Murine Model of Leptomeningeal Dissemination Human Medulloblastoma Cells
Using
Masanobu YAMADA, Keiji SHIMIZU, Kazuyoshi TAMURA, Shusuke MORIUCHI, Eiichiro MABUCHI, Kae Chang PARK, Yasuyoshi MIYAO and Toru HAYAKAWA Department
of Neurosurgery,
Osaka University Medical School, Osaka
Abstract
An experimental model of meningeal dissemination was developed by intracisternal inoculation of human medulloblastoma (ONS-76) cells into nude mice. All mice died within 65 days after inoculation of 1 × 107 tumor cells. The median survival time was 56 days. Clinical signs and histological findings were similar to those in medulloblastoma patients with meningeal dissemination. Immunohisto chemical studies showed that ONS-76 cells in the subarachnoid space expressed major histocom patibility complex (MHC) class I antigens until 20 days after inoculation. After 30 days, expression of MHC class I antigens decreased and cells began to proliferate rapidly. Expression of MHC class I antigens on tumor cells may result in effective recognition by the host immune system. Key words:
meningeal
tumor,
cell line,
medulloblastoma,
Introduction The 5-year survival among medulloblastoma patients has improved to 70% in the last 15 years. 7,9,13)This dramatic improvement is mainly due to advances in radiation therapy. 2,1,12)Medulloblastomas are cer tainly radiosensitive, but are not always radiocurable because dosages are limited by adverse effects on the whole neural axis.') Eventually, tumor regrowth oc curs and cerebrospinal fluid (CSF) metastasis often causes death in medulloblastoma patients. To further advance the treatment of medullo blastoma, we have developed a murine model of leptomeningeal dissemination using human medullo blastoma cells. Here, we describe our histological results and the biology of disseminated cells in our murine model. Materials
and
Methods
I.
Cell line and animals This study used a human medulloblastoma cell line (ONS-76). The establishment and biological characterization were described previously. 16,11)The Received 1991
January
23,
1991;
Accepted
April
11,
experimental
model
ONS-76 cells demonstrate neuron-like characteris tics, expressing neuron-specific enolase and neuro filament proteins (145,200 kd). Flow cytometry showed that most ONS-76 cells continuously express ed major histocompatibility complex (MHC) class I antigens, and MHC class II antigens could be in duced in vitro after coculture with recombinant hu man interferon-gamma (200 U/ml) for 48 hours. 16) ONS-76 cells were maintained in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum at 37°C in a humid incubator under 5% CO2, and passaged in vitro more than 100 times. Twenty-five female athymic nude mice (BALB/ c, nu/nu, 8-12 weeks old; Japan SLC Inc., Hamamatsu, Shizuoka) were used. II.
Leptomeningeal dissemination model ONS-76 cells were harvested and suspended in phosphate buffer saline (PBS) at concentrations of 1 x 10', 5 x 106, and 1 x 106 cells/ 100 ,ul. Each 100 ,ul of cell suspension or saline was percutaneously transplanted into the cisterna magna of nude mice (n = 5) under ether anesthesia. The general condition and neurological signs were checked daily, and the median survival time (MST) calculated. The brain and spinal cord with the skull and vertebral column intact from dead mice were
fixed in 10% formalin. After decalcification, cor onal, transverse, and longitudinal blocks were cut and embedded in paraffin. Histological sections were prepared from each block and stained with HE. In another experiment, five mice inoculated with 1 x 10' tumor cells were sacrificed at 7, 20, and 30 days after inoculation to investigate the expression of MHC antigens on disseminated medulloblastoma cells. Animals were transcardially perfused with 100 ml PBS and the brains removed. Cryostat sections (8-10µm) were cut and fixed with cold acetone for 10 minutes. MHC class I antigens on disseminated cells were evaluated using the indirect immunoperox idase method with mouse monoclonal antibodies against human leukocyte antigens (HLA)-A, B, C (Serotec, Oxford,
U.K.). Results
I.
Leptomeningeal dissemination model Animals demonstrated severe anorexia and weight loss, and gradually became less active 30 days after tumor inoculation. Pareses of the hind limbs and urinary and rectal incontinence then developed. In the terminal stage, animals were prone with the back hyperflexed and the hind limbs extended. All five mice died within 65 days of intracisternal inoculation of 1 x 10' ONS-76 cells (Fig. 1). MST was 56 days. Only one of the five mice receiving 5 X 106 cells had died 50 days after tumor inoculation.
Fig. 2
Macroscopic appearance of coronal section of the brain from experimental animals, which died of meningeal dissemination. The whole ventricular system including the fourth ventri cle (arrow) was severely dilated. There was a tumor mass at the cranial base (arrowheads).
