(CANCER
RESEARCH
50. 6364-6370.
October 1. 1990]
Differential Spectrum of Expression of Neural Cell Adhesion Molecule Isoforms
and LI Adhesion Molecules on Human Neuroectodermal Tumors1
Dominique F. Figarella-Branger,
Pascale L. Durbec, and GenevièveN. Rougon2
URA 202 CNRS, 1CB, 3, Place V. Hugo. Marseille 13331 ¡P.L, D.. G. N. R.J, and Laboratoire Anatomie Pathologique et Neuropathologie. Hôpitalde la Timone. 27,
BdJ. Moulin. Marseille I338S [D. F. F-B.j. France
ABSTRACT
A series of four medulloblastomas, seven neuroblastomas (Nb), two
ependymomas, and three gliomas, human neuroectodermal tumors, were
screened for their expression of adhesion molecules LI, carcinoembryonic
antigen, neural cell adhesion molecule isoforms (N-CAM) and HNK1
epitope by Western blotting and double immunofluorescence labeling.
All seven neuroblastomas, whatever their differentiated state, ex
pressed LI, a neural cell surface developmental antigen, whereas all other
tumors tested were negative. All tumors expressed N-CAM; however, a
large diversity was observed among the ¡soforms.Low sialylated N-CAM
140 was present, with different intensity, in ependymomas and astrocytomas. High sialylated isoforms were detected by a monoclonal antibody
(anti-MenB) specifically recognizing high polymers of o2-8 linked neuraminic acid. They were expressed in all medulloblastomas studied (4 of
4), and in 4 of 7 Nbs examined. Negative cases corresponded to tumors
having undergone chemotherapeutic treatment or to ganglioneuroma. The
interconversion from high to low sialylated forms might reflect changes
which are critical for the conversion of Nbs into benign ganglioneuromas.
HNK1 epitope was expressed on a large diversity of molecules by nearly
all tumors except two Nbs which were also negative with anti-MenB
antibody. This simultaneous loss of carbohydrate epitopes could correlate
with higher maturation states of the tumors. None of the tumors ex
pressed carcinoembryonic antigen. Therefore, anti-Li and anti-MenB
antibodies define differentiation-related antigens that could differentiate
between Nbs and other tumors and may prove helpful in diagnosis and
understanding of the biological nature of neuroectodermal tumors.
An immunodot assay was set up and allowed to titrate the presence of
polysialic acid units in cerebrospinal fluid from patients presenting meningeal spread of medulloblastomas. It could help to assess metastasis
and to monitor the effects of chemotherapeutic treatment on polysialylated N-CAM positive tumors.
in glycosylation which are differently expressed depending on
the cell type and differentiation stage of the tissue considered
(9, 10). For example, N-CAM has been found to be expressed
in embryonic tissues under a polysialylated form (11, 12).
Moreover a variable percentage of these immunoglobulin su
perfamily adhesive molecules shared the carbohydrate epitope
HNK1. (13). Carbohydrate moieties had been shown to play an
active role in modulating adhesion (14, 15).
A considerable body of data shows that neuroectodermal
tumors can be distinguished from each other and from other
types of malignancies according to their antigenic profiles (16,
17); however, the biochemical structure and the function of
molecules considered as immunological markers is rarely
known. We postulated that the description of a pattern of
expression of developmentally regulated adhesive molecules by
tumors could represent an alternative approach in defining
markers; in particular, because tumors frequently express anti
gens found at a precise stage of development of the tissue from
which they are derived ( 18). Such markers should be informative
for phenotypic typing and definition of differentiation stage of
a given neuroectodermal tumor. This could be particularly
interesting for Nbs which are known to differentiate toward
mature neuronal phenotype (19) either spontaneously or follow
ing chemotherapeutic treatment and for Mbs which could
evolve toward either a glial or a neuronal phenotype (20). Mbs
frequently become metastatic via CSF, consequently monitor
ing the presence of metastasis in this fluid should help both
diagnosis and evaluation of chemotherapeutic treatment.
MATERIALS
INTRODUCTION
AND METHODS
Human Neuroepithelial Tumors
Adhesiveness is clearly one central property for tumor behav
ior and in particular for metastatic process (for review see Refs.
1-3). This has been particularly well investigated for groups of
tumors such as melanomas (4). However, little is known con
cerning other neuroectodermal tumors. In this report we de
scribe the expression of adhesion molecules of the Immunoglobulin superfamily on a tumor panel including Nb,1 Mb,
astrocytomas, and ependymomas. This family of cell surface
molecules is involved in basic cell surface recognition events
and many of its members have been reported to be specifically
expressed in normal brain tissues (for review see Ref. 5). Their
expression is developmentally regulated, and for some of them
as LI (6, 7) or the M, 180,000 isoform of N-CAM (8), neuron
specific. Their molecular diversity is due in part to differences
Received 2/12/90; accepted 7/5/90.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grants ARC N'6895 and AFM to G. N. R.,
Centre National de Recherche Scientifique, and University of Provence.
