Journal of Neuro-Oncology 12: 1-11, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.
Monoclonal antibody against ependymoma-derived cell line Yosuke Mihara, Yasuhiko Matsukado, Satoshi Goto, Yukitaka Ushio, Shin-ichi Tokumitsu I and Kiyoshi Takahashi 1
Department of Neurosurgery and 1Department of Pathology, Kumamoto University Medical School, Kumamoto 860, Japan
Key words: monoclonal antibody, ependymoma, glioma cell lines, enzyme-linked immunosorbent assay Summary
Mouse myeloma cells were fused with spleen cells from mice that had been immunized with a human ependymoma derived cell line, KMS II. Hybridomas producing monoclonal antibodies (MAbs) were screened and cloned. Specificity of the antibody was determined by enzyme-linked immunosorbent assay (ELISA) and/or indirect immunofluorescence assay. One of the MAbs, designated Ep-C4 (subclass = IgG1), reacted with two cell lines derived from ependymoma but did not react with 17 cell lines derived from other types of brain tumor nor with 4 neuroblastoma cell lines or 19 cell lines derived from carcinoma, hematopoietic tumors and amnion. Indirect immunofluorescence and immuno-electron microscopy studies revealed that the antigen recognized by MAb Ep-C4 was located on cell surface membrane. The membrane antigen of KMS II cells, immunoprecipitated by MAb Ep-C4, was a protein of 81,000 dalton. The reactivity of M A b Ep-C4 was further examined using immunofluorescence and/or immunoperoxidase methods and frozen sections and short-term cultures of various types of brain tumors. No cross-reactivity with normal adult or fetal brain tissues was detected by absorption assay and immunoperoxidase staining. Our results suggest that the antigen defined by MAb Ep-C4 is specific for ependymoma cells, and different from the antigens of glioma cells or other neuroectodermal-derived cells previously described.
Since the hybridoma technique was introduced by K6hler and Milstein , many investigators have produced monoclonal antibodies (MAbs) against various human tumor-associated antigens [2-10], using a variety of tumor cell lines as immunogens. The production of MAbs against malignant gliomaassociated antigens or astrocyte differentiation antigens has been reported [6, 8-10] and these MAbs seem to be useful for the diagnosis, developmental study, and immunotherapy of brain tumors. However, little information is available on the reactivity of these MAbs with ependymoma cells. Furthermore, no MAbs against ependymoma cells have yet been described. This may be due to a lack
of available ependymoma cell lines [11, 12] in contrast to glioblastoma and anaplastic astrocytoma cell lines [12-17]. Recently, we have established a human ependymoma derived cell line, KMS II . We now report that one of the MAbs raised against KMS II cells, Ep-C4, recognizes an ependymomaassociated antigen.
Materials and methods
Ependymoma cell line KMS H A human ependymoma derived cell line, KMS II, was established from a recurrent ependymoma developed in the fourth ventricle of a 4-year-old boy
. KMS II cells were maintained in Falcon plastic flasks (25 cm 2) in a humidified atmosphere of 95% air and 5% CO2 in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin G, and 100/~g/ml streptomycin. During the experiment Mycoplasma contamination was ruled out by a fluorescent method [18[.
Other cell lines The human cell lines used in this study (Table 1) were grown in RPMI 1640 medium supplemented with 10% FCS. Human ependymoma cell line NU 2-26  was a gift from Dr. Y. Nakagawa, Department of Neurosurgery, Kyoto Prefectural University of Medicine. The oligodendroglioma cell line KG-1  was a gift from Dr. E. Miyake, Miyake Neurosurgical Clinic. The glioblastoma cell line U-251 MG [13, 14], was a gift from Dr. J. Yoshida, Nagoya University Medical School, U-118 MG [13, 14] was from Dr. Tabuchi, Saga Medical College. The medulloblastoma cell line TE-671, and glioblastoma cell lines U-105 MG and U-373 MG [13, 14] were purchased from American Type Culture Collection. Anaplastic astrocytoma cell lines KMG-2, KMG-3 and KMG-15 and glioblastoma cell lines KMG-4, 5, 8, 9, 14, 16 and 17 were established in our laboratory . A short-term culture of fibroblast FF was also prepared in our laboratory. The other cell lines used in this study were kindly provided by several laboratories at Kumamoto University Medical School.
