The chemokine stromal cell-derived factor-1 (CXCL12/SDF-1) and its monogamous receptor CXCR4 are involved in trafficking of B cells and hematopoietic progenitors. CXCR4 expression was found in the large majority of non-Hodgkin’s lymphoma (NHL) cell lines and primary cells, and CXCR4 neutralization by monoclonal antibodies had profound in vitro effects on NHL cells including inhibition of transendothelial/stromal migration, enhanced apoptosis, decreased proliferation, and inhibition of pseudopodia formation. In a nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mouse model of human high-grade NHL, CXCR4 neutralization had an impressive efficacy. In a first tumor-challenge trial, CXCR4 neutralization of Namalwa cells injected i.p. delayed tumor growth and reduced tumor weight. In a second tumor-challenge trial, NOD/SCID mice received Namalwa cells i.v. All of the controls died of neoplasia within day 36, whereas 83% of mice injected with cells incubated with anti-CXCR4 were still alive and disease-free >150 days after transplant. The crucial role of CXCR4 in tumor cell extravasation was confirmed by the finding that CXCR4 neutralization before i.v. injection of Namalwa cells in NOD/SCID mice increased the number of cancer cells circulating 24 h after injection. In additional preclinical trials, the therapeutic effect of anti-CXCR4 antibodies was evaluated in mice bearing Namalwa cells injected 3 days before. Tumor growth was abrogated in the majority of treated mice and significantly delayed in the remaining group. Taken together, these data support clinical studies on CXCR4 neutralization in NHL patients by monoclonal antibodies or CXCR4 antagonists.

Stromal cell-derived factor-1 (CXCL12/SDF-1) is a chemokine involved in development and trafficking of B cells and hematopoietic progenitors (1, 2). Recent evidences also suggest CXCL12/SDF-1 involvement in breast cancer cell pseudopodia formation and in invasive breast cancer metastasis (3). The responses of hematopoietic, B, and breast cancer cells to CXCL12/SDF-1 appear to be mediated by its receptor CXCR4. CXCL12/SDF-1 seems to play a relevant role also in some B-cell malignancies. In fact, CXCL12/SDF-1 enhances migration of follicular NHL3 cells (4), and the CXCR4-CXCL12/SDF-1 circuitry appears to be crucial for migration of chronic lymphocytic leukemia (5) and acute lymphoblastic leukemia B cells (6).

In the present study, we evaluated a panel of malignant lymphoid cell lines and primary NHL cells, and found CXCR4 expression in the large majority of malignant cells. CXCR4 neutralization by monoclonal antibodies had profound in vitro effects on NHL cells including inhibition of transendothelial/stromal migration, enhanced apoptosis, decreased proliferation, and inhibition of pseudopodia formation. In preclinical models, CXCR4 neutralization demonstrated remarkable efficacy in either tumor challenge and therapy trials in the absence of overt short- or long-term toxicity. Furthermore, CXCR4 neutralization increased the number of lymphoma cells circulating 24 h after i.v. injection, suggesting a crucial role of CXCR4 in tumor cell extravasation. Taken together, our data indicate that the CXCR4-CXCL12/SDF-1 circuitry may be an useful target for NHL therapy.

Cells and Cell Lines.

We evaluated a panel of 12 malignant lymphoid cell lines and 19 primary NHL cells. NHL cell lines were Namalwa (Burkitt’s NHL), HS-Sultan (Burkitt’s NHL), DoHH2 (transformed follicular NHL), Granta-519 (mantle cell NHL), and RAP1-EIO (T cell-rich B-cell NHL) from patients with B-cell NHL; L363 from a patient with plasma cell leukemia; Karpas 299 from a patient with T-cell NHL; Jurkat, CEM, and MOLT-4 from patients with T-cell leukemia-NHL; and JJN3 and IM9 from patients with multiple myeloma. After informed consent, primary NHL cells were collected from the bone marrow or peripheral blood of 19 NHL patients (purity >87%). Diagnoses were diffuse large B-cell NHL (n = 3), mantle cell NHL (n = 5), follicular NHL (n = 4), peripheral blood T-cell NHL (n = 2), lymphocytic NHL (n = 3), and marginal zone NHL (n = 2). Cells were cultured in RPMI-10% FBS with the exception of the Granta-519 (DMEM-10% FBS) cell line.

Detection of Chemokine Receptors and CXCL12/SDF-1 mRNA.

CXCR1, 2, 3, and 4, and CCR1, 2, 3, 4, and 5 mRNA expression was evaluated by multiplex RT-PCR. Total RNA was isolated from cell lines and primary cells by QIAamp RNA kit (Quiagen, Hilden, Germany), and treated with a reverse transcriptase enzyme (SuperScript II; Life Technologies, Inc., Gaithersburg, MD). The cDNA generated following this approach was amplified by multiplex PCR using commercially available kits Cytoexpress hCXCR and hCCR (Biosource, Camarillo, CA) according to manufacturer’s instructions. PCR-amplified products were stained with ethidium bromide and evaluated by 2% agarose-gel electrophoresis. The Quantikine colorimetric assay (R&D, Minneapolis, MN) was used according to manufacturer’s instructions for quantitative evaluation of CXCL12/SDF-1 mRNA. Positive controls (Cytoexpress) and reagents to generate a calibrator curve (Quantikine) were obtained by manufacturers, and the appropriate null control reactions always remained negative.

