Download Cell Line Derived from a Metastasis of a Human

[CANCER RESEARCH 40. 2500-2506.
0008-5472/80/0040-OOOOS02.00
July 1980]
Cell Line Derived from a Metastasis of a Human Testicular Germ Cell
Tumor1
David L. Bronson,2 Peter W. Andrews, Davor Solter, Jaroslav Cervenka,
Paul H. Lange, and Elwin E. Fraley
Departments of Urologie Surgery [D. L. B.. P. H. L., E. E. F.] and Oral Pathology ¡J.C.¡,University of Minnesota
Minnesota 55455. and the Wistar Institute. Philadelphia, Pennsylvania 19104 [P. W. A.. D. S.J
College of Health Sciences,
Minneapolis.
ABSTRACT
sue, which suggests the formation by teratoma cells of fully
A cell line, designated 833K-E, has been established from a differentiated elements.
Much of the information about these tumors was obtained
metastasis of a human testicular germ cell tumor that consisted
from studies of mouse testicular teratomas, which were first
of four histological types of tumor cells. The 833K-E cells have
observed by Stevens and Little (28) and were described in
morphological and ultrastructural characteristics of epithelial
detail by Stevens (25) and by Pierce (22). Testicular teratomas
cells and a hyperdiploid karyotype indicative of their human
occur spontaneously at high frequency in strain 129 mice and
male origin. The cells grow in agar cultures and produce in
can be induced by grafting genital ridges of 12-day embryos
nude mice tumors which have the histological features of
or whole embryos into the testes of syngeneic males (26, 27).
embryonal carcinoma without differentiated elements. Many of
These
teratomas consist of a variety of tissues representing
the cells express a stage-specific mouse embryonic antigen,
derivatives of all 3 germ layers.
and low levels of the major histocompatibility antigens and ß2Some of the induced and spontaneous mouse teratomas can
microglobulin also were detected on a large percentage of the
be serially transplanted in syngeneic adult animals. The transcells. A lymphoblastoid cell line (833K-LC) established from
plantable tumors also contain EC cells and thus are classified
the same tumor specimen expresses major histocompatibility
as teratocarcinomas. Several EC cell lines have been estab
antigens and /?2-microglobulin but does not express the embry
lished in vitro from these tumors. Some are nullipotential lines,
onic antigen.
which exhibit little or no tendency to differentiate, whereas
others are multipotential lines, which differentiate readily in
INTRODUCTION
vitro and in vivo. These EC cells express cell surface (embry
onic) antigens that also are detected on undifferentiated cells
Carcinoma of the testis causes 11 to 13% of the deaths from
from other species, including humans, but are not expressed
cancer in North American men between the ages of 15 and 34
by differentiated cells (reviewed in Rets. 10, 13, and 19).
years, and more than 90% of the testicular tumors in this age
We are aware of reports of only 4 cell lines, Tera-1, Tera-2,
group are classified as germ cell tumors (18). Current evidence
SuSa, and NEC-8, that were derived from human testicular
suggests that these tumors arise from the primordial germ
germ cell tumors (9, 11, 30). In a previous communication (4),
cells, which line the seminiferous tubules and are the direct
we described ultrastructural observations of the production,
precursors of sperm.
although at low frequency, of particles morphologically identi
In the histological classification system proposed by the
cal to the human placental retrovirus (6, 16, 29) by cells
World Health Organization, testicular germ cell tumors are
classified further as EC,3 teratoma, teratocarcinoma (EC with
(designated 833K-E cells) established in vitro from an abdom
inal metastasis of a human testicular germ cell tumor. This
teratoma), choriocarcinoma,
yolk sac tumor, and seminoma
report describes some additional properties of the 833K-E cell
(20). EC consists of anaplastic, undifferentiated cells that re
line and compares these properties with those of other human
semble undifferentiated normal cells of an embryo in the earli
est stages of development. Approximately 40% of testicular
and mouse cell lines of similar classification.
germ cell tumors contain elements of more than one (e.g.,
teratocarcinoma), or even all, of the histological types of these
MATERIALS AND METHODS
tumor cells, and it is thought that the other nonseminomatous
Clinical History. A right radical orchiectomy was performed
germ cell tumors are formed by the differentiation of EC cells
in September 1975 on a 19-year-old Caucasian male. Histo(14, 18, 20). Thus, malignant transformation of the germ cells
leads to the development of seminoma or, by a separate
pathological examination of the testis revealed teratoma, EC,
seminoma, and foci of choriocarcinoma.
