Download Ultrastructural, Cell Membrane, and Cytogenetic Characteristics of B

[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
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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¿-"Ì-^*
I
'
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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
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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•-AÎA
*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
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RESEARCH
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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.
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4165
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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
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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.
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