Flow Cytometry Studies.

The expression of CXCR4 on the surface of cell lines and primary NHL cells was evaluated by four-color flow cytometry using a FACScalibur (BD, Mountain View, CA), anti-CD45, -CD19, -κ, -λ, and -CXCR4 monoclonal antibodies (BD), annexin V, and 7AAD to depict apoptotic or dead cells as described previously (7).

In Vitro Studies.

Sodium azide-free monoclonal antibodies anti-CXCR4 (clones MAB171 from R&D and 12G5 from BDPharMingen, San Diego, CA) and polyclonal anti-SDF-1 (R&D) were used to neutralize the CXCR4-CXCL12/SDF-1 circuitry. Appropriate irrelevant antibodies (sodium azide-free 2007OD and anti-CD19; BDPharMingen) were used as control in vitro and in vivo. After 5-h culture in RPMI-10% FBS at 37°C, the extent of cell proliferation was evaluated by a standard MTT assay (Sigma, St. Louis, MO) and by cell proliferation reagent WST-1 (Boehringer Mannheim, Mannheim, Germany; Ref. 8), and cell viability measured by flow cytometry. Apoptosis was investigated by flow cytometry and commercially available multiplex RT-PCR kits (Biosource) able to detect caspases, Fas, FasL, FLICE, FADD, and TRADD.

We used an approach similar to Burger et al.(5) and Poznansky et al.(9) with slight modifications to study the effect of CXCR4 neutralization in NHL cell transendothelial/stromal migration in transwell (diameter, 6.5 mm; pore, 5 μm; Costar, Cambridge, MA) culture. A layer consisting of 2 × 104 human microvascular endothelial cells (Cascade Biologics, Portland, OR) or bone marrow-derived stromal cell lines L87/4 and L88/5 (10) was seeded in the upper chamber and cultured for 48 h in RPMI-10% FBS. A total of 2 × 105 Namalwa NHL cells were preincubated for 30 min in 100 μl migration buffer containing different concentrations of neutralizing anti-CXCR4 monoclonal antibodies or control antibodies. Cells were seeded in the upper chambers coated with endothelial or stromal cells. After 30-min incubation at 4°C, chambers were transferred to wells containing medium with or without CXCL12/SDF-1 (125 ng/ml; R&D) as a chemoattractant and incubated for 2 h at 37°C. Cells that migrated to the lower chamber were counted in triplicates by flow cytometry.

Pseudopodia formation in Namalwa and Granta 519 cells was evaluated as described by Muller et al.(3). Cells were incubated at 37°C in RPMI supplemented with 125 ng/ml CXCL12/SDF-1 (or CX3CL1/fractalkine as negative control) in the presence of anti-CXCR4, anti-CD19, or control (irrelevant) antibodies. After a 20-min culture, cells were fixed by paraformaldehyde, and pseudopodia formation was observed and enumerated by microscopy.

In Vivo Studies.

CXCR4- and CXCL12/SDF-1 neutralization were evaluated in a model of human NHL generated in our laboratory by transplanting Namalwa cells in NOD/SCID mice (11, 12). This NHL cell line was found to be the most aggressive one in terms of efficiency of engraftment, speed of engraftment, and tumor size in a panel of lymphoid malignant cell lines tested in i.p. (11, 12) or s.c. (13) xenotransplants. To generate a disease similar to human high-grade B-cell NHL, we transplanted NOD/SCID mice i.p. rather than s.c., and Namalwa cells generated measurable i.p. tumors in the injection site in 100% of injected animals. Tumor volume was measured by calipers and the formula [width2 × length × 0.52] applied for approximating the volume of a spheroid (12).

In a first tumor-challenge trial, 2 × 105 Namalwa cells were preincubated with 10 μg of sodium azide-free anti-CXCR4, anti-CXCL12/SDF-1, or control antibodies before i.p. injection (n = 6/study group). In a second tumor-challenge trial, mice were injected i.v. with 2 × 105 Namalwa cells preincubated with 10 μg of sodium azide-free anti-CXCR4, anti-CXCL12/SDF-1, or control antibodies (n = 12/study group). In both tumor-challenge trials, tumor cells were washed before injection.

To investigate the therapeutic potential of CXCR4-neutralization, mice injected i.p. with 2 × 105 Namalwa cells (not preincubated by anti-CXCR4) were treated in a site different from tumor injection with 3 weekly i.p. injections of 100 μg of sodium azide-free anti-CXCR4 or control antibodies. Animals (n = 12/study group, in two replicate trials involving a total of 12 treated animals and 12 controls) were treated on days 3, 10, and 17 after tumor injection.

Tumor-bearing mice were sacrificed by CO2 inhalation, and tumor engraftment confirmed by histology, immunohistochemistry, and flow cytometry. Tumor weight was evaluated after complete removal of the i.p. tumor bulk. For histology and immunohistochemistry evaluation, tumor samples were fixed in 10% formalin and embedded in paraffin. Sections (4 μm-thick) were stained with H&E and Giemsa for conventional histology. For immunohistochemistry, sections were immunostained with the anti-CD10 and -CD20 monoclonal antibodies by DAKO (Glostrup, Denmark). In flow cytometry, tumor expression of human CD19 and CD20 antigens was evaluated by BD monoclonal antibodies.