3To whom requests for reprints should be addressed. Present address: URA
179 CNRS, Facultédes sciences Luminy, Case 901. Marseille 13288, France.
1The abbreviations used are: Nb, neuroblastoma; Mb, medulloblastoma; NCAM. neural cell adhesion molecule; CSF. cerebrospinal fluid; NSE, neuron
specific enolase; GFAP, glial fibrillary acid protein; mAb, monoclonal antibody:
CEA, carcinoembryonic antigen; PBS. phosphate buffered saline.
Eighteen surgically removed neuroepithelial tumors were diagnosed
and studied.
Of 4 Mbs (Mb 1-4), histológica!examination showed one medullomyoblastoma (Mb 1), a poorly differentiated Mb with no HomerWright rosette (Mb 2), and two classic Mbs (Mb 3 and 4). Immunocytological studies were performed with NSE, neurofilament, vimentin,
desmin, and GFAP antibodies. Results are summarized in Table 1.
Of 7 Nbs (Nb 1-7), histological examination showed a mature
ganglioneuroblastoma (Nb 1), four poorly differentiated Nbs (Nb 2-5),
and two necrotic ganglioneuroblastomas (Nbs 6 and 7). In 3 cases (Nb
2-4) surgery preceded chemotherapy and radiotherapy. Staging was as
follows: Nb 3, stage II; Nbs 1, 2, and 4, stage III; Nbs 5-7, stage IV.
Two ependymomas originated from the fourth ventricle of young
children.
Four gliomas were malignant astrocytomas at grade III or IV. Benign
infiltrating gliomas exhibiting mature neurons were excluded from our
samples.
A normal frontal area from human normal adult brain was taken as
control.
Antibodies
Rabbit polyclonal anti-NSE and anti-GFAP antibodies were from
Ortho Diagnostic Systems (Roissy, France), mouse mAb anti-NF 70,
M, 160,000 and 210,000 isoforms (clone DP5-43-12); mouse mAb antidesmin (clone D300) and mouse mAb anti-CEA (clone 35B) were from
6364
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1990 American Association for Cancer Research.
ADHESION MOLECULES ON BRAIN TUMORS
Table 1 Expression ofNSE, neurofilament, vimentin, desmin. and GFAP in Mbs
RESULTS
Antigen
Mb
NSE
Neurofilament
Vimentin
Desmin
GFAP
2
3
4
" Very few positive cells were observed.
Immunotech (Marseilles, France). Mouse mAb anti-vimentin (clone
V9) was from DakoPatts (Versailles, France). Anti-Li polyclonal an
tibody was a kind gift from F. Rathjen (Hamburg, Federal Republic of
Germany). Rabbit site directed anti-N-CAM polyclonal antibody rec
ognizing NH2 terminal domains of all N-CAM ¡soforms(21); P61 (22),
a rat mAb recognizing the transmembranar N-CAM isoforms, and antiMenB (12), a mouse mAb recognizing the highly sialylated N-CAM
isoforms, were prepared in our laboratory. HNK1 (23), a mouse mAb,
was from the American Type Culture Collection (Rockville, MD).
Affinity purified anti-isotype antibodies were rabbit anti-mouse IgM
purchased from Immunotech; goat anti-rat and goat anti-rabbit immunoglobulin antisera were prepared in our laboratory.
Tissue Extracts and Immunoblot Detection
Tissue extracts were prepared as described previously (9). Briefly
tissues were homogenized in 10 mM Tris-HCI, pH 7.8, containing 2%
(w/v) Nonidet P-40, 1 mM EDTA, 10 MMleupeptin, l /IM a2-macroglobulin, and 0.1 mM phenylmethylsulfonyl chloride as protease inhib
itors. In some instances tissues were delipidated by treatment with
CHClj/methanol (2/1, v/v). The homogenates were centrifuged at
100,000 x g for l h at 4°C.The supernatants were collected and protein
concentration was determined (24). The protein concentration was
adjusted to 10 mg/ml; samples were boiled for 3 min in reducing
electrophoresis buffer. Aliquots containing equivalent amounts (250
Mg) of proteins were then resolved on 7.5% sodium dodecyl sulfatecontaining polyacrylamide gels. Proteins were transferred to nitrocel
lulose sheets (Nitroscreen West, NEN, England) for 4 h at 0.5 Amper.
