Lymphoproliferation and heterotransplantation in nude mice: tumor cells in Hodgkin's disease.

Abstract

In the last 3 years we were able to establish four long-term cultures from Hodgkin-derived material [pleural effusions (2), bone marrow (1), and peripheral blood (1)], consisting of cells which represent morphologic and cytochemical as well as cytogenetic features of their in vivo ancestors. Two of these cell lines are described in this paper. These two lines share the same features: non-B-T lymphocytes, non-macrophages, non myeloid cells, EBV genome negative, monoclonality, multiple numerical and structural chromosome aberrations, and tumor formation upon intracranial xenotransplantation in nude mice. The two remaining lines are being characterized at the moment. The common characteristics expressed synonymously in the two described lines suggest that the Hodgkin tumor cell does not seem to share the features of marker-carrying lymphocytes, macrophages, or myeloblasts. The cellular origin of these cells is not clear. The loss of cellular differential markers during the process of possible dedifferentiation is discussed.

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Haematology and Blood Transfusion Vol. 26
Modern Trends in Human Leukemia IV
Edited by Neth, Gallo, Graf, Mannweiler, Winkler
© Springer-Verlag Berlin Heidelberg 1981
Lymphoproliferation and Heterotransplantation in Nude Mice: Tumor
Cells in Hodgkin's Disease*
V. Diehl, H. H. Kirchner, M. Schaadt, C. Fonatsch, and H. Stein
A. Summary
In the last 3 years we were able to establish
four long-term cultures from Hodgkin-derived
material [pleuraleffusions (2), bone marrow
(1), and peripheral blood (1)], consisting of
cells which represent morphologic and cyto-
chemical as well as cytogenetic features of their
in vivo ancestors. Two of these cell lines are
described in this paper.
These two lines share the same features:
non-B-T lymphocytes, non-macrophages, non
myeloid cells, EBV genome negative, mono-
clonality, multiple numerical and structural
chromosome aberrations, and tumor forma-
tion upon intracranial xenotransplantation in
nude mice.
The two remaining lines are being characte-
rized at the moment. The common characteri-
stics expressed synonymously in the two des-
cribed lines suggest that the Hodgkin tumor
cell does not seem to share the features of
marker-carrying lymphocytes, macrophages,
or myeloblasts. The cellular origin of these
cells is not dear. The loss of cellular differen-
tial markers du ring the process of possible de-
differentiation is discussed.
B. Introduction
The "Sternberg-Reed" (SR) and "Hodgkin"
(H) cells are considered to be the neoplastic
cells in Hodgkin's disease. Their cellular origin
* This work was supported by the Deutsche For-
schungsgemeinschaft Bad Godesberg (Di 184-
5/6)
is still subject of considerable controversy.
There are arguments for a histiocytic origin of
SR and H cells (Rappaport 1966; Mori and
Lennert 1969) as wen as for their lymphoid
origin (Dorfman et al. 1973; Papadimitriou et
al. 1978).
Because of the fragility of freshly isolated
Hand SR cells and the contamination with
numerous reactive cells the precise analysis of
their cell type is difficult. These limitations can
be overcome by the establishment of in vitro
proliferating cell populations with neoplastic
properties from patients with Hodgkin's disea-
se. The present report describes the features
of two established in vitro cell lines from the
pleural effusions of two patients with histologi-
cally proven Hodgkin's disease.
c. Patients, Material, and Methods
The pleural effusions were obtained from a 37-year-
old woman (E.M.) with histologically proven Hodg-
kin's disease, which was of the nodular sclerosing
type, stage IV B, and was primarily diagnosed in
1972 (L 428), and from a 36-year-old man (HR.)
with Hodgkin's disease, nodular sclerosing type,
which was diagnosed in 1976 from an inguinallymph
node biopsy (L 439).
I. Establishment of the In Vitro Cultures
The heparinized pleural effusion fluid was centrifu-
ged at 150 g for 10 min and the pellet was
resuspended in an 0.84%NH4Cl solution to desinte-
grate the remaining erythrocytes. After two was-
hings cells were incubated in RPMI 1640 medium
supplemented with 20% fetal calf serum, glutamin,
and penicillin/streptomycin at 37° C in a 5 % CO2 air
atmosphere.
229