In separate studies (n = 6), Namalwa cell extravasation was evaluated in vivo injecting NOD/SCID mice i.v. with 2 × 105 Namalwa cells preincubated with 10 μg of sodium azide-free anti-CXCR4 or control antibodies. Mice were sacrificed 24 h after injection, and the frequency and viability of Namalwa cells circulating in the peripheral blood evaluated by flow cytometry. A minimum of 100,000 circulating cells were evaluated.

All of the procedures involving animals were done in accordance with national and international laws and policies.

Statistical Analysis.

Statistical comparisons were performed using the t test and ANOVA when data were normally distributed, and the nonparametric analyses of Spearman and Mann-Whitney when data were not normally distributed. All of the Ps were two-sided and considered statistically significant at <0.05.

Expression of CXCR4, Other Chemokine Receptors, and CXCL12/SDF-1 in NHL Cells.

Strong CXCR4 mRNA expression was found in 10 of 12 lines and in 18 of 19 primary NHL cells, respectively. As indicated in Table 1, other chemokine receptors were less frequently expressed in cell lines and primary NHL cells. Flow cytometry studies confirmed CXCR4 expression in all of the RT-PCR-positive NHL lines and in primary cells, and indicated high levels of CXCR4 expression when compared with other normal lymphoid cells (Fig. 1).

Using the RT-PCR approach, low levels (faint bands) of CXCL12/SDF-1 mRNA expression were found in 4 of 12 lines and 6 of 19 primary NHL cells. Using the more sensitive quantitative colorimetric assay, CXCL12/SDF-1 mRNA was found to be 2–4-fold higher in NHL primary cells and cell lines compared with lymphocytes obtained from 5 healthy controls (Fig. 2 a; P < 0.01). When compared with NHL cell lines, the amount of CXCL12/SDF-1 mRNA expressed by bone marrow-derived stromal cell lines L87/4 and L88/5 was slightly increased but not significantly higher.

CXCR4 Neutralization Inhibits NHL Cell Transendothelial/Stromal Migration, Increases the Frequency of Apoptotic Cells, Reduces Cell Proliferation, and Inhibits Pseudopodia Formation.

In transwell cultures, anti-CXCR4 antibodies inhibited Namalwa cell migration across endothelial or stromal cell layers (n = 4/group; Fig. 2,b). When compared with control antibodies, mean inhibition ranged from 7% (1 μg anti-CXCR4 in 100 μl culture medium and endothelial cell layers) to 61% (10 μg anti-CXCR4 in 100 μl culture medium and stromal cell layers). In four independent experiments, the addition of 10 μg anti-CXCR4 in 100 μl culture medium significantly enhanced Namalwa cell apoptosis (evaluated by flow cytometry enumeration of annexin V-positive cells; Fig. 2,c) from 2.0 ± 0.9% (i.e., 1.8, 3.2, 0.9, and 2.1% apoptosis) to 6.8 ± 1.1% (i.e., 6.7, 7.1, 5.3, and 8.0% apoptosis; P = 0.005). Moreover, mRNA expression of death-domain- related proteins FADD and TRADD significantly enhanced in CXCR4-neutralized cells (Fig. 2,c). As expected, anti-CXCR4 also reduced the mean channel of CXCR4-related fluorescence (Fig. 2 c).

Using MTT and WST-1 assays (8), we observed that the addition of 1–10 μg anti-CXCR4 or neutralizing anti-CXCL12/SDF1 antibodies to Namalwa cell culture inhibited cell proliferation (n = 4/group; Fig. 2 d). When compared with controls, mean inhibition of cell proliferation ranged from 2% (1 μg anti-CXCL12/SDF1) to 32% (10 μg anti-CXCR4), and data from the MTT and WST-1 assays were superimposable.

As showed in Fig. 3, culture in the presence of CXCL12/SDF1 (but not in the presence of CX3CL1/fractalkine used as a negative control) was associated with distinct pseudopodia formation in 20–30% of Namalwa cells (expressing high levels of CXCR4 mRNA) as well as in Granta 519 cells (expressing low levels of CXCR4 mRNA). In both cell lines, CXCR4 neutralization by monoclonal antibodies inhibited pseudopodia formation (Fig. 3).

CXCR4 Neutralization Prevents in Vivo NHL Growth and Inhibits Tumor Cell Extravasation.

In the first tumor-challenge trial, preincubation of Namalwa cells with anti-CXCR4 or anti-CXCL12/SDF-1 antibodies before i.p. injection significantly delayed tumor growth (n = 6/study group; Fig. 4,a). Furthermore, mean tumor weight was significantly reduced by 32% (anti-CXCL12/SDF-1) and 48% (anti-CXCR4; Fig. 4,b). In a second tumor-challenge trial, mice were injected i.v. with Namalwa cells (n = 12/study group). All of the control mice died of neoplasia by day 36, all of the mice injected with NHL cells preincubated with 10 μg anti-CXCL12/SDF-1 died of neoplasia by day 60, and, remarkably, 83% of mice injected with NHL cells incubated with 10 μg anti-CXCR4 were still alive and disease-free >150 days after transplant (P < 0.0001; Fig. 4 c).

