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[CANCER RESEARCH 41, 4162-4166, 0008-5472/81 /0041-OOOOS02.00 October 1981] Ultrastructural, Cell Membrane, and Cytogenetic Characteristics of B-Cell Leukemia, a Murine Model of Chronic Lymphocytic Leukemia1 Shimon Slavirv Lola Weiss, Shoshana Morecki. Hannah Ben Bassat, Rachel Leizerowitz, Aviva Korkesh, Ruth Voss, and Aaron Polliack Immunobiology Research Laboratory, Department of Medicine A [S. S., L. W., S. M.], Department Department of Genetics [R. V.]. Hadassah University Hospital, Jerusalem, Israel ABSTRACT A murine model of a spontaneous, transplantable BALB/c B-cell leukemia (BOLO is described. Extreme leukemia and splenomegaly develop in /-/^-compatible recipients of tumor cells. Tumor cells are medium to large lymphocytes that can be transformed into plasmacytoid cells following in vitro stim ulation with lipopolysaccharide. Karyotypic analysis of trans formed tumor cells reveals 36 chromosomes with several monosomies and 7 marker chromosomes. The ultrastructure of the tumor cells was studied using transmission and scanning elec tron microscopy. Although the appearance of tumor cells seems normal by morphological criteria, an impaired capping ability was documented using the fluorescein-conjugated concanavalin A-binding test. Impaired capping ability was docu mented before leukemia was overt as early as 1 to 3 days following inoculation of tumor cells. The B-cell leukemia (BCD provides a useful murine model for the study of various aspects of human bone marrow-derived malignant disorders. INTRODUCTION BCLi3 is a spontaneous B-cell leukemia originally described in 1978 by Slavin and Strober (22) in a 2-year-old female BALB/cKa (H-2d) mouse. The original tumor bearing mouse had high leukocyte counts (up to 200,000 lymphocytes/cu mm) and marked splenomegaly. Leukemic cells resembled human CLL cells, and uniform mature, medium to large lym phocytes were found in the spleen and peripheral blood. Pos itive cell surface determinants included monomeric IgM and IgD with monoclonal X light chain and a single idiotype, Fc receptors, H-2d alloantigens including H-2D and H-2K, and la antigens encoded by the E subregion (13, 22, 24, 27). Tumor cells responded in vitro to LPS, a typical B-cell mitogen, with marked proliferation (22) and secretion of monoclonal IgM bearing the same idiotype as the cell surface IgM (27). No responsiveness was obtained to T-cell mitogens including phytohemagglutinin and concanavalin A (22). The BCL, has been successfully passaged in syngeneic BALB/c mice by s.c., ¡.p., or i.v. injection of tumor cells. The vast majority of spontaneous lymphomas and leukemias described in inbred mice are of the T-cell lineage (20). Although B-lymphocyte tumors have been reported in mice, these neo plasms were induced experimentally, using carcinogens or ' Supported by NIH Grant A1 15387, NCI CA 30313. and United States-Israel Binational Science Foundation Grant 1786/79. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: BCL,, B-cell leukemia; LPS, lipopolysaccharide; PBL, peripheral blood lymphocytes; F-Con A, fluorescein isothiocyanate-conjugated concanavalin A; PBS. phosphate-buffered saline (0.1 M). Received October 27, 1980; accepted July 7, 1981. 4162 of Hematology Haim Gamliel, [H. B. B., R. L., H. G., A. K.. A. P.], and Abelson virus (1, 4, 9, 18). To the best of our knowledge, BCL, is the first animal model of a spontaneous CLL-like B-cell tumor. Since most human lymphocytic lymphomas and leuke mias appear to derive from the B-cell lineage (15), the BCL, may provide a useful animal model for the study of normal and neoplastic characteristics of B-cell immunobiology and B-cell tumor therapy. In the present report, we have further charac terized the tumor cell ultrastructure, growth kinetics, and biol ogy including studies on cell membrane characteristics and BCL,-specific cytogenetic markers. MATERIALS AND METHODS Mice. Inbred male or female BALB/c (H-2d/d). C57BL/6 (H-26/i>), BALB/c x C57BL/6 F, (H-2a/1)), and C3H/He (H-2""") mice, 6 to 8 weeks old, were used throughout this study. All mice were kept in conventional animal facilities. BCL, Tumor Maintenance. The BCL, tumor was passaged and maintained in vivo by i.v. inoculation of 5 x 106 PBL obtained from leukemic mice into syngeneic BALB/c