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11. Cytochemistry
The following enzyme staining methods were perfor-
med: Naphthol-AS-D.chloracetate-esterase, pero-
xidase, acid-a-naphthyl acetate esterase, and alkali-
ne posphatase.
111. Surface Markers
Membrane-bound immunoglobulins were investiga-
ted as described earlier. Ia -like antigen and lysozyme
was detected by specific anti sera in an indirect
immunofluorescence procedure.
1. Rosette Assays
The E-rosette technique is described by Jondal et al.
(1972). Rosette assays to demonstrate C3b andC3d
receptors (EAC rosette assays) and IgG receptors
were performed as described by Stein (1978).
Intracellular antigens were demonstrated by immu-
nostaining for k, A, and lysozyme by means of the
PAP technique (Stein and Kaiserling 1974).
2. Immune Phagocytosis
From the L 428 and L 439 line 1 X 106 cells were
mixed with either IgG-EoxA or EAC 3b, pelleted by
centrifugation at 200 g for 5 min, and incubated at
37° C for 2 h. The pellet was resuspended by gentle
shaking and centrifuged onto glass slides. The slides
were stained with Grünwald Giemsa and the propor-
tion of cells with ingested indicator cells was
counted.
IV. EBV-Specific Antigens (EBNA, EA, and
VCA)
The detection of EBV -associated nuclear antigen
(EBNA) was performed according to Reedman and
230
Klein (1973). For the demonstration of the viral
capsid antigen (VCA) and the early antigen (EA)
the method described by Henle and Henle (1966)
was applied.
V. Cytogenetic Studies
The cytogenetic techniques are described elsewhere
(Hell riegel et al. 1977). For a detailed analysis of
metaphases the Giemsa-banding method was per-
formed (Sumner et al. 1971).
VI. Heterotransplantation in Nude Mice
Details of breeding and colony maintenance of the
Balb C nu/nu mice have been described previously
(Krause et al. 1975). Cell suspensionof 1 X 106/0.01
ml were injected intracranially or subcutaneously
into 4-week-old mice. Transplantation of cells em-
bedded in a plasma dot was performed according to
the technique described by Lozzio et al. 1976.
D. Results
From the pleural effusions both celliines ( 428,
L 439) could be established after a lag phase of
4 to 6 weeks. Morphologically the L 428
cultures varied greatly in size and structure
(Fig. 1). Beside large (50-80Jml) multinuc1ea-
ted cells with smooth membrane surfaces,
medium sized (30-50 Jml) rnono- or binuc1ea-
ted cells with "hairy" membrane protrusions
becarne obvious. A third variant consisted of
rather small cells (15-30 Jml).
The second culture (L 439) contained round
mono- or multinuc1eated cells with prominent
nucleoli and few villi on the surface membrane.
Fig. 1. L 428 cultures using
Pappenheim. X 613

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The findings of the evaluation of surface
membrane and cytoplasmic constitutents are
summarized in Table 1. The only structurally
defined surface antigen demonstrable in both
lines was the Ia-like antigen. Membrane recep-
tors capable of binding T-lymphocytes were
formed in more than 90% of the L 428 and
L 439 cells. By enzyme cytochemistry the
Properties/reagents
Surtace staining tor:
Polyvalent Ig
IgM
IgA
IgG
K
A
Ia-like antigen
Lysozyme
Binding of aggr. IgG
Rosette assays with:
EAC3b
EAC3d
IgG-EA
Sheep E"
Human T - cells
Cytoplasmic Staining tor:
IgG
K
A
Lysozyme
Immunophagocytosis ot:
C3b-coated E
IgG-coated E
Enzyme cytochemical staining
Naphthol chloracetate
esterase
Peroxidase
Acid a-naphthyl acetate
esterase
Alkaline phosphatase
Epstein-Barr virus specitic antigens
EBNA
EA
VCA
EBV receptors
L428cells
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Pos. (60%-80%)
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Pos. (90%)
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Pos.
Neg.
Neg.
Neg.
Neg.
Pos.
Heterotransplantation in nude mice
Primary material
Intracranial
Cultured cells
Intracranial
Subcutaneous (suspension)
Subcutaneous (plasma dot)
2/2
5/6
0/1
2/2
reactions for acid phophatase and acid esterase
were found positive in two celllines.
The L 428 and L 439 cells did not exhibit
EBV-specific antigens (EBNA, VCA, EA).
EBV -receptors could be demonstrated on the
L 428 cells.
Cytogenetic evaluation revealed various
structural and numerical chromosome aberra-
L439cells
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Pos. (70%-95%)
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Pos. (90%)
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Pos.
Neg.
Neg.
Neg.
Neg.
Not tested
Not tested
4/4
0/3
Not tested
Table 1. Characteristics of the
Hodgkin cell lines L 428 and
L439
a Untreated and neuraminidase-treated sheep erythrocytes
231