As showed in Fig. 5, CXCR4 neutralization had profound effects in NHL cell extravasation. In fact, circulating Namalwa cells were found 24 h after i.v. injection in mice given CXCR4-neutralized cells but not in mice given cells preincubated with control antibodies. Interestingly, 32–47% of CXCR4-neutralized Namalwa cells circulating 24 h after injection expressed the apoptosis marker annexin V (Fig. 5 d). Variance in the percentage of apoptotic cells reflects results of several independent experiments. Increased apoptosis in circulating cells was clearly attributable to CXCR4 neutralization, because annexin V staining was not observed in normal mouse cells.

CXCR4 Neutralization Has Therapeutic Potential in Preclinical NHL Model.

NOD/SCID mice injected i.p. with Namalwa cells (not preincubated by anti-CXCR4) were treated on days 3, 10, and 17 with i.p. injections of 100 μg anti-CXCR4 or control antibodies (n = 12/study group). Therapeutic treatment with anti-CXCR4 antibodies abrogated tumor growth in 7 of 12 treated mice and significantly inhibited tumor growth in the remaining group (P < 0.001; Fig. 6, a and b). In tumor-challenge and therapy trials, no toxicity was observed in long-term survivor mice. At autopsy, liver, kidney, bladder, stomach, gut, lung, heart, brain, spleen, or bone marrow toxicity was not observed.

Chemokines are a group of structurally related molecules that regulate leukocyte trafficking (14). Mice lacking the CXCL12/SDF-1 chemokine die perinatally and have defects in cerebella, cardiac septum, gastrointestinal vessels, and B-cell development (1, 15). In these mice, disorganization in the marrow stromal environment leading to failures in localization and retention of progenitors seems to be the cause of defective B-cell development. Despite frequent redundancy and binding promiscuity between other chemokines and their ligands, the finding that mice lacking the CXCR4 receptor have defects overlapping with CXCL12/SDF-1 knockout mice suggests a monogamous relationship between CXCR4 and CXCL12/SDF-1.

Cell migration along chemokine gradients involves a number of steps including establishment of cell polarity, directional cell locomotion through cytoskeletal rearrangements, and adhesive interactions with extracellular matrix (16). Breast cancer cell migration and metastasis usually follows a distinct pattern involving regional lymph nodes, bone marrow, lung, and liver. This pattern shares relevant similarities with leukocyte trafficking and seems to be mediated through CXCR4 or CCR7 signaling (3, 17). Similarly, the CXCR4-CXCL12/SDF-1 circuitry seems to be involved in migration of chronic lymphocytic leukemia (5) and acute lymphoblastic leukemia B cells (6). Regarding NHL, CXCL12/SDF-1 has been found recently to enhance migration of follicular NHL cells but not of their normal counterpart, germinal center B cells (4). Moreover, Sei et al.(18) reported recently increased CXCL12/SDF-1 mRNA levels in circulating mononuclear cells from AIDS-related NHL pediatric patients.

In the present study we observed that CXCR4 neutralization significantly impairs transendothelial/stromal NHL cell migration. Moreover, CXCR4 neutralization enhanced NHL cell apoptosis and reduced NHL cell proliferation. Because quantitative studies indicated that CXCL12/SDF-1 mRNA was frequently expressed by NHL cells at levels significantly higher than normal lymphocytes, enhanced apoptosis and reduced proliferation in the presence of anti-CXCR4 or -CXCL12/SDF-1 might be because of inhibition of an autocrine loop. Burger et al.(19) have described recently a novel antiapoptotic role of the CXCR4-CXCL12/SDF-1 circuitry in B-cell chronic lymphocytic leukemia. In this disease, CXCL12/SDF-1 seems to provoke an antiapoptotic effect on malignant cells through a novel population of blood-derived nurse-like cells. We are currently investigating whether a similar pattern also exists in different NHL types. Along this line, our finding that neutralization of the CXCR4-CXCL12/SDF-1 circuitry results in some degrees of inhibition of Namalwa cell proliferation fits well with the original identification of CXCL12/SDF-1 as a B-cell progenitor growth factor (20). We have observed distinct pseudopodia formation in NHL cells stimulated by CXCL12/SDF-1. Previous studies in lymphoma cells have demonstrated that pseudopodia formation is crucial for tissue invasion and metastases formation (21). Again, CXCR4 neutralization dramatically inhibited pseudopodia formation in cells stimulated by CXCL12/SDF-1.

These in vitro observations, together with reports of impaired human hematopoietic stem cell engraftment in immunodeficient mice treated with anti-CXCR4 antibodies (2), prompted us to study the effect of CXCR4 neutralization in a preclinical NHL model developed in our laboratory by injecting Namalwa cells in NOD/SCID mice. When compared with a panel of lymphoid malignant cells, this line was the most aggressive in terms of efficiency of engraftment, speed of engraftment, and tumor size (11, 12, 13). In NHL tumor-challenge trials, CXCR4 neutralization of i.p.-injected Namalwa cells delayed tumor growth and reduced tumor weight. The effect of CXCR4-neutralization of i.v.-injected Namalwa cells was particularly remarkable, because all of the controls died of NHL within day 36, whereas 83% of mice injected with cells incubated with anti-CXCR4 were still alive and disease-free >150 days after transplant.

