EphB4/ephrinB2 ephrinB1 Interaction Mediated Chronic Myelogenous Leukemia Mesenchymal Stromal Cells Osteogenic Differentiation in Vitro and In Vivo

Background and Objective: Osteoblasts, important of stromal cells in bone marrow microenvironment, maintain HSCs in resting state and protect its' functions. Osteoblasts derived from mesenchymal stem cells (MSCs), which can be differentiated into osteoblast in bone marrow under the regulation of cytokines. Recent studies have indicated that EphB4/ephrinB2 protein participates in the regulation of osteogenesis differentiation of MSCs in bone marrow microenvironment.

Our previous study found that EphB4 receptor was over expressed in CML patients and cell lines, which played an important role to change characterize of Imatinib(IM)-resistant in chronic myeloid leukemia cells. Furthermore, we performed experiments to prove that osteogenic differentiation in MSCs from CML-initial patient significantly higher in contrast to normal human MSCs and the change of EphB4 molecules on leukemia cells may transform MSCs functions in vitro. However, the mechanism of these transformations of MSCs in vitro and what is change in vivo were still unclear. Therefore, we hypothesis that the change of EphB4 molecules on leukemia cells might play an important role to osteogenic differentiate in MSCs in vitro and in vivo, which support to leukemia progression and disruption of normal hematopoiesis.

Methods and Results: MSCs were prepared from bone marrow mononuclear cells isolated from normal human or CML- chronic phase (CCP) patients' BM and cultures in Cyagen Bone marrow culture medium at 37 °C, 5% CO2 incubator. In vitro, after stimulated with different concentrations of EphB4-Fc (0, 5, 8, 10 ug/ml) for 21 days, visualized by Alizarin Red staining, MSCs (CCP) produced maximum calcium nodules (P<0.05, n=3) in EphB4-Fc (8 ug/ml) group in contrast with other groups, accompanied by increased ephrinB1 and STAT3 phosphorylation. In vitro osteogenesis condition, after treatment with EphB4-Fc (0, 8 ug/ml) 14 days, MSCs (CCP) incubated with K562 cells. After 48 h, the IC50 (0.842±0.065, P<0.05, P<0.05 ANOVA, n=4) of K562 cells in MSCs+EphB4-Fc (8ug/ml) group increased, S phase cells percentage(56.6±4.01, P<0.05, P<0.01, ANOVA, n=4) increased and cells apoptosis rate(P<0.01, P<0.001, LSD, n=4) declined compared with K562 (control group) and K562+MSCs+EphB4-Fc (0 ug/ml). In vivo, K562-R, K562-R+MSCs (normal) (5:1), K562-R-EphB4-sh, K562-R-EphB4-sh+MSCs (normal) (5:1), MSCs (normal) cells were injected respectively into bone cavity of NOG rat (NOD/SCID/ɣ c-/-, n=12) rat and blank control group were also established. Examined peripheral blood in rats while hCD45⁺ cells > 1% is considered as leukemia model. K562-R+MSCs mice were earliest to establish leukemia model (31.75±1.26d) and had the shortest survival time(4.25±1.71d) than other groups. After treatment with IM, survival times of K562-R+MSCs mice were not significantly extended (4.7±3.055 d, pared-samples T test, P>0.05). In bone marrow of K562-R+MSCs mice, RUNX2 mRNA (0.654±0.0278; P < 0.001) over expressed in contrast to other groups. After treatment with IM, expression level of RUNX2 mRNA was significantly increased than non-treatment group. Among four leukemia groups of mice, expression levels VEGF mRNA in bone marrow were no significantly difference and there was no statistical difference existed in treatment group and non-treatment group. The same cells lines above were subcutaneously injected to establish subcutaneous transplantation tumor, respectively, in NOG rat (NOD/SCID/ɣ c-/-, n=8) rat. K562-R+MSCs tumors were earliest to appear (17.333±1.154 d) and had the biggest tumors volume (13116.27±165.502 mm3, P<0.001) compared to other groups. After mice treated by IM, compared with non-treatment group, K562-R+MSCs tumors had significantly increased in volume (14703.14±309.333mm3, pared-samples T test, P<0.01). VEGF mRNA (0.861±0.0648; P<0.01) in K562-R+MSCs tumor over express than other groups. After treatment, the expression level was no significantly declined (0.796±0.0688, P>0.05). The level expression of RUNX2 mRNA in four groups of subcutaneous transplantation tumors are low and had no statistical difference.

Conclusion: Our experiments in vitro and in vivo illustrated that EphB4 molecule on leukemia cells may transform MSCs osteogenic differentiation to change characterize of Imatinib(IM)-resistant in CML through ephrinB1 and STAT3 phosphorylation.