Production of monoclonal antibodies (MAbs) BALB/c mice were immunized with 3 intraperitoneal injections of 1 x 107 KMS II cells 2 week apart. Spleen cells from the immunized mice were fused with the mouse myeloma cell line P3-X63Ag8-U (P3U1) 3 days after the last injection according to the method described previously . Hybridomas producing MAbs were screened by enzyme-linked immunosorbent assay (ELISA) (see below), and cloned 3 times by limiting dilutions.
ELISA ELISA was performed according to the method described previously  with minor modifications. Briefly, target cells (5 x 10a/well) were incubated overnight in 96-well plates. The adherent cells were fixed in 0.05% glutaraldehyde for 10 minutes at 4 ° C, and washed 3 times with phosphate buffered saline (PBS, pH7.4). To avoid non-specific binding, the fixed cells were incubated with 1% bovine serum albumin (BSA) in PBS for 30 minutes at room temperature. Culture fluid (50/xl) from hybridomas was added, and the plates were incubated for 2 hours at room temperature. After 3 washes with PBS, 50/zl of peroxidase-conjugated F(ab')2 fragment of rabbit anti-mouse immunoglobulin (Zymed Laboratories Inc., South San Francisco, CA) at a 1 : 1000 dilution was added to each well. After 2 hour incubation at room temperature, the plates were washed 5 times with PBS, and the substrate reaction using o-phenylenediamine was assessed at 492 nm in a Titertek Multiskan spectrophotometer (Flow Laboratories, McLean, VA). Binding activity of a MAb was expressed as BR, which was determined by the following formula: BR = value of specific absorbance with a MAb/ value of non-specific absorbance with an irrelevant MAb.
Indirect immunofluorescence For culture, 50/zl of cell suspension (5 x 10 6) in PBS were added to 50/zl of MAb and incubated for 30 minutes at room temperature. After washing, the cells were incubated for 30 minutes with fluorescein-conjugated goat anti-mouse immunoglobulin (Zymed Laboratories Inc.). The cells were washed and then observed under a Nikon epifluorescence microscope. Adherent cells, unfixed or fixed with various fixatives on Lab-Teck chamber slides (Miles Scientific, Naperville, IL) were also examined with selected MAbs. For histological study, frozen sections were fixed with acetone and stained.
Immunoperoxidase staining Immunoperoxidase staining for electron microscopy was according to a modified method of Louvard et al. . Cells grown on Lab-Teck chamber slides were fixed with 2% formaldehyde, 0.05% glutaraldehyde in 0.1M PBS for 30 minutes at 4°C. The cells were then washed 3 times in PBS containing 50raM NH4C1, incubated with a MAb for 1 hour at room temperature, and washed in PBS for 30 minutes. Then they were incubated with peroxidase-conjugated F(ab')2 fragment of rabbit antimouse immunoglobulin diluted 1 : 100 for 1 hour at room temperature, and washed in PBS at 4°C. They were then fixed with 1% glutaraldehyde in 0.1M cacodylate buffer (pH7.4) for 10 minutes, and washed in 0.1M Tris-Hcl (pH7.6). Detection of peroxidase was performed in DAB (0.2rag/ ml) + 0.003% H202. Then the cells were washed with PBS and postfixed in 1% OsO4 for 30 minutes. The cells were dehydrated in graded ethanol series, embedded in Epon and thin sections were cut. For light microscopy, cryostat sections of brain tumors, normal brains, and a spinal cord were fixed in cold acetone for 5 minutes, incubated with a MAb overnight at 4° C, and subsequently stained by the ABC procedure , using ABC system (Vector Laboratories Inc., Burlingame, CA).