To better elucidate the role of CXCR4 neutralization in NHL cell extravasation, we enumerated tumor cells circulating in the peripheral blood of mice injected i.v. with Namalwa cells. The day after tumor injection, in recipients of Namalwa cells preincubated with control antibodies, circulating tumor cells were below the sensitivity threshold of the flow cytometry procedure. Conversely, in mice injected with CXCR4-neutralized Namalwa cells the frequency of circulating tumor cells was always >0.5%. Taken together with the observation of impaired transendothelial/stromal Namalwa cell migration after CXCR4 neutralization, our data suggest a pivotal role of the CXCR4-CXCL12/SDF-1 circuitry in NHL cell extra- and intravasation.

In additional preclinical trials, we investigated the therapeutic effect of anti-CXCR4 antibodies in mice bearing NHL cells (not preincubated by anti-CXCR4) injected 3 days before. CXCR4 neutralization had a clear therapeutic potential, because tumor growth was abrogated in the majority of mice, and tumor growth was significantly delayed in the remaining group. As already reported by Muller et al.(3), no cytotoxicity for either anti-CXCR4 antibodies (or control antibodies) could be detected in vitro. The evidence that CXCR4 neutralization by monoclonal antibodies is active in vivo against human NHL despite a severely B-cell-, T-cell-, natural killer-cell-, myeloid cell-, and complement-deficient host mice (22) is of particular interest. In fact, it suggests that most of anti-CXCR4-induced biological responses may not be because of antibody-dependent cellular cytotoxicity or complement-dependent cell lysis. We are now investigating whether this therapeutic effect of CXCR4 neutralization is mostly because of impaired NHL cell trafficking, in vivo NHL cell apoptosis, targeting of nurse cell activity, decreased angiogenesis, or other, still unknown mechanisms. In the meantime, it is interesting to note that despite the wide spectrum of CXCL12/SDF-1 and CXCR4 expression in different tissues and organs (14), mice given anti-CXCR4 antibodies showed no major short- or long-term toxicity.

In conclusion, present data on CXCR4 expression in most NHL primary cells, in vitro effects of CXCR neutralization in NHL cells, and the efficacy of anti-CXCR4 antibodies observed in either tumor-challenge and therapy preclinical NHL trials indicate that the CXCR4-CXCL12/SDF-1 circuitry is a very attractive target for NHL therapies. Because the HIV-1 virus may use CXCR4 as a coreceptor (23), a number of CXCR4 antagonists have been discovered and are entering clinical trials for AIDS patients (24, 25, 26). Our data suggest that these novel molecules, together with anti-CXCR4 antibodies, might be considered for clinical trials in NHL patients.

Fig. 1.

Representative flow cytometry evaluation of CXCR4 expression in primary NHL cells infiltrating the bone marrow of a mantle cell NHL patient (top panels) and in B cells form a healthy control (bottom panels). Panels on the left show size and forward scatters used to design the lymphocyte acquisition gates; panels in the middle show CD19 and CD45 expression used to gate on B cells; and the histogram on the right shows higher CXCR4 expression in malignant cells from the patient (faint line) compared with normal B cells from the healthy control (bold line).

Fig. 1.

Representative flow cytometry evaluation of CXCR4 expression in primary NHL cells infiltrating the bone marrow of a mantle cell NHL patient (top panels) and in B cells form a healthy control (bottom panels). Panels on the left show size and forward scatters used to design the lymphocyte acquisition gates; panels in the middle show CD19 and CD45 expression used to gate on B cells; and the histogram on the right shows higher CXCR4 expression in malignant cells from the patient (faint line) compared with normal B cells from the healthy control (bold line).

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Fig. 2.

A, quantitative CXCL12/SDF-1 mRNA evaluation in lymphocytes from 5 healthy controls, 19 NHL primary cells, 12 NHL cell lines, and bone marrow-derived stromal cell lines L87/4 and L88/5. Data are quantitative, because mRNA was extracted from 500,000 cells for each cell type. Bars and lines represent mean values and 95% CIs; ∗ means P < 0.01 versus healthy controls. B, in cultures supplemented by control antibodies, a median of 16% of Namalwa cells migrated in response to 125 ng/ml CXCL12/SDF-1. Anti-CXCR4 antibodies (1–10 μg) in 100 μl culture medium inhibited up to 61% of Namalwa cell migration across endothelial or stromal cell layers. No cell aggregation was observed by microscopy. Bars and lines represent mean values and 95% CIs of four replicated studies. C, representative flow cytometry evaluation of Namalwa cell apoptosis (depicted by annexin V staining) in the presence of control (top panel) or anti-CXCR4 (bottom panel) antibodies. Culture in the presence of 10 μg anti-CXCR4 in 100 μl culture medium increased the frequency of apoptotic cells from 2 ± 0.9 to 6.8 ± 1.1; P = 0.005). As expected, preincubation with anti-CXCR4 reduced the mean channel of CXCR4-related fluorescence. mRNA expression of death- domain-related proteins FADD and TRADD significantly enhanced in CXCR4-neutralized cells. D, inhibition of Namalwa cell proliferation (evaluated by MTT and WST-1 assays) in the presence of 1–10 μg anti-CXCR4 or -CXCL12/SDF-1 antibodies in 100 μl culture medium. In control cultures supplemented by irrelevant and/or anti-CD19 antibodies, cell doubling time was 20–23 h. Bars and lines represent mean values and 95% CIs.

