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. Author manuscript; available in PMC: 2015 Apr 21.
Published in final edited form as: Br J Haematol. 2011 Jul 12;155(1):53–64. doi: 10.1111/j.1365-2141.2011.08794.x

Bone Marrow Stromal Cells Protect Lymphoma B-cells from Rituximab-Induced Apoptosis and Targeting Integrin alfa-4-beta-1 (VLA-4) with Natalizumab can Overcome this Resistance

Marek Mraz 1,3, Clive S Zent 1, Amy K Church 1, Diane F Jelinek 2, Xiaosheng Wu 2, Sarka Pospisilova 3, Stephen M Ansell 1, Anne J Novak 1, Neil E Kay 1, Thomas E Witzig 1, Grzegorz S Nowakowski 1
PMCID: PMC4405035  NIHMSID: NIHMS301497  PMID: 21749361

Abstract

Rituximab improves the outcome of patients with non-Hodgkin lymphoma, but does not completely eradicate residual B-cell populations in the microenvironment of the bone marrow and lymph nodes. Adhesion to stromal cells can protect B-cells from apoptosis induced by chemotherapy drugs (cell adhesion-mediated drug resistance; CAM-DR). A similar mechanism of resistance to rituximab has not, to our knowledge, been described. We tested the hypothesis that the microenvironment protects malignant B-cells from rituximab-induced apoptosis, and that blocking these interactions with natalizumab, an antibody targeting VLA-4 (integrin alfa-4-beta-1/CD49d), can overcome this protection. VLA-4 is an adhesion molecule constitutively expressed on malignant B-cells and is important for pro-survival signalling in the bone marrow and lymph node microenvironment. The human bone marrow stromal cell line HS-5 was shown to strongly protect B-cell lymphoma cells from rituximab cytotoxicity, suggesting the existence of a stromal cell adhesion-mediated antibody resistance (CAM-AR) mechanism analogous to CAM-DR. Natalizumab decreased B-lymphocyte adherence to fibronectin by 75-95% and partially overcame stromal protection against rituximab and cytotoxic drugs. These pre-clinical findings suggest that the addition of stromal adhesion-disruptive drugs to rituximab-containing therapy could improve treatment efficacy.

Keywords: lymphoma, stromal cells, cell adhesion-mediated drug resistance, rituximab, natalizumab

Introduction

Rituximab monotherapy and chemoimmunotherapy have significantly improved the outcome of patients with B-cell non-Hodgkin lymphoma. Rituximab has been shown to mediate antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct induction of apoptosis, and can also sensitize malignant B-cells to the effects of chemotherapy (Smith 2003). The exact contributions of each of these mechanisms to the clinical efficacy of rituximab in vivo remain uncertain. Rituximab-induced apoptosis of malignant B-cells appears to be related to reorganizing the CD20 molecules in lipid rafts, which is followed by pro-apoptotic signalling (Deans, et al 2002) which is independent of immune effector mechanisms and Fc function (Vega, et al 2009). These data suggest that rituximab-induced apoptosis could be an important mechanism of action for rituximab cytotoxicity in some B-cell malignancies.

While the mechanisms explaining the resistance of CD20+ B-cells to CDC and ADCC, including increased expression of complement control proteins, exhaustion of complement components, blockade of ADCC by deposited C3, loss of CD20 expression and the expression of the low affinity polymorphisms of FcγR have been explored (Taylor and Lindorfer 2010), mechanisms by which malignant B-cells are able to resist direct rituximab cytotoxicity are less well understood. Rituximab appears to be less effective in patients with bulky lymphoma and extensive bone marrow involvement (Coiffier, et al 1998, van Oers, et al 2010) and some B-cells surviving rituximab treatment appear to acquire resistance to subsequent rituximab therapies (Davis, et al 2000, Martin, et al 2008). The role of the microenvironmental stromal cells in mediating the resistance of B-cells to rituximab has not been extensively studied.

The microenvironment of B-cell lymphomas is similar to that which supports the growth and maturation of normal B-cells. In this regard, B-cell malignancies are dependent on the signals from this niche for survival and proliferation (Burger, et al 2009). The critical role of the microenvironment in the pathophysiology of lymphoma is illustrated by the finding that the survival of patients with follicular lymphoma correlates with the molecular features of non-malignant cells present in the lymph node (Dave, et al 2004). Moreover, the architecture and gene expression of lymph node stromal cells in diffuse large cell lymphoma correlates with outcome following treatment with a rituximab-containing regiment (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone)(Lenz, et al 2008). Therefore, microenvironmental interactions appear to be an important prognostic factor for B-cell lymphomas in the rituximab era.