Quantitative absorption test Cells (2 to 16 x 106) were suspended in 200/zl of a MAb. After 1 hour incubation at room temperature the cells were centrifuged, and the antibody remaining in the supernatant was estimated by ELISA using 5 x 10 4 KMS II cells as targets.
Absorption with brain homogenates A fetal brain (16-week-gestation) was obtained from a prostaglandin-induced abortion from a rubella-infected mother. Normal adult brain tissues were obtained at lobectomy for arteriovenous mal-
formations, cerebellum tissue was obtained at lobectomy for acoustic neurinomas and spinal tissue was obtained at autopsies. As a control, a KMS II-tumor grown in a nude mouse was used. These tissues were mechanically homogenized in Hanks' solution, centrifuged at 100,000g for 30 minutes, and washed 3 times with Hanks' solution. The pellets were used for the absorption assays. A MAb was mixed with various volumes of tissue homogenates and incubated for 1 hour at room temperature. After centrifugation, the antibody remaining in the supernatant was estimated by ELISA using 5 x 10 4 KMS II cells as targets.
Immunoprecipitation procedure Cell surface proteins of KMS II cells were labeled with a25Iby the method of Mitchell et al. . The ceils were dissolved in NET buffer (0.15 M NaC1, 0104 M Tris-HC1, 0.2 mM phenylmethylsulphonyl fluoride, pH 7.0) containing 0.5% Nonidet P40 for 30 minutes at 4° C. After centrifugation at 105,000g for 1 hour, aliquots of the supernatant (2 x 106cpm) were incubated with 5/xl normal mouse serum, 20/xl rabbit anti-mouse immunoglobulin (DAKO Corporation, Santa Barbara, CA), and then 300/xl of 10% formalin-fixed Cowan I strain of Staphylococcus aureus (SAC) suspension. After centrifugation, the precleared extracts were mixed with 100/xl of a MAb and incubated overnight at 4° C. To this mixture were added 15/xl of rabbit anti-mouse immunoglobulin; the mixture was incubated further for 6 hours at 4° C. The resultant antigen/antibody complexes were precipitated by adding 300/xl of 10% SAC suspension. After washing with NET buffer containing 0.5% Nonidet P40 and 0.1% BSA, the antigens were eluted from the complexes by boiling them with 100/xl buffer solution containing 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% acrylamide gel was performed by the method of Laemmli . Autoradiography was carried out at - 80° C using Kodak X-Omat AR film.
Table 1. Reactivity of MAb Ep-C4 to human cell lines and human cells Cells
Neuroectodermal origin KMS II NU 2-26 TE-671 KG-1 KNS-42 U-118 MG U-105 MG U-251 MG U-373 MG KMG-2 KMG-3 KMG-4 KMG-5 KMG-8 KMG-9 KMG-14 KMG-15 KMG-16 KMG-17 SK-N-BE(2) SK-N-AS SK-N-DZ SK-N-FI Non-neuroectodermal origin KOIMC-011 MCF-7 KMK-2 KATO III GaCa GBK-1 CAR-1 SUIT-2 HeLa $3 MOLT-4 RPMI-8402 HPB -ALL CCRF-CEM HUT 102 Raji EBV-transformed NALM-16 Reh Non-neoplastic origin WISH FF PBL
Ependymoma Ependymoma Medulloblastoma Oligodendroglioma Glioblastoma Glioblastoma Glioblastoma Glioblastoma Glioblastoma Anaplastic astrocytoma Anaplastic astrocytoma Glioblastoma Glioblastoma Glioblastoma Glioblastoma Glioblastoma Anaplastic astrocytoma Glioblastoma Glioblastoma Neuroblastoma Neuroblastoma Neuroblastoma Neuroblastoma
Maxillary sinus carcinoma Breast carcinoma Gastric carcinoma Gastric carcinoma Gastric carcinoma Gall bladder carcinoma Rectal carcinoma Pancreatic carcinoma Cervical carcinoma T-cell T-cell T-cell T-cell T-cell B-cell B-cell non-T, non-B-cell non-T, non-B-cell Amnion Skin fibroblast Healthy donors
Reactivity of MAb Ep-C4 was determined by ELISA and/or indirect immunofluorescence. + = positive reaction with MAb Ep-C4 and = negative results for same. -
Fig. 1. Indirect immunofluorescent staining of EDTA-dispersed KMS II cells with MAb Ep-C4 (left). Right: phase contrast picture of the same field. All the cells show membrane fluorescence.