Fig. 2.

A, quantitative CXCL12/SDF-1 mRNA evaluation in lymphocytes from 5 healthy controls, 19 NHL primary cells, 12 NHL cell lines, and bone marrow-derived stromal cell lines L87/4 and L88/5. Data are quantitative, because mRNA was extracted from 500,000 cells for each cell type. Bars and lines represent mean values and 95% CIs; ∗ means P < 0.01 versus healthy controls. B, in cultures supplemented by control antibodies, a median of 16% of Namalwa cells migrated in response to 125 ng/ml CXCL12/SDF-1. Anti-CXCR4 antibodies (1–10 μg) in 100 μl culture medium inhibited up to 61% of Namalwa cell migration across endothelial or stromal cell layers. No cell aggregation was observed by microscopy. Bars and lines represent mean values and 95% CIs of four replicated studies. C, representative flow cytometry evaluation of Namalwa cell apoptosis (depicted by annexin V staining) in the presence of control (top panel) or anti-CXCR4 (bottom panel) antibodies. Culture in the presence of 10 μg anti-CXCR4 in 100 μl culture medium increased the frequency of apoptotic cells from 2 ± 0.9 to 6.8 ± 1.1; P = 0.005). As expected, preincubation with anti-CXCR4 reduced the mean channel of CXCR4-related fluorescence. mRNA expression of death- domain-related proteins FADD and TRADD significantly enhanced in CXCR4-neutralized cells. D, inhibition of Namalwa cell proliferation (evaluated by MTT and WST-1 assays) in the presence of 1–10 μg anti-CXCR4 or -CXCL12/SDF-1 antibodies in 100 μl culture medium. In control cultures supplemented by irrelevant and/or anti-CD19 antibodies, cell doubling time was 20–23 h. Bars and lines represent mean values and 95% CIs.

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Fig. 3.

Pseudopodia formation in Namalwa (left panels) and Granta 519 (right panels) NHL cells. Stimulation with CXCL12/SDF1 (but not stimulation with CX3CL1/fractalkine used as a negative control) was associated with distinct pseudopodia formation (indicated by arrows in top and middle panels) in 20–30% of cells. In both cell lines, CXCR4-neutralization by monoclonal antibodies inhibited pseudopodia formation (bottom panels).

Fig. 3.

Pseudopodia formation in Namalwa (left panels) and Granta 519 (right panels) NHL cells. Stimulation with CXCL12/SDF1 (but not stimulation with CX3CL1/fractalkine used as a negative control) was associated with distinct pseudopodia formation (indicated by arrows in top and middle panels) in 20–30% of cells. In both cell lines, CXCR4-neutralization by monoclonal antibodies inhibited pseudopodia formation (bottom panels).

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Fig. 4.

A–C, tumor volume (A) and weight (B) in NOD/SCID mice injected i.p. with 2 × 105 Namalwa cells preincubated with 10 μg anti-CXCR4, anti-CXCL12/SDF-1, or control antibodies (n = 6/study group). Bars and lines represent mean values and 95% CIs; ∗ indicate P < 0.01. C, survival in NOD/SCID mice injected i.v. with 2 × 105 Namalwa cells preincubated with 10 μg anti-CXCR4, anti-CXCL12/SDF-1, or control antibodies (n = 12/study group).

Fig. 4.

A–C, tumor volume (A) and weight (B) in NOD/SCID mice injected i.p. with 2 × 105 Namalwa cells preincubated with 10 μg anti-CXCR4, anti-CXCL12/SDF-1, or control antibodies (n = 6/study group). Bars and lines represent mean values and 95% CIs; ∗ indicate P < 0.01. C, survival in NOD/SCID mice injected i.v. with 2 × 105 Namalwa cells preincubated with 10 μg anti-CXCR4, anti-CXCL12/SDF-1, or control antibodies (n = 12/study group).

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Fig. 5.

A–D, representative evaluation of tumor cells circulating in the peripheral blood of NOD/SCID mice 24 h after i.v. injection of Namalwa cells. A, shows the acquisition gate used to exclude platelets and cell debris. As showed in B, in recipients of Namalwa cells preincubated with control antibodies, circulating tumor cells (human CD19+) were not detected. Bottom panels show the analysis gate (according to side and forward scatter, C) of tumor cells circulating in the peripheral blood of mice given CXCR4-neutralized Namalwa cells. The frequency of circulating tumor cells was always >0.5%, and annexin V staining of human CD19+ cells (D) indicated an apoptotic process in 32–47% of circulating Namalwa cells.

Fig. 5.

