Chemokines have been implicated as key contributors of non–small cell lung cancer (NSCLC) metastasis. However, the role of CXCR7, a recently discovered receptor for CXCL12 ligand, in the pathogenesis of NSCLC is unknown. To define the relative contribution of chemokine receptors to migration and metastasis, we generated human lung A549 and H157 cell lines with stable knockdown of CXCR4, CXCR7, or both. Cancer cells exhibited chemotaxis to CXCL12 that was enhanced under hypoxic conditions, associated with a parallel induction of CXCR4, but not CXCR7. Interestingly, neither knockdown cell line differed in the rate of proliferation, apoptosis, or cell adherence; however, in both cell lines, CXCL12-induced migration was abolished when CXCR4 signaling was abrogated. In contrast, inhibition of CXCR7 signaling did not alter cellular migration to CXCL12. In an in vivo heterotropic xenograft model using A549 cells, expression of CXCR4, but not CXCR7, on cancer cells was necessary for the development of metastases. In addition, cancer cells knocked down for CXCR4 (or both CXCR4 and CXCR7) produced larger and more vascular tumors as compared with wild-type or CXCR7 knockdown tumors, an effect that was attributable to cancer cell–derived CXCR4 out competing endothelial cells for available CXCL12 in the tumor microenvironment. These results indicate that CXCR4, not CXCR7, expression engages CXCL12 to mediate NSCLC metastatic behavior.

Implications: Targeting CXCR4-mediated migration and metastasis may be a viable therapeutic option in NSCLC. Mol Cancer Res; 12(1); 38–47. ©2013 AACR.

This article is featured in Highlights of This Issue, p. 1

Non–small cell lung cancer (NSCLC) is the most commonly diagnosed cancer worldwide and is the leading cause of cancer death (1, 2). Most patients with NSCLC die of metastatic disease, typically involving the mediastinal lymph nodes, other areas of the lungs, adrenal glands, brain, liver, and the bone marrow. Understanding the biology that underlies NSCLC metastasis formation is therefore important.

The CXCL12–CXCR4 biological axis is an evolutionarily preserved mechanism essential to homing of progenitor cells during organogenesis, retention of precursor cells in the bone marrow, and their recruitment to sites of tissue repair. In addition to its physiologic roles, this pathway has been implicated in metastasis formation in diverse cancers: many human cancer cells, including NSCLC squamous cell and adenocarcinoma, express CXCR4. In addition, the expression of CXCR4 on cancer cells is upregulated in hypoxia, as might be encountered in the tumor microenvironment (3, 4). CXCL12 is constitutively expressed in tissues that are targets of metastases, and a substantial body of literature supports the notion that interrupting this ligand–receptor interaction inhibits cancer cell migration and metastasis formation (5, 6). CXCR4 and CXCL12 were thought to be an exclusive ligand–receptor pair until the discovery of CXCR7, an alternative receptor for CXCL12 that also binds CXCL11 (7). CXCR7 is expressed on subsets of T and B cells, activated endothelial cells, fetal hepatocytes, as well as many cancer cells (6). In the context of malignancy, the role of CXCR7 and its functional relationship to CXCR4, CXCL12, and CXCL11 are incompletely defined. In diverse experimental settings and using different cancer models, some studies have reported that CXCR7 acts exclusively as a decoy receptor (8), others report that it mediates migration to CXCL12 or CXCL11 (9–11), and still others indicating that it mediates cancer proliferation, apoptosis or adhesion (12–15), or angiogenesis (15, 16).

We sought to define the relative contribution of CXCR4 and CXCR7 in NSCLC migration in vitro and metastasis formation in vivo by generating human A549 and H157 cell lines that are stably knocked down for CXCR4, CXCR7, or both. We report that migration and metastasis of NSCLC cells is dependent on the interaction of CXCL12 with CXCR4, but is independent of the interaction of CXCL11 or CXCL12 with CXCR7.

Cell culture

A549 and H157 cell lines were maintained in RPMI 1640 media (Invitrogen) supplemented with 1 mmol/L l-glutamine, 25 mmol/L HEPES, 1× Pen/Strep, and 10% FBS, hereafter referred to as RPMI complete media. In some experiments, cells were transferred to RPMI with 1% FBS (hereafter referred to as starvation media) and incubated in modular incubator chambers (Billups-Rothenberg) under normoxic (21% O2, 74% N2, 5% CO2) or hypoxic (1% O2, 94% N2, 5% CO2) culture conditions. In some experiments, 10 μg/mL anti-human CXCR4 (MAB170; R&D systems) or CXCR7 polyclonal antibodies were added to the media. Parental cell lines were purchased from American Type Culture Collection at the beginning of the project. At the conclusion of the experiments, all parental and modified cell lines were authenticated by short tandem repeat DNA profiling and comparison to published ATCC profiles (Genetica).

Reagents

Polyclonal rabbit anti-human CXCR7 was produced by the immunization of a rabbit with a 21-mer peptide (MDLHLFDYAEPGNFSDISWPC) constituting the amino terminus of human CXCR7 (hCXCR7). The rabbit was immunized in multiple intradermal sites with CFA followed by 3 boosts in IFA. Direct ELISA was used to evaluate antisera titers, and serum was used for Western blot, and neutralization assays when titers had reached greater than 1:1,000,000. The specificity of anti-hCXCR7 Ab was assessed by Western blot analysis against cells expressing hCXCR7 and a panel of other human and murine recombinant cytokines. Polyclonal goat anti-human CXCL12 antibodies were produced as previously described (17).

Flow cytometry

Cells were stained with anti-hCXCR4 or anti-hCXCR7 antibodies or appropriate controls followed by APC-conjugated secondary antibodies (A-21463; BD Bioscience). In some experiments, cells were also stained with CD45-PerCP (557513; BD Bioscience) and rabbit anti-Factor VIII–related antigen or appropriate control (F3520 and I-8140; Sigma), followed by secondary Alexa Fluor 610-R-phycoerythin goat anti-rabbit immunoglobulin G (IgG; A20981; Invitrogen). To quantify metastases, organs were digested as previously described (3, 18–20). Organs from animals inoculated with Green fluorescence protein (GFP)-transfected cancer cells were compared with organs from healthy animals, and the proportions of GFP-expressing cells in organ digests were quantified. Samples were analyzed on a fluorescence-activated cell sorting (FACS) Canto II flow cytometer (Becton Dickinson) using FACS Diva software.

