Microenvironmental-Mediated Regulation of L-Selectin in Chronic Lymphocytic Leukaemia

Chronic Lymphocytic Leukaemia (CLL) is characterized by the accumulation of CD5/19+ve B cells in the blood, bone marrow and lymph nodes. The tumour microenvironment of the bone marrow and secondary lymphoid organs promotes cell proliferation, survival and protection from drug induced apoptosis. Selectins, integrins and chemokines mediate the trafficking of CLL cells to these protective niches. An important adhesion molecule in this process is L-selectin (CD62L) which is involved in initial tethering and rolling of CLL cells in the high endothelial venules, allowing migration between the blood and the secondary lymphoid organs. It has been shown that CLL cell expression of CD62L increases during in vitro culture and blocking antibodies cause an increase in apoptosis, suggesting a role for CD62L in cell survival (Burgess et al., Clin Cancer Res. 2013). The B-cell receptor (BCR) signalling pathway is the most important pathway involved in micro-environmental crosstalk and CLL cell survival, and has recently been shown to interact with the STAT3 signalling pathway (Rozovski et al., Blood 2014).

In this study, Hs5 Bone Marrow Stromal Cell (BMSC), Human Umbilical Vein Endothelial cells (HUVEC) and patient derived BMSC co-cultures were used to mimic pro-survival microenvironmental signals and investigate the effect on the expression of a panel of cell surface adhesion molecules. These coculture models resulted in a significant increase in CD62L positive CLL cells (p<0.01, n=7). Stimulation of the BCR using immunoglobulin F(ab´)₂fragments induced tyrosine phosphorylation of STAT3 and resulted in a significant increase in CD62L expression, which was abrogated by pre-treatment with the Bruton tyrosine kinase (btk) inhibitor Ibrutinib prior to BCR stimulation. (p<0.05, n=5).  siRNA mediated STAT3 knockdown and treatment with STAT3 inhibitors, including Cucurbitacin I, resulted in a significant decrease in CD62L positive CLL cells (p<0.0001, n=14).  Co-culture with Hs5 cells, HUVECs, patient derived BMSC or BCR stimulation did not overcome Cucurbitacin I induced downregulation of CD62L.

The effect of STAT3 inhibition on CLL cell adhesion and chemotaxis was also investigated. A microfluidic system including a neMESYS Low Pressure syringe pump system (Cetoni GmbH) and Cellix Vena8Endothelial+biochips coated with Human Umbilical Vein Endothelial cells (HUVECs) or Human Dermal Lymphatic Endothelial cells (HDLEC) was utilised to investigate CLL cell adhesion under fluid shear flow conditions. Flow rates of 0.5 dynes/cm² for the HUVEC-coated biochips and 0.08 dynes/cm² for the HDLEC coated biochips were used to model the fluid shear of blood and lymphatic flow respectively. Treatment with Cucurbitacin I resulted in a mean decrease of 50% in CLL cell adhesion to HUVEC but not HDLEC in this system (p<0.05, n=3). Chemotaxis of CLL cells was investigated using Neuroprobe 96-well ChemoTx plates. Treatment with Cucurbitacin I resulted in a mean decrease of 87% of CLL cells migrated in response to the chemokine CXCL12 compared to control (p<0.0001, n=4).

In conclusion, this study shows microenvironmental signals mediate the expression of CD62L in CLL cells. Our data suggest that the STAT3 pathway regulates the expression of CD62L and the adhesion and chemotaxis of CLL cells. The down-regulation of CD62L expression through inhibition of BCR signalling or direct inhibition of STAT3 may have implications for the trafficking of CLL cells and treatment of CLL.