Leukemia Microenvironment-Specific Galectin-3 Expression of Leukemic Cells Promotes Malignant Niche Formation and Bone Marrow Lodgment of Leukemic Cells in Chronic Myelogenous Leukemia

Abstract 1678

We recently identifid that galectin-3 (Gal-3) is specifically induced in leukemic cells due to the support of the bone marrow microenvironment and promotes cell proliferation and resistance in leukemic cells to a variety of drugs, including imatinib mesylate, dasatinib, or genotoxic agents, especially in the case of chronic myelogenous leukemia (CML). In the current study, we continued and extended our study in order to identify the role of Gal-3 in the disease development of CML. First, we used a transmigration assay to investigate the role of Gal-3 in cell migration of leukemic cells. HS-5 conditioned medium (CM/HS-5) was used as the source of bone marrow stromal cells (BMSCs)-derived chemotactic stimuli. HS-5 is an immortalized human BMSC-derived cell line, which potently secretes G-CSF, GM-CSF, M-CSF, Kit ligand, MIP-1a, and interleukin (IL)-6, IL-8, or IL-11. When MYL cells, a CML cell line, were compared with Gal-3 overexpressing MYL cells (MYL/G3), which were generated by means of gene introduction, the latter showed a greater capacity for cell migration induced by CM/HS-5. Next, we investigated the involvement of Gal-3 in malignant niche formation, since recent studies have hypothesized that leukemic cells excrete growth factors which stimulate, via paracrine and autocrine loops, the growth of adjacent leukemic cells as well as of bone marrow supporting cells such as BMSCs or endothelial cells. When MYL cells and MYL/G3 cells were cultured with media containing conditioning medium (CM) from MYL cells and MYL/G3 cells in various ratios (designated as CM/MYL and CM/MYL/G3, respectively), both MYL cells and HS-5 cells proliferated more at higher concentrations of CM/MYL/G3, indicating that MYL/G3 cells excrete more growth factors for the MYL cells themselves as well as BMSCs. These findings were the same for cases with CML cell line K562 and Gal-3 overexpressing K562. We finally examined the in vivo role of Gal-3 in CML. Approval was obtained from the institutional review board at Kyoto University Hospital for the use of mice for this study which was conducted in accordance with the ethical principles of the Declaration of Helsinki. Fourteen male NOD/SCID mice at 6 weeks of age were sublethally irradiated (2 Gy) and 1.0×10⁶ MYL cells (Group A) or 1.0×10⁶ MYL/G3 cells (Group B) were transplanted intravenously via their tail veins into seven mice each. Body weight (BW) and the percentage of leukemic cells in peripheral blood (PB) were monitored at least twice a week. Although transplanted leukemic cells increased in a similar manner in the PB of both groups during the first three weeks, the number of PB leukemic cells of Group A mice then gradually decreased, while those of Group B mice remained the same until death. Although we had initially hypothesized that Group B might have a shorter survival than Group A, the actual result was the opposite, with the survival of Group A being significantly shorter than that of Group B (p =0.025). Surprisingly, the sites of disease involvement at death showed major differences between the two groups. Most mice from Group A showed extensive extramedullary involvement, such as intra-abdominal, mediastinal and/or subcutaneous tumors isolated from BM, while only one of seven mice showed BM involvement at the time of death. In contrast, all mice in Group B showed BM involvement, which sometimes outgrew BM, but none showed tumors isolated from BM. These results indicate that Gal-3 overexpression may facilitate BM homing and lodgment of CML cells. We also speculate that the reason for the shorter survival of Group A is that the tumors expanded much faster once leukemic cells had advanced outside BM and that this may have had a significantly more deleterious effect on the mice in Group A than on those in Group B. In conclusion, the findings of our study suggest that BMME-induced Gal-3 in leukemic cells plays a crucial role in disease development of CML, especially in the BM milieu.