Previous studies have shown that adhesion to cultured stromal cells or ligand-coated surfaces can protect malignant B-cells from apoptosis induced by chemotherapy drugs (cell adhesion-mediated drug resistance; CAM-DR) (Dalton 2002, Damiano, et al 1999, Kay, et al 2007, Lwin, et al 2007, Taylor, et al 1999). Importantly, adhesion-mediated resistance could be a therapeutic target. One potential candidate for targeted disruption of this protective stroma-B-cell interaction is VLA-4 (integrin alfa-4-beta-1/CD49d). Integrins are cell surface receptors that mediate both cell-cell adhesion and cell-extracellular matrix adhesion and can signal “inside out” and “outside in” to confer protection against drug-induced apoptosis (Hood and Cheresh 2002). VLA-4 is a heterodimer of alfa-4 and beta-1integrin that has an important role in the adhesion of B-cells to both the endothelium and stroma and provides pro-survival signalling (Koopman, et al 1994, Matsunaga, et al 2003, Weekes, et al 2001, Zucchetto, et al 2009). VLA-4 is highly expressed by most primary lymphoma cells (Baldini, et al 1992, Jacob, et al 1999, Lúcio, et al 1998) as well as a subset of patients with aggressive CLL (Rossi, et al 2008, Shanafelt, et al 2008). Therapeutic targeting with VLA-4 could be achieved using natalizumab. Natalizumab is a humanized IgG4 monoclonal antibody currently used in the treatment of Crohn’s disease and multiple sclerosis (Ghosh, et al 2003, Ransohoff 2007), where its benefit is related to a decrease in homing of lymphocytes to sites of inflammation (Rice, et al 2005). It is a blocking antibody and the IgG4 isotype was chosen, because the IgG4 subclass does not activate complement or ADCC and persists longer in the circulation than other subtypes of IgG (Mountain and Adair 1992).

This study demonstrated for the first time that interactions with the microenvironment protect malignant B-cells from rituximab-induced apoptosis and that this protection is comparable to CAM-DR seen with cytotoxic agents. We also demonstrate that this stromal protection against rituximab-induced cytotoxicity can be overcome by blocking VLA-4 with natalizumab, which also disrupts the stroma-mediated resistance of B-cells against cytotoxic drugs. These data provide strong pre-clinical evidence that the efficacy of rituximab-containing regimens could be enhanced by the inhibition of malignant B-cell-stroma interactions with natalizumab.

Materials and methods

Cell cultures

B-cell lines (Karpas-422, Raji, DOHH2, HT, RL, Granta-519, Mino, Jeko-1, SUDHL-6, MEC-1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) or the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and were cultured under standard conditions (5% CO2, 37°C) for all experiments using the recommended media, RPMI-1640 (Karpas-422, Raji, DOHH2, HT, RL, Mino, Jeko-1, SUDHL-6), IMDM (MEC-1), DMEM (HS-5, Granta-519), with 10% fetal bovine serum, which was heat inactivated to prevent CDC. These cells were selected for this study after testing for VLA-4 expression by flow cytometry (Becton Dickinson, San Jose, CA, USA) using a mouse anti-cd49d antibody (BD PharMingen, San Diego, CA, USA). All the cell lines expressed VLA-4 except for SUDHL-6, which was used as the negative control for experiments with natalizumab.

Primary B-cell lymphoma samples were prepared as previously described (Witzig, et al 1990). Briefly, lymphoma tissue were minced over a fine mesh screen (60 μm) to obtain a single-cell suspension, and then centrifuged over Ficoll-Paque (Sigma-Aldrich, St. Louis, MO, USA) to isolate mononuclear cells. The median percentage of malignant B-cell was 71% in studied B-cell malignancy samples (range of 45%-86). The VLA-4 expression in primary malignant B-cell was 63% and 72% in 2 patients with chronic lymphocytic leukemia/small lymphocytic lymphoma and was over 90% (91-97%) for other lymphoma subtypes including mantle cell lymphoma.

Target cells were cultured (0.5 × 106 cells/1ml/well) on either a plastic surface, a confluent monolayer of the bone marrow stromal cell line HS-5 (obtained from ATCC), or a plastic surface coated with fibronectin (20 μg/ml; Invitrogen, Carlsbad, CA, USA) purified by affinity chromatography. This fibronectin molecule contains all sites known to be important for VLA-4 dependent cell adhesion including the CS1 site, C-terminal heparin-binding domain (Hep II), and the RGD (Arg-Gly-Asp) sequence (Domínguez-Jiménez, et al 1996, Mould and Humphries 1991, Sánchez-Aparicio, et al 1994, Wayner, et al 1989). Additional experiments were performed by culturing cells on surfaces coated with the VLA-4 ligand vascular cell adhesion molecule 1 (VCAM-1; 5 -40ug/ml; R&D Biosystems, Minneapolis, MN) as described previously (Buchner, et al 2010a).