Growth and selection of hybridomas A total of 310 hybridomas producing MAbs which reacted with KMS II cells but not with KMG-2 cells were screened by ELISA and 9 were selected for study. In indirect immunofluorescence studies one
Table 3. Immunohistochemical reactivity of MAb Ep-C4 with brain tumors and neuronal tissues Tissue tested
No. positive/No, tested
Neoplastic origin Table 2. Reactivity of MAb Ep-C4 with short-term cultures of various types of brain tumors Tumor tested*
No. positive/No, tested
Glioblastoma Anaplastic astrocytoma Mixed glioma Ependymoma Oligodendroglioma Medulloblastoma Cerebral neuroblastoma Meningioma Pituitary adenoma
0/7 0/4 0/1 4/4 0/2 0/3 0/1 0/4 0/3
* Cells were examined at passage 1 to 3.
Glioblastoma Anaplastic astrocytoma Astrocytoma Mixed glioma Ependymoma Oligodendroglioma Medulloblastoma Cerebral neuroblastoma
0/8 0/5 0/4 0/1 2/3* 0/2 0/2 0/1
Non-neoplastic origin Adult tissue Cerebrum Cerebellum Spinal cord Fetal brain (16-week-old)
0/2 0/2 0/2 0/1
* The negative ependymoma did not show typical ependymal rosettes.
reacted strongly with the cell membrane of KMS II cells; the other 8 MAbs mainly reacted with cytoplasm. The former was cloned by limiting dilutions and designated Ep-C4. A MAb secreted in the culture fluid by clone Ep-C4 was pooled and used at a 1 : 20 dilution in the present study. The immunoglobulin subclass determined by immunodiffusion technique was IgG1. As a control, the culture fluid of a mouse hybridoma producing an irrelevant MAb of the IgG1 subclass was used.
Reactivity of MAb Ep-C4
Fig. 2. Indirect immunofluorescence staining of methanol-fixed KMS II cells with MAb Ep-C4.
The reactivity of MAb Ep-C4 with adherent cells was determined by ELISA and indirect immunofluorescence techniques. For non-adherent cells, the indirect immunofluorescence technique was used. The results are summarized in Table 1. The binding activity (BR) of MAb Ep-C4, determined by ELISA, was 21 for KMS II cells and 25 for NU 2-26 cells. Non-specific absorbance at 492 nm was between 0 and 0.1. The other cell lines showed absorbance of essentially the same value as that of an irrelevant MAb and the BR was below 1.1,
Fig. 3. Electron microgram of KMS II cells immunostained with MAb Ep-C4. The cell membrane is positively stained. Bar = 1/xm.
Fig. 4. Indirect immunofluorescence staining of an EDTA-dispersed primary culture of an ependymoma (left). Right: phase contrast picture of the same field. One of the cells (center) shows membrane fluorescence.
Fig. 5. Immunohistochemical staining of two different ependymomas. A positive reaction was mainly observed in rosette-forming cells. Left: Immunoperoxidase staining (ABC method), methyl green counterstain. Right: Indirect immunofluorescence staining.
z < 0.5 m
z m ¢ 0.5 0
0 (n m