No mouse inoculated within 3 months.
with 1 X 106 ONS-76
cells died
II.
Histological findings Macroscopic examination found various degrees of hydrocephalus and leptomeningeal thickening at the cranial base in all models (Fig. 2). Microscopic ex amination revealed tumor layers and nodules within the subarachnoid or epidural spaces from the cerebral hemisphere to the cauda equina. In par ticular, thick tumor layers occurred at the skull base involving major cranial nerves, such as the optic
Fig. 1
Survival curves for murine model after in tracisternal inoculation of ONS-76 cells (n = 5). All mice inoculated with 1 x 10' cells (0) died within 65 days of inoculation. MST was 56 days. Only one mouse receiving 5 x 106 (•) or less (1 x 106, A) tumor cells died within 3 months. o : mice injected with saline.
(Figs. 3A and 4A) or trigeminal nerves (Fig. 3B). Heavy infiltration of tumor cells was observed especially in the cistern around the brainstem. The spinal root ganglions were also preferentially in volved (Fig. 4B). The tumor cells extended into the perivascular space of the penetrating vessels (Vir chow-Robin's space) (Fig. 4C). Many mitotic figures were observed in the infiltrating cells (Fig. 4D). These histological findings were similar to those in medulloblastoma patients.
III. MHC class I antigen expression on inoculated ONS-76 cells MHC class I antigens (HLA-A, B, C) were detected on transplanted cells until 20 days after tumor inoculation (Fig. 5A, B). About 30 days after inoculation, expression of MHC class I antigens became almost undetectable (Fig. 5C). Discussion Fig. 3
Coronal sections through the frontal lobe and optic nerves (A), and the midbrain and trigeminal nerves (B). A: The tumor cells in filtrated the skull base, and the bilateral optic nerves (arrows) were encased by many tumor cells. Severe hydrocephalus was also observed. HE stain, x 10. B: The tumor cells encased the midbrain and filled the surrounding cis terns. Bilateral trigeminal nerves (arrows) were surrounded by thick layers of tumor cells. HE stain, x 10.
Fig. 4
Tumor cell dissemination along the CSF pathways is nearly always fatal in medulloblastoma patients despite improved irradiation techniques. The development of an effective therapeutic strategy to prevent meningeal ma requires animal
dissemination of medulloblasto models to investigate the patho
physiology. However, few animal models for menin geal dissemination using human medulloblastoma cells have been developed because of the difficulties in establishing a medulloblastoma cell line. Present culture techniques can now culture human medullo blastoma cells in vitro, and several useful cell lines
Photomicrographs of tumor cells. A: Massive tumor cells encased the optic nerve. HE stain, x 100. B: The spinal meninges were infiltrated by tumor cells (arrowhead) and the dorsal nerve root ganglions were also involved (arrow). HE stain, x 100. C: Perivascular extension of tumor cells. Virchow-Robin's space was markedly distended. HE stain, x 250. D: Many mitotic figures (arrow) could be observed in the infiltrating cells. HE stain, x 500.
Fig. 5
have
been
Immunohistochemical study of MHC class I antigens on the transplanted cells at days 7 (A), 20 (B), and 30 (C) after tumor inoculation. The tumor cells in the subarachnoid space positively stained for class I antigens (HLA-A, B, C) at days 7 and 20, but only a few cells expressed the antigens at day 30. x 200.
reported.4,s.lo,ls)
Friedman
et al.',')
re
ported experimental models for human medullo blastoma using TE-671 cells, which, however, were demonstrated to be a subline of human rhabdo myosarcoma.15) In 1988, Friedman et al.4) reported the first animal models with meningeal involvement using human medulloblastoma cell line (D341 Med). We have further developed murine models for meningeal dissemination using human medulloblas toma (ONS-76) cells. The neurological signs and histological findings in our models were very similar to those in medulloblastoma patients. ONS-76 cells expressed MHC class I antigens on their cell surface, and also in the subarachnoid space. MHC class I antigens were present until 20 days after tumor inoculation, but expression marked ly decreased after 30 days. Around this time, animals began to demonstrate anorexia and weight loss. This suggests that the inoculated tumor cells proliferated rapidly when autologous MHC antigen expression was reduced. In contrast, ONS-76 cells developed with difficulty as subcutaneous xenografts in athymic nude mice. ONS-76 cells easily propagated in the subarachnoid space, which is thought to be an im munologically privileged site. These results suggest that expression of MHC class I antigens results in effective recognition of the disseminating tumor cells by the host immune system,' 1,8,14,17) although im munological reactions in the subarachnoid space may be different from systemic host reactions."
There
may
be a relationship
between
the tumor
cell
phenotype, such as MHC antigen expression, and oc currence of meningeal metastasis. This model should be useful in investigating the clinical outcome of human medulloblastoma with meningeal dissemina tion.
