Abstract

We have recently developed a circulating model of CLL that more accurately mimics the transient interactions that take place between lymphocytes and the vascular endothelium. Normal and malignant lymphocytes actively undergo transendothelial migration in our system in the absence of a chemoattractant. We have now refined our model to include a CXCL12 gradient and evaluated the ability of CXCR4 antagonists to modulate lymphocyte migration. Initially we demonstrated that a single deposition of CXCL12 (100ng/ml) into the extravascular space (EVS) of our model significantly increased CLL cell migration (P = 0.049, mean circulating CXCL12 level = 254pg/ml). Subsequently, we created a stable chemokine gradient by seeding CXCL12-secreting MRC5 cells into the EVS. Although this resulted in lower peak circulating levels of CXCL12 (as measured by ELISA, mean 23pg/ml), it significantly increased migration (P = 0.024) presumably because of the constant nature of the chemokine gradient. As previously shown CXCR4 expression is up-regulated on circulating CLL cells when compared to static cultures (P=0.023). Basal CXCR4 expression and expression after circulation for 48h, correlated with the degree of CLL cell migration into the EVS (r2=0.40, P=0.003; r2=0.31, P=0.009 respectively). It is worthy of note that as a proportion of all circulating peripheral blood mononuclear cells, CD3+ T-cells migrated significantly more than CD19+ CLL cells (T-cells: 12.72% ±13.47, CLL cells: 1.16% ±1.23 P =0.02, n = 10). This may be partly the result of the known defects in CLL cell migration but is also likely to be promoted by CLL cell secretion of T-cell attracting chemokines CCL3 (298pg/ml ±197.5, n=13) and CCL4 (56pg/ml ±115.6, n=13) in our model. Furthermore, migrated T-cells and CLL cells recovered from the EVS showed a marked increase in Ki-67 expression (P<0.0001 and P<0.0001 respectively) confirming that both subsets of cells are activated. We next explored whether we could block the CXCR4/CXCL12 axis using the CXCR4 antagonist ONO-7161. In transwell chambers (3μm pores) 30nM ONO-7161 resulted in significantly decreased CLL cell migration (54.1% ±25.53 of control, P=0.0003) and was more potent than Plerixafor (1μM) (P=0.06). Additionally, in the circulating system, ONO-7161 resulted in significantly reduced CLL cell migration into the EVS at 48h (P=0.0006). T-cell migration was also reduced by ONO-7161 but not significantly. In summary, we have enhanced our circulating model system of CLL by adding a physiologically relevant CXCL12 chemokine gradient. This then allowed us to dissect the role of the CXCR4/CXCL12 axis in modulating CLL cell migration. Finally we demonstrated that the CXCR4 antagonist ONO-7161 was a potent inhibitor of CLL cell migration in our model. Given that ONO-7161 was non-toxic to CLL cells at concentrations up to 10μM, it seems likely that it might be usefully combined with chemotherapy and biologically targeted agents in the treatment of this and other diseases.

Disclosures

Yoshizawa:Ono Pharmaceutical Co., Ltd.: Employment. Fegan:ONO PHARMA: Honoraria.

Author notes

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Asterisk with author names denotes non-ASH members.