Establishment of stable knockdowns of CXCR4, CXCR7, and both CXCR4 and CXCR7 in A549 and H157 NSCLC cell lines

GFP was transfected into parental A549 and H157 cell lines using EmGFP vector (K493600; Invitrogen). GFP-transfected cell lines had comparable migratory capacity to CXCL12 as compared with control cells that were not transfected with the vector. Block-iT Pol II miR RNAi Expression Vector Kit (K494300; Invitrogen) was used to generate knockdown cells. For CXCR4 knockdown cells, human CXCR4 miRNA oligo was designed (top strand, 5′-TGC TGT TGA CTG TGT AGA TGA CAT GGG TTT TGG CCA CTG ACT GAC CCA TGT CAT ACA CAG TCA A-3′; bottom strand, 5′-CCT GTT GAC TGT GTA TGA CAT GGG TCA GTC AGT GGC CAA AAC CCA TGT CAT CTA CAC AGT CAA C-3′) using Invitrogen miRNA Designer software. These 2 RNAi oligos were annealed (20 μmol/L condition, 95°C for 4 minutes) followed by ligation into pcDNA 6.2-GW/miR vector. Ligated product was transformed into TOP10 competent Escherichia coli. Spectinomycin (50 μg/mL)-selected transformant was grown in Lysogeny broth (LB) media to purify CXCR4 miRNA-containing pcDNA 6.2-GW/miR (pcDNA 6.2-GW/CXCR4) vector. Purified pcDNA 6.2-GW/CXCR4 vector was mixed with pDONR 221 vector and pLenti6/V5-DEST vector to generate pLenti6/V5-GW/CXCR4 expression construct. Lentiviral stock, containing the packaged pLenti6/V5-GW/CXCR4 expression construct, was produced by cotransfecting the ViraPower Packaging Mix and pLenti6/V5-GW/CXCR4 expression construct into 293FT Producer cell line. Transduction of this lentiviral construct into A549 and H157 cell line was performed in the presence of polybrene, followed by selection with blasticidin. For CXCR7 knockdown cells, CXCR7 miRNA oligo was designed (top strand, 5′-TGC TGA AGA TGA GGT GTG TGA CTT TGG TTT TGG CCA CTG ACT GAC CAA AGT CAC ACC TCA TCT T-3′; bottom strand, 5′-CCT GAA GAT GAG GTG TGA CTT TGG TCA GTC AGT GGC CAA AAC CAA AGT CAC ACA CCT CAT CTT C-3′) and processed for lentiviral transduction as described earlier. To produce cell lines with knockdown of both CXCR4 and CXCR7, CXCR4 knockdown cells were further transfected with CXCR7 miRNA followed by selection by reverse transcriptase (RT)-PCR. The resulting knockdown cell lines had greatly diminished expression of CXCR4 and CXCR7 mRNA and protein as compared with nontransfected parental cell lines (Supplementary Fig. S1A–S1D). On flow cytometry, both A549 and H157 cell lines displayed cell surface expression of CXCR4 and CXCR7. CXCR4 expression was enhanced under hypoxic conditions as previously reported (3) whereas CXCR7 expression was unaffected. In both A549 and H157 CXCR4 knockdown lines, cell surface expression of CXCR4 was abolished without detectable effect on CXCR7 under both normoxic and hypoxic conditions; similarly, A549 and H157 CXCR7 knockdown lines had little detectable cell surface expression of CXCR7 but intact CXCR4 expression. A549 and H157 lines with both CXCR4 and CXCR7 knockdown had little detectable cell surface expression of either molecule under normoxic and hypoxic culture conditions (Supplementary Fig. S1E–S1H).

Chemotaxis assays

Chemotaxis assays were performed using 12-well chemotaxis chambers and 5 μm pore size filters (Neuroprobe) coated with 5 μg/mL fibronectin. Human CXCL12 or CXCL11 (300-46 and 300-28B; Peprotech) were added to the lower wells and 105 cells added to the upper wells and incubated for 6 hours at 37°C. Filters were then removed, fixed in methanol, stained in 2% Toluidine blue, and the cells that had migrated through to the underside of the filters was counted in 5 separate fields of view under ×400 magnification. For transendothelial migration assays, human lung microvascular endothelial cells were first seeded onto transwell plate inserts with 8 μm pore size, CXCL12 was added to the lower well, and 105 A549 or H157 cells added to upper wells. After incubation for 6 hours at 37°C, migrated cells were counted under fluorescence microscope. For the competitive chemotaxis assay, 105 human lung microvascular endothelial cells were seeded onto 8 μm pore size transwell inserts. The cells were allowed to adhere over night, the media was changed to starvation media, and 30 ng/mL of CXCL12 was added to the lower wells containing the A549 cells. The inserts were placed in the lower wells and incubated for 6 hours at 37°C. The inserts were then removed, fixed in methanol, stained in 2% Toluidine blue, and the cells that had migrated through to the underside of the insert was counted in 5 separate fields of view under ×400 magnification.

Western blotting

Immunoblotting for CXCR4, CXCR7, and factor VIII–related antigen were performed as previously described (17).

Heterotopic human NSCLC-SCID mouse chimeras

We used a heterotopic model of human NSCLC in severe combined immunodeficient (SCID)-beige mice, as previously described (17). Age-matched female 6- to 8-week-old C57BL/6 mice were purchased from Jackson Laboratories and were maintained under pathogen-free conditions and in compliance with institutional animal care regulations at the University of Virginia animal care facility. Briefly, 106 A549 cells in 100 μL were injected subcutaneously into one flank of 6-week-old female CB17-SCID beige mice (Charles River) and tumors size monitored weekly. In some experiments, tumor-bearing SCID mice were injected intraperitoneally with 500 μL of neutralizing anti-CXCL12 or control serum, containing 10 mg goat IgG, 3 times per week, starting at the time of xeno-engraftment. This amount of anti-CXCL12 serum was found to specifically neutralize 1 μg of murine CXCL12 protein in bioassays without cross-reacting to a panel of other chemokines (17). Animals were euthanized at a designated times, and organs and blood samples were processed as described previously (3, 18–20). At the time of tissue harvest, primary tumors were removed from animals, their orthogonal diameters measured using Thorpe calipers (Biomedical Research Instruments), and tumor volume calculated as [volume = (d1 × d2 × d3) × 0.5236], where dn represents orthogonal diameter measurements.