For co-culture experiments with stromal cells, HS-5 cells were plated 2 days before the experiment onto 24-well plates (~5 × 104 cells/1ml/well). After confirming the confluence of the stromal layer by phase contrast microscopy, the media were aspirated and target cells were added in fresh media onto the HS-5 layer. The human stromal cell line HS-5 was used because it is a well characterized model for the bone marrow microenvironment (Ghosh, et al 2009, Graf, et al 2002, Roecklein and Torok-Storb 1995).

Adhesion assay

The ability of natalizumab to disrupt cell adhesion was assessed by staining 0.25 × 106 lymphocytes with calcein (Invitrogen) according to the manufacturers protocol for 30 minand then incubating them in 24-well plates with a fibronectin-coated surface (20 μg/ml) in the recommended media with 10% FBS containing natalizumab (10 μg/ml, Biogen IDEC, Cambridge, MA, USA), or control immunoglobulin (IgG; 10 μg /ml, Sigma-Aldrich) or medium alone. After 2 h of culture, the non-adherent cells were washed off twice with 1 ml of phosphate-buffered saline (PBS) and the number of adherent cells was measured with a fluorometer (CytoFluor, Applied Biosystems, Foster City, CA, USA). The percentage of adherent cells was calculated by comparing the measured absorbance with that of 0.25 × 106 cells.

Apoptosis assays

Rituximab (Genentech, San Francisco, CA, USA), natalizumab or control IgG was used at a concentration of 10 μg/ml. Doxorubicin (Bedford Laboratories, Bedhord, OH, USA) and F-ara-A (Sigma-Aldrich), the active metabolite of fludarabine, were used in escalating doses as described below.

To assess the effect of rituximab on B-cell apoptosis, cells were directly treated for 24 h in suspension culture (plastic surface) or in co-culture with HS-5. For experiments utilizing cytotoxic drugs, lymphocytes were pre-treated for 16 h with doxorubicin or F-ara-A (plastic surface), plated on confluent layer of HS-5 or plastic surface and then cultured for an additional 24 hours. The pre-incubation of target lymphocytes with doxorubicin or F-ara-A was followed by washing to remove any residual cytotoxic drugs and thus prevent toxicity to HS-5 stromal cells. The effect of natalizumab on doxorubicin-, F-ara-A-, or rituximab-induced apoptosis was compared to control IgG in these experiments. Cell viability was measured by flow cytometry using annexin V and propidium iodide staining (BD PharMingen, San Diego, CA, USA) according to the manufacturer’s instructions. For each assay, 10,000 CD19+ cells were analysed using CellQuest software (BD Biosciences, Mountain View, CA, USA).

To test if HS-5 cells have any phagocytic activity we utilized a flowcytometer that works with defined volume of sample (Cell LabQuanta SC, Beckman Coulter) and counted the viability and absolute amount of CD19+ cells per well over time (as specified below).

Confocal microscopy

For confocal microscopy, natalizumab and rituximab were labelled according to the manufacturer’s protocol with Alexa 488 and Alexa 568, respectively (Alexa Fluor Antibody Labeling Kit, Invitrogen). Cells were incubated with antibodies for 20 min in the dark (10 μg/ml; 5% CO2, 37°C), washed, fixed with 4% paraformaldehyde, and plated on glass slides coated with 0.1% polyethanolamine (Sigma-Adrich). Slides were then examined by confocal microscopy (Zeiss LSM510, Carl Zeiss MicroImaging, Thornwood, NY, USA).

Statistical analyses

Statistical analyses were done using 2-tailed paired t-test and 2-factorial analysis of variance (ANOVA) with post-hoc Tukey test (Statistica 6.0, StatSoft, Tulsa, OK, USA) and graphically visualized by GraphPad Prism Software v. 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). All data shown represent the results of at least two independent experiments and p-values <0.05 were considered significant.

