Identification of Leukemic Stem Cells and Their Niche in Adult T Cell Leukemia Using the Tax-Transgenic Mouse Model

Abstract 1877

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 these cells from chemotherapy.

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 in the spleen, bone marrow (BM), thymus and lymph node (LN) is still unclear.

To identify the ATL-LSC niche in vivo, we performed a homing assay. Lymphoma cells isolated from a Tax-Tg mouse were GFP transduced by a lentivirus, and then sorted GFP⁺ cells (2×10⁶) were transplanted intraperitoneally into a non-irradiated NOD/SCID mouse. The homing of GFP⁺ cells to tissues was traced by flow cytometry (FCM) at 16 hours and 3, 7, 14 and 21 days after transplantation. At 16 hours after transplantation, GFP⁺ lymphoma cells were detected in the spleen and BM. 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 were AT-LSCs (GFP⁺/CD117⁺ cells). From day 3 to 7, more than 40% of proliferating cells in the BM and spleen were ATL-LSCs. At day 3, only a few non-ATL-LSCs (GFP⁺/CD117^–cells) were detected in the thymus, LN and peripheral blood. The number of GFP+ cells was drastically increased at day 14 in the spleen. These data indicate that ATL-LSCs prefer to colonize and proliferate in the spleen and BM.

To identify the specific niche of ATL-LSCs in the spleen and BM, we performed imaging analysis of ATL-LSCs. ATL-LSCs (GFP⁺/CD117⁺ and CD38^–/CD71^–/CD117⁺cells) were mainly localized near the vascular region in the spleen and endosteal region of trabecular bone in the BM. We found that some ATL-LSCs were attached to reticular cells (RC) in the spleen. In the BM, ATL-LSCs cells were localized at the endosteal region of the trabecular bone. Interestingly, similar to the spleen, RCs were observed at the endosteal region and contacted ATL-LSCs in the BM. FCM analysis confirmed that the number of reticular cells and mesenchymal stem cells (MSCs), were increased in the ATL BM and spleen. These data suggest that RCs are a possible candidate for the ATL-LSC niche and may be a new target of therapy.

Finally, to characterize the ATL-LSC niche, we isolated osteoblastic cells, blood endothelial cells, lymphatic endothelial cells and reticular cells from normal and ATL BM to compare the gene expression profiles of each niche cell type. Here, together with DNA microarray analysis of ATL-LSCs both in the BM and spleen, we have characterized ATL-LSC niche cells both in the spleen and BM.