Immunolocalization

Immunohistochemistry of factor VIII–related antigen, Ki67 and GFP were performed as previously described (18, 21). Briefly, paraffin-embedded tumor sections were deparaffinized, subjected to antigen retrieval (HK080-9K; Biogenex), fixed for 20 minutes in 1:1 absolute methanol and 3% H2O2, rinsed in PBS, and then blocked (HK085-5K; Biogenex) at room temperature for 30 minutes followed by Avidin/Biotin blocking (SP-2001; Vector Laboratories). Then, a 1:800 dilution of anti-factor VIII–related antibody, anti-Ki67 Ab-4 rabbit polyclonal antibody (RB-1510; Thermo Scientific), or control rabbit IgG or goat anti-GFP (Vector Laboratories; SP-0702), or control goat IgG (Sigma; I5356) were added and slides were incubated for 30 minutes. Slides were then rinsed with PBS, overlaid with biotinylated goat anti-rabbit IgG (PK-6101; Vector Laboratories), and incubated for an additional 30 minutes. Slides were rinsed twice with PBS, treated with streptavidin-conjugated peroxidase for 30 minutes, washed 3 times with PBS, incubated for 5 to 10 minutes in the substrate chromogen 3,3′-diaminobenzidine solution (SK-4100; Vector Laboratories) to allow color development and then rinsed with water. Finally, the slides were counterstained with hematoxylin. For GFP immunofluorescent staining, a 1:50 dilution of either mouse IgG or anti-GFP (SC-9996; Santa Cruz) was added and slides were incubated for 30 minutes. Slides were then rinsed with PBS, overlaid with Alexa Fluor 488 anti-mouse IgG (A11001; Invitrogen), and incubated for an additional 30 minutes. Slides were again washed 2 times with PBS, and then coversliped using aqueous mounting media. For TUNEL staining, DeadEnd Colorimetric TUNEL System (G7130; Promega) was used, according to manufacturer specifications. For image analysis, we prepared 5 histological sections per tumor, from 5 mice per condition, and photographed 10 different fields from each section at ×200 magnification using a Zeiss Image A1 microscope with an inline AxioCam HRc camera interfaced with a PC. Images were captured with AxioVision software (v.4.8) and analyzed using NIH ImageJ software. Data were expressed as percent of positive pixels per field.

Statistical analysis

The animal studies involved 5 to 10 mice bearing A549 tumors for each group. Data were analyzed on a personal computer using the Statview 5.0 statistical package (Abacus Concepts). Comparisons were evaluated by the ANOVA test with the post hoc analysis Bonferroni/Dunn. Data were expressed as mean ± SEM and differences were considered statistically significant if P values were less than 0.05.

CXCR4 mediates chemotaxis of NSCLC cells to CXCL12

We began by examining the relative contribution of CXCR4 and CXCR7 to chemotaxis of A549 and H157 cells to varying concentrations of CXCL12. Under normoxic culture conditions, both cell lines displayed chemotaxis to concentration of CXCL12 between 10 to 300 ng/mL; this effect was significantly attenuated in both cell lines with CXCR4 knockdown and combined CXCR4/7 knockdown but was unaffected in CXCR7 knockdown lines (Fig. 1A and D). Under hypoxic culture conditions, chemotaxis of cell lines with unmanipulated CXCR4/7 expression to CXCL12 was enhanced to concentrations as low as 1 ng/mL; this effect was again greatly diminished in cell lines knocked down for CXCR4 and CXCR4/7 but was unaffected in lines knocked down for CXCR7 alone (Fig. 1B and E).

Figure 1.

CXCR4 mediates migration of NSCLC cells. A549 (A–C) and H157 (D–E) cells were serum-starved and cultured in normoxic or hypoxic culture conditions for 24 hours before assessing chemotaxis to indicated concentrations of CXCL12 for 6 hours under normoxic (A and D) or hypoxic (B and C and E and F) culture conditions. Results are representative of 3 experiments; data shown represent mean ± SEM of cells per high-power field of n = 3 for each group. WT, nontransfected cells; R4KD, cells with stable knockdown of CXCR4; R7KD, cells with stable knockdown of CXCR7; DbKD, cells with stable knockdown of CXCR4 and CXCR7; *, P < 0.05 in comparison of CXCR4 knockdown and CXCR4/CXCR7 knockdown cells as compared with nontransfected cells; #, P < 0.05 compared with chemotaxis to CXCL12 without anti-CXCR4 exposure.

Figure 1.

CXCR4 mediates migration of NSCLC cells. A549 (A–C) and H157 (D–E) cells were serum-starved and cultured in normoxic or hypoxic culture conditions for 24 hours before assessing chemotaxis to indicated concentrations of CXCL12 for 6 hours under normoxic (A and D) or hypoxic (B and C and E and F) culture conditions. Results are representative of 3 experiments; data shown represent mean ± SEM of cells per high-power field of n = 3 for each group. WT, nontransfected cells; R4KD, cells with stable knockdown of CXCR4; R7KD, cells with stable knockdown of CXCR7; DbKD, cells with stable knockdown of CXCR4 and CXCR7; *, P < 0.05 in comparison of CXCR4 knockdown and CXCR4/CXCR7 knockdown cells as compared with nontransfected cells; #, P < 0.05 compared with chemotaxis to CXCL12 without anti-CXCR4 exposure.

Close modal

To confirm that CXCR4 is necessary for migration of NSCLC cell lines, we tested the effect of Ab-mediate neutralization of CXCR4 on chemotaxis of the cancer cells to CXCL12. As observed previously, both A549 and H157 lines exhibited brisk chemotaxis to 10 ng/mL CXCL12 under hypoxic culture conditions; this effect was diminished in cell lines knocked down for CXCR4 and CXCR4/7, but not lines knocked down for CXCR7 alone (Fig. 1C and F). In addition, chemotaxis of A549 and H157 lines with unmanipulated CXCR4/7 expression and those of lines knocked down for CXCR7 was abolished in the presence of neutralizing anti-CXCR4 antibodies, again demonstrating the critical role of CXCR4 in migration of these cells to CXCL12.

CXCR7 is dispensable for migration of NSCLC cell lines to CXCL11 and CXCL12

We used several strategies to assess the contribution of CXCR7 to migration of NSCLC cells. First, we tested the effect of a neutralizing anti-CXCR7 antibody on migration of NSCLC cells in chemotaxis studies. Neutralization of CXCR7 did not influence the chemotaxis of A549 or H157 cells with unmanipulated CXCR4/7 expression to CXCL12, whereas cell lines knocked down for CXCR4 had diminished chemotaxis to CXCL12, again unaffected by CXCR7 neutralization (Supplementary Fig. S2). We next used a transient siRNA approach to attenuate CXCR7 expression and assess its role in cell migration. Nontransfected A459 and H157 cells and cells with stable CXCR4 knockdown were subjected to CXCR7 siRNA transfection and were found to have efficient inhibition of CXCR7 expression (Supplementary Fig. S2B), but showed no change in their migratory activities toward CXCL12 (Supplementary Fig. S2C).

Because CXCL11 is another ligand for CXCR7, we next tested the effect of CXCL11 on chemotaxis of NSCLC cells. For both A549 and H157 lines, CXCL11 had no detectable influence on the migration of cells under normoxic and hypoxic culture conditions; this finding was unaffected in cells with stable knockdowns of CXCR4, CXCR7, or both. Furthermore, concentrations of CXCL11 between 1 and 300 ng/mL did not interfere with CXCR4-dependent migration of A549 and H157 cells induced by 10 ng/mL CXCL12 (Fig. 2). These results suggest that CXCR7 does not contribute to the migration of NSCLC cells under our experimental conditions.

Figure 2.