Results

Stromal cells mediate protection against rituximab-induced apoptosis

To study stroma-mediated resistance to rituximab-induced apoptosis, we first tested 10 CD20-positive lymphocyte cell lines derived from malignant B-cells for sensitivity to rituximab induced apoptosis at 24 h in suspension culture (Fig. 1A). In accordance with previously published studies, these cell lines showed a wide range of sensitivity (0.3 – 43% cytotoxicity) to rituximab-induced apoptosis (Bonavida 2007, Semac, et al 2003, Shan, et al 2000). Subsequent experiments used the Karpas-422, Raji, and DOHH2 cell lines, as they were most sensitive to rituximab-induced apoptosis and are representative of diffuse large B-cell lymphoma, Burkitt lymphoma, and follicular lymphoma, respectively. Co-cultures of all these rituximab-responsive lymphoma cell lines with HS-5 resulted in a significant reduction of rituximab-induced apoptosis compared to cells cultured on a plastic surface (18% vs. 31% for DOHH2, 2% vs. 19% for Raji, 8% vs. 29% for Karpas-422; p<0.05; Fig. 1B). We also compared the protective effects of HS-5 on rituximab-induced death to CAM-DR. Each cell line was treated with a doxorubicin dose (50 ng/ml-Karpas-422; 100 ng/ml-Raji; 10 ng/ml-DOHH2) that induced a similar decrease in viability to that achieved by 10 μg/ml of rituximab. This experiment demonstrated that the protective effect of stromal cells on rituximab cytotoxicity was very similar in magnitude to the cell adhesion-mediated resistance to doxorubicin (Fig. 1B).

Figure 1.

Figure 1

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(A) Responsiveness of 10 CD20-positive cell lines to rituximab-induced apoptosis. Lymphocyte cell lines were treated with rituximab (10μg/ml) in suspension culture for 24 h. Changes in the percentage of apoptotic cells were measured by comparison with control untreated cells. (B) Stroma-mediated rituximab resistance. Cells were co-cultured on a confluent monolayer of HS-5 stromal cells for 24 h after which rituximab (Rit) was added and the co-culture continued for an additional 24 hours. For experiments with doxorubicin (Dox), cells were treated with a doxorubicin concentration that caused similar decreases in cell viability as rituximab (50 ng/ml-Karpas-422, 100 ng/ml-Raji, 10 ng/ml-DOHH2) and co-cultured for 24 h with HS-5. All cells were then harvested and assessed for viability. Ctrl, control. (C) Stroma-mediated rituximab resistance of primary lymphoma samples. Primary samples were obtained from tissue samples (as described in methods) and cells were plated on a plastic surface or co-cultured on a confluent monolayer of HS-5 stromal cells for 24 h. This was followed by addition of rituximab (10 μg/ml) to both cultures (HS-5 co-culture and to cells cultivated on plastic) for an additional 24 h. After a total co-culture time of 48 h all cells were harvested and assessed for viability. SLL, small lymphocytic variant of chronic lymphocytic leukaemia; MCL, mantle cell lymphoma; FL, follicular lymphoma; MZL, marginal zone lymphoma; T-cell NHL, T-cell non-Hodgkin lymphoma. The viability of control untreated cells is set as zero.

* p<0.05 (t-test), Error bars represent standard error of the mean.

To test if stroma can protect against rituximab-induced apoptosis, we studied 8 primary B-cell malignancy samples (3 CLL and 5 other B-cell lymphomas) and 1 control T-cell lymphoma sample. The complex process of obtaining single cell suspensions of these malignant lymphocytes from lymphoma samples causes a high level of induction of apoptosis. We first assessed the effect of co-culturing primary lymphoma cells with stromal cells and observed that the level of post-processing apoptosis was significantly lower in the lymphoma cells cultured on HS-5 stroma compared to a plastic surface (17% vs. 44%; p=0.03). The induction of apoptosis by the isolation procedure together with a relatively lower responsiveness to rituximab complicates the assessment of rituximab effects and the interpretation of results. However, we next assessed the impact of stroma on rituximab-induced apoptosis (Fig. 1C). Significant stroma protection against rituximab-mediated apoptosis was observed for the 4 most rituximab-responsive primary B-cell malignancy samples (difference in apoptosis 7% vs. 25%; 2% vs. 16%; 5% vs. 12%; 0% vs. 12%; stroma vs. plastic, respectively; p<0.05; Fig. 1C). However, because of the limited supply of tissue and the high rates of spontaneous apoptosis in the primary cells, we conducted the subsequent assays using B-cell lymphoma cell lines.