A–D, representative evaluation of tumor cells circulating in the peripheral blood of NOD/SCID mice 24 h after i.v. injection of Namalwa cells. A, shows the acquisition gate used to exclude platelets and cell debris. As showed in B, in recipients of Namalwa cells preincubated with control antibodies, circulating tumor cells (human CD19+) were not detected. Bottom panels show the analysis gate (according to side and forward scatter, C) of tumor cells circulating in the peripheral blood of mice given CXCR4-neutralized Namalwa cells. The frequency of circulating tumor cells was always >0.5%, and annexin V staining of human CD19+ cells (D) indicated an apoptotic process in 32–47% of circulating Namalwa cells.

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Fig. 6.

A, survival in NOD/SCID mice injected i.p. with 2 × 105 Namalwa cells (not preincubated by anti-CXCR4) and treated in two replicate trials (n = 12/study group) with three weekly i.p. injections of 100 μg anti-CXCR4 or control antibodies on days 3, 10, and 17 after Namalwa cell injection. Mice were sacrificed when tumor weight exceeded 10% of body weight. On day 70, 7 of 12 mice treated with anti-CXCR4 were alive and well without signs of tumor growth. B, tumor volume in control NOD/SCID mice and in the cohort of 5 of 12 mice treated with anti-CXCR4 showing tumor growth. Bars and lines represent mean values and 95% CIs; ∗ indicate P < 0.01.

Fig. 6.

A, survival in NOD/SCID mice injected i.p. with 2 × 105 Namalwa cells (not preincubated by anti-CXCR4) and treated in two replicate trials (n = 12/study group) with three weekly i.p. injections of 100 μg anti-CXCR4 or control antibodies on days 3, 10, and 17 after Namalwa cell injection. Mice were sacrificed when tumor weight exceeded 10% of body weight. On day 70, 7 of 12 mice treated with anti-CXCR4 were alive and well without signs of tumor growth. B, tumor volume in control NOD/SCID mice and in the cohort of 5 of 12 mice treated with anti-CXCR4 showing tumor growth. Bars and lines represent mean values and 95% CIs; ∗ indicate P < 0.01.

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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

Supported in part by AIRC (Associazione Italiana per la Ricerca sul Cancro) e FIRC (Fondazione Italiana per la Ricerca sul Cancro). F. B. is a scholar of the United States National Blood Foundation.

3

The abbreviations used are: NHL, non-Hodgkin’s lymphoma; NOD/SCID, nonobese diabetes/severe combined immunodeficiency; RT-PCR, reverse transcription-PCR; 7AAD, 7-Aminoactinomycin D; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FADD, Fas-associated death domain; CI, confidence interval.

Table 1

Chemokine receptors mRNA expression in a panel of 12 malignant lymphoid cell lines and 19 primary NHL cells

Gray boxes indicate positive RT-PCR.

Chemokine receptors mRNA expression in a panel of 12 malignant lymphoid cell lines and 19 primary NHL cells
Chemokine receptors mRNA expression in a panel of 12 malignant lymphoid cell lines and 19 primary NHL cells
a

DLBC, diffuse large B-cell NHL; FL, follicular NHL; PBTC, peripheral blood T-cell NHL; LL, lymphocytic NHL; MZL, marginal zone NHL.

We thank Aron Goldhirsch for critical reading of the manuscript.