recipients. Evaluation of Leukemogenesis. The appearance of leukemia was monitored by weekly PBL counts and evaluation of splenomegaly. Peripheral blood obtained from the retroorbital veins of tumor-bearing mice was collected in heparinized capillaries and diluted in 2% acetic acid. The leukocyte count was determined using a hemacytometer. Mice were sacrificed at different time intervals following BCL, inocula tion for kinetic evaluation of spleen size by weight and cell content. Light Microscopy, Histochemistry. The following stains were used for cytocentrifuge preparations and smears of the leukemic cells: oil red O (3), Sudan black, peroxidase, periodic acid-Schiff (10), acid phosphatase (2), nonspecific esterase (a-naphthyl acetate method with and without fluoride inhibition), AS-D chloroacetate esterase (26), ßglucuronidase (7), and muramidase (lysozyme) (11 ). Transmission Electron Microscopy. Cell pellets (6 to 8 x 106 cells) were fixed with cacodylate-buffered 1.25% glutaraldehyde (pH 7.3, 4°)for at least 1 hr, rinsed with 0.2 M cacodylate buffer, postfixed in cacodylate-buffered osmium tetroxide for 1 hr at 4°, dehydrated through a graded series of ethanols, embedded in low-viscosity epoxy resin embedding medium according to the method of Spurr (23), and sectioned with a MT-2 Porter Blum microtome equipped with a diamond knife. Thin sections were mounted on uncoated copper grids, stained with uranyl acetate and lead citrate, and viewed with a Phillips EM-300 electron microscope. Scanning Electron Microscopy. Six million cells were fixed in sus pension in 1% phosphate-buffered glutaraldehyde (pH 7.3, 310 mosmol) for at least 1 hr at room temperature and then collected onto glass coverslips covered by poly-L-lysine as described by Sanders et al. (19). Coverslips with monolayers of cells were fixed for 1 hr longer at room temperature, then prepared for scanning electron microscopy by further dehydration in a graded series of ethanol and Freon 113, and critical point dried using Freon 13 as described in earlier studies (17). The specimens were then coated with a thin layer of gold:palladium and examined with a Jeol SM-35X scanning electron microscope. CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1981 American Association for Cancer Research. VOL. 41 Murine Differential counts were performed from random areas on 500 cells. Cells were classified according to the presence of surface ridges and ruffles, microvilli, and blebs. Assay for Binding of F-Con A. Peripheral blood was collected into heparinized glass tubes. Spleen cells were obtained by cutting spleen tissue into small pieces and teasing the cells into 0.1 M PBS, pH 7.2. Lymphocyte suspensions were layered on a Ficoll-Hypaque gradient (5). Contaminating erythrocytes were lysed with ammonium chloride, and the cells were washed twice in PBS. F-Con A was prepared at a fluorescein: protein molar ratio of 1.86 (Miles-Yeda, Rehovot, Israel). From 1 to 2 x 106 cells were incubated with F-Con A at 100 /tg/ml for 30 min at 37°. The cells were washed in PBS, and the percentage of F-Con A capping was determined after counting a total of 300 to 400 cells using a Zeiss microscope with UV light source. Only single cells and small clumps (2 to 5 cells) were counted in order to determine the percentage of caps. Preparation of Cells for Cytogenetic Studies. Spleen cells for cytogenetic analysis were obtained by teasing spleen fragments through a steel mesh into PBS. Cells were washed twice in F-10 medium (Grand Island Biological Co., Grand Island, N. Y.) and put into 0.075 M KCI for 30 min. In some instances, Colcemid (0.05 ¿ig/ml) was added to the KCI solution simultaneously with the addition of the spleen cells. Harvesting for chromosomal analysis was done by hypotonie treat ment with 0.075 M KCI and fixation with methanol:acetic acid (3:1) using standard procedures. Chromosome preparations were done on wet slides and dried for a few sec on a hot plate (63°). For chromosome counts, cells were stained with 3% Giema's stain (Gurr improved R66) in phosphate buffer, pH 6.8. Chromosome G-banding was performed in trypsin using a modification of the technique described by Klinger (12). Chromosomes were arranged according to the standard mouse karyotype (6). Marker analysis and specific breakpoints were deter mined using the