Chemotherapy was
pathway, of EC, and the EC cells differentiate to form chorio
initiated with methotrexate, cyclophosphamide, and actinomycarcinoma, yolk sac tumor, or teratoma. In addition, some
teratomas contain cartilage, epithelium, and neural tis- cin D, but EC was discovered in a left periaortic lymph node in
February 1976, and the patient died 2 months later with wide
' This investigation was supported in part by Grants CA 15551. CA 10815,
spread
métastases.
and CA 21069 from the National Cancer Institute; T32-GM 07511-03 from the
Tissue Culture. Tissue from an abdominal metastasis, con
National Institute of General Medical Sciences; HD 12487 from the National
Institute of Child Health and Human Development; and 1-301 from the National
sisting of choriocarcinoma with elements of EC, teratoma, and
Foundation-March of Dimes.
seminoma, was obtained at autopsy. The specimen was placed
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: EC. embryonal carcinoma; HLA, human histo
in culture by the coverslip method (8), and cultures were
compatibility leukocyte antigens: SSEA-1, stage-specific embryonic antigen;
incubated at 35° in Roswell Park Memorial Institute Medium
PBS, phosphate-buffered
saline (0.9 ITIM CaCI2, 2.7 mM KCI, 1.5 RIM KH2PO4.
138 mM NaCI. and 9.6 mM Na2HPO«.pH 7.4).
Received August 21. 1979; accepted April 10. 1980.
2500
1640 containing 10% tryptose phosphate broth, 15% heatinactivated (56°,30 min) fetal bovine serum, 2 mM L-glutamine,
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Cell Line from Germ Cell Testicular Cancer
100 units of penicillin per ml, and 100 /ig of streptomycin per
ml.
Cells were detached from culture vessels by incubation for
7 min at 37°with trypsin:citrate diluted with an equal volume
of Grand Island Biological Co. Salt Solution A. Trypsinxitrate
contains 0.25% trypsin (Difco Laboratories, Detroit, Mich.; 1:
250) in Salt Solution A and, per liter, 10 g of sodium citrate,
5.5 g of sodium chloride, and 0.02 g of phenol red (pH 7.4;
formula provided by H. T. Holden, National Cancer Institute).
After several passages by this method, cells were subcultured
with undiluted trypsin:citrate.
Assays for Mycoplasma were done by in vitro cultivation
techniques (Flow Laboratories, Inc., Rockville, Md.) and by
ultrastructural examination of the cells. No Mycoplasma were
detected.
Tumorigenicity.
The methods for seeding cells in agar cul
tures have been described (7). Athymic (nude) mice were given
s.c. inoculations of 5 to 10 x 106 cells, and tumors were
removed for histological examination when approximately 5
mm in diameter.
Cytogenetics. Chromosome analyses were done by conven
tional staining methods as well as by Q- and G-banding tech
niques (7).
Antisera. Monoclonal antibody W6/32, specific for a deter
minant common to the 43,000-dalton chains of HLA-A, -B, and
-C (2), and monoclonal Antibody B.BM1, reactive with human
/?2-microglobulin (3), were kindly provided by Colin Barnstable,
University of Oxford. The production and specificity of a mono
clonal antibody, anti-SSEA-1, which recognizes a stage-spe
cific mouse embryonic antigen and which reacts with an anti
gen expressed by human teratocarcinoma cells and sperm,
has been described (24).
Two monospecific HLA-typing sera with specificities for HLAA2 (Pinquette, No. FD 89 2-50-6-03-15-03)
and HLA B12
(O'Driscoll, No. P3672B) were supplied by NIH and by Julia
Bodmer, University of Oxford.
Serology. Cells in monolayer cultures were harvested with
trypsin, which does not affect the antigens studied." The mono
clonal antibodies were assayed by indirect radioimmunobinding as described previously (24). Briefly, diluted antibody was
incubated for 60 min at 4°with 2 x 10s target cells in a total
volume of 100 /J of PBS containing 2.5% fetal bovine serum.
After being pelleted and washed 3 times with PBS containing
2.5% fetal bovine serum and 0.1% sodium azide, the cells were
suspended in 50 /il of the same washing solution containing
50,000 cpm of 125l-labeled rabbit anti-mouse immunoglobulin
(it-specific, or heavy- and light-chain-specific,
as appropriate).