CXCL11 does not interfere with the CXCL12/CXCR4 chemotactic behavior of NSCLC cells. A549 (A and B) and H157 (C and D) cells were serum-starved and cultured in normoxic or hypoxic culture conditions for 24 hours before chemotaxis assays. Cells were then subjected to chemotaxis assays in response to the indicated concentrations of CXCL11 (1–300 ng/mL) with or without 10 ng/mL CXCL12 for 6 hours under normoxic (A and C) or hypoxic (B and D) culture conditions. Results are representative of 3 experiments; data shown represent mean ± SEM of cells per high-power field of n = 3 for each group. *, P < 0.05 in comparison of CXCR4 knockdown and CXCR4/CXCR7 knockdown cells as compared with nontransfected cells.

Figure 2.

CXCL11 does not interfere with the CXCL12/CXCR4 chemotactic behavior of NSCLC cells. A549 (A and B) and H157 (C and D) cells were serum-starved and cultured in normoxic or hypoxic culture conditions for 24 hours before chemotaxis assays. Cells were then subjected to chemotaxis assays in response to the indicated concentrations of CXCL11 (1–300 ng/mL) with or without 10 ng/mL CXCL12 for 6 hours under normoxic (A and C) or hypoxic (B and D) culture conditions. Results are representative of 3 experiments; data shown represent mean ± SEM of cells per high-power field of n = 3 for each group. *, P < 0.05 in comparison of CXCR4 knockdown and CXCR4/CXCR7 knockdown cells as compared with nontransfected cells.

Close modal

CXCR4 but not CXCR7 mediates the migration of NSCLC cells across endothelial barriers

A key feature of metastasis formation is the ability of cancer cells to cross endothelial barriers. We therefore tested the role of CXCR4 and CXCR7 in migration of NSCLC cells in modified in vitro chemotaxis assays that incorporate migration across a human microvascular endothelial cell barrier. As expected, A549 and H157 cells migrated across the endothelial barrier to CXCL12 under normoxic conditions and trans-endothelial migration was enhanced under hypoxic culture conditions (Fig. 3). In addition, migration of A549 and H157 cell lines knocked down for CXCR4 and CXCR4/7 toward CXCL12 was significantly diminished, but lines knocked down for CXCR7 alone had comparable migration to CXCL12 as nontransfected cells (Fig. 3). Similar to findings of chemotaxis in the absence of endothelial cells, Ab-mediated neutralization of CXCR4 inhibited the migration of A549 and H157 cells with intact CXCR4 expression to CXCL12 (Fig. 3C and F).

Figure 3.

CXCR4 mediates migration of NSCLC cells across endothelial cell barriers. A549 (A–C) and H157 (D–F) cells were serum-starved and cultured in normoxic or hypoxic culture conditions for 24 hours before assessing trans-endothelial migration to indicated concentrations of CXCL12 for 6 hours under normoxic (A and D) or hypoxic (B–C and E–F) culture conditions. Results are representative of 3 experiments; data shown represent mean ± SEM of cells per high-power field of n = 3 for each group. *P < 0.05 in comparison of CXCR4 knockdown and CXCR4/CXCR7 knockdown cells to cells with intact expression of CXCR4 and CXCR7; #P < 0.05 compared with chemotaxis to CXCL12 without anti-CXCR4 exposure.

Figure 3.

CXCR4 mediates migration of NSCLC cells across endothelial cell barriers. A549 (A–C) and H157 (D–F) cells were serum-starved and cultured in normoxic or hypoxic culture conditions for 24 hours before assessing trans-endothelial migration to indicated concentrations of CXCL12 for 6 hours under normoxic (A and D) or hypoxic (B–C and E–F) culture conditions. Results are representative of 3 experiments; data shown represent mean ± SEM of cells per high-power field of n = 3 for each group. *P < 0.05 in comparison of CXCR4 knockdown and CXCR4/CXCR7 knockdown cells to cells with intact expression of CXCR4 and CXCR7; #P < 0.05 compared with chemotaxis to CXCL12 without anti-CXCR4 exposure.

Close modal

CXCR4-deficient NSCLC lines produce large vascular primary tumors with reduced metastases in vivo

To assess the biological relevance of our in vitro findings, we tested GFP-transfected A549 cells and A549 cells with stable knockdowns of CXCR4, CXCR7, and CXCR4 and CXCR7 in a heterotropic xenotransplantation animal model. Mice bearing CXCR4 and CXCR4/7 knockdown cancers were unexpectedly found to have dramatically larger primary tumors after only 4 weeks, necessitating animal euthanasia per animal welfare guidelines. In contrast, cells with intact CXCR4/CXCR7 expression and CXCR7 knockdown cells produced tumors that did not differ significantly in size; the tumors were at the limit of in vivo detection after 4 weeks and, after 9 weeks, were approximately a quarter of the size of CXCR4 and CXCR4/7 knockdowns tumors at 4 weeks (Fig. 4). We reasoned that the observed differences in tumor size may be the result of differences in the rate of proliferation or apoptosis of cancer cells with disrupted CXCR4 or CXCR7 expression. However, knockdown of CXCR4 or CXCR7 in A549 and H157 lines did not result in altered proliferation of the cell lines as compared with respective control cell lines, irrespective of the presence of CXCL12 in the media (Supplementary Fig. S3A and S3B). Similarly, rate of apoptosis of tumor cells in response to staurosporine and methotrexate was unaffected by knockdown of CXCR4, CXCR7, or both in A549 or H157 cells and was unrelated to the presence of CXCL12 (Supplementary Fig. S3C and S3D).

Figure 4.

Role of CXCR4 and CXCR7 expression on primary A549 tumor size in a heterotopic xenograft murine cancer model. Data represent the mean ± SEM of n = 7 to 9 mice per group. *P < 0.05 in comparison of CXCR4 knockdown and CXCR4/CXCR7 knockdown cells to cells with intact expression of CXCR4 and CXCR7; NS, no statistically significant difference.

Figure 4.

Role of CXCR4 and CXCR7 expression on primary A549 tumor size in a heterotopic xenograft murine cancer model. Data represent the mean ± SEM of n = 7 to 9 mice per group. *P < 0.05 in comparison of CXCR4 knockdown and CXCR4/CXCR7 knockdown cells to cells with intact expression of CXCR4 and CXCR7; NS, no statistically significant difference.

