Heterotransplantation and Maturation of a Chronic Myelogenous Leukemia Cell line (KCL22) In Vivo

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1 International Journal of Cell Cloning 2: (1984) Heterotransplantation and Maturation of a Chronic Myelogenous Leukemia Cell line (KCL22) In Vivo Ichiro Kubonishi, Yuji Ohtsuki, Shizuo Yoshimoto, Isao Miyoshi Departments of Medicine and Pathology, Kochi Medical School, Kochi, Japan Key Words. CML. Differentiation. Heterotransplantation. Granulocytic sarcoma. Ph1 chromosome Abstract. Heterotransplantation was performed to study the maturational capacity of a new Ph1 chromosome-positive chronic myelogenous leukemia cell line (KCL-22). Five million KCL-22 cells of undifferentiated blasts were implanted intraperitoneally into 15 immunosuppressed newborn hamsters. Of the 15 hamsters, 10 that survived developed granulocytic tumors days after implantation. The tumor cells were composed of a mixed population of undifferentiated blasts, neutrophils, and eosinophils and had a human female karyotype with double Phl chromosomes identical to that found in the KCL-22 cell line used for transplantation. These findings indicate that Ph' chromosome-positive cells can be induced to mature along the neutrophil-eosinophil lineage after growth in the heterologous hosts. Introduction The heterotransplantation studies have been valuable for investigating the biologic characteristics of human leukemia cells [ In a previous report, we described the cytologic and cytogenetic properties of a new Phl chromosome-positive cell line (KCL-22) that was established from a patient with chronic myelogenous leukemia (CML) in blast crisis [4]. This paper reports on the morphologic, cytogenetic, and histochemical features of granulocytic tumors produced in newborn hamsters by transplantation of the KCL-22 cell line, with special reference to their maturation in vivo. Correspondence: Dr. Ichiro Kubonishi, Department of Medicine, Kochi Medical School, Kochi (Japan). Received January 30, 1984; accepted March 8, Alpha Med Press

2 Phl-Positive Granulocytic Sarcoma 244 Materials and Methods Cell Line The KCL-22 cell line used was derived from the pleural effusion cells of a female patient with CML in blast crisis [4]. This cell line consisted of undifferentiated blasts with no azurophilic granules (Fig. 1A) and had double Phl chromosomes, one of which arose from a translocation between chromosomes 9 and 22. KCL-22 cells were maintained in liquid suspension culture with RPMI 1640 medium (Flow Laboratories, McLean, VA) supplemented with 15% fetal calf serum (FCS; Flow Laboratories) at 37 C in a humidified atmosphere of 5% COz in air. Heterotransplantation Five million KCL-22 cells in 0.1 ml of saline were implanted intraperitoneally into 15 newborn hamsters (bred in our laboratory) within 24 h of birth. The same volume of anti-human lymphocyte serum (ALS), which was prepared by the method of Levey and Medawar [5], was inoculated into the peritoneal cavity immediately after cell graft, and twice a week thereafter. Histologic and Cytologic Examination The hamsters were sacrificed by ether inhalation. Histologic sections were prepared from tumors and pertinent organs such as the liver, spleen, kidneys, lungs, lymph nodes, and bone marrow. The sections were stained with hematoxylin and eosin (H & E) and tested for naphthol AS-D chloroacetate esterase. Imprint smears of the tumors were stained with May-Grunwald-Giemsa and evaluated for peroxidase, naphthol AS-D chloroacetate esterase, a-naphthyl butyrate esterase, Sudan black B, acid phosphatase, and alkaline phosphatase. Chromosome Analysis Chromosome analysis of the tumor cells was performed as described previously [6, 71. Immunologic Examination To identify the cellular origin of the tumors, immunologic examination was performed using anti-human and anti-hamster lymphocyte sera. Twice every two weeks, a rabbit was injected intravenously with 1.0 x 10s cells from a human non-t, non-b acute lymphoblastic leukemia (ALL) cell line PALL-1) [S]. After absorption with hamster spleen cells and thymocytes, the anti-nall- 1 serum was rendered specific for human cells. ALS was also rendered specific for hamster cells after absorption with KCL-22 and human T, B, and non-t, non-b ALL cell lines [S]. These two antisera were used to stain the tumor cell suspensions by indirect membrane immunofluorescence. Electron Microscopy Small pieces of the tumors were fixed in 3% glutaraldehyde and postfixed in 1% osmium tetroxide. After dehydration with a graded series of ethanol, the tissues were embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a Hitachi H-300 electron microscope (Hitachi Seisakusho, Hitachi, Japan).

