunofluorescence (IF) images of inducible nitric oxide synthase (iNOS)-F4/80 expression in colorectal tissues of controls and high-fat diet (HFD)-fed mice (scale bars represent 50 μm); B: Double IF images of inducible nitric oxide synthase CD206-F4/80 expression in colorectal tissues of controls and HFD-fed mice (scale bars represent 50 μm); C: Double IF images of CD206-oxidized low-density lipoprotein (ox-LDL) expression in colorectal tissues of controls and HFD-fed mice (scale bars represent 50 μm); D: Double IF images of CD206-LOX-1 expression in colorectal tissues of controls and HFD-fed mice (scale bars represent 50 μm); E: Quantification of iNOS positive macrophages in colorectal tissues based on IF results in Figure 3A; F: Quantification of CD206 positive macrophages in colorectal tissues based on IF results in Figure 3B; G: Quantification of CD206 and ox-LDL positive cells in colorectal tissues based on IF results in Figure 3C; H: Quantification of CD206 and LOX-1 positive cells in colorectal tissues based on IF results in Figure 3D. Data are shown as the mean ± SD. Statistical analyses were conducted using an unpaired t test. The boxed area is enlarged in the bottom right corner. ^aP < 0.05, ^cP < 0.001. ox-LDL: Oxidized low-density lipoprotein; HFD: High-fat diet; iNOS: Inducible nitric oxide synthase.

Figure 4 Continuous stimulation with oxidized low-density lipoprotein promotes CD206+ macrophages. A: Quantitative polymerase chain reaction (qPCR) detection of mRNA expression of CD206 and inducible nitric oxide synthase (iNOS) in THP-1 cells after oxidized low-density lipoprotein (ox-LDL) stimulation; B: Quantification of CD206-positive cells in RAW 264.7 cells based on IF results in Figure 4C; C: Cell fluorescence images of CD206 expression in RAW 264.7 cells after ox-LDL stimulation (scale bars represent 25 μm); D: qPCR detection of the expression of LOX-1 mRNA in THP-1 cells after transfection with LOX-1 small interfering RNA of different sequences; E: qPCR detection of the expression of iNOS and CD206 mRNA in THP-1 cells with low expression of LOX-1 stimulated with ox-LDL; F: qPCR detection of the expression of interleukin IL-4, IL-10, IL-13, and tumor necrosis factor-β mRNA in THP-1 cells after 72 h of ox-LDL stimulation. Data are shown as the mean ± SD. Statistical analyses were conducted using an unpaired t test (Figure 4A, 4D, 4E, and 4F) or one-way analysis of variance followed by Dunnett’s multiple comparison test (Figure 4B). The boxed area is enlarged in the bottom right corner. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. ox-LDL: Oxidized low-density lipoprotein; iNOS: Inducible nitric oxide synthase; IL: Interleukin; TNF: Tumor necrosis factor; si-NC: Small interfering RNA negative control.

Figure 5 Macrophages promote the expression of CD44 in LoVo cells in an oxidized low-density lipoprotein-stimulated hyperlipidemic microenvironment. A: Flow diagram of the experimental result in Figure 5C; B: Flow diagram of the experimental result in Figure 5E; C: Protein expression of CD44 and CD133 in LoVo cells after oxidized low-density lipoprotein (ox-LDL) or THP-1 + ox-LDL stimulation detected by western blot; D: Quantification of CD44 and CD133 expression in LoVo cells based on western blot results in Figure 5C; E: Protein expression of CD44 and CD133 in LoVo cells after THP-1 + ox-LDL stimulation detected by western blot. The THP-1 cells were transfected with si-LOX-1 or small interfering RNA negative control; F: Quantification of CD44 and CD133 expression in LoVo cells based on western blot results in Figure 5E. Data are shown as the mean ± SD. Statistical analyses were conducted using an unpaired t test. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. ox-LDL: Oxidized low-density lipoprotein; si-NC: Small interfering RNA negative control.