Close modal

We next assessed the composition of the in vivo primary tumors. Because of the dramatic differences in the growth rate of the tumors in different cell types, it was not possible to examine CXCR4 and CXCR4/7 knockdown cancers beyond week 4; conversely, primary tumors caused by CXCR7 knockdown cells and cells with unmanipulated CXCR4/7 expression were at the limit of detection on week 4. We therefore harvested the tumors on week 4 from animals with CXCR4 knockdown and CXCR4/7 knockdown tumors and on week 9 from animals with CXCR7 knockdown tumors and tumors with unmanipulated CXCR4/7 expression. Grossly, tumors produced by CXCR4 and CXCR4/7 knockdown cells were friable and hemorrhagic as compared with the more compact tumors produced by A549 cells with intact CXCR4/CXCR7 expression and CXCR7 knockdown cells. We also found CXCR4 and CXCR4/7 knockdown tumors to have a lower proportion of cancer cells (detected on the basis of expression of GFP), but increased proportion of host-derived endothelial cells (defined as CD45-negative cells expressing factor VIII-related antigen; Fig. 5A and B). Consistent with this finding, CXCR4 and CXCR4/7 knockdown tumors had higher factor VIII–related antigen protein content and lower GFP protein content as compared with cells with intact CXCR4/CXCR7 expression or CXCR7 knockdown tumors (Fig. 5C). To better assess the vascular mass of the tumors, we also examined the resected tissue histologically. Immunohistochemical localization of endothelial cells revealed greatly increased vascularity within tumors generated by CXCR4 knockdown, as compared with nontransfected, A549 cells (Fig. 5D). Histology also revealed that, in addition to being much larger and more vascular, the CXCR4 and CXCR4/7 knockdown tumors did contain many more necrotic cells than wild-type and CXCR7 knockdown tumors in vivo.

Figure 5.

Role of CXCR4 and CXCR7 expression on primary A549 tumor composition and vascularity. GFP-expressing cancer cells (A) and CD45-factor VIII related antigen (F8RA+) endothelial cells (B) were quantified in tumor cell suspensions by flow cytometry. Tumor F8RA and GFP protein content was quantified by Western blot and intensity of the corresponding band was quantified by densitometer and normalized to sample β-actin protein (C). Panel D shows representative immunohistochemical data (original magnification ×100 for main panels, ×400 for insets) of primary tumors of CXCR7 knockdown A549 cells and cells with intact CXCR4 and CXCR7 expression, stained with anti-F8RA antibody (right) or the control IgG (left). Bar graphs represent mean ± SEM of n = 5 mice per group (A and B) and 3 replicates representative of 3 separate experiments (C). In all panels, R4KD and DbKD were obtained on week 4 and WT and R7KD samples were obtained on week 9. *P < 0.05 in comparison of CXCR4 knockdown and CXCR4/CXCR7 knockdown cells to cells with intact expression of CXCR4 and CXCR7; NS, no statistically significant difference.

Figure 5.

Role of CXCR4 and CXCR7 expression on primary A549 tumor composition and vascularity. GFP-expressing cancer cells (A) and CD45-factor VIII related antigen (F8RA+) endothelial cells (B) were quantified in tumor cell suspensions by flow cytometry. Tumor F8RA and GFP protein content was quantified by Western blot and intensity of the corresponding band was quantified by densitometer and normalized to sample β-actin protein (C). Panel D shows representative immunohistochemical data (original magnification ×100 for main panels, ×400 for insets) of primary tumors of CXCR7 knockdown A549 cells and cells with intact CXCR4 and CXCR7 expression, stained with anti-F8RA antibody (right) or the control IgG (left). Bar graphs represent mean ± SEM of n = 5 mice per group (A and B) and 3 replicates representative of 3 separate experiments (C). In all panels, R4KD and DbKD were obtained on week 4 and WT and R7KD samples were obtained on week 9. *P < 0.05 in comparison of CXCR4 knockdown and CXCR4/CXCR7 knockdown cells to cells with intact expression of CXCR4 and CXCR7; NS, no statistically significant difference.

Close modal

To confirm that the increased size of primary tumors in CXCR4-knockdown cancers is attributable to the interaction of CXCR4 with CXCL12, we also assessed the effect of CXCL12 immunoneutralization on the size and composition of primary tumors. CXCL12 neutralization resulted in reduced tumor size in CXCR4 and CXCR4/7 knockdown tumors associated with reduction of CD45 F8RA+ endothelial cells, but did no affect the size or vascularity of CXCR7 knockdown tumors or tumors with intact CXCR4 and CXCR7 expression (Supplementary Fig. S4). These data indicate that primary tumors generated by CXCR4 knockdown A549 cells have greatly increased vascular mass, which contributes to their increased size; conversely, CXCR7 expression has no detectable effect on the size, cellularity, or vascularity of primary tumors.

CXCR4-deficient NSCLC tumors have reduced metastatic potential in vivo

We next assessed extent of metastases from different cancer cells by evaluating the proportion of GFP-expressing cells in the lungs, liver, bone marrow, brain, adrenal glands, and peripheral blood. As expected, the extent of metastases increased between 4 and 9 weeks in both A549 cells with intact CXCR4/CXCR7 expression and in CXCR7 knockdown cells (Fig. 6 and Supplementary Fig. S5). Despite their larger size, we found significantly fewer metastatic cells in cancers caused by A549 cells knocked down for CXCR4 and CXCR4/7 as compared with A549 cells knocked down for CXCR7 alone or cells with intact CXCR4 and CXCR7 expression 4 weeks after cancer implantation. Similar to the observations in vitro (Supplementary Figs. S3A–S3D), we found no difference in the proportion of proliferating cells in the tumors based on Ki67 staining (22.5 ± 1.9% in A549 cancer cells, 20.8 ± 1.7% in CXCR4 knockdown cells, 19.1 ± 3.1% in CXCR7-knockdown cells, and 22.8 ± 2% in CXCR4 and CXCR7 knockdown cells) or the proportion of apoptotic cells in the tumors based on TUNEL staining (5.8 ± 1.2% in A549 cancer cells, 4.7 ± 1.0% in CXCR4 knockdown cells, 7.0 ± 0.85% in CXCR7-knockdown cells, and 4.5 ± 0.92% in CXCR4 and CXCR7 knockdown cells). Given that cancer cell adhesion to matrix is critical to metastasis formation and that CXCR7 had previously been implicated in cell adhesion (7, 22), we also examined the role of CXCR4 and CXCR7 in adhesion of A549 and H157 cells to plastic in vitro. We found that neither the presence of CXCL12 nor the expression of CXCR4 or CXCR7 had any detectable effect on adhesion of either NSCLC line (Supplementary Fig. S3E and S3F). Thus, CXCR4 but not CXCR7 seems to be critical to metastasis formation in NSCLC.

Figure 6.

Role of CXCR4 and CXCR7 expression on A549 tumor metastasis. GFP-expressing cancer cells were quantified in various organ suspensions by flow cytometry. GFP-expressing metastatic cells were also detectable in the brain histologically (Supplementary Fig. S5). Data represent the mean ± SEM of n = 5 mice per group. *, P < 0.05 in comparison to cells with intact expression of CXCR4 and CXCR7 at 4 weeks; #, P < 0.05 in comparison to cells with intact expression of CXCR4 and CXCR7 at 9 weeks; NS, no statistically significant difference.

Figure 6.

