Stromal cell-derived factor-1 (CXCL12/SDF-1) is a chemokine involved in development and trafficking of B cells and hematopoietic progenitors. Recent evidences also suggest CXCL12/SDF-1 involvement in breast cancer cell pseudopodia formation and in invasive breast cancer metastasis . 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 , and the CXCR4-CXCL12/SDF-1 circuitry appears to be crucial for migration of chronic lymphocytic leukemia  and acute lymphoblastic leukemia B cells .

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, -, -, and -CXCR4 monoclonal antibodies (BD), annexin V, and 7AAD to depict apoptotic or dead cells as described previously .

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. and Poznansky et al. 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 x 10⁴ human microvascular endothelial cells (Cascade Biologics, Portland, OR) or bone marrow-derived stromal cell lines L87/4 and L88/5  was seeded in the upper chamber and cultured for 48 h in RPMI-10% FBS. A total of 2 x 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.  . 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 x length x 0.52] applied for approximating the volume of a spheroid (12) .

In a first tumor-challenge trial, 2 x 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 x 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 x 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 x 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

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