After saturation for 2 h in PBS containing 3% (w/v) defatted milk, the
sheets were reacted with primary antibodies (1:1000 dilution for antiLi, anti-N-CAM polyclonal antibodies and anti-MenB. anti-HNKl
mAb ascites fluids). Rat mAb P61 was hybridoma culture supernatant,
used 1:2 diluted. Anti-CEA mouse mAb was used at 1Mg/ml. Incubation
was conducted for 20 h at 4°Cunder shaking. Afterwards, rinsed blots
were incubated with the appropriate immunopurified secondary anti
bodies (2 Mg/ml) for 4 h at 24°C.Bound antibodies were revealed by
reaction with iodinated protein A (0.5 x IO6 cpm/ml). Sheets were
subsequently autoradiographed.
Immunodot Procedures
Two-fold step serial dilutions of CSF were made in PBS; 0.5 p\ of
each was spotted onto nitrocellulose (Schleicher and Schüll,Federal
Republic of Germany). After saturation with PBS-milk, the sheets were
reacted with anti-MenB mAb and anti-N-CAM polyclonal antibody
and treated as described above for immunoblots.
Immunocytochemical Procedures
Immunoperoxidase. Five-Mm serial paraffin sections were cut from
Mbs. NSE, neurofilament, GFAP, desmin, and vimentin antigens were
visualized by the peroxidase-antiperoxidase technique (25).
Immunofluorescence. Four-Mm cryostat sections were prepared from
4 Nbs (Nbs 2-5), 2 Mbs (Mbs 2 and 3), and 2 gliomas. They were
incubated with a mixture of rabbit anti-N-CAM polyclonal antibody
(1:500 dilution) and mouse IgM anti-MenB antibody (1:1000 dilution)
for 12 h at 4°C.Bound antibodies were revealed by incubation with
fluorescein labeled goat anti-rabbit F(ab)2 antibodies and tetramethylrhodamine labeled goat anti-mouse F(ab)2 antibodies diluted 1:50.
Staining was also performed on serial sections with rabbit anti-Li and
anti-N-CAM polyclonal antibodies diluted 1:500. Sections were ex
amined under a Zeiss epifluorescence microscope equipped with appro
priate filters.
Immunoblot Detection of LI Molecules on Brain Tumors. In
normal tissues, LI cell surface glycoprotein has been detected
on developing postmitotic and mature neurons. It plays a role
in neurite fasciculation and is believed to be absent from astrocytes (6, 7). LI, in vertebrate brain tissue, showed either a single
band or a doublet band at M, 200,000 and, on some occasions,
additional bands at M, 140,000 and 80,000 (26). We investi
gated its expression on tissue extracts from adult normal human
brain and from neuroectodermal tumors. Results are shown in
Fig. 1. Strikingly, the expression of this molecule seemed to be
specific to Nbs. Mbs as well as ependymomas and gliomas were
consistently negative. The molecular weights observed were
similar to the ones of LI molecules in normal brain.
Immunoblot Detection of N-CAM Isoforms on Brain Tumors.
N-CAM is the name given to a class of plasma membrane
sialoglycoproteins that mediate cell-cell contacts or cell-sub
strate interactions. The adhesive properties of N-CAM are
modulated by the differential expression of isoforms in highly
regulated pattern (27). N-CAM isoform diversity is generated
by both alternative splicing (28) and a variety of posttranslational modifications including glycosylation (9, 10, 13). NCAM from embryonic tissues migrate in sodium dodecyl sulfate
gels as a broad band above M, \ 80,000 whereas N-CAM from
adult tissues shows three prominent proteins with apparent
molecular weights of 180,000, 140,000, and 120,000 (hereafter
called N-CAM 180,140, and 120). The two largest polypeptides
span the membrane. We took advantage of the availability of
antibodies recognizing various isoforms to monitor their
expression in our panel of neuroectodermal tumors. The same
extracts as above were reacted with a site directed rabbit poly
clonal antibody recognizing N-CAM NH2 terminal domains
which was able to reveal all N-CAM isoforms inasmuch as they
share identical NH: sequences (21). N-CAM was expressed by
every tumor tested with great heterogeneity in the variety of
isoforms expressed (Fig. 2). Ependymomas and gliomas ex
pressed only N-CAM 140; it is noteworthy that N-CAM 120
was never detected in such tumors. Normal brain displayed NCAM 180, N-CAM 140, and N-CAM 120.
A mAb P61, recognizing an intracytoplasmic epitope shared
by all transmembrane isoforms, was also used (22). Results
obtained for gliomas and ependymomas, confirmed that the M,
140,000 band revealed by anti-N-CAM polyclonal is a transmembrane isoform (not shown).