Role of CXCR4 and CXCR7 expression on A549 tumor metastasis. GFP-expressing cancer cells were quantified in various organ suspensions by flow cytometry. GFP-expressing metastatic cells were also detectable in the brain histologically (Supplementary Fig. S5). Data represent the mean ± SEM of n = 5 mice per group. *, P < 0.05 in comparison to cells with intact expression of CXCR4 and CXCR7 at 4 weeks; #, P < 0.05 in comparison to cells with intact expression of CXCR4 and CXCR7 at 9 weeks; NS, no statistically significant difference.

Close modal

CXCR4 expression by A549 cells inhibits endothelial cell chemotaxis to CXCL12

To explain the unexpected finding of larger and more vascular primary tumors in the absence of CXCR4, we hypothesized that NSCLC CXCR4 competes with endothelial cells for CXCL12 in the niche of primary tumor. Because a major means of clearing chemokines, including CXCL12, is receptor-mediated cellular uptake, we first assessed the role of CXCR4 and CXCR7 on A549 in clearing exogenous CXCL12 from the media. We found that tumor cells with intact expression of CXCR4/7 and cells knocked down for CXCR7 showed rapid clearance of exogenous CXCL12, whereas cells that were knocked down for CXCR4 alone or both CXCR4/7 had little detectable clearance of CXCL12 (Fig. 7A). We next assessed the effect of this process on the in vitro chemotaxis of endothelial cells to CXCL12 is the presence of A549 cancer cells. We found chemotaxis of endothelial cells to CXCL12 was markedly attenuated in the presence of nontransfected A549 cells and CXCR7 knockdown A549 cells (Fig. 7B). In contrast, chemotaxis of endothelial cells to CXCL12 was not influenced by the presence of A549 cells that were knocked down for CXCR4 or CXCR4/7. These data suggest that cancer cell CXCR4 outcompetes endothelial cells for available CXCL12; as a result, CXCR4 promotes angiogenesis only in the absence of cancer cell CXCR4.

Figure 7.

Effect of NSCLC cell expression of CXCR4 on CXCL12. (A) concentration of CXCL12 in the supernatant was measured after 24 hours culture of A549 cells in hypoxic conditions in the presence of 30 ng/mL CXCL12 (represented by the dashed line). (B) after 24 hours culture in hypoxic conditions, chemotaxis of human microvascular endothelial cells (placed in the upper chambers of transwell plates) to 10 ng/mL CXCL12 in the lower chambers was quantified, in the presence or absence of A549 cells in the lower chambers. Results from each panel are representative of 3 experiments; data shown represent mean ± SEM of n = 3 for each group. *, P < 0.05 in comparison to R4- and double-knockdown cells (A) and compared with chemotaxis of endothelial cells in the absence of A549 cells (B); NS, no statistically significant difference in comparison to chemotaxis of endothelial cells in the absence of A549 cells.

Figure 7.

Effect of NSCLC cell expression of CXCR4 on CXCL12. (A) concentration of CXCL12 in the supernatant was measured after 24 hours culture of A549 cells in hypoxic conditions in the presence of 30 ng/mL CXCL12 (represented by the dashed line). (B) after 24 hours culture in hypoxic conditions, chemotaxis of human microvascular endothelial cells (placed in the upper chambers of transwell plates) to 10 ng/mL CXCL12 in the lower chambers was quantified, in the presence or absence of A549 cells in the lower chambers. Results from each panel are representative of 3 experiments; data shown represent mean ± SEM of n = 3 for each group. *, P < 0.05 in comparison to R4- and double-knockdown cells (A) and compared with chemotaxis of endothelial cells in the absence of A549 cells (B); NS, no statistically significant difference in comparison to chemotaxis of endothelial cells in the absence of A549 cells.

Close modal

In this study, we used NSCLC cell lines with stable knockdowns of CXCR4, CXCR7, or both receptors to investigate the contribution of CXCR4 and CXCR7 to NSCLC cell migration. CXCR4, but not CXCR7, was found to dictate the migration of NSCLC cells toward CXCL12 in vitro and to mediate metastases in vivo. We also unexpectedly discovered that tumor growth was accelerated in vivo in the absence of cancer cell CXCR4 expression, which was associated with increased tumor vascularity that was dependent on CXCL12. Finally, CXCR4 on A549 cells gave the cells the ability to outcompete endothelial cells in binding to CXCL12, indicating that CXCR4 expression in NSCLC has important roles in cell migration and metastasis but paradoxically attenuates vascular mass in the tumor microenvironment.

In the context of cancer biology, the CXCL12/CXCR4 molecular pathway has been shown to regulate the migration of tumor cells to metastatic sites in many cancers (23–29). In addition, several reports have suggested that a decrease in CXCR4 expression inhibits CXCL12-induced migration of prostate cancer or lung cancer cells (30–32). The discovery of CXCR7 as a promiscuous receptor for CXCL11 and CXCL12 ended the previously accepted exclusive association between CXCL12 and CXCR4. Although CXCR4 and CXCR7 have been reported to play diverse roles in the migration, proliferation and apoptosis of different cell types, our data confirm the exclusive association of CXCL12 with CXCR4 in migration and metastasis of NSCLC cells, and that CXCR7 or CXCL11 were not involved in this process. In this context, the literature suggests that CXCR7 is involved in the migration of some but not other cells: CXCR7 is crucial for proper migration of primordial germ cells or of interneurons toward their targets by attaining regulation of CXCL12 distribution or CXCR4 expression level (33, 34). However, in MCF breast cancer cells and in engrafted neural stem cells, CXCR7 does not mediate migration of these cells (7, 35). In addition, CXCR7 functions as a decoy receptor that does not activate G protein-mediated signaling in breast cancer cells and vascular smooth muscle cells (7, 33). Furthermore, expression of CXCR4 or CXCR7 did not influence apoptosis, proliferation, or adhesion, in agreement with our previous report that CXCL12 did not significantly affect proliferation or apoptosis of NSCLC cells in vitro (17).