nomenclature of Nessbitt and Francke (16). periodic acid-Schiff, Sudan black, peroxidase BCL, esterases, ß- glucuronidase, acid phosphatase, and muramidase were neg ative. Kinetic Growth of BCL, Cells in the Blood. Leukemia de veloped in all BALB/c mice inoculated with as few as 10 BCL, cells. The kinetics of leukemia development in the peripheral blood as monitored by serial venous aspirates was clearly cell dose dependent as demonstrated in Chart 1. Maximal periph eral blood counts reached 540 x 106/cu mm. Kinetics of leukemia development was uniform in all groups receiving a similar BCL, inoculum. Kinetic Development of BCL, in the Spleen. A significant increase in spleen weight occurred as early as 3 days following inoculation of 107 cells, before any peripheral leukemic infiltra tion was apparent (0.89 ±0.01 (S.D.) as compared to 0.14 ± 0.01 g in normal mice). The spleen size has occasionally increased up to 50-fold reaching a cell content of up to 5 x 109 cells. Hematological Status of Leukemic Mice. The hematological status of BCL,-inoculated mice revealed progressive anemia and thrombocytopenia concomitantly with the development of advanced leukemia. Anemia became more severe in long-term survivors as documented in Table 1. No overt tendency to bleeding or infections could be noted during the early stages of BCL,. Spontaneous bleeding (rectal) was occasionally noted during terminal stages. Histocompatibility-related Resistance to Leukemogenesis. BCL, tumor cells developed originally in BALB/c mice maintain surface H-2d alloantigens including H-2D, H-2K, and la antigen (data not shown). Semiallogeneic BALB/c x C57BL/6 F, (H2d/b) mice were somewhat more resistant to BCL, inoculation and showed a protracted disease course. Nevertheless, all F, recipients of 107 tumor cells developed a typical leukemia as RESULTS Morphology. Light microscopy showed that many of these cells resembled normal large and medium-sized lymphocytes. Some of them had a more delicate nuclear chromatin, and nucleoli were evident. Most cells had a high nuclearcytoplasmic ratio and a rim of basophilic cytoplasm. After incubation with LPS, cells appeared larger, and some had plasmacytoid features with more abundant basophilic cytoplasm. Transmission Electron Microscopy. Typical ultrastructural features of lymphocytes were seen (Fig. 1). Most cells had nuclei with marginated heterochromatin and evenly distributed euchromatin. In some cells, nuclear pockets and nucleoli were clearly evident, particularly after stimulation with LPS. The cytoplasm showed polyribosomes, some short strands of rough endoplasmic reticulum, and relatively large numbers of mito chondria. The mitochondria, which were frequently clustered together were large and often showed "ballooning" of the matrix with disruption of the cristae. This finding may, however, be due in part to fixation artifact. After LPS stimulation, a small proportion of the larger cells, estimated to be between 5 to 10% of the overall population showed plasmacytoid features with a well-developed Golgi apparatus and rough endoplasmic reticulum. These cells had eccentric nuclei with more delicate euchromatin and nucleoli (Fig. 2). Scanning electron microscopy revealed that the cells were spherical, with varying numbers of short microvilli. Most cells had moderate to markedly villous surfaces and lacked ridgelike profiles and ruffles (Fig. 3). Cytochemical stains of unstimulated BCLi cells including OCTOBER 1981 shown in Fig. 3. Completely allogeneic mouse strains (C57BL/ 6-H-2"; C3H/HeJ-H-2k) uniformly rejected the histoincompatible H-2" tumor cells (Chart 2). ti3 // f^BCli r.(IU ""»u**io ; •¿ I •¿iliI Ett 3£ 1 1 *-". O¡/ |* o¡i I A8-É100IOSO(020»i-•? P*r 10^ •¿ —¿ ' / 10 * ^ 1QÕO——¿ / 'O' o I¿-"ÃŒ-^* I ' ' Chart 1. Kinetics of leukemia development in BALB/c mice as a function of the dose of the initial BCLi inoculum. Each group consisted of 6 mice. Hematological Table 1 status of mice given injections of 107 BCL, cells at different time intervals following inoculation Time inter val follow ing BCL, in oculationControls mm6,220 (g/100ml)14.6 mm708,800 ±1.0 808,000 ±212 .683 15.8 ±1.8 7,460 ± 1 ,053 15 days ,(j37 224,000 ±57108,830 10.2 ±0.9 73.800 ± 27144,360a ,869 30 days 264,000± ±13 ,166 112 daysWBC/cu 189,667±±3 ,223Hemoglobin7.0 ±1.0Platelets/cu a Mean ±S.D. 4163 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1981 American Association for Cancer Research. S. Slavin et al. 300 Karyotypic Analysis of BCLi. Chromosomal analysis of spontaneous and LPS-induced mitoses were carried out in cultured whole blood, PBL, and spleen cells. No spontaneously dividing cells were found in the peripheral blood. Seventy-four metaphase plates were counted following in vitro stimulation of Ficoll-Hypaque-purified PBL with LPS. Five cells had 40 chro IBALB/c »C57BL/61F, ».