The most striking patterns observed with polyclonal anti-NCAM on Mbs and Nbs were fuzzy large bands, migrating above
M, 200,000; this migration is usually associated with highly
sialylated forms of N-CAM or with lipids tightly associated
with the molecule and perturbating gel separation. In addition,
in some cases (Mbs 2 and 4 for instance), bands at M, 140,000
and 120,000 were also easily discernible. In some instances
delipidation of tissues prior to membrane preparation removed
the fuzziness of the band. This is shown for Nb 1 (Fig. 2, Lane
1') for which bands at M, 180,000, 140,000, and 120,000
become easily discernible. By contrast, Nb 2 exhibited the same
profile after this treatment (Fig. 2, Lane 2').
Immunoblot Detection of Polysialylated N-CAM on Brain
Tumors. We used anti-MenB, a mAb directed against W-acetylneuraminic acid («2-8) polymers consisting of at least eight
residues and known to react only with highly sialylated isoforms
(12), to probe the glycosylation state of N-CAM expressed by
tumors.
As expected from results obtained with the site directed
6365
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1990 American Association for Cancer Research.
ADHESION
MOLECULES ON BRAIN TUMORS
S
Mr xlO
N
200
^
66
^
42
^
1
2
3
1
1
1
Fig. 1. Western blotting analysis of LI molecules. Membrane extracts from various tumors and normal brain tissue (A') were applied (250 *ig/lane), under reducing
conditions, to a ~' polyacrylamidegel and then electroblotted and incubated with anti-LI polyclonal antiscrum at 1:1000 dilution. The bound antibody was visualized
by incubation with I25l-labeled protein A. No bands were detected by incubation with protein A alone (not shown).
/
MrxUr
200
».
66
^
42
^
•
1
3
*
1
1
2
2'
3
4
5
6
7
12
1234
Fig. 2. Western blotting analysis of overall N-CAM isoforms. Same extracts as in Fig. 1 were analyzed for their expression of N-CAM, Lanes /' and 2' were,
respectively. Nb l und Nb 2 after the delipidation procedure; a rabbit polyclonal recognizing the \H •
terminal domain common to all the N-CAM was used at 1:1000
dilution. Procedure was the same as in Fig. 1. Arrows, position of N-CAM 180. 140. and 120 in normal adult brain (N). Positive bands observed in Mb 2 and NB 1'
are likely degrádame products from N-CAM.
6366
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1990 American Association for Cancer Research.
ADHESION MOLECULES ON BRAIN TUMORS
*~
MrxlO
fp
200
42
1
1
1
Fig. 3. Western blot analysis of polysialylated N-CAM isoforms. Membrane extracts were processed as in Fig. 1 and probed for their content in polysialic acid
polymers using anti-MenB mouse IgM mAb. Note that normal adult brain (A') was negative. Nylon sheets were first incubated with immunopurified
rabbit antimouse IgM prior to application
of protein A.
MrxlO"3
200
66
N
^
^
1
3
4
1
2
3
1
1
Fig. 4. Western blotting analysis of HNK1 positive molecules. Membrane extracts were processed as in Fig. I and reacted with a mouse IgM mAb recognizing
HNK1, a carbohydrate epitope shared by various adhesion molecules. For revelation the same procedure was used as in Fig. 3. Arrows, position of high molecular
weight positive molecules in Mb«,of M, 140.000 positive molecule in ependy momas, and of M, 90.000 molecule in normal adult brain (N).
6367
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1990 American Association for Cancer Research.
ADHESION
MOLECULES ON BRAIN TUMORS
Fig. 5. Double immunofluorescence staining of cryostat tumor sections. Mb sections labeled with anti-N-CAM rabbit polyclonal antibody I I) and mouse antiMenB mAb (B) (final dilution. 1:1000 for each). Nb serials sections labeled with anti-Li polyclonal antibody (C) and anti-N-CAM polyclonal antibody (D) (final
dilution 1:1000 for each). Incubation was conducted for 24 h at 4 ( . Anti-MenB was revealed with anti-isotype conjugated to rhodamin, anti-Li, and anti-N-CAM
antibodies with anti-isotype antibody conjugated to fluorescein.
polyclonal antibody, N-CAM expressed by ependymomas and dymomas for example in which a band at M, 140,000 was
gliomas as well as by normal adult brain was not highly sialy- visible). However, other strong bands with molecular weight
around 200,000-250,000 (Mb 4, gliomas 2 and 4) and M,
lated (Fig. 3). By contrast, all Mbs showed high sialylation state
of N-CAM. Interestingly, although most Nbs also showed
90,000 (N) were also seen. According to its molecular weight,
highly sialylated N-CAM, three of them (Nbs 1, 6, and 7) did the M, 90,000 band is likely to correspond to myelin associated
not. This is also in agreement with the patterns observed in glycoprotein which is known to be expressed by neurons (13).
Fig. 2 for N-CAM after delipidation of tissues. They corre
On glioma cells, the dispersed high molecular weight band
sponded (Nbs 6 and 7) to Nbs whose patients had received
could be cytotactin, a cell substrate adhesion molecule shown
chemotherapeutic treatment prior to surgery. Thus, anti-MenB
to be synthesized by glial cells (29).