The cells were incubated at 4°for 60 min, pelleted, and washed
3 times as above, and the 125Ibound to the cell pellet was
determined as cpm in a gamma counter. F9 cells served as
targets for anti-SSEA-1, and 833K-LC cells served as targets
for W6/32 and B.BM1 antibodies. Determinations were made
in triplicate and the variation between the triplicate samples did
not exceed 5%. Quantitative absorptions were performed by
incubating increasing numbers of cells at 4°for 60 min with
200 fil of monoclonal antibody that was diluted (anti-SSEA-1,
1:16,000; W6/32, 1:2,000; B.BM1, 1:10,000) to give 90% of
maximum binding. After the cells were pelleted, residual anti
body in the supernatant was assayed by the indirect radioim' P. W. Andrews and D. Seller, unpublished data.
JULY
munobinding assay described above. The negative controls for
these absorptions of W6/32 and B.BM 1 antibodies were Daudi
cells, which express neither HLA nor /J2-microglobulin (23).
The lymphoid cells were typed for HLA by standard tech
niques in the Tissue Typing Laboratory, University of Pennsyl
vania. Expression of HLA alloantigens by 833K-E cells was
determined by incubating the cells with 12 jul of alloantiserum
(Pinquette or O'Driscoll) on ice for 60 min. The cells were then
pelleted in a Beckman microfuge, and the residual activity of
the serum was titered on peripheral blood lymphocytes from
donors of established HLA type [A1, A2, B8; and A3, A11, B12
(B5, B18, BW35)] by the microcytotoxicity
assay. Another
human testicular tumor cell line, designated 1156Q-E,5 of
known HLA type (A1, A3, B7, B15) was used as a negative
control.
For indirect immunofluorescence
assays, cells were incu
bated with antibody and then with rabbit anti-mouse immuno
globulin of appropriate specificity (Cappell Laboratories, Cochranville, Pa.) conjugated with fluorescein isothiocyanate. Incu
bations were at 4°in the presence of 0.1 % sodium azide. After
the cells had been washed 3 times, they were suspended in
50% glycerol in PBS and examined under UV epiillumination
with a BP 390-490 violet-blue exciting filter and a No. 515
suppression filter.
RESULTS
Tissue Culture. Numerous small islands of epithelial cells
were present in plates and on coverslips a few days after the
tissue fragments were placed in culture. Secondary cultures
were established with free-floating cells and by transferring
coverslips with areas of epithelial cell growth to 35-mm plates.
Subsequent passages were made with trypsin:citrate. The cells
exhibited scant cytoplasm, large nuclei, and prominent nucleoli
(Fig. 1). Ultrastructural examination revealed microvilli, desmosomes, and cytoplasmic tonofibrils (Fig. 2), which are char
acteristics of epithelial cells. Dome formation, another charac
teristic of epithelial cells (21), is observed frequently in aged
833K-E cultures. The cells have been subcultured more than
100 times and thus constitute an established cell line.
In addition, lymphocytes persisted in a few of the primary
cultures, and proliferation of these cells was noted within 5 to
6 weeks after the tissue was explanted. Each time the medium
was replaced ¡nthese plates, floating cells were pelleted and
seeded in a separate vessel. The continued growth of these
cells resulted in establishment of a lymphoblastoid (833K-LC)
cell line. The 833K-LC cells grow in suspension (i.e., they do
not attach to the vessel growth surface) as single cells or small
clusters, are highly pleomorphic, and exhibit uropods. A
herpes-type virus was detected by ultrastructural examinations
of these cells in early culture, and all cells examined expressed
Epstein-Barr virus antigens.6 Of the lymphoid cells, the EpsteinBarr virus selectively infects and transforms B-lymphocytes;
thus, the expression of these viral antigens suggests that the
833K-LC are derivatives of polyclonal B-lymphocytes (15).
Cytogenetics. Chromosomal analyses were performed with
833K-E cells in passages 5 and 59 with virtually identical
results. The cells have a hyperdiploid number of chromosomes,
5 D. L Bronson, unpublished data.
6 G. R. Dreesman, personal communication.
1980
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2501
D. L. Branson et al.
with a modal number of 56 to 61 in 38 of 58 mitotic spreads
counted. Five karyotypes were constructed by G-banding, and
20 mitotic spreads were analyzed. In each of the karyotypes,
the A-group chromosomes were overrepresented, whereas the
number of chromosomes of the other groups did not deviate
consistently from diploid constitution. In all metaphases, 2 or
3 isochromosomes of F-group size were observed, as were 3
to 8 chromosomes of abnormal morphology that could not be
identified with certainty. No marker chromosome was found
that would identify this cell line specifically. The Y chromosome
was detected by Q-banding in all mitotic figures (Fig. 3).
