Leukemia (2002) 16, 1541–1548  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Growth of human T cell acute lymphoblastic leukemia lymphoblasts in NOD/SCID mouse fetal thymus organ culture F Ma 1 , A Manabe 1 , D Wang 1 , M Ito 1 , A Kikuchi 3 , M Wada 1 , M Ito 4 , A Ohara 5 , R Hosoya 6 , S Asano 2 and K Tsuji 1 1 Division of Cellular Therapy, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; 2 Division of Molecular Therapy, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; 3 Department of Bone Marrow Transplantation, Saitama Children’s Medical Center, Saitama, Japan; 4 Central Institute for Experimental Animals, Kawasaki, Japan; 5 Department of Pediatrics, Toho University School of Medicine, Tokyo, Japan and 6 St Luke’s International Hospital, Tokyo, Japan The in vitro proliferation of T cell acute lymphoblastic leukemia (T-ALL) cells in its entirety has not been well delineated because of a lack of an appropriate culture system that mimics the growth pattern in a living body. We applied a NOD/SCID mouse fetal thymus organ culture (FTOC) for leukemic cells from fresh (one case) and frozen (seven cases) bone marrow (BM) samples of children with T-ALL. Cell growth was observed in all seven samples in the culture, reaching a proliferational peak at 4 weeks, and it was calculated that the proliferation potential was 212-to 319-fold. The FTOC-derived T-ALL cells showed similarity to the original cells morphologically and immunophenotypically, still possessed clonalities and were able to regenerate overt leukemia in NOD/SCID mice. These FTOC-derived T-ALL cells differed from ordinary cell lines because they always need FTOC support. Thus, we established a new in vitro culture for T-ALL cells. A comparison of the orig- inal and FTOC-derived T-ALL cells revealed that the proportion of cells expressing IL-7R increased in all seven cases. Sorting and re-seeding of FTOC-derived IL-7R+ and IL-7R cells into secondary FTOC resulted in a predominant generation of IL- 7R+ cells from both fractions, while IL-7R cells proliferated more potently than did IL-7R+ cells, suggesting that a pathway for the conversion of IL-7R to IL-7R+ exists during the pro- liferation of T-ALL lymphoblasts. Addition of exogenous IL-7 or neutralization with anti-IL-7 antibody did not influence the growth pattern of T-ALL cells in FTOC. The current study pro- vides a unique assay system for the exploration of the hier- archy within human T-lymphoid leukemic cells, and should facilitate the establishment of novel therapeutic modalities. Leukemia (2002) 16, 1541–1548. doi:10.1038/sj.leu.2402547 Keywords: human; T-ALL; leukemia; IL-7 receptor; FTOC Introduction T cell acute lymphoblastic leukemia (T-ALL), like other lymph- oid malignancies, is a consequence of the malignant trans- formation of clonal abnormal progenitor cells capable of expansion with infinite self-renewal potential. 1 This disease covers about 15% of ALL cases and has been associated with a comparatively poor prognosis based upon clinical data. 2 Because T-ALL has been subclassified using monoclonal anti- bodies which also react with surface antigens for normal T cell differentiation and maturation, 3,4 leukemic cells sharing certain characteristic with their normal counterparts may respond similarly to stimulation for growth. An understanding of in vitro growth pattern of T-ALL cells in its entirety will furnish important knowledge about the proliferation and dif- ferentiation stages, dependence on particular microenviron- ments, and responsiveness to various cytokines of the leu- Correspondence: A Manabe, Division of Cellular Therapy, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4–6–1 Shirokanedai, Minato-ku, Tokyo 108– 8639, Japan; Fax: 81–3–5449–5428 Received 13 July 2001; accepted 16 January 2002 kemic cells. Short-term cultures have been used to explore conditions for the proliferation and cytoreduction of T-ALL cells. 5–9 Although immortalized cell lines derived from T-ALL cells have also been useful in characterizing the cell growth pattern, 10,11 they may not be suitable for an observation of T- ALL cells that mimic the overall growth pattern in a living body. The receptor for IL-7 (IL-7R) has proved a critical surface molecule for the development of normal T lymphocytes. 12 Several recent reports demonstrated that IL-7R was also expressed on T-ALL cells and might play an important role in the regulation of cell growth. 5,6,13,14 However, the behavior of T-ALL cells that express IL-7R is not clear. We recently established a NOD/SCID mouse fetal thymus organ culture (NOD/SCID FTOC) that enabled us to explore the early development of T-lineage cells from human CD34 + hematopoietic progenitors. 15 By using NOD/SCID FTOC, we found that T-ALL cells from both fresh and cryopreserved samples were able to proliferate continuously in the culture system. Thus, NOD/SCID FTOC provides a novel assay system for research on T-ALL cells. Materials and methods Patients and leukemia cell samples Freshly obtained or cryopreserved BM cell samples from six children with T-ALL at onset and one at relapse (male three, female three, median age 6.5 years old, range 1 to 12) were taken before treatment. Informed consents were obtained from the guardians by physicians who treated patients. The median initial white blood cell (WBC) count was 207.6  123.3  10 9 /l (range 75 to 404). After separation by Ficoll–Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation, immunophenotyping of all leukemic cell samples was carried out using a panel of monoclonal anti- bodies specific for cell surface and intracytoplasmic antigens. These samples were confirmed to contain more than 90% leu- kemic cells based on morphological, cytochemical and immu- nophenotypic criteria. The viability of either fresh or thawed cell samples was always more than 92% when determined by trypan blue exclusion. Table 1 shows the clinical character- istics of the seven cases investigated. Mice Experimental NOD/Shi-scid (NOD/SCID) mice possessing dysfunctional mature lymphocytes and macrophages and lacking circulating complements 16 were provided by the Cen- tral Institute for Experimental Animals (Kawasaki, Japan). The