Mantle cell lymphoma cells express high levels of CXCR4, CXCR5, and VLA-4 (CD49d): importance for interactions with the stromal microenvironment and specific targeting

Abstract

Mantle cell lymphoma (MCL) is characterized by an early, widespread dissemination and residual disease after conventional treatment, but the mechanisms responsible for lymphoma cell motility and drug resistance are largely unknown. There is growing evidence suggesting that chemokine receptors and adhesion molecules are critical for malignant B-cell trafficking and homing to supportive tissue microenvironments, where they receive survival and drug resistance signals. Therefore, we examined chemokine receptor and adhesion molecule expression and function in MCL cells and their importance for migration and adhesion to marrow stromal cells (MSCs). We found that MCL cells display high levels of functional CXCR4 and CXCR5 chemokine receptors and VLA-4 adhesion molecules. We also report that MCL cells adhere and spontaneously migrate beneath MSCs in a CXCR4- and VLA-4-dependent fashion (pseudoemperipolesis). Moreover, we demonstrate that MSCs confer drug resistance to MCL cells, particularly to MCL cells that migrate beneath MSC. To target MCL-MSC interactions, we tested Plerixafor, a CXCR4 antagonist, and natalizumab, a VLA-4 antibody. Both agents blocked functional responses to the respective ligands and inhibited adhesive interactions between MCL cells and MSCs. These findings provide a rationale to further investigate the therapeutic potential of these drugs in MCL.

Figure 1

MCL cell lines demonstrate a high level of CXCR4 and CXCR5 expression. Overlay histogram plots depict the relative CXCR4 and CXCR5 fluorescence intensity (bold line) of CD19⁺ MCL cells in comparison with isotype control stain (thin line). The MFIRs are displayed next to each histogram. The MCL lines SP-53, MINO, and JeKo-1 demonstrate high levels of CXCR4 and CXCR5 expression, whereas in the EBV⁺ cell line Granta 519 expression of these chemokine receptors is low.

Figure 2

Chemotaxis and actin polymerization in primary MCL cells in response to CXCL12 and CXCL13. (A) Actin polymerization of MCL cells in response to CXCL12 and CXCL13. Intracellular F-actin content in MCL cells was determined at the time points indicated on the horizontal axis after the addition of 200 ng/mL CXCL12 or CXCL13. Inhibition of CXCL12-induced actin polymerization was detected after preincubation with Plerixafor or pertussis toxin. The relative F-actin content compared with samples before chemokine stimulation (100%) is displayed on the vertical axis and is the mean plus or minus SEM of 4 MCL samples from different patients. (B) Effect of Plerixafor on actin polymerization in response to CXCL12 stimulation. Remarkable inhibition of actin changes mediated by CXCL12 was demonstrated after pretreatment with Plerixafor. The histograms show representative results from one of 4 experiments with MCL B cells from different patients. (C) Displayed is the mean relative chemotaxis of primary MCL cells toward the chemokines CXCL12 or CXCL13 under the conditions displayed on the vertical axis. MCL cells display relatively high levels of spontaneous migration toward wells without chemokine (control). CXCL12 and CXCL13 both induced chemotaxis of MCL cells, and CXCL12-induced chemotaxis is inhibited by pretreatment with pertussis toxin or Plerixafor. Results are percentages of migrated cells relative to input and are mean ± SEM of 4 experiments. *Significant difference compared with control sample (P < .05).