CEA Detection by Immunoblot. In searching for expression
antibody is able to demonstrate a heterogeneity of glycosylation
among N-CAM expressed by Nbs.
of potential adhesion molecules, the CEA antigen, another
HNK1 Detectin by Immunoblot. Polysialic acids are not the membrane of the immunoglobulin superfamily (30), had shown
only glycosylated epitopes borne by N-CAM. It is well known
consistently negative results in all the tumors examined (not
that in normal tissues a variable percentage of N-CAM mole
shown).
cules share with other adhesion molecules such as LI and
Immunohistochemical Analysis. In immunoblot experiments,
in some instances, mature forms of N-CAM appeared super
myelin associated glycoprotein the HNK1 epitope (13).
We examined the expression of this epitope in normal as imposed with highly sialylated isoforms. Thus it was interesting
well as tumor tissues (Fig. 4). It was present in all Mbs as well to monitor at the histológica! level whether this corresponded
as ependymomas and gliomas tested; the patterns were very to heterogeneous cell populations with cells expressing only the
heterogeneous even in a given group of tumors. Here again a low sialylated isoforms, or whether cells always coexpressed
heterogeneity was seen among Nbs, because two (Nbs 1 and 6) both forms.
Double immunofluorescence labeling using anti-MenB mAb
were negative and some showed weak immunoreactivity. It is
very likely that a portion of the positive molecules corresponded
and site directed polyclonal antibodies were used. A typical
to N-CAM based on their position of migration of gels (epen
result is shown in Fig. 5 (A and B) for a Mb. All the cells
6368
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1990 American Association for Cancer Research.
ADHESION
MOLECULES ON BRAIN TUMORS
DILUTION
'•"
.1:2000
A
•
»
B
•
•
C
•
•
D
I
C
•
D
*
B
Fig. 6. Immunodol analysis of polysialylated N-CAM in CSF. Two-fold step
serial dilutions (0.5 «Dof CSF from patients presenting Mb metastasis (I, /!} or
in a remissive phase (C) and from normal subjects (D, E, F) were spotted on
nitrocellulose using an Hamilton syringe. After incubation with anti-Menu anti
body (A) or with anti-N-CAM polyclonal antibody (A), the revelation procedure
was conducted as for immunoblots.
appeared labeled with anti-MenB, not revealing heterogeneity
of N-CAM positive cells in the tumor. It is most likely that the
same cells could coexpress both mature and highly sialylated
isoforms.
We also showed that LI immunoreactivity was superimposed
with N-CAM immunoreactivity and expressed by all the cells
in the Nbs examined by immunofluorescence labeling of serial
sections [Fig. 5 (C and D)].
Immunodot Detection of Polysialylated Molecules in CSF. As
an alternative to cytological analysis of CSF to detect the
presence of metastatic cells, we set up an immunodot assay.
Preliminary data (Fig. 6) conducted with anti-MenB mAb (Fig.
6A) and polyclonal anti N-CAM antibody (Fig. 6B) on CSF
from normal subjects and patients exhibiting metastasis are
shown. The metastasis was assessed by the presence of Mb cells
in CSF examined after centrifugation and staining with MayGrunwald-Giemsa reagent. Experiments have been conducted
as described in "Materials and Methods" on serial dilutions of
CSF. Normal subjects (Fig. 6, Lanes D, E, F) exhibited only
very weak reactivity that was lost at dilution higher than 1:8 of
the sample. Patients diagnosed with acute Mb metastasis (Fig.
6, Lanes A, B) showed high reactivities inasmuch as a positivity
was still detectable at a dilution of 1:2048. In agreement with
diagnostic data, a patient in a remission phase (Fig. 6C) shows
much lower reactivity. It is noteworthy that anti-MenB and
anti-N-CAM immunoreactivity correlates for every sample
tested; this is in agreement with the fact that polysialylated
molecules detected are indeed N-CAM.
DISCUSSION
Characteristic features of tumors are (a) the presence of
structural elements resembling those found during embryonic
development of the tissue from which they originated (18) and
(b) the role played by adhesive properties in metastatic process.
These observations prompted us to investigate neuroectodermally derived tumors for the expression of LI and N-CAM
that are neural cell developmentally regulated adhesion mole
cules. Although expression of N-CAM on such tumors has
already been reported (31), information concerning the nature
of isoforms expressed was missing. The availability of probes
allowing discernment between the various isoforms, and in
particular anti-polysialic acid polymer antibody, was instrumen
tal in this study.