This work also provides new insights into the biological significance of the CXCL12–CXCR4 axis in NSCLC. A striking finding in our study was that knockdown of CXCR4 in A549 cells resulted in greatly accelerated development of primary tumors in SCID mice that was associated with greatly increased tumor vasculature, suggesting that the absence of tumor CXCR4 expression resulted in enhanced recruitment or differentiation of endothelial cells or progenitors in the tumor. Indeed, substantial evidence supports the contention that CXCL12 is chemotactic to endothelial cells in vitro and is angiogenic in some circumstances (36–38). In contrast, previous work has demonstrated that CXCL12 concentrations are very low in CXCR4-expressing cancers and that immunoneutralization of CXCL12 in this setting attenuates metastases and prolonged survival in animal models without influencing primary tumor angiogenesis (17, 24, 39). In contrast, this article shows that, when CXCR4 is absent only on cancer cells but is present on endothelial cells, the abundant CXCL12 in the tumor milieu results in potent angiogenesis and accelerated tumor growth. Based on these observations and our data showing that cancer cell CXCR4 is sufficient to clear extracellular CXCL12 and to inhibit endothelial cell chemotaxis to CXCL12, we propose the following model: In the context of normal host where CXCR4 is expressed by both malignant and noncancerous cells, the tumor microenvironment is devoid of extracellular CXCL12 because of uptake of the ligand by CXCR4 on the cancer cells; as a result, the CXCL12–CXCR4 axis does not participate in recruitment of endothelial cells and angiogenesis in the tumor, but plays a key role in recruiting cancer cells that have escaped into circulation to tissues with high levels of CXCL12 expression, such as the lungs, meninges, and bone marrow. The selective lack of CXCR4 on cancer cells, however, results in high levels of CXCL12 in the tumor microniche, resulting in robust recruitment of endothelial cells, enhanced angiogenesis, and development of large, vascular tumors. Interestingly, in addition to being larger and more vascular, CXCR4 knockdown tumors were found to contain more areas of necrosis in vivo. Because CXCR4 knockdown cancer cells did not display altered proliferation or death in vitro, we speculate that increased necrosis may be the result of formation of abnormal microvasculature. Alternatively, it is possible that the increased necrosis is an indirect consequence of altered tumor microenvironment in vivo.

In summary, our results demonstrate that CXCL12 and CXCR4 have a single ligand–single receptor relationship in mediating NSCLC migration and metastasis, and in the context of NSCLC that expresses CXCR4, the CXCR4–CXCL12 axis is not involved in angiogenesis of the primary tumor.

R.M. Strieter is employed as a Global Head of Translational Medicine for Respiratory Diseases in Novartis Institutes of Biomedical Research. No potential conflicts of interest were disclosed by the other authors.

Conception and design: R.M. Strieter

Development of methodology: Y.H. Choi, M.D. Burdick, R.M. Strieter

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y.H. Choi, M.D. Burdick, B.A. Strieter, R.M. Strieter

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y.H. Choi, M.D. Burdick, B. Mehrad, R.M. Strieter

Writing, review, and/or revision of the manuscript: Y.H. Choi, B. Mehrad, R.M. Strieter