Figure 3

Effects of natalizumab, Plerixafor, pertussis toxin, and CS-1 peptide on the migration of MCL cells beneath stromal cells (pseudoemperipolesis). To demonstrate the effect of blocking VLA-4 integrins on pseudoemperipolesis, a confluent layer of M2-10B4 marrow stromal cells was established (A), and untreated (B) or pretreated (with natalizumab in panel C, with CS-1 peptide in panel D) MCL cells were seeded onto the stromal cell layer. After 6 hours, nonmigrated cells were removed by vigorous washing. Migrated cells are characterized by a dark appearance, whereas nonmigrated cells remain bright. The stromal cell layer containing the migrated MCL cells was photographed (100× magnification). These photomicrographs illustrate that pseudoemperipolesis of MCL cells after treatment with natalizumab (C) and CS-1 peptide inhibitor (D) was greatly reduced compared with untreated controls (B). Cells were imaged using a phase contrast microscope (Model ELWD 0.3; Nikon, Garden City, NY) with a 10×/0.25 NA objective lens. Images were captured with a Nikon D40 digital camera (Nikon, Tokyo, Japan) using Camera Control Pro software (Nikon) and processed with Adobe Photoshop 9.0 software (Adobe Systems, San Jose, CA). The MCL cell lines SP-53 (E) and MINO (F) were left untreated (controls) or pretreated with the agents displayed on the horizontal axis and then incubated on confluent MSC layers. After incubation, the nonmigrated cells were vigorously washed off, and the MCL cells that had migrated into the MSC layer were quantified by FACS. The mean ± SEM relative pseudoemperipolesis is shown for 3 independent experiments for each cell line. *Significant inhibition of pseudoemperipolesis compared with control sample (P < .05).

Figure 4

Marrow stromal cells protect MCL cells from F-ara-A-induced apoptosis. (A,B) Mean viability of SP-53 cells (A) or MINO cells (B) treated with F-ara-A at the time points displayed on the horizontal axis. MCL viability was higher when MCL cells were cocultured with M2-10B4 MSC cells (▨) compared with MCL without stromal cell support ([graphic024]). Results are presented as mean relative viability compared with untreated controls (100%) and are the mean ± SEM values of triplicates. *Significant protection of MCL cells from F-ara-A cytotoxicity compared with control sample (P < .05). (C) Cell viability of MCL cells was determined by staining with DiOC₆ and PI. Contour plot represents supernatant and migrated fractions of pretreated with 10 μM F-ara-A SP-53 cells after 72 hours of cultivation on M2-10B4. The predominance of vital cells (DiOC₆^bright, PI^exclusion) was detected for migrated fraction (right panel) compared with supernatant fraction (left panel). The percentage of vital cells is displayed in each contour map. (D) Viability of migrated and supernatant fractions of MCL cells pretreated with 10 μM F-ara-A after 24, 48, and 72 hours of cultivation on stromal cells. Results are represented relative to untreated controls and are mean ± SEM values of 3 different experiments.

Figure 5

Marrow stromal cells protect MCL cells from 4-HC-induced cytotoxicity. The mean viability of SP-53 cells (A) and MINO cells (B) treated with 4-HC is presented at the different time points displayed on the horizontal axis. MCL cell viability was increased when MCL cells were cocultured with M2-10B4 (), compared with MCL in suspension and without stromal cell support (). Results are presented as mean relative viability compared with untreated controls (100%) and are the mean ± SEM of triplicates. *Protection of MCL cells from 4-HC-induced cytotoxicity with significantly higher viabilities compared with controls (P < .05). (C) Contour plots that depict the viability of MINO MCL cells, as detected by staining with DiOC₆ and PI after 24 hours of culture with 10 μM 4-HC, in the presence or absence of M2-10B4 stromal cells, as indicated above each of the plots. The proportion of viable cells is indicated above each of the gates that define viable cells by bright DiOC₆ staining and PI exclusion.

Figure 6

Effects of CXCL12 and CXCL13 stimulation on p44/p42 MAPK activation. SP-53 cells were stimulated with 250 ng/mL CXCL12 or 1 μg/mL CXCL13 for different times. To block CXCR4-derived signaling, cells were preincubated for 1 hour with 100 μg/mL Plerixafor and then stimulated with CXCL12. MCL cell lysates were probed with antiphospho p44/p42, antiβ-actin mAbs, and (on separate gels) with anti-p44/p42 mAbs. As indicated, maximum p44/p42 activation occurs within 2 minutes of stimulation with a subsequent decline in signal intensity. Pretreatment with Plerixafor effectively inhibits CXCL12-mediated p44/p42 MAPK activation. Results shown are representative of 3 experiments.