The Interaction of CXCR4/SDF-1 and VLA-4 Regulates Adhesion and Transendothelial Migration in Waldenstrom Macroglobulinemia.

Background. Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM). Adhesion of malignant cells to the BM microenvironment induces proliferation and resistance to therapy. We previously showed that SDF-1/CXCR4 axis regulates migration and adhesion of WM cells in vitro indicating a potential role in homing. Homing of malignant cells to the BM niches requires active navigation through the endothelial cell barrier where tumor cells must adhere, roll and transmigrate. We hypothesized that CXCR4 and its interaction with VLA-4 is critical for this 3-step process.

Methods. The level of CXCR4 and VLA-4 was determined using flow cytometry and RT-PCR in patient samples and WM cell lines (BCWM.1 and WM-WSU). The CXCR4 inhibitor AMD3100 (10–100uM, Sigma, MO), Gi protein inhibitor pertussis toxin, PTX (10–200ng/ml, Sigma, MO) were used to inhibit CXCR4 signaling. These studies were confirmed using CXCR4 knockdown using shRNA with lentivirus infection. Adhesion assay was done with either plates coated with the VLA-4 ligand fibronectin for adhesion of tumor cells in presence and absence of SDF1 (30nM), or coated with endothelial cells (HUVEC) and BMSCs for cell-cell adhesion experiments. Transmigration assay was performed using the transwell system with either SDF1 in the lower chamber to study migration towards SDF1, or with the upper chamber coated overnight with endothelial cells (HUVEC) and BMSCs to study transmigration. Experiments with HUVEC cells were performed in absence or presence of TNF-alpha that activates the HUVEC cells.

Results. WM tumor cells from patients and cell line expressed high surface levels of CXCR4 (mean 70%) and VLA-4 (mean 95%). Adhesion of WM cells to fibronectin was significantly increased with SDF1 compared to BSA control, and AMD3100 20uM inhibited adhesion by 50% compared to control. Similar results were obtained with PTX 200ng/mL and using CXCR4 knockdown in WM cells. Furthermore, WM cells adhesion was greatly enhanced in co-culture with BMSCs and endothelial cells, and was significantly inhibited using AMD3100 and PTX. To further dissect the mechanisms of interaction of CXCR4 and VLA-4, we showed that AMD3100 inhibited surface expression of CXCR4 but not of VLA-4. Immunoprecipitation studies showed that CXCR4 and VLA-4 directly interact in response to SDF-1. Downstream signaling pathways showed that CXCR4 regulates migration and adhesion through the PI3K/Akt and ERK/MAPK pathways. We next studied whether CXCR4 was involved in transmigration through the endothelial barrier. We demonstrated that BCWM.1 cells transmigrated through the barrier of endothelial cells, that was increased when SDF1 was added to the lower chamber, and that AMD3100 inhibited the transmigration of BCWM.1 cells. Studies of the role of CXCR4 in rolling on endothelial cells is ongoing.

Conclusion. We showed CXCR4/SDF1 axis regulates migration and adhesion of WM cells to the BM microenvironment indicating a potential role in homing. Moreover, we demonstrate that CXCR4/SDF1 axis is critical for transendothelial migration, a critical step of homing and egression of tumor cells in and out the BM niches.