Study supervision: R.M. Strieter

This work is supported by the National Heart, Lung, and Blood Institute HL073848 (B. Mehrad), HL098329 (B. Mehrad, R.M. Strieter), HL066027 and HL098526 (R.M. Strieter).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Ferlay
J
,
Shin
HR
,
Bray
F
,
Forman
D
,
Mathers
C
,
Parkin
DM
. 
Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008
.
Int J Cancer
2010
;
127
:
2893
917
.
2.
Siegel
R
,
Naishadham
D
,
Jemal
A
. 
Cancer statistics, 2012
.
CA Cancer J Clin
2012
;
62
:
10
29
.
3.
Phillips
RJ
,
Mestas
J
,
Gharaee-Kermani
M
,
Burdick
MD
,
Sica
A
,
Belperio
JA
, et al
Epidermal growth factor and hypoxia-induced expression of CXC chemokine receptor 4 on non–small cell lung cancer cells is regulated by the phosphatidylinositol 3-kinase/PTEN/AKT/mammalian target of rapamycin signaling pathway and activation of hypoxia inducible factor-1α
.
J Biol Chem
2005
;
280
:
22473
81
.
4.
Schioppa
T
,
Uranchimeg
B
,
Saccani
A
,
Biswas
SK
,
Doni
A
,
Rapisarda
A
, et al
Regulation of the chemokine receptor CXCR4 by hypoxia
.
J Exp Med
2003
;
198
:
1391
402
.
5.
Burger
JA
,
Kipps
TJ
. 
CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment
.
Blood
2006
;
107
:
1761
7
.
6.
Sun
X
,
Cheng
G
,
Hao
M
,
Zheng
J
,
Zhou
X
,
Zhang
J
, et al
CXCL12/CXCR4/CXCR7 chemokine axis and cancer progression
.
Cancer Metastasis Rev
2010
;
29
:
709
22
.
7.
Burns
JM
,
Summers
BC
,
Wang
Y
,
Melikian
A
,
Berahovich
R
,
Miao
Z
, et al
A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development
.
J Exp Med
2006
;
203
:
2201
13
.
8.
Luker
KE
,
Steele
JM
,
Mihalko
LA
,
Ray
P
,
Luker
GD
. 
Constitutive and chemokine-dependent internalization and recycling of CXCR7 in breast cancer cells to degrade chemokine ligands
.
Oncogene
2010
;
29
:
4599
610
.
9.
Grymula
K
,
Tarnowski
M
,
Wysoczynski
M
,
Drukala
J
,
Barr
FG
,
Ratajczak
J
, et al
Overlapping and distinct role of CXCR7-SDF-1/ITAC and CXCR4-SDF-1 axes in regulating metastatic behavior of human rhabdomyosarcomas
.
Int J Cancer
2010
;
127
:
2554
68
.
10.
Miekus
K
,
Jarocha
D
,
Trzyna
E
,
Majka
M
. 
Role of I-TAC-binding receptors CXCR3 and CXCR7 in proliferation, activation of intracellular signaling pathways and migration of various tumor cell lines
.
Folia Histochem Cytobiol
2010
;
48
:
104
11
.
11.
Tarnowski
M
,
Liu
R
,
Wysoczynski
M
,
Ratajczak
J
,
Kucia
M
,
Ratajczak
MZ
. 
CXCR7: a new SDF-1-binding receptor in contrast to normal CD34(+) progenitors is functional and is expressed at higher level in human malignant hematopoietic cells
.
Eur J Haematol
2010
;
85
:
472
83
.
12.
Hattermann
K
,
Held-Feindt
J
,
Lucius
R
,
Müerköster
SS
,
Penfold
ME
,
Schall
TJ
, et al
The chemokine receptor CXCR7 is highly expressed in human glioma cells and mediates antiapoptotic effects
.
Cancer Res
2010
;
70
:
3299
308
.
13.
Miao
Z
,
Luker
KE
,
Summers
BC
,
Berahovich
R
,
Bhojani
MS
,
Rehemtulla
A
, et al
CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature
.
Proc Natl Acad Sci U S A
2007
;
104
:
15735
40
.
14.
Singh
RK
,
Lokeshwar
BL
. 
The IL-8-regulated chemokine receptor CXCR7 stimulates EGFR signaling to promote prostate cancer growth
.
Cancer Res
2011
;
71
:
3268
77
.
15.
Zheng
K
,
Li
HY
,
Su
XL
,
Wang
XY
,
Tian
T
,
Li
F
, et al
Chemokine receptor CXCR7 regulates the invasion, angiogenesis and tumor growth of human hepatocellular carcinoma cells
.
J Exp Clin Cancer Res
2010
;
29
:
31
.
16.
Kollmar
O
,
Rupertus
K
,
Scheuer
C
,
Nickels
RM
,
Haberl
GC
,
Tilton
B
, et al
CXCR4 and CXCR7 regulate angiogenesis and CT26.WT tumor growth independent from SDF-1
.
Int J Cancer
2010
;
126
:
1302
15
.
17.
Phillips
RJ
,
Burdick
MD
,
Lutz
M
,
Belperio
JA
,
Keane
MP
,
Strieter
RM
. 
The stromal derived factor-1/CXCL12-CXC chemokine receptor 4 biological axis in non–small cell lung cancer metastases
.
Am J Respir Crit Care Med
2003
;
167
:
1676
86
.
18.
Addison
CL
,
Daniel
TO
,
Burdick
MD
,
Liu
H
,
Ehlert
JE
,
Xue
YY
, et al
The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity
.
J Immunol
2000
;
165
:
5269
77
.
19.
Arenberg
DA
,
Kunkel
SL
,
Polverini
PJ
,
Glass
M
,
Burdick
MD
,
Strieter
RM
. 
Inhibition of interleukin-8 reduces tumorigenesis of human non–small cell lung cancer in SCID mice
.
J Clin Invest
1996
;
97
:
2792
802
.
20.
Arenberg
DA
,
Kunkel
SL
,
Polverini
PJ
,
Morris
SB
,
Burdick
MD
,
Glass
MC
, et al
Interferon-gamma-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non–small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases
.
J Exp Med
1996
;
184
:
981
92
.
21.
Keane
MP
,
Arenberg
DA
,
Lynch
JP
 3rd
,
Whyte
RI
,
Iannettoni
MD
,
Burdick
MD
, et al
The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis
.
J Immunol
1997
;
159
:
1437
43
.
22.
Dai
X
,
Tan
Y
,
Cai
S
,
Xiong
X
,
Wang
L
,
Ye
Q
, et al
The role of CXCR7 on the adhesion, proliferation and angiogenesis of endothelial progenitor cells
.
J Cell Mol Med
15
:
1299
309
.
23.
Geminder
H
,
Sagi-Assif
O
,
Goldberg
L
,
Meshel
T
,
Rechavi
G
,
Witz
IP
, et al
A possible role for CXCR4 and its ligand, the CXC chemokine stromal cell-derived factor-1, in the development of bone marrow metastases in neuroblastoma
.
J Immunol
2001
;
167
:
4747
57
.
24.
Muller
A
,
Homey
B
,
Soto
H
,
Ge
N
,
Catron
D
,
Buchanan
ME
, et al
Involvement of chemokine receptors in breast cancer metastasis
.
Nature
2001
;
410
:
50
6
.
25.
Pan
J
,
Mestas
J
,
Burdick
MD
,
Phillips
RJ
,
Thomas
GV
,
Reckamp
K
, et al
Stromal derived factor-1 (SDF-1/CXCL12) and CXCR4 in renal cell carcinoma metastasis
.
Mol Cancer
2006
;
5
:
56
.
26.
Scotton
CJ
,
Wilson
JL
,
Scott
K
,
Stamp
G
,
Wilbanks
GD
,
Fricker
S
, et al
Multiple actions of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer
.
Cancer Res
2002
;
62
:
5930
8
.
27.
Smith
MC
,
Luker
KE
,
Garbow
JR
,
Prior
JL
,
Jackson
E
,
Piwnica-Worms
D
, et al
CXCR4 regulates growth of both primary and metastatic breast cancer
.
Cancer Res
2004
;
64
:
8604
12
.
28.
Taichman
RS
,
Cooper
C
,
Keller
ET
,
Pienta
KJ
,
Taichman
NS
,
McCauley
LK
. 
Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone
.
Cancer Res
2002
;
62
:
1832
7
.
29.
Vandercappellen
J
,
Van Damme
J
,
Struyf
S
. 
The role of CXC chemokines and their receptors in cancer
.
Cancer Lett
2008
;
267
:
226
44
.
30.
Hong
JS
,
Pai
HK
,
Hong
KO
,
Kim
MA
,
Kim
JH
,
Lee
JI
, et al
CXCR-4 knockdown by small interfering RNA inhibits cell proliferation and invasion of oral squamous cell carcinoma cells
.
J Oral Pathol Med
2009
;
38
:
214
9
.
31.
Xing
Y
,
Liu
M
,
Du
Y
,
Qu
F
,
Li
Y
,
Zhang
Q
, et al
Tumor cell-specific blockade of CXCR4/SDF-1 interactions in prostate cancer cells by hTERT promoter induced CXCR4 knockdown: a possible metastasis preventing and minimizing approach
.
Cancer Biol Ther
2008
;
7
:
1839
48
.
32.
Yang
J
,
Zhang
B
,
Lin
Y
,
Yang
Y
,
Liu
X
,
Lu
F
. 
Breast cancer metastasis suppressor 1 inhibits SDF-1alpha-induced migration of non–small cell lung cancer by decreasing CXCR4 expression
.
Cancer Lett
2008
;
269
:
46
56
.
33.
Boldajipour
B
,
Mahabaleshwar
H
,
Kardash
E
,
Reichman-Fried
M
,
Blaser
H
,
Minina
S
, et al
Control of chemokine-guided cell migration by ligand sequestration
.
Cell
2008
;
132
:
463
73
.
34.
Sanchez-Alcaniz
JA
,
Haege
S
,
Mueller
W
,
Pla
R
,
Mackay
F
,
Schulz
S
, et al
Cxcr7 controls neuronal migration by regulating chemokine responsiveness
.
Neuron
2011
;
69
:
77
90
.
35.
Carbajal
KS
,
Schaumburg
C
,
Strieter
R
,
Kane
J
,
Lane
TE
. 
Migration of engrafted neural stem cells is mediated by CXCL12 signaling through CXCR4 in a viral model of multiple sclerosis
.
Proc Natl Acad Sci U S A
2010
;
107
:
11068
73
.
36.
Salvucci
O
,
Yao
L
,
Villalba
S
,
Sajewicz
A
,
Pittaluga
S
,
Tosato
G
. 
Regulation of endothelial cell branching morphogenesis by endogenous chemokine stromal-derived factor-1
.
Blood
2002
;
99
:
2703
11
.
37.
Tachibana
K
,
Hirota
S
,
Iizasa
H
,
Yoshida
H
,
Kawabata
K
,
Kataoka
Y
, et al
The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract
.
Nature
1998
;
393
:
591
4
.
38.
Takabatake
Y
,
Sugiyama
T
,
Kohara
H
,
Matsusaka
T
,
Kurihara
H
,
Koni
PA
, et al
The CXCL12 (SDF-1)/CXCR4 axis is essential for the development of renal vasculature
.
J Am Soc Nephrol
2009
;
20
:
1714
23
.
39.
Schrader
AJ
,
Lechner
O
,
Templin
M
,
Dittmar
KE
,
Machtens
S
,
Mengel
M
, et al
CXCR4/CXCL12 expression and signalling in kidney cancer
.
Br J Cancer
2002
;
86
:
1250
6
.