Fig 2: The effects of CXCL12 on CCR7 homodimerization, ligand binding, and the plasma membrane localization. A, CCR7 homodimer formation after treatment with CXCL12 in MDA231 cells. The levels of bioluminescence signals are shown for cells transfected with combinations of CCR7-CGLuc and CCR7-NGLuc in the presence of BSA or CXCL12. A representative experiment from at least three independent experiments is shown. Data represent mean ± SD (n = 4). *p < 0.05 by Student's t test. B, Confocal microscopic images of MDA-R7/X4 cells treated with or without CXCL12 for 30 min, fixed, and stained with recombinant CCL19-Fc, biotin-anti-human IgG, and Alexa Fluor 647–conjugated streptavidin. The expressions of CCR7-EGFP (green) and CCL19-Fc (magenta) are shown. Insets show high magnification of membrane ruffles. The images were analyzed to obtain the percentage of the cells with CCL19-Fc binding. Scale bar, 30 µm. ***p < 0.01 by Student's t test. C, Luciferase complementation assay using CCR7-Nluc and ß-arrestin 2-Cluc. CCL21 was added 30 min after treatment of cells with CXCL12 or a solvent, and the luminescence signal was measured at each time point indicated. Error bars indicate standard error of the mean. D, MDA-R7/X4 cells were stimulated with or without CXCL12 for 30 min, fixed, and stained with Alexa647-phalloidin. Fluorescence images were captured by a confocal microscopy. The expression of CCR7 (green), CXCR4 (red), and F-actin (white) are shown. White arrowheads show CXCR4- and CCR7-enriched membrane ruffles. Scale bar, 10 µm. E, The average score of CXCR4/CCR7-enriched membrane ruffles of each cell. The cells were treated with the indicated concentrations of CXCL12 in the presence or absence of CXCR4-specific antagonist AMD3100. The ruffling index was evaluated as 0 = no ruffles, 1 = ruffles covering less than 25% of the peripheral area, and 2 = ruffles covering more than 25% of the peripheral area. The statistical difference was determined by one-way ANOVA and depicted with ***p < 0.01

Fig 3: The effects of CXCL12 on CCR7 ligand–dependent MDA231 cell migration. A, CXCL12-induced cell migration assay was performed using MDA231 cells in the presence or absence of CXCL12 at indicated concentrations. The number of migrated cells to the lower wells in response to CXCL12 was analyzed. Graphs represent means ± SD of the number of cells in eight random fields per membrane in triplicate assay. * p < 0.05 by Student's t test. B, CCL21-induced cell migration of MDA-MB-231. The assay was performed in the presence of CCL21 in the lower wells at indicated concentrations. One-way ANOVA; *p < 0.05, **p < 0.01. C, The effect of CXCL12 on MDA231 cell migration induced by a suboptimal concentration of CCL21 (50 ng/mL). The assay was performed under the indicated concentrations of CXCL12 and CCL21 added to the upper or the lower wells. One-way ANOVA; **p < 0.01. D, The effect of CXCL12 on CCL21-induced cell migration in the presence of anti-CXCR4 neutralizing antibody. CXCL12 (2.5 µg/mL) was added to the upper wells with or without anti-CXCR4 mAb or an isotype control immunoglobulin (10 µg/mL). Mean ± SD (Student's t test; *p < 0.05, **p < 0.01, n = 3)

Fig 4: Expression of CXCL12 and CCL21 in MDA231-derived tumor. A, Parental MDA231 cells (1 × 106) were injected into the thoracic mammary fat pad of female SCID mice at 9 weeks of age (n = 3). A sham operation was performed on the contralateral side. Twelve weeks after injection, primary tumors were dissected. Serial sections of fresh-frozen tumor specimens from tumor xenograft were subjected to immunohistochemical staining with anti-CXCL12 (green, upper panel) and anti-human p53 mAb (red, upper panel) or anti-LYVE-1 (green, lower panel) and anti-CCL21 (red, lower panel) antibodies. Scale bar, 100 µm. B, Upper row: RT-PCR analysis of CXCL12 and CCL21 in the parental MDA231 cell line, MDA231-derived tumor tissue, and C57BL/6 mouse lymph node using oligonucleotide primer pairs common to mouse and human sequences. GAPDH were used as an endogenous control. Bottom: the tumor-derived nucleotide sequence of the RT-PCR products was determined and compared with the species-specific nucleotide positions of the CXCL12 and CCL21 genes. C, Localization of CXCL12 expression (red) and alpha-smooth muscle actin (a-SMA)-positive cancer-associated fibroblast (green) in MDA231-derived tumor tissue