1
Nagasawa T., Hirota S., Tachibana K., Takakura N., Nishikawa S., Kitamura Y., Yoshida N., Kikutani H., Kishimoto T. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/ SDF-1.
Nature (Lond.)
,
382
:
635
-638,  
1996
.
2
Peled A., Petit I., Kollet O., Magid M., Ponomaryov T., Byk T., Nagler A., Ben-Hur H., Many A., Shultz L., Lider O., Alon R., Zipori D., Lapidot T. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.
Science (Wash. DC)
,
283
:
845
-848,  
1999
.
3
Muller A., Homey B., Soto H., Ge N., Catron D., Buchanan M. E., McClanahan T., Murphy E., Yuan W., Wagner S. N., Barrera J. L., Mohar A., Verastegui E., Zlotnik A. Involvement of chemokine receptors in breast cancer metastasis.
Nature (Lond.)
,
410
:
50
-56,  
2001
.
4
Corcione A., Ottonello L., Tortolina G., Facchetti P., Airoldi I., Guglielmino R., Dadati P., Truini M., Sozzani S., Dallegri F., Pistoia V. Stromal cell-derived factor-1 as a chemoattractant for follicular center lymphoma B cells.
J. Natl. Cancer Inst.
,
92
:
628
-635,  
2000
.
5
Burger J. A., Burger M., Kipps T. J. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells.
Blood
,
94
:
3658
-3667,  
1999
.
6
Bradstock K. F., Makrynikola V., Bianchi A., Shen W., Hewson J., Gottlieb D. J. Effects of the chemokine stromal cell-derived factor-1 on the migration and localization of precursor-B acute lymphoblastic leukemia cells within bone marrow stromal layers.
Leukemia (Baltimore)
,
14
:
882
-888,  
2000
.
7
Philpott N. J., Turner A. J., Scopes J., Westby M., Marsh J. C., Gordon-Smith E. C., Dalgleish A. G., Gibson F. M. The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques.
Blood
,
87
:
2244
-2251,  
1996
.
8
Liu S. Q., Saijo K., Todoroki T., Ohno T. Induction of human autologous cytotoxic T lymphocytes on formalin-fixed and paraffin-embedded tumour sections.
Nat. Med.
,
1
:
267
-271,  
1995
.
9
Poznansky M. C., Olszak I. T., Foxall R., Evans R. H., Luster A. D., Scadden D. T. Active movement of T cells away from a chemokine.
Nat. Med.
,
5
:
543
-548,  
2000
.
10
Thalmeier K., Meissner P., Reisbach G., Falk M., Brechtel A., Dormer P. Establishment of two permanent human bone marrow stromal cell lines with long-term post irradiation feeder capacity.
Blood
,
83
:
1799
-807,  
1994
.
11
Fusetti L., Pruneri G., Gobbi A., Rabascio C., Carboni N., Peccatori F., Martinelli G., Bertolini F. Human myeloid and lymphoid malignancies in the non-obese diabetic/severe combined immunodeficiency mouse model: frequency of apoptotic cells in solid tumors and efficiency and speed of engraftment correlate with vascular endothelial growth factor production.
Cancer Res.
,
60
:
2527
-2534,  
2000
.
12
Bertolini F., Fusetti L., Mancuso P., Gobbi A., Corsini C., Ferrucci P. F., Martinelli G., Pruneri G. Endostatin, an antiangiogenic drug, induces tumor stabilization after chemotherapy or anti-CD20 therapy in a NOD/SCID mouse model of human high-grade non-Hodgkin’s lymphoma.
Blood
,
96
:
282
-287,  
2000
.
13
Hudson W. A., Li Q., Le C., Kersey J. H. Xenotransplantation of human lymphoid malignancies is optimized in mice with multiple immunologic defects.
Leukemia (Baltimore)
,
12
:
2029
-2033,  
1998
.
14
Zlotnik A., Yoshie O. Chemokines: a new classification system and their role in immunity.
Immunity
,
12
:
121
-127,  
2000
.
15
Zou Y. R., Kottman A. H., Kuroda M., Taniuchi I., Littman D. R. Function of the chemokine receptor CXCR4 in haematopoiesis and cerebellar development.
Nature (Lond.)
,
393
:
595
-599,  
1998
.
16
Sanchez-Madrid F., del Pozo M. A. Leukocyte polarization in cell migration and immune reactions.
EMBO J.
,
18
:
501
-511,  
1999
.
17
Scotton C. J., Wilson J. L., Milliken D., Stamp G., Balkwill F. R. Epithelial cancer cell migration: a role for chemokine receptors?.
Cancer Res.
,
61
:
4961
-4965,  
2001
.
18
Sei S., O’Neill D. P., Stewart S. K., Yang Q., Kumagai M., Boler A. M., Adde M. A., Zwerski S. L., Wood L. V., Venzon D. J., Magrath I. T. Increased level of stromal cell-derived factor-1 mRNA in peripheral blood mononuclear cells from children with AIDS-related lymphoma.
Cancer Res.
,
61
:
5028
-5037,  
2001
.
19
Burger J. A., Tsukada N., Burger M., Zvaifler N. J., Dell’Aquila M., Kipps T. J. Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1.
Blood
,
96
:
2655
-2663,  
2000
.
20
Nagasawa T., Kikutani H., Kishimoto T. Molecular cloning and structure of a pre-B-growth-stimulating factor.
Proc. Natl. Acad. Sci. USA
,
91
:
2305
-2309,  
1994
.
21
Verschueren H., Van der Taelen I., Dewit J., De Braekeleer J., De Baetselier P. Metastatic competence of BW5147 T-lymphoma cell lines is correlated with in vitro invasiveness, motility and F-actin content.
J. Leukoc. Biol.
,
55
:
552
-556,  
1994
.
22
Greiner D. L., Hesselton R. A., Shultz L. D. SCID mouse models of human cell engraftment.
Stem Cells
,
16
:
166
-177,  
1998
.
23
Feng Y., Broder C. C., Kennedy P. E., Berger E. A. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane. G protein-coupled receptor.
Science (Wash. DC)
,
272
:
872
-877,  
1996
.
24
Doranz B. J, Grovit-Ferbas K., Sharron M. P., Mao S. H, Goetz M. B., Daar E. S., Doms R. W., O’Brien W. A. A small-molecule inhibitor directed against the chemokine receptor CXCR4 prevents its use as an HIV-1 coreceptor.
J. Exp. Med.
,
186
:
1395
-1400,  
1997
.
25
Hendrix C. W., Flexner C., MacFarland R. T., Giandomenico C., Fuchs E. J., Redpath E., Bridger G., Henson G. W. Pharmacokinetics and safety of AMD-3100, a novel antagonist of the CXCR-4 chemokine receptor, in human volunteers.
Antimicrob. Agents Chemother.
,
44
:
1667
-1673,  
2000
.
26
Murakami T., Zhang T. Y., Koyanagi Y., Tanaka Y., Kim J., Suzuki Y., Minoguchi S., Tamamura H., Waki M., Matsumoto A., Fujii N., Shida H., Hoxie J. A., Peiper S. C., Yamamoto N. Inhibitory mechanism of the CXCR4 antagonist T22 against human immunodeficiency virus type 1 infection.
J. Virol.
,
73
:
7489
-7496,  
1999
.