Abstract
Mantle cell lymphoma (MCL) is an aggressive lymphoma that generally is associated with a rapid dissemination of the malignant B-cells and a high risk of relapse. Chemokine receptors and adhesion molecules play key roles in normal B cell migration and homing to distinct microenvironments, but their expression and function in MCL is largely unknown. In this study, we profiled the expression and function of chemokine receptors and adhesion molecules (CXCR4, CXCR5, CXCR3, CD49d/VLA-4, CD44, and CD62L) in MCL cell lines (SP-53, MINO, JeKo-1 and Granta-519) and primary MCL cells. Except for the EBV-positive cell line Granta 519, all MCL lines displayed high levels of CXCR4, CXCR5, CD49d and CD44. Primary MCL cells from different patients (n=6) displayed a similar immunophenotype. We then analyzed chemotaxis of MCL cells towards CXCL12 (200 ng/ml) and CXCL13 (1μg/ml) in transwell assays. 35.7±5.7% of input SP-53, 25.8±2.8% of MINO, and 6.7±0.9% of JeKo-1 cells migrated towards CXCL12 within 3 hours (mean±SEM, triplicates). 43.4±9.8% of input SP-53, 16.7±2.8% of MINO, and 4.3±0.5% of JeKo-1 cells migrated toward CXCL13. Granta-519 did not demonstrate any significant chemotaxis. Pre-treating the cells with a CXCR4 antagonist (AMD3100, Plerixafor) effectively blocked chemotaxis towards CXCL12. Marrow stromal cells (MSC) constitutively secrete CXCL12 and provide ligands for VLA-4 integrins. These two molecules now can be targeted clinically using CXCR4 antagonists or monoclonal antibodies (mAbs), respectively. In co-cultures with MSC, SP-53 and MINO displayed abundant spontaneous migration beneath MSC (pseudoemperipolesis/PEP). Therefore we examined whether blocking of CXCR4 or VLA-4 affects the PEP of MCL cells. Pre-incubation of SP-53 or MINO cells with AMD3100 reduced PEP to levels that were 49.4±1.5% (p<.07) or 11.7±1.7% (p<.04) of controls. Pretreatment with anti-VLA-4 antibodies (Natalizumab) also resulted in significant decrease of PEP to levels that were 5.8±1.4% (p<.02) in SP-53 and 2.2±0.6% (p<.01) in MINO cells. Pre-incubation with a cyclic peptide inhibitor with the minimal VLA-4 binding motif “LDV” also significantly reduced PEP of MCL cells to 22.1±4.0% (p<.006) in SP-53 and 7.1±1.9% (p<.005) in MINO cells. MCL cells treated with 10 μM fludarabine (F-ara-A) in suspension cultures resulted in high levels of apoptosis in MCL cells within 24 to 72 hrs. We found that co-culture with MSC significantly reduced F-ara-A-induced apoptosis, a primary drug resistance mechanism termed cell adhesion-mediated drug resistance (CAM-DR). Interestingly, the MCL cell fraction that had migrated into the stromal cell layer was particularly protected from the cytotoxic effect of F-ara-A. As such, clinical targeting of CXCR4 and VLA-4 integrins in MCL may not only antagonize the migration and homing associated with the dissemination of the disease, but also disrupt the adhesion to stromal cells, help to overcome CAM-DR, and thus make MCL cells more accessible to conventional drugs. Collectively, our studies provide a rationale to further explore the efficacy of combinations of CXCR4- and/or VLA-4 antagonists with conventional drugs in patients with this disease. This approach may lead to new therapeutic avenues for MCL patients.
BMP7 Expression Correlates with Secondary Drug Resistance in Mantle Cell Lymphoma