-*.. 200 u 100 CD 50C57BL/6 C3H/HtJ \ r=r*sf= 3 5 9 TI Weeks 13 15 17 Chart 2. Kinetics of BCLi in the blood of histoincompatible C3H/HeJ), syngeneic (BALB/c), and semiallogeneic (BALB/c recipients (10' cells/mouse). BLOOD •¿ leukemic o normal W30_uOÃ-Ci 19 (C57BL/6 and x C57BL/6 F,) mosomes, 62 cells had 36 chromosomes, 6 cells had 35 chromosomes including one robertsonian translocation, and one cell had only 34 chromosomes. Spontaneous mitoses were easily demonstrated in the spleen. Altogether, 139 cells were counted of which 23 had 40 chromsomes, 99 cells had 36 chromosomes, 11 cells had 35 chromosomes, and 6 cells had only 34 chromosomes. These results indicate that the majority of the cells representing the BCL, tumor cells consisted of 36 chromosomes. Only 27 chromosomes of the BCL, cells showed banding patterns corresponding to the normal banded mouse karyotype. Seven marker chromosomes were defined in addi tion to 2 deleted chromosomes, Nos. 14 and 15. A detailed analysis of the karyotype was published separately (25). A few residual normal diploid cells were also present. DISCUSSION A leukemic .i normal The present investigations further characterize the sponta neous BALB/c B-cell leukemia which we described previously ovAA (22). The cytological features of the BCL, cells are essentially the same as those seen in human disorders of well-differen tiated lymphocytic lymphoma and CLL. The main features of the BCL, include leukemia and splenomegaly with lesser in AA-A-OA0Û*v0 OA* A•-AÃŽA *t•AOoA volvement of lymph nodes, bone marrow, and other visceral 200o.W0" Ai*A0AAo•AAAOOA• organs (21). The neoplastic cells are mature, relatively large with a moderate amount of cytoplasms. The morphology, the tissue distribution, and the immunological features of the tumor AliAAOOo cells (high density of surface IgM, low density of surface IgD, and response to LPS) (14) all suggest that the BCL, represent a clonal expansion of early B-cells. In that respect, the BCL, 71-21 2 3 5 10 disease closely resembles that of a subset of human CLL Days after Inoculation described by Galton era/. (8) as prolymphocytic leukemia. This Chart 3. Cap formation with F-Con A by PBL and spleen lymphocytes obtained group of patients in general shows a much more malignant from BALB/c mice at different time intervals following i.v. inoculation of BCLi type of CLL with marked splenomegaly and extremely high PBL cells. counts. Interestingly, splenectomy seems to have a positive Cap Formation with F-Con A in Lymphocytes Obtained therapeutic effect in both the BCL, disease and in prolympho from Mice Inoculated with BCLi Cells. Mice were inoculated cytic CLL (14, 25). with BCL, cells (107/mouse), at selected intervals beginning The BCLi tumor cells appear to be unique in their vigorous 18 to 20 hr after inoculation they were sacrificed, and lympho response to in vitro stimulation with LPS. We have demon cytes from peripheral blood and spleen were obtained. The strated that, following LPS stimulation, the BCL, cells undergo cap-forming ability of the lymphocytes with F-Con A (100 /¿g/ further proliferation and differentiation into larger cells that ml) was determined and compared to that of normal controls. show plasmacytoid features with an eccentric immature nu The results clearly indicated a decrease in the cap-forming cleus and well-developed endoplasmic reticulum (Fig. 2). ability with F-Con A of the lymphocytes from BCL,-inoculated The proliferative response of BCL, cells to LPS was also mice beginning as early as 1 to 3 days after inoculation (Chart used for the study of cytogenetic characteristics of tumor cells. 