Of particular interest was that LI was found expressed only
by the Nbs examined. The consistent pattern of expression of
this antigen, known to appear later than N-CAM in develop
ment (6), is in agreement with the fact that Nbs originate from
neuronal cells. These cells are presumably neuroblasts at a
unique state of normal differentiation, when cells are particu
larly susceptible to malignant transformation. Conceivably, the
stem cell phenotype, including cell surface antigen pattern, is
retained by the tumor cells. The recent demonstration that LI
and N-CAM are engaged in a close functional association
within the cell surface (15, 32) might result in particular adhe
sive behavior of LI/N-CAM positive tumors.
We found that N-CAM was expressed by every tumor tested,
in agreement with their neuroectodermal origin. Immunoblot
examination with antibodies of various specificities showed that
ependymomas and gliomas expressed only N-CAM 140. Survey
of more cases is necessary to ascertain that this is a common
feature of such tumors. Interstingly, N-CAM 120, known to
occur later in development (33), was never detected. For Mbs,
N-CAM patterns were more complex. Bands migrating at M,
140,000 and 120,000 were clearly discernible (Mb 2), together
with higher molecular weight fuzzy bands. We cannot decide
whether this is attributable to astrocytic or neuronal differen
tiation. However, in every case, GFAP immunoreactivity was
either absent or expressed in very few cells.
Polysialylation occurred in every Mb tested; this result led us
to investigate the presence of polysialylated N-CAM in the CSF
from patients suffering from CSF spreading of Mbs. We set up
an immunodot assay requiring only 10 n\ of CSF. Preliminary
data showed that CSF metastasis of Mbs was correlated with
high titers in polysialylated N-CAM, whereas CSF from control
subjects or from a patient in a remissive phase gave much lower
titers. Larger systematic assays should determine whether this
quantitation of polysialylated N-CAM could constitute a test
to detect metastasis at early stages and also monitor the effect
of chemotherapeutic treatment. More generally it can also be
useful for assessing the malignant potential and the aggressive
ness of Mb growth and help clinical staging and prognosis.
MenB immunoreactivity was also frequently exhibited by Nbs.
This finding corroborates with the data (31, 34) showing that
human Nb cell lines IMR32 and CHP-134 express extended
polysialic acid chains. However, three of the Nbs tested were
negative with anti-MenB antibody and expressed mature forms
of N-CAM. They corresponded to patients having received
chemotherapeutic treatment. Nbs 6 and 7 showed necrotic areas
and large ganglionic cells. Nbl showed a histológica! pattern
of ganglioneuroma. Conceivably, the embryonic toward mature
N-CAM form interconversion reflects cellular changes critical
for the conversion of some Nbs into benign ganglioneuromas.
This was also correlated with the loss of the HNK1 epitope
expression, therefore with other changes affecting adhesion
molecules. Interestingly, in normal brain, this epitope was
6369
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1990 American Association for Cancer Research.
ADHESION
MOLECULES ON BRAIN TUMORS
reported to be more heavily expressed at early stages of devel
opment (13).
Expression of highly sialylated forms of N-CAM had also
been reported on tumors of presumed neuroectodermal origin
such as Ewing's (35) and small cell lung cancers (36). Its
expression on Wilm's tumor is more enigmatic (37).
It is particularly interesting that polysialic units are part of a
primary cell adhesion molecule of the immunoglobulin superfamily (28), that is proposed to have a regulatory role in cell
growth and differentiation (38). Changes in cell surface carbo
hydrates accompanying malignant transformation have been
implicated in the altered growth and behavior of tumor cells.
In many instances, carbohydrate moieties that were expressed
during certain stages of embryonic development, but suppressed
later on were found to be reexpressed in tumors. However, in
very few cases could this be correlated with a specific role of
the carbohydrate in cell growth and differentiation. We have
shown that, in Mbs and Nbs, the highly sialylated form of NCAM that is transiently and regionally expressed during nerv
ous system development and nearly absent from mature brain
(39) and other tissues is present. Further experiments must be
aimed at clarifying its possible significance in tumor growth
and its usefulness as a tumor marker.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
ACKNOWLEDGMENTS
We thank Dr. F. Rathjen for his kind gift of the anti-Li antibody,
Dr. C. Scheiner for providing neuroblastoma frozen samples, and Dr.
D. Gambarelli for a medulloblastoma specimen. N. Bianco provided
expert technical assistance. We thank Professor J. Marvaldi and Pro
fessor J. P. Pellissier for constant support and Dr. J. Barbet for critical
comments on the manuscript.
25.
26.
27.
28.
REFERENCES
1. Hart, I. R. Immune profile in metastasis. Curr. Opin. Imm.mol,. 1:900-903.
1989.
2. Berg. F. L., Goldstein. L. A.. Jutila. M. A.. Nakache. M.. Picker. L. J., and
Streeter, P. R. Homing receptors and vascular addressins: cell adhesion
molecules that direct lymphocyte traffic. Immunol. Rev., 108: 5-18, 1989.
