In Vitro Culture Reveals Microenvironment-Dependent Growth, Heterogeneity and Hierarchical Structure of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells

Abstract 1483

In Vitro culture reveals microenvironment-dependent growth, heterogeneity and hierarchical structure of primary human pediatric pre-B cell acute lymphoblastic leukemia (pre-B ALL) cells.

J.-H. Shieh¹, P. Steinherz², J Shieh³ and M. A.S. Moore¹. ¹Moore Laboratory. Cell Biology Program and ²Leukemia and Lymphoma Studies, Department of Pediatrics, Memorial-Sloan Kettering Cancer Center, New York, NY. ³Department of Biology, Brandeis Univ., Waltham, MA

Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children. Although this pediatric pre-B ALLs are treatable, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. To address this issue, we systemically evaluated the effects of various tissue culture parameters to the growth of primary pre-B ALL cells. A serum-free MS-5 cells (a murine bone marrow stromal cell line) co-culture system is capable of expanding the pre-B ALL CD34⁺CD19⁺ cells and supporting their differentiation to CD34⁻CD19⁺ B cells. This expansion requires a contact between the stromal cells and the pre-B ALL cells, and is inhibited by fetal bovine serum and IL-6 in a dose-dependent manner. c-Kit ligand and Flt3 ligand can reverse the IL-6 inhibition. Expansion of individual CD34⁺CD19⁺ cells revealed a hierarchical structure with respect to CD34 antigen expression and an heterogeneity in cell proliferation. When the pre-B ALL cells were sorted into CD34^dim and CD34^bright populations, the CD34^dim cells were capable of a faster proliferation but gradually lost their CD34 antigen. In contrast, the CD34^bright cells were more slowly proliferating and retained their CD34 antigen. We transduced the B-ALL cells with a fusion gene expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL). These GFP-Lu-pre-B ALL cells display the similar in vitro characteristics and in vivo xenograftment to NOD/SCID IL2R gamma null (NSG) mice as the non-transduced pre-B ALL cells. One hundred, 10³, 10⁴ or 10⁵ GFP-LU-pre-B ALL CD34⁺ cells were i.v. transplanted to NSG mice. Both 10⁴ and 10⁵ cells resulted in the engraftment of the leukemia cells in limbs and cranium as judged by imaging after 6 weeks, and 10³ cells engrafted after 13 weeks. When the 10⁵ cells-transplanted mice were sacrificed after 14 weeks, the harvested peripheral blood, spleen (3–4×10⁸cells/spleen) and bone marrow (5−10×10⁶ cells/femur) displayed 2–3%, 51–55% and 75–81% of human CD34⁺CD19⁺ cells, respectively. Human CD34⁻CD19⁺ cells were 1–2%, 12–13% and 15–21%, respectively. Therefore, our stromal culture system supports leukemic stem cell/leukemia initiating cell proliferation and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo, and reveals the heterogeneity and hierarchical structure of human pre-B ALL cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides powerful tools to dissect the pathophysiology of human pre-B ALL, and to screen new drugs for pre-B ALL therapy.