Natalizumab affects B-cell adhesion and morphology

We tested if targeting VLA-4 could inhibit the adhesion of malignant B-cells and disrupt stroma-mediated protection against cytotoxic therapy. Although natalizumab was designed purely as a blocking antibody and should not induce apoptosis in VLA-4 expressing B-cells, this has not been tested in lymphoma cells. We therefore first tested the effect of natalizumab on lymphoma cells. Target lymphoma cells were incubated in media with natalizumab (10 μg/ml) for 24 h and then assessed for viability. As expected, natalizumab had no effect on the viability of cells cultivated on a plastic surface or on stromal cells (average change in cell viability 2%; Fig. S1). To visualize the binding of natalizumab to B-cells, the antibody was labelled with Alexa 488. Cells were washed and incubated with the antibody (10 μg/ml) for 20 min (37°C), and visualized using confocal microscopy. Fluorescently-labelled natalizumab bound specifically to VLA-4-positive cell lines with striking co-localization to the rituximab binding sites on the B-cell membrane (Fig. 2). The co-localization of VLA-4 and CD20 corresponded with previously published data showing that VLA-4 is a component of lipid raft-dependent signalling of B-cells in the lymph node and bone marrow microenvironment. VLA-4 is likely to be functionally associated with the B-cell receptor, CD38, CD40 and CD20 on the B-cell membrane (Buggins, et al 2011, Deaglio, et al 2008, Kheirallah, et al 2010, Silvy, et al 1997, Zucchetto, et al 2009). To test if natalizumab affects CD20 expression or rituximab binding, cells were incubated with natalizumab (10 μg/ml) for 30 min or 24 h under standard conditions on plastic surface. These time points were selected as representatives of possible short term (formation of lipid raft clusters containing CD20 or rituximab) and long-term effects (gene expression changes). Cells were harvested at given time points and CD20 expression or rituximab binding (assessed by anti-CD20 antibody or labelled rituximab, respectively) was compared to control cells. This experiment demonstrated that natalizumab did not have a significant effect on CD20 expression or rituximab binding (Fig. S2,S3). Natalizumab also did not affect the complement fixation (Fig. S4).

Figure 2.

Figure 2

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A representative immunofluorescence micrograph demonstrating co-localization of natalizumab and rituximab binding sites on the B-cell membrane. Natalizumab and rituximab were labelled with Alexa 488 and Alexa 568, respectively. Cells were incubated with antibodies for 20 min (10 μg/ml; 5% CO2, 37°C), washed, fixed and then examined by confocal microscopy.

We then investigated the ability of natalizumab to inhibit the adhesion of lymphoma cells to the VLA-4 ligand fibronectin. Seven VLA-4-positive cell lines (Raji, Karpas-422, DOHH2, Mino, Granta-519, Mec-1, Jeko-1) and a VLA-4-negative control cell line (SUDHL-6) were plated on a fibronectin coated surface and treated for 2 h with natalizumab or control IgG. Natalizumab decreased lymphocyte adherence to fibronectin for all VLA-4 positive cell lines by 75-95% (p<0.05; Fig. 3A). The morphology of adherent lymphocytes (formation of pseudopodia) was lost upon treatment with natalizumab (Fig. 3B). Very similar changes in cell adhesion morphology were previously observed after treatment with CXCR-4 inhibitors (Bertolini, et al 2002). Treated cells did not re-adhere to fibronectin even after a prolonged cultivation time of 72 h. Similar results were obtained when cells were first pre-incubated for 2 h with natalizumab, then washed and plated in fresh media on fibronectin or VCAM-1 coated wells (data not shown). The VLA-4 negative cell line SUDHL-6 had only weak adherence to a fibronectin-coated surface, which was not affected by natalizumab (Fig. 3A).

Figure 3.

Figure 3

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(A) The effect of natalizumab on B-cell lymphoma cell line adhesion. Cells were stained with calcein and plated in media containing natalizumab (10 μg/ml) or control IgG (10 μg/ml) or no added antibody on a fibronectin (VLA-4 ligand) coated surface. After 2 h of culture the non-adherent cells were washed twice with 1 ml of phosphate-buffered saline and the number of adherent cells was measured. All cell lines except for SUDHL-6 are positive for expression of VLA-4. The percentage of adherent cells was calculated based on the absorbance of total cells. (B) Natalizumab effect on cell adhesion morphology. Cell line Karpas-422 was plated on a fibronectin-coated surface and treated with natalizumab or control IgG. The cell morphology following natalizumab treatment is compared to cells plated in non-adherent conditions (plastic surface).

* p<0.05 (t-test), Error bars represent standard error of the mean.

