CXCL12 promotes CCR7 ligand-mediated breast cancer cell invasion and migration toward lymphatic vessels

Abstract

Chemokines are a family of cytokines that mediate leukocyte trafficking and are involved in tumor cell migration, growth, and progression. Although there is emerging evidence that multiple chemokines are expressed in tumor tissues and that each chemokine induces receptor-mediated signaling, their collaboration to regulate tumor invasion and lymph node metastasis has not been fully elucidated. In this study, we examined the effect of CXCL12 on the CCR7-dependent signaling in MDA-MB-231 human breast cancer cells to determine the role of CXCL12 and CCR7 ligand chemokines in breast cancer metastasis to lymph nodes. CXCL12 enhanced the CCR7-dependent in vitro chemotaxis and cell invasion into collagen gels at suboptimal concentrations of CCL21. CXCL12 promoted CCR7 homodimer formation, ligand binding, CCR7 accumulation into membrane ruffles, and cell response at lower concentrations of CCL19. Immunohistochemistry of MDA-MB-231-derived xenograft tumors revealed that CXCL12 is primarily located in the pericellular matrix surrounding tumor cells, whereas the CCR7 ligand, CCL21, mainly associates with LYVE-1⁺ intratumoral and peritumoral lymphatic vessels. In the three-dimensional tumor invasion model with lymph networks, CXCL12 stimulation facilitates breast cancer cell migration to CCL21-reconstituted lymphatic networks. These results indicate that CXCL12/CXCR4 signaling promotes breast cancer cell migration and invasion toward CCR7 ligand-expressing intratumoral lymphatic vessels and supports CCR7 signaling associated with lymph node metastasis.

Keywords: breast cancer; chemokine; invasion; lymph node; metastasis.

FIGURE 1

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)

FIGURE 2

The effects of CXCL12 on CCR7‐mediated MDA231 cell invasion to the collagen gel. A, Parental MDA231 cells, Meta‐1, and Meta‐2 cells were pretreated with 1 μg/mL CXCL12 and subjected to a collagen gel cell invasion assay under a suboptimal concentration (10 ng/mL) of CCL21. After 15 h incubation, the number of cells on the surface of the collagen matrix and those migrated into the matrix was counted, and the percentage of cell invasion was analyzed. Data represent the mean ± SD of three independent experiments, each performed in triplicate wells (Student's t test; *p < 0.05). B, Meta‐1 cells were pretreated with CXCL12 in the presence of 10 μg/mL anti‐CCR7 mAb (left panel), anti‐CXCR4 mAb, or isotype control and were then applied to a collagen gel containing CCL21

FIGURE 3

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 (α‐SMA)‐positive cancer‐associated fibroblast (green) in MDA231‐derived tumor tissue

FIGURE 4

The effects of CXCL12 on MDA231 cell migration toward lymphatic vessels. A, The fluorescent images of CCR7‐1 cells (green) invading into tissues are shown. Lymphatic networks were visualized by immunostaining with anti‐CD31 antibody (red). Scale bar, 50 µm. B, Distribution of CCR7‐1 cells’ distance from the nearest lymphatic network is shown. In each sample, images including 100–150 cells (n = 5) were captured and subjected to the analysis. The results shown are representative of three independent experiments. C, The percentages of CCR7‐1 cells located less than 10 µm away from the nearest network are analyzed. Data represent the mean ± SD percentage from triplicate images of three independent experiments (one‐way ANOVA; *p < 0.05, **p < 0.01)

FIGURE 5

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 β‐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

FIGURE 6

Localization of CCR7 and CXCR4 in the lipid rafts of ruffling membrane. A, CCR7 (green) and CXCR4 (red) were detected in cells expressing CCR7‐EGFP and CXCR4‐mCherry. GM1 (blue) was detected with Alexa Fluor647‐labeled CTxB. B, CD44 (blue) was detected with a biotinylated anti‐human CD44 mAb followed by Alexa Fluor647–conjugated streptavidin. White arrowheads represent ruffling membrane