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Xenotransplantation of Human Lymphoid Malignancies Is Optimized in Mice with Multiple Immunologic Defects WA Hudson1,Qli1,Cle2 and JH Kersey1

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Xenotransplantation of Human Lymphoid Malignancies Is Optimized in Mice with Multiple Immunologic Defects WA Hudson1,Qli1,Cle2 and JH Kersey1 Leukemia (1998) 12, 2029–2033 1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu BIO-TECHNICAL METHODS SECTION (BTS) BTS Leukemia Xenotransplantation of human lymphoid malignancies is optimized in mice with multiple immunologic defects WA Hudson1,QLi1,CLe2 and JH Kersey1 1Cancer Center, Departments of Pediatrics and Laboratory Medicine/Pathology and 2Division of Biostatistics, University of Minnesota, Minneapolis, MN, USA While it is known that mice with genetic immune defects are phomas.6–11 A mouse strain with another genetic mutation useful for establishing durable engraftment of human tumor involving the Rag-1 gene has been developed12 and most xenografts, the relative role of components of host innate and adoptive immunity in engraftment has not been determined. We recently the SCID mutation was backcrossed into the NOD/Lt directly compared the ability of four strains of genetically strain resulting in an animal with multiple immunologic immunodeficient mice (NOD/SCID, SCID, Nude and Rag-1- defects.13 Several studies have compared growth of human deficient) to successfully engraft and support the human cell tumors in SCID and Nude mice.14–19 To our knowledge, to lines Daudi, Raji, Namalwa and Molt-4 as subcutaneous tumors. date no studies have directly compared xenogeneic transplan- We additionally examined the effect of further immunosuppres- tation in NOD/SCID, SCID, nude and Rag-1 mice. The present sion of the mice by whole body irradiation at a dose of 600 cGy for Nude and Rag-1 and 300 cGy for SCID mice and by adminis- study’s purpose was to compare the ability of mice with tration of anti-natural killer (asialo-GM1) antibody on tumor NOD/SCID, SCID, Nude, or Rag-1 genetic defect to permit growth. Mice with each of the defects supported xenografts to the growth of selected human lymphoma and leukemia cell varying degrees. We found differences in growth character- lines. This study permitted further evaluation of the addition istics in the cell lines tested, with Namalwa consistently pro- of whole body irradiation and additional immunosuppression ducing the largest tumors. With all cell lines studied, optimal with asialo-GM1, an antibody directed against murine natural growth was achieved using NOD/SCID mice. Overall, tumor growth was somewhat enhanced by pretreatment with radiation killer (NK) cells. with little additional benefit from the addition of anti-asialo-GM1 antibody. The importance of multiple components of the innate and adoptive immune system in xenotransplantation were best Materials and methods demonstrated when results in untreated NOD/SCID mice were compared to SCID, nude and RAG-1-deficient mice. The NOD/SCID mouse with or without additional immunosuppres- Animals sion provides the optimal model for the study of the biology and treatment of human leukemias and lymphomas. Four strains of immune-deficient mice were used for these Keywords: NOD/SCID mice; xenografts; leukemia/lymphoma; studies: athymic nude (NCr nu/nu) male mice from the immunosuppression National Cancer Institute; CB-17 severe combined immuno- deficient (C.B-17/IcrTac-scidfDF) male mice from Taconic (Germantown, NY, USA); Rag-1 (B6, 129-Rag1tm1Mom) male Introduction and female mice from Jackson Laboratories (Bar Harbor, ME, USA) were obtained at 5–7 weeks of age; and NOD/LtS2-scid Mice with genetically determined immunodeficiencies are mice were originally obtained from the Jackson Laboratory potentially useful for studying the growth and behavior of and locally bred. All mice were maintained in autoclaved foreign tissues, including human tumors. Since the 1960s, micro-isolator cages in a specific pathogen-free area. mice with the Nude mutation have been the standard for establishing in vivo models of human malignancies. This strain’s value in growing human leukemias and lymphomas Human cell lines has been well described,1,2 but residual immunity can inter- fere with transplantability. A mouse strain with a genetic The human B cell lines, Raji, Daudi and Namalwa (Burkitt’s defect causing severe combined immunodeficiency (SCID) lymphomas), one T cell line, Molt-4 (acute lymphoblastic was developed in the 1980s.3–5 Previous studies from this and other laboratories have confirmed the ability of the SCID leukemia) and the fibrosarcoma HT1080 were obtained from mouse to grow a variety of human leukemias and lym- the American Type Culture Collection, Rockville, MD, USA and placed in culture in our laboratory. The Raji, Daudi and Namalwa lines were maintained in RPMI 1640 (Gibco