Immune Netw.  2014 Dec;14(6):296-306. 10.4110/in.2014.14.6.296.

Simultaneous Inhibition of CXCR4 and VLA-4 Exhibits Combinatorial Effect in Overcoming Stroma-Mediated Chemotherapy Resistance in Mantle Cell Lymphoma Cells

• Kim YR
• Eom KS

• KMID: 2150820
• DOI: http://doi.org/10.4110/in.2014.14.6.296

Abstract

There is growing evidence that crosstalk between mantle cell lymphoma (MCL) cells and stromal microenvironments, such as bone marrow and secondary lymphoid tissues, promotes tumor progression by enhancing survival and growth as well as drug resistance of MCL cells. Recent advances in the understanding of lymphoma microenvironment have led to the identification of crucial factors involved in the crosstalk and subsequent generation of their targeted agents. In the present study, we evaluated the combinatory effect of blocking antibodies (Ab) targeting CXCR4 and VLA-4, both of which were known to play significant roles in the induction of environment-mediated drug resistance (EMDR) in MCL cell line, Jeko-1. Simultaneous treatment with anti-CXCR4 and anti-VLA-4 Ab not only reduced the migration of Jeko-1 cells into the protective stromal cells, but also enhanced sensitivity of Jeko-1 to a chemotherapeutic agent to a greater degree than with either Ab alone. These combinatorial effects were associated with decreased phosphorylation of ERK1/2, AKT and NF-kappaB. Importantly, drug resistance could not be overcome once the adhesion of Jeko-1 to the stromal occurred despite the combined use of Abs, suggesting that the efforts to mitigate migration of MCLs should be attempted as much as possible. Our results provide a basis for a future development of therapeutic strategies targeting both CXCR4 and VLA-4, such as Ab combinations or bispecific antibodies, to improve treatment outcomes of MCL with grave prognosis.

Keyword

CXCR4; VLA-4; Mantle cell lymphoma; Microenvironment; Drug resistance

MeSH Terms

Antibodies, Bispecific
Antibodies, Blocking
Bone Marrow
Cell Line
Drug Resistance
Drug Therapy*
Integrin alpha4beta1*
Lymphoid Tissue
Lymphoma
Lymphoma, Mantle-Cell*
NF-kappa B
Phosphorylation
Prognosis
Stromal Cells
Antibodies, Bispecific
Antibodies, Blocking
Integrin alpha4beta1
NF-kappa B

Figure

• 

  Figure 1 Expression of CXCR4 and VLA-4. Surface expression of CXCR4 (A) and VLA-4 (B) was determined by FlowJo analysis. Most of Jeko-1 expressed CXCR4 and VLA-4.

• 

  Figure 2 Chemotaxis in MCL cell line in response to SDF-1α and anti-CXCR4 Ab. (A) Migration of Jeko-1 cells increased in response to SDF-1α, peaked at 100 ng/ml, but without statistical significance. (B) SDF-1α-induced chemotaxis was inhibited by pretreatment with anti-CXCR4 Ab.

• 

  Figure 3 Pseudo-emperipolesis of MCL cell. (A) Control, with marrow stromal cells only. (B) Stromal cells with MCL cells. Migrated cells are characterized by a dark appearance, whereas non-migrated cells remain bright. (C) Stromal cells and MCL cells with anti-VLA-4 Ab. Reduced migration beneath marrow stromal cells (pseudo-emperipolesis) was observed in the presence of anti-VLA-4 Ab. Original magnification ×20.

• 

  Figure 4 Effects of combined blockage by anti-CXCR4 and VLA-4 antibody. The migration was inhibited by preincubation of anti-CXCR4 (7.5µg/ml) and VLA-4 Ab (2.5µg/ml). *denotes statistical significance at p<0.05, compared with control sample.

• 

  Figure 5 Effects of blocking antibodies on the protective effect of MSCs. In 24 well plate, MCL cells were cultured in the absence or presence of MSCs with Ara-C (A and C, MCL cells without MSC: B and D, MCL with MSC). Results are percentages of cells of apoptosis and apoptosis and cell death. **denotes statistical significance at p<0.01, compared with the control sample.

• 

  Figure 6 Phosphorylation of signaling pathways in MCL cells in response to chemotherapy Ara-C. MCL cells pre-treated with antibodies in the absence (A, B, and C) or presence (D, E, and F) of MSCs with Ara-C in a 24 well plate for 48 hours. Phosphorylation of ERK1/2, AKT and NF-κB was examined by flow cytometry. Phosphorylation of each signaling protein was markedly suppressed with the addition of antibodies, especially in the presence of both antibodies. *denotes statistical significance at p<0.05, compared with control sample.

• 

  Figure 7 Effects of migration of MCL cells on apoptosis and cell death. MCL cells pretreated with Abs in the upper chamber were analyzed by annexin V (A) and PI (B) flow cytometry and compared with the cells migrated to the lower chamber. Results are expressed as percentages of apoptotic, necrotic and dead MCL cells. *denotes statistical significance at p<0.05, compared with control sample.

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