Natalizumab inhibits stroma-mediated protection against cytotoxic drugs

It was previously demonstrated that the adhesion of B-cells to stroma can protect cells from cytotoxic drugs and inhibition of this adhesion can sensitize the B-cells to apoptosis (Burger et al 2009). To investigate the effects of natalizumab on doxorubicin-induced apoptosis, B-cells were pre-treated for 16 h with various concentrations of doxorubicin, plated on confluent layers of HS-5 or a plastic surface and treated with natalizumab or control IgG. The target cells pre-incubated with doxorubicin were washed prior to incubation with HS-5 stromal cells to prevent doxorubicin toxicity to the stromal cells. After 24 h of culture on HS-5 or a plastic surface (total cultivation time of 40 h), all cells were harvested and assessed for viability. We showed that HS-5 stroma cells strongly protected B-cell lines from doxorubicin-induced death (Fig 4; p<0.001). The number of apoptotic cells induced by higher doses of doxorubicin was: 68% vs. 94% (Karpas-422), 63% vs. 87% (DOHH2), 29% vs. 83% (Raji) for cells cultivated on HS-5 vs. plastic, respectively. The protective effect of HS-5 observed in these experiments was of long duration (Fig. S5A and S5B) and determination of these effects was not affected by phagocytic activity of stromal cells (Fig. S5C).

Figure 4.

Figure 4

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(A, B, C, D) The effect of natalizumab on doxorubicin/F-ara-A induced apoptosis. Cells were pre-treated for 16 hours with various concentrations of doxorubicin/F-ara-A (plastic surface), plated on confluent layer of HS-5 or plastic surface and treated with natalizumab/control IgG. After 24 h of culture on an HS-5/plastic surface (total cultivation time 40 h), all cells were harvested and assessed for viability. The pre-incubation of target cells with doxorubicin/F-ara-A was followed by washing to prevent any damage of HS-5 stromal cells by cytotoxic drugs. (E) Natalizumab does not affect the VLA-4 negative cell line SUDHL-6. This experiment was performed as described for Fig. 4A,B,C,D.

** p<0.001 (Anova), Error bars represent standard error of the mean.

Natalizumab significantly (p<0.001) inhibited stroma-mediated protection by ~30% for both Karpas-422 (Fig. 4A) and Raji (Fig. 4C). Surprisingly, natalizumab did not have any effect on doxorubicin-induced apoptosis for DOHH2 cells (Fig. 4D) despite being effective at inhibiting the adhesion of DOHH2 cells to fibronectin (Fig. 3A). As expected, natalizumab did not have any effect on the control VLA-4 negative SUDHL-6 cell line (Fig. 4E). The stroma-mediated protection from doxorubicin and F-ara-A cytotoxicity were similar with comparable reversal of protection by natalizumab (illustrated for Karpas-422; Fig.4AB).

To investigate the lack of natalizumab effect on DOHH2 we tested the cell dependency on VLA-4 induced signalling. In this experiment cell lines Karpas-422 and DOHH2 were pre-treated for 16 h with doxorubicin (50 μg/ml), washed and plated on plastic- or VCAM-1-coated surfaces using three different concentrations of VCAM-1. After 24 h of culture on VCAM-1 or a plastic surface (total cultivation time of 40 h) all cells were harvested and assessed for viability. VCAM-1 coating (VLA-4 ligand) protected Karpas-422 cells from doxorubicin-induced apoptosis, but was not sufficient for a significant protection of DOHH2 cells (Fig. 5A). We then titrated the concentration of doxorubicin to test if DOHH2 protection by VCAM-1 occurred at only specific levels of doxorubicin. As shown in Fig. 5C, there was no observed protection by VCAM-1. We concluded that VCAM-1 was not an important ligand mediating DOHH2-stomal interactions and the limited dependency of this cell line on VLA-4 signalling thus explains why natalizumab does not inhibit stromal protection.

Figure 5.

Figure 5

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(A) The effect of VCAM-1 on doxorubicin-induced apoptosis for Karpas-422 cell line. Cells were pre-treated for 16 h with doxorubicin (50 μg/ml), washed and plated on plastic or VCAM-1 coated surface (3 different concentrations of VCAM-1). After 24 h of culture on VCAM-1 or a plastic surface (total cultivation time of 40 h) all cells were harvested and assessed for viability. (B) The lack of VCAM-1 effect on doxorubicin-induced apoptosis for the DOHH2 cell line at increasing doses of VCAM-1. This experiment was performed as described for Fig. 5A (C) The lack of VCAM-1 effect on doxorubicin-induced apoptosis for the DOHH2 cell line using different doses of doxorubicin. DOHH2 cell line was pre-treated for 16 h with escalating doses of doxorubicin, washed and plated on plastic or VCAM-1 coated surface (5 μg/ml). After 24 h of culture on VCAM-1 or a plastic surface (total cultivation time of 40 h) all cells were harvested and assessed for viability.

* p<0.05 (t-test), Error bars represent standard error of the mean.