BRL, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) Correspondence: JH Kersey, University of Minnesota Cancer Center, University of Minnesota, Box 86, 420 Delaware St SE, Minneapolis, from Hyclone (Logan, UT, USA), 50 U/ml penicillin and MN 55455, USA; Fax: 612 626 3069 50 ␮g/ml streptomycin (Gibco). Molt-4 was cultured in the Received 2 October 1997; accepted 9 September 1998 same media, with the addition of 1% sodium pyruvate and Bio-technical methods section (BTS) WA Hudson et al 2030 1% non-essential amino acids (Gibco). HT1080 was main- Tumor measurement tained in Earl’s MEM (Gibco) with 10% FBS. All cultures were ° incubated at 37 C in 5% carbon dioxide. Mice were observed to note the beginning of visible tumor growth. From that point on, measurements of two diameters of the tumor (length and width) were made with calipers every Pretreatment of mice 2 days until the mice were moribund. Total tumor volume was estimated using the previously reported formula length × 1/2 Treatment groups were made up of five to nine mice each. width2.22 The mean tumor volume was calculated for each For precise numbers in each group, see Table 1. Nude and group, and these numbers were arrayed over time. Morbidity Rag-1 mice were pretreated with three doses of 200 cGy in mice given the Namalwa tumor determined the experi- 137 whole body irradiation from a Cs source 1 week apart prior ment’s endpoint. to cell injection. SCID and NOD/SCID mice were given only 100 cGy per week due to their known increased sensitivity to radiation.20,21 Half of the mice receiving radiation were also treated with injections of anti-asialo-GM1 (Wako, Richmond, Statistical analysis VA, USA), a rabbit anti-NK cell antibody. The antibody was administered in doses of 25 ␮l per mouse, i.p., immediately Statistical analysis of results was performed using analysis of prior to cell injection, and again on days 8 and 16 following variance for repeated measures as implemented in the SAS injection. One-third of the mice in each strain was given no system’s PROCGLIM.23 pretreatment. Results Cell injection In order to optimize tumor growth, our tumor cells of interest Influence of cell type on tumor growth were mixed with HT1080 fibrosarcoma cells prior to injection.1 The HT1080 cells were trypsinized, spun and Study results are shown graphically in Figure 1 and in tabular resuspended. They were then given a lethal dose of 6000 cGy form in Table 1. As expected, the results demonstrated highly radiation from a 137Cs source and re-suspended in serum-free significant time effects (Wilk’s lambda P = 0.0001). As shown media at a concentration of 50 × 106 cells/ml. Daudi, Raji, in Figure 1 and Table 1, cell type significantly influences cell Molt-4 and Namalwa were checked for viability by trypan growth (P Ͻ 0.0001). Note that Namalwa tumors grew to the blue dye exclusion, spun, and re-suspended at a concen- largest size in every treatment group, and that the scale for tration of 50 × 106 cells/ml. Each cell line was mixed 2:1 with Namalwa is therefore different from that of the other cell lines. HT1080 and injected subcutaneously into the flanks of mice, In general, Raji tumors grew larger than Molt-4 tumors, which for a total dose of 10 × 106 cells of interest and 5 × 106 in turn were larger than Daudi tumors although differences HT1080 cells in 0.3 ml media. between each of the three latter tumors were not significant. Table 1 Effect of cell type, mouse strain, and additional immunosuppression on mean tumor growth Mouse Ab + Radiation Radiation No treatment strains Cell type: Daudi SCID 390.9 (302.9, 478.9) (n = 8) 270.4 (202.6, 338.2) (8) 72.9 (50.2, 95.7) (8) Nude 46.3 (17.2, 75.5) (8) 77.3 (28.4, 126.2) (8) 26.9 (22.9, 30.9) (8) RAG1 24.9 (6.3, 43.6) (5) 57.6 (11.5, 103.6) (6) 82.1 (62.4, 101.7) (6) NSCID 427.1 (328.2, 526.1) (6) 444.3 (223.2, 665.5) (6) 338.6 (182.6, 494.6) (6) Cell type: Molt-4 SCID 807.2 (353.6, 1260) (8) 569.2 (348.2, 790.2) (8) 115.9 (61.9, 169.9) (5) Nude 61.3 (45.4, 77.1) (8) 51 (31.3, 70.8) (7) 39.4 (25.2, 53.6) (9) RAG1 30.7 (19.1, 42.4) (6) 43.3 (16.1, 70.6) (6) 31.3 (21.1, 41.6) (6) NSCID 364.2 (273.7, 454.8) (5) 354.2 (298.7, 409.6) (6) 214.8 (129.5, 300.2) (6) Cell type: Namalwa SCID 2346 (1956, 2736) (8) 1702.3 (1434, 1970) (8) 555.8 (237.3, 874.4) (5) Nude 504.3 (263.5, 745.1) (8) 836.2 (513.1, 1159.3) (7) 394.1 (146.1, 642) (9) RAG1 685.9 (325.4, 1046) (6) 386.3 (141.8, 630.8) (6) 187.7 (25.1, 350.4) (6) NSCID 2885 (2403, 3368) (6) 3784 (2703, 4865) (6) 3688 (2438, 4939) (6) Cell type: Raji SCID 547.2 (385.2, 709.4) (8) 334.3 (237.5, 431.2) (8) 100.3 (62.5, 138.2) (8) Nude 260.9 (124.9, 397.1) (8) 133.4 (56.2, 210.7) (8) 27.2 (11.6, 42.7) (8) RAG1 68.35 (41.2, 95.5) (5) 53.98 (18.8, 89.1) (6) 44.2 (34.6, 53.7) (6) NSCID 511.3 (276.9, 745.7) (6) 363.7 (236.5, 490.9) (6) 433.5 (222.4, 644.8) (6) Number shown is means of tumor volumes followed by 95% confidence intervals in parentheses.
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