3 Kubonishi/Ohtsuki/Yoshimoto/Miyoshi 245

4 Ph1-Positive Granulocytic Sarcoma 246 Results Heterotransplantation Of the 15 hamsters transplanted with KCL-22 cells, five were lost by cannibalism within the first week, and the ten surviving hamsters developed inguinal tumors after 10 days. When sacrificed days after transplantation, whitish tumors, 1-4 cm in diameter, were present in the inguinal and upper abdominal regions of the animals (Fig. IB). Histologic and Cytologic Examination of Tumors The tumors consisted predominantly of immature cells with round or ovoid nuclei having prominent nucleoli. Granulocytes in various stages of differentiation were scattered among these cells (Fig. 1C); some granulocytes were positive for naphthol AS-D chloroacetate esterase. Imprint smears of the tumor cells showed a mixture of immature undfierentiated cells and granulocytes (neutrophils and eosinophils) of different maturity (Fig. ld, E). These neutrophils were positive for peroxidase, naphthol AS-D chloroacetate esterase, and acid phosphatase. The eosinophils were positive for peroxidase but negative for naphthol AS-D chloroacetate esterase. The cytoplasmic granules of the eosinophils were distinctly stained with H & E stains, while those of the neutrophils were not. Both of these neutrophils and eosinophils were negative for a-naphthyl butyrate esterase, Sudan black B, and alkaline phosphatase (Table I). No tumor cell infiltration was observed in other organs. Fig. 1. (A) Smear of the KCL-22 cell line. The cytoplasm contains vacuoles but no azurophilic granules. X 500. (B) Autopsy findings of hamster No. 3. Tumors are present in the left inguinal and right upper abdominal regions, protruding into the abdominal cavity. (C) Histologic section of the tumor shown in (B). Note features of granulocytic sarcoma consisting of a mixed cell population of undifferentiated blasts with scattered granulocytes. X 400. (D) Smear of tumor cell from hamster No. 7. Neutrophilic and eosinophilic promyelocytes and myelocytes are present. X 500. (E) Same smear as shown in (D). Neutrophilic metamyelocytes and eosinophilic band cells are seen between immature cells. X 800.

5 Kubonishi/Ohtsuki/Yoshimoto/Miyoshi 247 Table I. Cytochemical characteristics of KCL-22 and KCL-22-derived tumor cells KCL-22 KCL-22-derived tumor cells in hamsters Blasts Neutrophils Eosinophils Peroxidase - - f-b+ + Naphthol AS-D chloroacetate esterase a-naphthyl butyrate es terase Sudan black B Acid phosphatase Alkaline phosphatase Hematoxylin and eosin -a +b a Cytoplasmic granules were negative. b Cytoplasmic granules were positive. Table II. Distribution of chromosome numbers in cells from tumors produced in hamsters by transplantation of KCL-22 Hamster Chromosome numbers Total cells analyzed Chromosome Analysis Chromosome analysis of the tumor cells showed a hyperdiploid human female karyotype with double Phl chromosomes (Table 11). A representative chromosome karyotype was 52,XX, + lp-, + 6, + 8, + 8, + 8,t (9q + ;22q-), + 22q- which was completely identical to that of the KCL-22 cell line (Fig. 2).

6 Ph1-Positive Granulocytic Sarcoma 248 t Pd Phf &XI xx Fig. 2. Representative karyotype of tumor from hamster No. 5: 52,XX, + Ip-, + 6, + 8, + 8, + 8,t(9q+ ;22q-), + 22q-. This karyotype was identical to that of the original KCL-22 cell line.

