Bone Marrow Transplantation, (1998) 22, 367–374 1998 Stockton Press All rights reserved 0268–3369/98 $12.00 http://www.stockton-press.co.uk/bmt Differential growth patterns in SCID mice of patient-derived chronic myelogenous leukemias J McGuirk1,2,5, Y Yan1,4, B Childs1,2, J Fernandez1, L Barnett1, C Jagiello1, N Collins1 and RJ O’Reilly1,2,3,4 1Bone Marrow Transplantation Service, 2Department of Medicine, 3Department of Pediatrics, and 4Research Animal Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Summary: The product of this rearrangement is a bcr-abl hybrid gene, which encodes a p210 protein product with tyrosine kinase The development of an in vivo model for the study of activity and is thought to have leukemogenic properties.4 CML would be of significant importance in studying its CML typically begins as an initial chronic phase (CP) biological behavior and developing novel therapeutic which lasts approximately 4–6 years with eventual pro- strategies. We examined the ability of human leukemic gression to blastic transformation commonly heralded by cells derived from patients in either chronic (CP), accel- the acquisition of additional chromosomal abnormalities by erated (AP) or blast phase (BP) CML to grow and dis- Ph+ stem cells including duplication of the Ph chromosome, seminate in CB17-SCID mice by subcutaneous (s.c.) isochromy 17q and trisomy of chromosomes 8, 19 or 21.5,6 inoculation without conditioning treatment or adminis- The relationship of these non-random chromosomal tration of cytokines. Additionally, samples derived from changes to the progression of this disease is not well under- patients with CP-CML were injected s.c. into CB17- stood. The majority of patient-derived CML cells have lim- SCID mice treated with anti-Asialo GM1 (an anti-NK ited proliferative capacity in vivo and available in vitro cell antibody) and NOD-SCID mice (absent NK cell assays are only capable of detecting progenitors with activity) to study the potential role of NK cell-mediated limited proliferative and replating potential.7,8 anti-leukemic activity in preventing the propagation of An in vivo model of CML would potentially allow for CP-CML cells. We observed a significant differential the development of novel therapeutic strategies in the treat- growth pattern of CML cells in the mice such that BP- ment of this disease as well as possibly lead to the eluci- CML grew rapidly as s.c. tumors and disseminated, dation of the molecular events involved in the evolution of while AP-CML or CP-CML cells grew temporarily as the CP to blastic phase (BP). small nodules that spontaneously regressed and did not Several recent studies have shown the growth of some disseminate. This differential growth pattern suggests human leukemia cells in the severe combined immunodefi- possible important biological differences. Furthermore, ciency (SCID) mouse model in a manner analogous to their no significant difference in s.c. growth or dissemination clinical characteristics in patients.9–12 This has allowed the of CP-CML samples derived from newly diagnosed propagation of human leukemic cells that in prior studies patients in untreated CB17-SCID mice and CB-17 SCID failed to grow in vitro or in nude mice. Although several mice treated with Anti-Asialo GM1 and NOD-SCID reports demonstrate the feasibility of consistently growing mice occurred, suggesting that factors other than NK human acute myeloid and lymphoid leukemias in SCID cell anti-leukemic activity may be important. mice the growth of CML cells in this model have mostly Keywords: microenvironment; blast phase; accelerated been limited to cell lines or samples derived from patients phase; chronic phase; subcutaneous in the BP of the disease.13–16 Only one group has been able to demonstrate even limited growth of CP-CML cells in SCID mice; this growth was limited to the bone marrow and Ph+ cells were identified in only a small minority of Chronic myelogenous leukemia (CML) is an acquired the animals studied.17 malignant disorder of the hematopoietic stem cell and is Recently, we developed a SCID mouse model that per- characterized by the proliferation of clonal myeloid cells mits the s.c. growth of primary human acute leukemia 1 and their progenitors. The hallmark of this disorder is the cells.9 Furthermore, the s.c. growth in this model is associa- presence of the Philadelphia (Ph) chromosome which ted with dissemination of the leukemia reflecting a pattern results from a specific reciprocal translocation, of growth similar to the usual clinical presentation. To t(9;22)(q34;q11), of the abl oncogene on chromosome 9 to determine whether and to what degree different phases of 2,3 the break point cluster region (bcr) on chromosome 22. CML would grow or disseminate within the SCID mouse model, we subcutaneously inoculated SCID mice with samples of cells derived from patients with CP, AP or BP- 5Correspondence at his present address: Dr JP McGuirk, Yale School