Natalizumab inhibits stroma-mediated protection against rituximab-induced apoptosis

To measure the effect of natalizumab on rituximab-induced apoptosis, B-cells were cultured on a plastic surface or confluent monolayer of HS-5 for 24 h in the presence of natalizumab or control IgG. Rituximab was then added, the cells cultured for an additional 24 hours and then harvested and assessed for viability. Natalizumab significantly inhibited the stroma-mediated protection against rituximab-induced apoptosis in Karpas-422 (50% decrease in protection) and Raji (73% decrease in protection) cell lines (p<0.05; Fig. 6AB). Natalizumab did not have any effect on rituximab- (Fig. 6C) or doxorubicin- (Fig. 4D) induced apoptosis in the DOHH2 cell line. The effect of natalizumab on rituximab-induced apoptosis was not observed in the VLA-4 negative control cell line SUDHL-6 (Fig. 6D).

Figure 6.

Figure 6

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(A, B, C) The effect of natalizumab on the stroma-mediated resistance to rituximab-induced apoptosis. B-cells were cultured on a plastic surface or confluent monolayer of HS-5 for 24 h in the presence of natalizumab or control IgG. Rituximab was then added and the cells were cultured for an additional 24 h. After a total cultivation time of 48 h, all cells on the HS-5/plastic surface were harvested and tested for viability. (D) Natalizumab does not affect the VLA-4 negative cell line SUDHL-6. This experiment was performed as described for Fig. 6A,B,C.

* p<0.05 (t-test), Error bars represent the standard error of the mean.

Discussion

Therapeutic antibodies have become an important part of lymphoma therapy. Unfortunately, similarly to cytotoxic agents, resistance to antibodies can develop in many patients. Interactions with the microenvironment were shown to be critical for mediating tumour resistance to cytotoxic therapy (Burger, et al 2009, Dalton 2002). To test if a similar mechanism could have a role in resistance to rituximab, we developed a malignant B-cell-stroma adhesion model utilizing the HS-5 stromal cell line derived from an immortalized human bone marrow culture of mesenchymal origin. HS-5 is a very well characterized model for the haematolymphopoietic microenvironment. HS-5 expresses adhesion molecules collagen I, III and IV, and VCAM-1, and secretes numerous cytokines (Graf, et al 2002, Roecklein and Torok-Storb 1995). Co-cultivation of B-cells with HS-5 cells for 48 h is sufficient for the up-regulation of pro-survival proteins including Bcl-2, Mcl-1, XIAP, p-STAT3 (Ghosh, et al 2009, Lwin, et al 2007) and is partly mediated by the interaction of integrins and their ligands expressed on stromal cells. HS-5 and stromal cells can protect primary CLL and different lymphoma subtype malignant B-cells from cytotoxic drugs (Ghosh, et al 2009, Gibson 2002, Kay, et al 2007, Lwin, et al 2007).

We have shown that the bone marrow stromal-cell line HS-5 protects both B-cell lymphoma cell lines and primary lymphoma cells from rituximab-induced apoptosis. Indeed, the protective effect of stromal cells against rituximab cytotoxicity was comparable to the cell adhesion-mediated resistance to cytotoxic drugs (Fig. 1B). To our knowledge, this is the first report of adhesion-mediated resistance to rituximab-induced apoptosis and a first example of cell adhesion mediated antibody resistance (CAM-AR). Whether a similar mechanism mediates resistance to other therapeutic antibodies remains to be established. It is also plausible that cell adhesion may play a role in resistance to rituximab-induced CDC, as recently suggested by a study using CLL cells (Buchner, et al 2010b). The presence of CAM-AR is also supported by indirect in vivo evidence. In this regard, it has been long established that therapeutic antibodies are more effective in eliminating circulating tumour cells than tumour cells from haematolymphopoietic organs (Osterborg, et al 1997). Importantly, the identification of this mechanism of resistance could have significant implications for lymphoma therapy, and is pre-clinical evidence supporting interventions targeting the malignant B-cells – microenvironment interface.

Disruption of malignant B-cell adhesion to stroma could be a potentially feasible approach to overcome CAM-AR. In this regard, VLA-4 is an interesting candidate for targeted disruption of the protective stroma-B-cell interaction and mobilization of malignant B-cells into the circulation, where they are likely to be more sensitive to treatment. The VLA-4 integrin is critically important for trafficking, activation and survival of B-cells in bone marrow and lymph nodes (Lo, et al 2003, Miyake, et al 1991). It is involved in the formation of immune synapses and interaction between B-cells and stromal cells in the germinal centres and lymphoid follicles (Rose, et al 2001, Rose, et al 2002). In addition to being an adhesion molecule, VLA-4 transmits pro-survival signals that result in up-regulation of Bcl-2 (Hayashida, et al 2000). VLA-4 is highly expressed by most primary lymphoma B-cells (Baldini, et al 1992, Johnson, et al 1993, Lúcio, et al 1998) and higher levels are associated with lymphadenopathy and a worse prognosis in CLL patients (Rossi, et al 2008, Shanafelt, et al 2008, Till, et al 2002).