7 Kubonishi/Ohtsuki/Yoshimoto/Miyoshi 249 Reactivities of Antisera The reactivities of rabbit anti-nall-1 serum and ALS with the tumor cells, KCL-22 and NALL-1; hamster spleen cells; and thymocytes are summarized in Table 111. Figure 3 shows that anti-nall-1 serum reacted with almost all (93-97%) of the tumor cells, KCL-22 and NALL-1 cells, but not with hamster spleen cells and thymocytes. In contrast, ALS reacted with hamster spleen cells and thymocytes, but not with KCL-22 and NALL-1 cells. Only 5-10% of the cells from the tumors were found to be positive for ALS. These positive cells were considered to be the strorrtal cells of hamster origin. Electron Microscopic Examination The tumors were composed of cells with round or bilobed nuclei and abundant cytoplasm. The nuclei frequently contained large conspicuous nucleoli and peripheral heterochromatin clumps. Nearly half of the tumor cells exhibited varying numbers of electron-dense membrane-bound granules less than 0.8 pm in diameter (Fig. 4A). The granules which were heterogeneous in electron density had homogeneous internal structures (Fig. 4B). There were no central crystalloids in these granules. The rough endoplasmic reticulum and Golgi apparatus were well developed in some cells; lipid droplets were seen occasionally. Discussion In the present study, transplantation of a newly established CML cell line (KCL-22) into immunosuppressed hamsters resulted in the production of tumors. Chromosome analysis of the tumor cells revealed Ph1 chromosomes and other abnormalities identical to those of the KCL-22 cell line, and they were also identified to be of human origin by membrane immunofluorescence. Histologically, the tumors consisted of a mixed cell population of primitive cells and granulocytes in various maturational levels. Electron microscopy demonstrated membrane-bound granules in the cytoplasm in approximately half of the tumor cells. These granules were cytologically consistent with those of immature and mature neutrophils and eosinophils. The original KCL-22 cell line used in this study consisted only of undifferentiated blasts with no evidence of maturation during continuous culture in vitro [4]. Therefore, the production of granulocytic tumors in hamsters was considered to be the result of differentiaton of the implanted

8 Phl-Positive Granulocytic Sarcoma 250 Table IU. Reactivities of rabbit anti-nall-1 serum and ALS with human and hamster cells Target cells Reactivities (%) anti-nall- 1 ALS Human cells KCL KCL-22-derived tumor cells a NALL Hamster cells Spleen cells Thymocytes a Reactivities with tumors from 10 tumor-bearing hamsters. Fig. 3. Indirect membrane immunofluorescence of tumor cells from hamster No. 7. The tumor cells, both immature blasts and granulocytes (A) gave a positive reaction with anti-nall-1 serum (B).

9 Kubonishi/Ohtsuki/Y oshimotolmiyoshi 25 1 Fig. 4. (A) Electron micrograph of the tumor shown in Figure 1B. The nucleus is oval and has a prominent nucleolus. The cytoplasm contains many membrane-bound granules with various electron densities. x 11,000. (B) Higher magnification of the cytoplasmic granules. Note the homogeneity of the individual granules and lack of central crystalloid structures. X 23,000. KCL-22 cells in vivo. Some fresh leukemia cells from blast crisis of CML are known to mature in vivo [7,9] as are chronic phase CML cells in vitro [ ; however, no cellular maturation has been previously observed following heterotransplantation of cultured CML cell lines [3, 13, 141. Cytochemical staining showed that the granulocytes in hamster tumors were positive for peroxidase but negative for Sudan black B; this is in contrast to normal granulocytes which are usually positive for both. Moreover, the ultrastructural features of these granulocytes indicated asynchronous maturation between the nucleus and cytoplasm. These observations suggest that the maturational pattern of the implanted KCL-22 cells is still abnormal or incomplete in vivo.