of Medicine, Bone Marrow Transplantation, 333 Cedar Street Room 211 CML. Additionally, several samples derived from patients WWW, PO Box 208032, New Haven, Connecticut 06520–8032, USA with CP-CML were injected subcutaneously into SCID Received 5 February 1998; accepted 20 April 1998 mice treated with anti-Asialo GM1 (an anti-NK cell CML growth in SCID mice J McGuirk et al 368 antibody) and NOD-SCID mice (absent NK cell activity) to growth was assessed by weekly measurements of the study the potential role of NK cell-mediated anti-leukemic dimensions of subcutaneous nodules. Tumor size was cal- activity in preventing the propagation of CP-CML in culated by taking the square of the mean radii and multiply- SCID mice. ing by ␲. Animals inoculated with BC-CML leukemic cells In this report we demonstrate a differential growth pat- were sacrificed by cervical dislocation when the dimensions tern of patient-derived CP and AP vs BP CML in a SCID of subcutaneous nodules were at least 1.5 cm2 or more than mouse by s.c. inoculation and present the characteristic and 3 months after inoculation. Animals inoculated with AP- biologic behavior of the human chronic myelogenous CML and CP-CML leukemic samples were sacrificed in leukemias in this model. the same manner when tumor nodule size reached maximal size or more than 3 months after inoculation. The gross anatomy was evaluated and samples from peripheral blood, Materials and methods sternum, femur, spleen, liver, lung–heart complex, kidney, brain and tumor nodules were subsequently removed for Leukemic samples analysis by flow cytometry (FACS), fluorescent in situ hybridization (FISH), polymerase chain reaction (PCR) for Samples were obtained, with informed consent, during rou- detection of bcr-abl transcripts, and histologic analysis. tine diagnostic blood studies or bone marrow (BM) aspir- ates from patients with newly diagnosed or previously diag- nosed and treated chronic myeloid leukemias at Memorial Analysis of tissue from xenografted animals Sloan-Kettering Cancer Center (MSKCC). Seventeen CP- Histopathology: Tissue sections from killed SCID mice CMLs, two AP-CMLs and eight BP-CMLs were studied. were fixed in 10% neutral-buffered formalin, dehydrated, Blast-enriched mononuclear cells were isolated by Ficoll– embedded in paraffin, sectioned and stained according to Hypaque density gradient separation and washed in RPMI standard histologic techniques. 1640 medium. After separation, most of the leukemic cells were freshly inoculated into SCID mice. In some patients, Immunophenotyping: Cell suspensions derived from leukemic cells were cryopreserved in liquid nitrogen before tissues of killed animals and primary CML cells were their injection into the animal. washed in phosphate-buffered saline (PBS) and stained with directly labeled fluorescein isothiocyanate (FITC)- SCID mice conjugated and phycoerythrin (PE)-conjugated monoclonal antibodies for 30 min at 4°C. The cells were then washed All SCID (CB17-SCID/SCID and NOD-SCID) mice were twice and analyzed by FACS (Becton Dickinson, Mountain purchased from Taconic Farms (Germantown, NY, USA) View, CA, USA). The following antibodies were used for and maintained in micro-isolator cages under sterile con- characterization of lineage surface markers: CD13, CD14, ditions with a specific pathogen-free environment without CD15, CD33, CD34, CD7, DR (Becton Dickinson). the use of any antibiotics. Female SCID mice between 6 Additionally, FACS analyses of samples of blood, BM, and 8 weeks of age were used. This work was approved spleen and liver from animals were performed by using by the MSKCC Animal Review Board and all animals were two-color immunofluorescence staining and pairwise com- sacrificed in accordance with MSKCC Animal Research binations of a selected panel of antibodies (CD3-PE, CD19- regulations. PE, CD33-PE and CD45-FITC) against human antigens and an antibody mCD45-FITC (Boehringer Mannheim, Indian- Inoculation of human leukemic cells into SCID mice apolis, IN, USA) against a murine antigen to discern the origins of the cells. A median of 2.0 × 107 (range 0.2–10 × 107) viable leukemic cells were resuspended in 200 ␮l of ice-cold Matrigel FISH: FISH was performed as previously described.9 In matrix (Becton Dickinson Labware, Bedford, MA, USA) brief, single-cell suspensions from tumor nodules or organs liquid and injected subcutaneously with a tuberculin were washed and fixed in a mixture of methanol and acetic syringe into the right flank of the SCID mice. acid (3:1 v/v). The cells were put on to slides and air-dried. The number of SCID mice inoculated and the cell dose Subsequently, the slides were washed at room temperature per mouse were dependent on the number of leukemic cells (RT) in successive jars containing 0.1 n HCl solution con- available from patient samples. The animals did not receive taining 0.5% Triton, PBS, 1% formaldehyde, PBS × 2, any conditioning treatment before inoculation.