3). By 3 to 5 days after injection, the decrease in cap formation Chromosomal deletions and 7 defined marker chromosomes of the lymphocytes from BCL,-inoculated mice was down to were found in most cells studied. the levels obtained with lymphocytes from leukemic mice (3 to Although the appearance of the BCL, cell surface seems to 4 weeks after inoculation). A significant decrease in cap for be normal by transmission and scanning electron microscopy, mation with F-Con A was observed simultaneously in cells we have characterized some defects in the capping properties of the cell membrane using F-Con A. The appearance of obtained from the peripheral blood and the spleen, showing lymphocytes with reduced cap-forming ability in the blood as 10.8 ±2.9 and 10.1 ±2.3 cells with caps, respectively, as compared to 24.8 ± 4.9 and 28.5 ± 3.5 cells with caps, early as 1 to 3 days after inoculation of tumor cells is not fully respectively, in healthy controls (p < 0.001 in the blood; p < understood. However, it was previously shown that lympho cytes from patients with Hodgkin's and non-Hodgkin's lympho0.001 in the spleen). 4164 CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1981 American Association for Cancer Research. VOL. 41 mas and CLL exhibited reduced cap-forming of Acute Leukemia, pp. 16-21. ability with F-Con A in the early stages of the disease and also during remission even though malignant cells were undetectable in the blood (28). Cell membrane defects may partially account for the unique homing patterns of B-cell tumors. We have previously demonstrated that BCL( cells home predominantly to the spleen (25), and others have reported that these tumor cells do not enter the thoracic duct circulation (14). The BCLi tumor described here may serve as a model for studying the immunology and immunopathology of human welldifferentiated lymphocytic lymphoma-leukemia variants. As a cell type of a particular clone with limited heterogeneity, the BOL, seems to be useful for biological and chemical analysis of various products associated with resting, proliferative, and secretory events following LPS stimulation. Also, the presence of a tumor-specific IgM \ idiotype on the surface of BCL^ cells makes it a useful model for studying new therapeutic modalities using the idiotypic marker as a target for selective immunotherapy with idiotype-specific antibodies. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. REFERENCES 1. Abelson. H. T., and Rabstein, L. S. Lymphosarcoma: virus-induced thymic independent disease in mice. Cancer Res., 30. 2213-2222, 1976. 2. Barka, T., and Anderson, P. J. Histochemical methods for acid phosphatase using hexazonium pararosanalin as coupler. J. Histochem. Cytochem., 10: 741-747, 1962. 3. Bennet. J. M., and Dutcher, T. F. The cytochemistry of acute leukemia. Observations on glycogen and neutral fat in bone marrow aspirate. 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Am sterdam: Elsevier North Holland. 1979. Zucker-Franklin, D., Liebes, F., and Silber, R. Differences in the behavior of the membrane-associated filamentous structures in normal and chronic lymphocytic leukemia (CLL) lymphocytes. J. Immunol.. 722. 97-103. 1979. 4165 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1981 American Association for Cancer Research. S. Slavin et al. Fig. 1. Electron micrograph of leukemic lymphocytes showing cf Ils with ample cytoplasm containing ribosomes and large mitochondria, which are at times clustered together with disruption of cristae. A few short strands of endoplasmic reticulum are seen, x 9,200. Fig. 2. Larger leukemic cell from LPS-stimulated culture with eccentric immature nucleus and ample cytoplasm showing ribosomes and relatively well-developed endoplasmic reticulum. The cell has plasmacytoid features, x 7,600. A. Details of endoplasmic reticulum. x 10,860. Fig. 3. Scanning electron microscopic photographs of spherical leukemic cells showing typical surface features, i.e., varying numbers of relatively short microvilli. x 8,700. 4166 CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1981 American Association for Cancer Research. VOL. 41 Ultrastructural, Cell Membrane, and Cytogenetic Characteristics of B-Cell Leukemia, a Murine Model of Chronic Lymphocytic Leukemia Shimon Slavin, Lola Weiss, Shoshana Morecki, et al. Cancer Res 1981;41:4162-4166. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/41/10/4162 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1981 American Association for Cancer Research.