The Osteoclast Targeting Therapy In Bone Metastasis For a Mouse Model Of Adult T Cell Leukemia

Adult T cell leukemia (ATL) is a lymphoproliferative disorder caused by infection with HTLV-I. Although various chemotherapies have shown significant complete remission rates, most of the treated patients relapse. These data indicate the existence of leukemic stem cells (LSCs) and a specific niche that regulates stemness and protects LSCs from various chemotherapies.

We have reported in previous studies that the ATL-LSCs isolated from a Tax-transgenic (Tax-Tg) mouse are enriched in the CD117⁺/CD38^–/CD71^– fraction of the lymphoma, and LSCs have the potential to reproduce the original tumor when transplanted into a NOD/SCID mouse (Yamazaki et al., Blood, 2009). However, the niche of ATL-LSCs is still unclear.

To identify the ATL-LSC niche in vivo, we performed a homing assay. Splenic lymphoma cells isolated from a Tax-Tg mouse were GFP transduced by a lentivirus, and then sorted GFP⁺ cells were transplanted intra-peritoneally into a non-irradiated NOD/SCID mouse. The homing of GFP⁺ cells to tissues was assessed by flow cytometry (FCM) at 16 hours and 3, 7, 14 and 21 days after transplantation. As a result, GFP⁺ lymphoma cells were first detected in the spleen and BM at 16 hours after transplantation. No GFP⁺ lymphoma cells were detected in the thymus and LN. Interestingly, more than 60% of first colonized cells in the spleen and BM at 16 hours after transplantation were AT-LSCs (GFP⁺/CD117⁺ cells). From day 3 to 7, more than 40% of colonizing cells in the BM and spleen were ATL-LSCs.

To identify the specific niche of ATL-LSCs in the BM, we performed imaging analysis of ATL-LSCs. ATL-LSCs (GFP⁺/CD117⁺ and CD38^–/CD71^–/CD117⁺ cells) were mainly localized near the endosteal region of trabecular bone in the BM. We found that ATL-LSCs were also attached to the reticular cells in the trabecular bone. In addition, we found the number of osteoclast was significantly increased at the trabecular region. Increasing number of osteoclasts correlates the increased the serum calcium concentration and decreased the mass of trabecular bone. FCM analysis and in vitro differentiation assay confirmed that the number of osteoclast precursors was increased in the ATL BM.

To clarify the role of osteoclast in the ATL BM, we treated osteoclast inhibitor Zoledronic acid (ZOL) to the ATL mouse model. As a result, ZOL itself significantly reduced the number of GFP+ ATL cells in the BM. When we treated ZOL with anti cancer drug, GFP+ ATL cells were dramatically reduced in the BM and extend the mouse survival rate significantly despite anti cancer drug does not reduced the number of ATL cells itself. In addition, abnormal trabecular bone morphology was completely recovered in the treated mouse. These data suggest that osteoclast may have a function to support leukemic stem cell niche.

To clarify the key signals to induce osteoclast in ATL BM, we checked the expression of RANKL and PTHrP. We found that RANKL was up-regulated both in the lymphoma cell and stromal cells in the bone marrow.

　　In this study, we found that ATL-LSC niche is located at the trabecular bone region in the BM and osteoclasts have a role to support ATL cell and develop LSCs niche in a mouse model of ATL. We conclude that osteoclast have a potential therapeutic target in the mouse model of ATL.