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L428
L439
Table 2. Growth characteri-
stics of cell lines L 428 and
L 439 in vitro
Cell size
(mean diameter)
Growth pattern
15-80 j..lm
40-60 j..lm
Doubling time
Max. concentration
(cells/ml)
Single cells
42-46 H
1.56 x 106
tions in both lines. In the L 428 line the total
number of chromosomes per cell amounted to
48-50. The monodonal origin was ascertained
by identifying several identical marker chro-
mosomes: 1 p +, 2 P + , 6 q +, 7 q +, 9 P +, 11
q-, 13 p+, and 21 q-. Each metaphase
evaluated showed an extra chromosome No.
12 and lacked one chromosome 13. Some cells
exhibited a 14 q + marker.
In the L 439 line likewise a variety of
identical marker chromosomes could be shown
which indicated its monodonal origin: 1 p + ,
1 q-, 1 p-, 2 q+, 10p-, 15 p+, 18 q+, and 21
q -. In addition an extra chromosome No. 5 and
No. 12 was constantly detected.
Intracranial heterotransplantation of L 428
and L 439 cells into nude mice resulted in
tumor formation. Intracerebral tumor growth
could be histologically confined (Fig. 2).
After subcutaneous inoculation both lines
failed to induce tumors. When the L 428 cells
(1 x 106) were embedded in a fibrin dot prior
to transplantation, infiltrative tumor growth
could be induced subcutaneously (Fig. 3).
232
Single cells with
small clumps
38-72 H
0.72 X 106
Detailed growth characteristics are shown in
Table 2.
E. Discussion
The L 428 line as well as the L 439 line have
been obtained from the pleural effusion of two
patients with histologically proven Hodgkin's
disease. The monodonal origin of the cul-
ture cells was demonstrated by identical mar-
ker chromosomes in each metaphase, de-
spite the morphologie variations among indivi-
dual cells. An additional argument for the
malignant nature of both lines is the lack of
EBV specific antigens, since hitherto EBV -ne-
gative lymphoid cell lines of a nonneoplastic
nature have not been described.
The tumorigenicity of L 428 and L 439 cells
after intracranial heterotransplantation in
nude mice does not provide proof of the
neoplastic nature, since this is a common
feature of LCL and lymphoma lines (Schaadt
et al. 1979). Tumor formation after subcutane-
Fig. 2. Intracerebral tumor-
growth of the celliine L 428.
Giemsa. X 244

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Fig. 3. Tumor grawth of L 428 cells after transplantation in a fibrin eIot into the subcutaneous tissue. Giemsa
x32
ous transplantation, which seems to be more
strongly correlated to other markers of malig-
nancy (Diehl et al. 1977), did not occur after
inoculation of a cell suspension from the cell
lines but did occur after embedding of L 428
cells in a fibrin eIot before subcutaneous
transplantation.
The histologie seetions of the mouse tumors
derived from the L 428 and the L 439 lines
showed eell individuals representing a mor-
phology very similar to that of "Sternberg-
Reed" and "Hodgkin" cells. The immunologie
and biologie eharacterization of both Hodgkin
lines revealed the identical pattern observed in
freshly obtained Hand SR cells (Papadimit-
riou et al. 1978): they laek SIgG, HTLA, and
receptors for C3b, C3d, IgFc, mouse E, and
sheep E; are devoid of lysozyme, peroxidase,
and chloracetate esterase ; and they express
Ia-like antigens and reeeptors for T cells and
contain acid phosphatase and acid esterase.
The origin of the cultured cells thus far is still
obscure. These eells, however resemble very
eIosely features of eultured Hodgkin cells
described by Kaplan et al. (personal communi-
cation).
Acknowledgments
The skilful tcchnical assistence of Ms. Anke Gleser,
Mrs. Elvira Lux, Mrs. Cristina Boie, and Ms.
Gabriele Seifert is gratefully acknowledged.
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