3. Roos, E. Cellular adhesion, invasion and metastasis. Biochim. Biophys. Acta.
738: 263-284, 1984.
4. Natali, P., Nicotra. M. R., Cavaliere, R., Bigotti. A.. Romano, G., Temponi.
M.. and Ferrone, S. Differential expression of intercellular adhesion molecule
1 in primary and metastatic melanoma lesions. Cancer Res.. 50:1271-1278,
1990.
5. Williams, A., and Barclay. A. N. The immunoglobulin superfamily, domains
for cell surface recognition. Annu. Rev. Immunol., 6: 381-405, 1988.
6. Rathjen. N.. and Schachner, M. Immunological and biochemical character
ization of LI antigen which is involved in cell adhesion. EMBO J., 3: 1-10,
1984.
7. Wolff, J. M., Rainer, F., Mujoo, K.. Spiro. R., Reifeid. R., and Rathjen. F.
A human brain glycoprotein related to the mouse cell adhesion molecule LI.
J. Biol. Chem.. 263: 11943-11947, 1988.
8. Schlosshauer. B. Purification of neuronal cell surface proteins and generation
of epitope-specific monoclonal antibodies against cell adhesion molecules. J.
Neurochem.. 52: 82-92. 1989.
9. Rougon, G., Deagostini-Bazin. H.. Hirn. M., and Goridis. C. Tissue and
developmental stage specific forms of neural cell surface antigen linked to
differences in glycosylation of a common polypeptide. EMBO J., /: 12391244, 1982.
10. Rothbard. J. B.. Brackenbury. R.. Cunningham, B.. and Edelman. G. Differ
ences in the carbohydrate structures of neural cell adhesion molecules from
adult and embryonic chicken brains. J. Biol. Chem.. 257: 11064-11069,
1982.
11. Finne, J., Finne, U., Bazin-Deagostini, H., and Gioridis, C. Occurrence of
«2-8linked polysialosyl units in a neural cell adhesion molecule. Biochem.
Biophys. Res. Commun., 112: 482-487. 1983.
12. Rougon. G., Dubois, C., Buckley, N., Magnani, J., and Zollinger, W. A
monoclonal antibody against Meningococcus group B polysaccharides distin
guishes embryonic from adult N-CAM. J. Cell Biol., 103: 2429-2437. 1986.
13. Kruse. J., Mailhammer. R.. W'ernecke. M., Faissner, A., Sommer. I.. Goridis,
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
C.. and Schachner, M. Neural cell adhesion molecules and myelin associated
glycoproteins share a common carbohydrate moiety recognized by monoclo
nal antibodies L2 and HNK1. Nature (Lond.), 311: 153-155, 1984.
Sadoul, R.. Hirn. M., Deagostini-Bazin, G.. Rougon. G., and Goridis, C.
Adult and embryonic mouse neural cell adhesion molecules have different
binding properties. Nature (Lond)., 304: 347-349, 1983.
Kadmon, G., Kovvitz, A., Alterogt. P.. and Schachner, M. Functional coop
eration between the neural adhesion molecules LI and N-CAM is carbohy
drate dependent. J. Cell Biol., HO: 209-218. 1990.
Kemshead. J. T.. Goldman, A.. Fritshy, J., Malpas. J. S.. and Pritchard, J.
Use of panels of monoclonal antibodies in the differencial diagnosis of
neuroblastoma and lymphoblastic disorders. Lancet. /: 12-15, 1983.
Helson. L., Weinberger, C., Helson, C., and Lieberman. P. Neuroectodermal
tumor and monoclonal antibodies. Ann. J. Pediatr. Hematol. Oncol., 6:227231, 1984.
Kennedy, P. G. E. Neural cell markers and their applications to neurology.
J. Neuroimmunol., 2: 35-53. 1982.
Russell. D. S., and Rubinstein. L. J. Medullo blastomas (including cerebellar
neuroblastoma). In: Pathology of Tumors of the Nervous System, pp. 251279. London: Edward Arnold Publishers (Ltd.). 1989.
Burger. P. C.. Grahmann. F. C.. Bliestle. A., and Kleihues, P. Differentiation
into the medulloblastoma: a histological and immunological study. Acta
Neuropathol., 73: 115-123, 1987.
Rougon. G., and Marshak, D. Structural and immunological characterization
of the amino terminal domain of mammalian neural cell adhesion molecules.
J. Biol. Chem., 265: 3396-3401, 1986.
Gennarini, G., Rougon, G., Deagostini-Bazin. H., Hirn, M., and Goridis, C.
