The CCL21/CCR7 pathway plays a key role in human colon cancer metastasis through regulation of matrix metalloproteinase-9

(A) Decreased CCR7 mRNA expression in SW480 cells 24h after transfection with CCR7-shRNA vectors. CCR7 mRNA levels were normalized to β-actin mRNA levels. (B) Left, flow cytometric analysis of the frequency of GFP⁺ cells among parental SW480 cells and stably transfected SW480/CCR7⁻ cells. Right, flow cytometric analysis of the frequency of GFP⁺ cells among parental SW480 cells and stably transfected SW480/control cells. (C) Immunoblotting analysis of inhibition of CCR7 protein expression by CCR7-shRNA vectors in stably transfected cell lines (lanes 1 and 2: parental SW480 cells; lanes 3 and 4: SW480/control cells; lanes 5 and 6: SW480/CCR7⁻ cells). Upper panel, quantification of immunoblots.

(A) Invasion assay in SW480/control cells treated with 100ng/ml CCL21. (B) Invasion assay in SW480/CCR7⁻ cells treated with 100ng/ml CCL21. (C) Quantification of invasion assays.

(A) Concentration of MMP-9 in CCL21-treated or untreated SW480/control and SW480/CCR7⁻ cell supernatants as measured by ELISA. (B) Concentration of MMP-9 in SW480/control and SW480/CCR7⁻ xenografted tissues as measured by ELISA. (C) MMP-9 activity in CCL-21 treated or untreated SW480/control and SW480/CCR7⁻ cell supernatants as measured by gelatin zymography (lane 1: untreated SW480/control cells; lane 2: SW480/control cells treated with 100ng/ml CCL21; lane 3: untreated SW480/CCR7⁻ cells; lane 4: SW480/CCR7⁻ cells treated with 100ng/ml CCL21). Lower panel, quantification of gelatin zymography. (D) MMP-9 activity in SW480/control and SW480/CCR7⁻ xenografted tissues as measured by gelatin zymography (lane 1: the SW480/control group; lane 2: the SW480/CCR7⁻ group). Lower panel, quantification of gelatin zymography.

(A) Immunohistochemical staining of CCR7 in xenografts from mice that received SW480/CCR7⁻ cells (SABC; original magnification, 400×). (B) H&E staining of xenografts from mice that received SW480/CCR7⁻ cells (original magnification, 400×). (C) Immunohistochemical staining of CCR7 in xenografts from mice that received SW480/control cells (SABC; original magnification, 400×). (D) H&E staining of xenografts from mice that received SW480/control cells (original magnification, 400×). (E) Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in regional lymph nodes (SABC; original magnification, 400×). (F) H&E staining of regional lymph nodes (SABC; original magnification, 400×). Immunostaining showed that CCR7 was located in the cell membrane and PCNA was located in the cell nucleus.

(A) Growth curve of xenografted tumours in mice that received SW480/control cells or SW480/CCR7⁻ cells. (B) Kaplan–Meier analysis showed that the SW480/control group did not survive as long as the SW480/CCR7⁻ group (group 1=SW480/control group; group 2=SW480/CCR7⁻ group).

(A) Fluorescence microscopy of SW480/control cells (Nikon, original magnification, 200×). (B) Fluorescence microscopy of SW480/CCR7⁻ cells (Nikon, original magnification, 200×). (C) Whole-body fluorescence image of a mouse that received SW480/control cells (Image Station 2000MM, original magnification, green fluorescence light). (D) Whole-body fluorescence image of a mouse that received SW480/CCR7⁻ cells (Image Station 2000MM, original magnification, green fluorescence light).