Tumorigenicity. 833K-E cells in passage 85 were inoculated
into 5 nude mice. Tumors at least 5 mm in diameter were
produced at the site of inoculation in 4 of the animals between
35 and 76 days after inoculation. No métastaseswere noted.
The tumor-free animal died on Day 59 after inoculation. Sec
tions of the tumors from all 4 animals showed cells arranged in
tightly packed, convoluted sheets. The tumor cells exhibited a
uniform morphology consistent with EC without differentiated
elements, although the convoluted areas suggested poorly
defined glandular structures (Fig. 4). No tumors formed in 9
nude mice within 44 to 141 days after inoculation (;'.e., at the
time of death of the animal) with 833K-LC cells.
The 833K-E cells also formed colonies in agar cultures with
an efficiency of 1 to 2%. Forty-four clones have been isolated
in 3 separate experiments and grown in mass culture for
additional characterization studies. The clones exhibit the cel
lular morphology and growth pattern of the parent 833K-E cell
line.
Antigen Expression. Quantitative absorptions of monoclonal
antibodies W6/32, B.BM1, and anti-SSEA-1 indicated that
cells of the 833K-E line express HLA, /8?-microglobulin, and
the cross-reacting mouse embryonic antigen, SSEA-1. The
latter antigen is absent from the 833K-LC cells (Chart 1). Like
other lymphoid cell lines, the 833K-LC cells express more HLA
and /i?-microglobulin than do peripheral blood lymphocytes.
However, the 833K-E cells express approximately 50% less
HLA and /i?-microglobulin on a per-cell basis than do peripheral
blood lymphocytes even though the 833K-E cells are much
larger (roughly 5:1 based on the volume of equivalent numbers
of packed cells).
Unotsorbed
Similarly, in immunofluorescence assays, the 833K-E cells
showed only a weak, stippled fluorescence after reaction with
W6/32 and B.BM1 antibodies, and some of the cells were
scored as negative (Table 1). However, the apparent lack of
HLA and /?2-microglobulin on those cells may reflect only the
technical difficulties of scoring a weak system. More certain
was the observation that some 833K-E cells did not express
SSEA-1, because reactive cells fluoresced strongly.
Absorption of HLA typing sera confirmed that HLA of both
the A and B loci are expressed by 833K-E and 833K-LC cells
(Chart 2). Similar results were obtained with 833K-E cells in
passages 34 and 117.
DISCUSSION
The 833K-E cell line was established from metastatic tumor
tissue consisting of 4 histological types of testicular tumor
cells, which raises the question of which type(s) of testicular
tumor cell is (are) represented by this cell line. As a general
observation, it can be stated that the 833K-E cells exhibit
ultrastructural and growth properties of epithelial cells and are
morphologically similar (i.e., have scant cytoplasm, large nu
clei, and prominent nucleoli) when continuously subcultured at
a high cell density. All clones derived from this cell line exhibit
Table 1
Indirect immunotluorescence assays
Cell suspensions were incubated with monoclonal antibody at 4°for 60 min.
pelleted, washed 3 times, and then incubated at 4°for 60 min with rabbit antimouse immunoglobulin conjugated with fluorescein isothiocyanate. The cells
were pelleted, washed 3 times, and resuspended in 50% glycerol in PBS for
examination under UV.
% of positive cells
Anti-SSEA-1
W6/32 (anti-HLA)
Cell line
)"0087(+
(+ + +
833K-E833K-LCOaudiF9LNSV667
f100(+
(+
+)0010080<
+
)100(++
+)00NTC
+
+)092+
+ + + , strong fluorescence; + , weak, stippled fluorescence.
1Human skin fibroblasts transformed with SV40 (5).
NT. not tested.
Serum Unorjsorbed
o
o
o.
x
15
30
Serum
O_
2
« 2
E
7.5
B.BMI (anti-ft-microglobulin)
o.