We investigated if targeting integrin VLA-4 by natalizumab, a clinically available VLA-4 blocking antibody, could overcome CAM-AR. Interestingly, natalizumab mobilizes lymphocytes and haematopoietic stem cells in patients treated for non-malignant disorders (Krumbholz, et al 2008) and has been proposed as a mobilizing agent in transplantation protocols (Neumann, et al 2009). We showed that natalizumab markedly decreased malignant B-cell adherence to fibronectin (by 75-95%) for all tested cell lines that involved the inhibition of adhesion to all VLA-4 binding sites present on the utilized fibronectin molecule (CS1, Hep II and RGD). Moreover, this was accompanied by a change in the morphology of the cells, which lost their adhesion phenotype, and led to a prolonged inability of cells to re-adhere. These results are compatible with published data showing that natalizumab treatment in vitro results in the inhibition of migration (pseudoemperipolesis) of cells from a MCL cell line beneath stromal cells (Kurtova, et al 2009).

We confirmed the specificity of natalizumab for VLA-4 positive B-cells by performing flow cytometry (data not shown) and confocal microscopy with fluorescent-labelled natalizumab. As expected, natalizumab binding to VLA-4 did not affect the viability of malignant B-cells cells cultivated on a plastic surface or on stromal cells (Fig. S1) and did not mediate CDC (data not shown). Natalizumab also did not significantly affect CD20 expression by B-cell lines or rituximab binding and complement activation (Fig. S2,S3,S4). Importantly, we have shown that natalizumab is effective at disrupting the stroma-mediated protection of lymphocytes from doxorubicin and fludarabine toxicity as well as rituximab-induced apoptosis. The protection from cytotoxic drugs and rituximab mediated by stroma cells was diminished with natalizumab by 30% (Fig. 4ABC) and >50% (Fig. 6AB) respectively. Thus, our data suggests that the inhibition of VLA-4-mediated stromal-tumor cell interactions overcomes cell adhesion-mediated resistance to antibodies and cytotoxic therapy. The effect of natalizumab on CAM-DR was similar to the data from CXCR4 inhibitors in chronic and acute leukemias (Burger, et al 2005, Zeng, et al 2006, Zeng, et al 2009). The exact role of VLA-4 inhibition on CAM-DR requires further study. This could include investigation of whether combination therapy using VLA-4 and CXCR4/CXCL12 blockade could increase the disruption of CAM-AR.

Integrins are dynamically activated and deactivated by conformational changes modifying their down-stream signalling (Hood and Cheresh 2002). Moreover, the stromal-malignant B-cell interactions include multiple interactions with extensive crosstalk of chemokines, growth factors and adhesion molecules. The redundancy of stroma-induced signalling was the most likely reason for the lack of natalizumab effect on DOHH2 cell lines, where the inhibition of VLA-4 did not have any effect on doxorubicin- and rituximab-induced apoptosis (Fig. 4D,6C) despite being effective at inhibiting VLA-4-dependent adhesion (Fig. 3A). We have shown that the DOHH2 cell line is less dependent on VLA-4 induced signalling (Fig. 5) and expresses lower levels of VLA-4 on the cell surface (mean fluorescence intensity [MFI] for CD49d of 158) compared to Karpas-422 or Raji (MFI for CD49d of 547 and 359 respectively). This further supports the notion that the inhibition of stroma-dependent resistance might require the targeting of multiple pathways.

In summary, we have shown that the human bone marrow stromal cell line HS-5 protects B-cell lymphoma cell lines from rituximab cytotoxicity, thus demonstrating the existence of a stromal cell adhesion-mediated antibody resistance (CAM-AR) mechanism analogous to CAM-DR. Natalizumab partially overcomes CAM-AR against rituximab cytotoxicity, suggesting that VLA-4 is an important component of the stroma-lymphoma cell interaction and that the disruption of this mechanism could be therapeutic. These pre-clinical findings provide a rationale for extended testing of natalizumab and adding drugs that inhibit stromal adhesion to rituximab-containing therapy to improve treatment efficacy.

Supplementary Material

Supp Fig S1
Supp Fig S2
Supp Fig S3
Supp Fig S4
Supp Fig S5

Acknowledgments

This research was supported in part by funding from the National Cancer Institute CA097274 (University of Iowa/Mayo Clinic Lymphoma Specialized Program of Research Excellence), Genentech (CSZ) and IGAMZCR NT11218-6/2010.

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Supplementary Materials

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