Studies on the transmembrane disposition of the neural cell adhesion mole
cules N-CAM; a monoclonal antibody recoginizing a cytoplasmic domain
and evidence for the presence of phosphoserine residues. Eur. J. Biochem..
142:57-64. 1984.
Abo. T.. and Balch, C. M. A differentiation antigen on human NK and K
cells identified by a monoclonal HNK-1 antibody. J. Immunol., 27: 10241028. 1981.
Dulley. J. R.. and Grieves. P. A. A simple technique for eliminating interfer
ence by detergent in the Lowry method of protein determination. Anal.
Biochem., 64: 136-141. 1975.
Hsu, S. M., Raine. L., and Fanger. H. Use of avidin biotin peroxidase
complex (ABC) in immunoperoxidase techniques. A comparison between
ABC and unlabelled antibody PAP procedures. J. Histochem. Cytochem. 29:
577-580. 1981.
Sadoul, K., Sadoul. R., Faisner, A., and Schachner, M. Biochemical charac
terization of different molecular forms of the neural cell adhesion molecule
LI. J. Neurochem., 50: 510-521. 1988.
Edelman, G. M. Cell adhesion molecules in neural histologenesis. Annu.
Rev. Physiol., 48: 417-430. 1986.
Walsh, F. S. The N-CAM gene is a complex transcriptional unit. Neurochem.
Int.. 12: 262-267. 1988.
Grumet. M.. Hoffman, K., Crossin, B., and Edelman, G. Cytotactin, an
extracellular matrix protein of neural and non-neural tissues that mediates
glia-neuron interactions. Proc. Nati. Acad. Sci. USA, «2:8075-8079. 1985.
Beuchimol. P.. Fuks. A., Jothy. S., Beauchemin, N., Shirota. K.. and Stanners, C. Carcinoembryonic antigen, a human tumor marker, functions as an
intercellular adhesion molecule. Cell. 57: 327-334, 1989.
Lipinsky, M., Hirsch, M. R.. Deagostini-Bazin, H.. Yamada. O.. Tursz, T.,
and Goridis, C. Characterization of neural cell adhesion molecules (N-CAM)
expressed by Ewing and neuroblastoma cell lines. Int. J. Cancer. 40: 81-86,
1987.
Kadmon, G., Kowitz, A., Altevogt, P., and Schachner, M. The neural cell
adhesion molecule N-CAM enhances LI-dependent cell-cell interactions. J.
Cell Biol., 110: 193-208. 1990.
Gennarini, G., Hirsch, M. R.. Hé,H. T., Hirn. M., Finne, J., and Goridis,
C. Differential expression of mouse neural cell adhesion molecules mRNA
species during brain development in neural cell lines. J. Neurosci., 6: 19831990, 1986.
Livingston, B.. Jacobs, J., Click, M. C., and Troy. F. Extended polysialic
chains (n > 55) in glycoproteins from human neuroblastoma cells. J. Biol.
Chem., 263: 9443-9448, 1988.
Lipinski, M., Braham. K.. Philip. T.. Goridis. C.. Lenoir. G., and Turz, T.
Neuroectoderm-associated antigens on Ewing's sarcoma cell lines. Cancer
Res.. 47: 183-187. 1987.
Kibbelaar. R. E.. Moolenaar. C., Michalides, R., Bitter-Suerman, D., Addis.
B.. and Mooi, W. Expression of the embryonal neural cell adhesion molecule
N-CAM in lung carcinoma: diagnostic usefulness of monoclonal antibody
735 for the distinction between small cell lung cancer and non-small cell lung
cancer. J. Pathol.. 159: 23-28. 1989.
Roth. J., Zuber. C.. Wagner, P., Taatjis, D., Weisgerber. C.. Heitz, P.,
Goridis. C.. and Bitter-Suerman, D. Reexpression of polysialic acids units of
the neural cell adhesion molecule in Wilms tumor. Proc. Nati. Acad. Sci. 85:
2999-3003. 1988.
Rutishauser, U., Acheson, A., Hall, A., Mann, D.. and Sunshine. J. The
neural cell adhesion molecule (N-CAM) as a regulator of cell-cell interactions.
Science (Washington, DC), 240: 53-57, 1988.
Aaron, L., and Chesselet, M. F. Heterogeneous distribution of poly sialylated
neural cell adhesion molecule during post-natal development and in the adult:
an immunohistological study in the rat brain. Neuroscience, 28: 701-710,
1989.
6370
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1990 American Association for Cancer Research.
Differential Spectrum of Expression of Neural Cell Adhesion
Molecule Isoforms and L1 Adhesion Molecules on Human
Neuroectodermal Tumors
Dominique F. Figarella-Branger, Pascale L. Durbec and Geneviève N. Rougon
Cancer Res 1990;50:6364-6370.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/50/19/6364
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 17, 2017. © 1990 American Association for Cancer Research.