10 Phl-Positive Granulocytic Sarcoma 252 CML is a pluripotent hematopoietic stem cell disorder [15]. Study of the G6PD isoenzyme in eosinophils from a G6PD heterozygote patient with CML by Koefler et al. [ 161 and the report of a case of eosinophilic blast crisis in a CML patient by Murinone et al. [17] showed that eosinophils are also involved in the malignant clone of CML. Our study demonstrated the presence of Phl chromosome abnormality in neutrophileosinophil precursor cells and confirmed their previous observations. Further studies are now in progress to clarify the maturational potential of the KCL-22 cell line to cell lineages other than neutrophils and eosinophils. References 1 Adams, R.A.; Pothier, L.; Hellerstein, E.E.; Boileau, G.: Malignant immunoblastoma: immunoglobulin synthesis and the progression to leukemia in heterotransplanted acute lymphoblastic leukemia, chronic lymphatic leukemia, lymphoma, and infectious mononucleosis. Cancer 31: (1973). 2 Nilsson, K.; Giovanella, B.C.; Stehlin, J.S.; Klein, G.: Tumorigenicity of human hematopoietic cell lines in athymic nude mice. Int J Cancer 19: (1977). 3 Machado, E.A.; Lozzio, B.B.; Lozzio, C.B.; Lair, S.V.; Aggio, M.C.: Development of myelosarcomas from human myelogenous leukemia cells transplanted in athymic mice. Cancer Res 37: (1977). 4 Kubonishi, I.; Miyoshi, I.: Establishment of a Ph1 chromosome-positive cell line from chronic myelogenous leukemia in blast crisis. Int J Cell Cloning I: (1983). 5 Levey, R.H.; Medawar, P.B.: Nature and mode of action of antilymphocytic antiserum. Proc Natl Acad Sci 56: (1966). 6 Seabright, M.: A rapid banding technique for human chromosomes. Lancet 2: (1971). 7 Miyoshi, I.; Kubonishi, I.; Uchida, H.; Hiraki, S.; Toki, H.; Tanaka, T.; Masuji, H.; Hiraki, K.: Direct implantation of Phl chromosome-positive myeloblasts into newborn hamsters. Blood 47: (1976). 8 Miyoshi, I.; Hiraki, S.; Tsubota, T.; Kubonishi, I.; Matsuda, Y.; Nakayama, T.; Kishimoto, H.; Kimura, I.: Human B cell, T cell and null cell leukaemic cell lines derived from acute lymphoblastic leukiiemias. Nature 267: (1977). 9 Kuroki, M.; Watanabe, S.; Shimosato, Y.; Nakajima, T.; Sato, Y.; Kitahara, T.: A new strain (KW- 1) of human chronic myeloid leukemia transplantable in lasat mice. Gan 73: (1982).

11 Kubonishi/Ohtsuki/Yoshimoto/Miyoshi Whang-Peng, J.; Peny, S.; Knutsen, T.A.: Maturation and phagocytosis by chronic myelogenous leukemia cells in vitro. A preliminary report. J Natl Cancer Inst 38: (1967). 11 Golde, D.W.; Byers, L.A.; Cline, M.J.: Chronic myelogenous leukemia cell growth and maturation in liquid culture. Cancer Res 34: (1974). 12 Miyoshi, I.; Uchida, H.; Tsubota, T.; Kubonishi, I.; Hiraki, S.; Kitajima, K.: Basophilic differentiation of chronic myelogenous leukaemia cells in vitro. Scand J Haematoll9: (1977). 13 Louio, B.B.; Machado, E.A.; Lozzio, C.B.; Lair, S.: Hereditary asplenicathymic mice: transplantation of human myelogenous leukemic cells. J Exp Med 143: (1976). 14 Kubonishi, I.; Freeman, A.I.; Minowada, J.: Heterotransplantation and clonal growth of human Phl-chromosome-positive leukemia-cell (NALM- 1) and B-cell leukemia-cell (BALM-2) lines. Cancer 45: (1980). 15 KoefBer, H.P.; Golde, D.W.: Chronic myelogenous leukemia-new concepts. N Engl J Med 304: (1981). 16 KoelYer, H.P.; Levine, A.M.; Sparkes, M.; Sparkes, R.S.: Chronic myelocytic leukemia: eosinophils involved in the malignant clone. Blood 55: (1980). 17 Marinone, G.; Rossi, G.; Verzura, P.: Eosinophilic blast crisis in a case of chronic myeloid leukaemia. Br J Haematol55: (1983).