O
3.8
7.5
15
30
3.8
75
15
30
Cells x 100,000
Cells x 100,000
Cells 100,000
Chart 1. A. absorption analysis of monoclonal antibody (anti-SSEA-1) reactivity. After absorption of antibody by 833K-E (A), 833K-LC (•).
or F9 (CDcells, residual
antibody in the clarified serum was tested by radioimmunoassay on F9 cells as described previously (24) Each point is the average of the results of triplicate assays.
B, absorption analysis of monoclonal antibody W6/32 (anti-HLA) reactivity on 833K-E (A). 833K-LC (•).
Daudi (O) cells, or peripheral blood lymphocytes O by
methods described in A. Residual antibody in the clarified serum was tested on 833K-LC cells. C. absorption analysis of monoclonal antibody B BM1 (anti-/i2microglobulin) reactivity on 833K-E (A). 833K-LC (•).
or Daudi (O) cells or peripheral blood lymphocytes (O) by methods described in A. Residual antibody in the
clarified serum was tested on 833K-LC cells.
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40
Cell Line from Germ Cell Testicular Cancer
12
14
Serum Dilution
II
12
14
Serum Dilution
Chart 2. Cytotoxicity assays of unabsorbed alloantisera (O) and of sera after
incubation with 833K-E (A), 833K-LC (O). or 1156Q-E (•)cells. C'C (A),
complement control. The activities of unabsorbed alloantisera. anti-HLA-A2 (A)
or anti-HLA-B12 (6) and of absorbed alloantisera were titered on peripheral
blood lymphocytes of known HLA type. The scores, representing the percentage
of lymphocytes that were lysed by antibody, are: 1 - 0 to 10%; 2 = 11 to 25%;
4 = 26 to 50%; 6 = 51 to 80%; 8 = 81 to 100%.
parison, SuSa cells also have an aneuploid karyotype (11 ), as
do the murine somatic cell hybrids (1). Thus, in some aspects
(origin, morphology, and expression of SSEA-1), the 833K-E
cells resemble other teratocarcinoma stem cells, and they also
produce EC in nude mice. In other aspects, such as expression
of major histocompatibility antigens, although weakly and in
karyotype, they are dissimilar from mouse teratocarcinoma
stem cells.
It is possible that 833K-E cultures contain more than one
type of malignant cell and, perhaps, variable numbers of differ
entiated cells (which could lack SSEA-1 but express HLA and
/?2-microglobulin), with only the EC cells capable of producing
tumors in nude mice. The cultures occasionally contain cells of
different morphology, depending in large part on culture con
ditions, which might represent in vitro differentiation, other
types of testicular tumor cells, or both. The immunofluorescence data also suggest heterogeneity of the cultures. The
developmental relationships between these possible subpopulations, and the question of whether the stem cells express
HLA and /?2-microglobulin, will become clearer after studies of
the clones derived from 833K-E and other human testicular
tumor cell lines.
these same properties and are morphologically indistinguish
able from the parent line. Chromosome analyses demonstrated
that the cells are of human male origin, and, although the
ACKNOWLEDGMENTS
chromosomal constitution is grossly abnormal, it is stable, as
The authors thank Peter Waldron and Hannelore Asmussen for excellent
is indicated by analyses of cells in widely different passages.
The tumors produced by inoculation of 833K-E cells into technical assistance, Donna Ritzi for photomicrography, and Judith Gunn Bronson for editing the manuscript.
nude mice were composed of undifferentiated small cells typi
cal of the EC cells seen in biopsies of human EC and of the
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CANCER
RESEARCH
VOL. 40
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Cell Line from Germ Cell Testicular Cancer
Fig. 1. Phase-contrast
photomicrograph
of 833K-E cells in culture, x 300.
Fig. 2. Electron micrograph of 833K-E cells with desmosomes (thick arrows) and tonofibrils (thin arrows), x 60,000
Fig. 3. Trypsin-Giemsa banding of chromosomes from 833K-E cells in passage 5 showing a hyperdiploid karyotype with a Y chromosome.
Fig. 4. Photomicrograph of a section of a tumor taken from a nude mouse inoculated with 833K-E cells. The tumor consisted of undifferentiated
into convoluted sheets. H & E, x 240.
JULY
cells organized
1980
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Cell Line Derived from a Metastasis of a Human Testicular Germ
Cell Tumor
David L. Bronson, Peter W. Andrews, Davor Solter, et al.
Cancer Res 1980;40:2500-2506.
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