References 1)
Alon Y, Hammerling GJ, Segal S, Bar EM: Associa tion in the expression of Kirsten-ras oncogene and the major histocompatibility complex class I antigens in fibrosarcoma tumor cell variants exhibiting different metastatic capabilities. Cancer Res 47: 2553-2557, 1987
2)
Dewit L, Van-Dam J, Rijnders A, Van-de-Velde G, Ang KK, Van-der-Schueren E: A modified radio therapy technique in the treatment of medulloblas toma. Int J Radiat Oncol Biol Phys 10: 231-241, 1984
3)
Friedman HS, Bigner SH, McComb RD, Schold SC Jr, Pasternak JF, Groothuis DR, Bigner DD: A model for human medulloblastoma. J Neuropathol Exp Neurol 42: 485-503, 1983 Friedman HS, Burger PC, Bigner SH, Trojanowski JQ, Brodeur GM, He XM, Wikstrand CJ, Kurtzberg J, Berens ME, Halperin EC, Bigner DD: Phenotypic and genotypic analysis of a human medulloblastoma cell line and transplantable xenograft (D341 Med) demonstrating amplification of c-myc. Am J Pathol 130: 472-484, 1988 Friedman HS, Burger PC, Bigner SH, Trojanowski
4)
5)
6)
7)
JQ, Wikstrand CJ, Halperin EC, Bigner DD: Establishment and characterization of the human medulloblastoma cell line and transplantable xenograft D283 medulloblastoma. J Neuropathol Exp Neurol 44: 592-605, 1985 Friedman HS, Schold SC Jr, Varia M, Bigner DD: Chemotherapy and radiation therapy of human medulloblastoma in athymic nude mice. Cancer Res 43: 3088-3093, 1983 Inoya H, Takakura K, Shitara N, Manaka S: Treat ment of medulloblastoma. Prog Exp Tumor Res 30: 91-99, 1987
8) Karre K, Ljunggren HG, Piontek G, Kiessling R: Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defense strategy. Nature 319: 675-678, 1986 9)
10)
11)
12)
13)
Levin VA, Vestnys PS, Edwards MS, Wara WM, Fulton D, Barger G, Seager M, Wilson CB: Improve ment in survival produced by sequential therapies in the treatment of recurrent medulloblastoma. Cancer 51: 1364-1370, 1983 McAllister RM, Isaacs H, Rongey R, Peer M, Au W, Soukup SW, Gardner MB: Establishment of a human medulloblastoma cell line. Int J Cancer 20: 206-212, 1977 Nicholas MK, Arnason BGW: Immunologic con siderations in transplantation to the central nervous system, in Seil FJ (ed): Neural Regeneration and Transplantation. New York, Alan R Liss, 1989, pp 239-284 Norris DG, Bruce DA, Byrd RL, Schut L, Littman P, Bilaniuk LT, Zimmerman RA, Capp R: Improved relapse-free survival in medulloblastoma utilizing modern techniques. Neurosurgery 9: 661-664, 1981 Park TS, Hoffman HJ, Hendrick EB, Humphreys PR, Becker LE: Medulloblastoma: Clinical presenta
tion
and management.
Experience
Sick Children, Toronto, 543-552, 1983
at the Hospital
1950-1980.
J Neurosurg
for 58:
14)
Plaksin D, Gelber C, Feldman M, Eisenbach L: Reversal of the metastatic phenotype in Lewis lung carcinoma cells after transfection with syngeneic H - 2Kb gene. Proc Natl Acad Sci USA 85: 4463-4467, 1988 15) Stratton MR, Darling J, Pilkington GJ, Lantos PL, Reeves BR, Cooper CS: Characterization of the human cell line TE671. Carcinogenesis 10: 899-905, 1989
16) Tamura K, Shimizu K, Yamada M, Okamoto Y, Matsui Y, Park KC, Mabuchi E, Moriuchi S, Mogami H: Expression of major histocompatibility complex on human medulloblastoma cells with neu ronal differentiation. Cancer Res 49: 5380-5384, 1989 17)
Wallich R, Bulbuc N, Hammerling GJ, Katzav S, Segal S, Feldman M: Abrogation of metastatic prop erties of tumor cells by de nove expression of H-2K antigens following H-2 gene transfection. Nature 315: 301-305, 1985 18) Yamada M, Shimizu K, Tamura K, Okamoto Y, Matsui Y, Moriuchi S, Park KC, Mabuchi E, Yamamoto K, Hayakawa T, Mogami H: Establish ment and biological characterization of human medulloblastoma cell lines. No To Shinkei 41: 695 -702, 1989 (in Japanese)
Address
reprint
ment School, 553,
requests
of
to:
K.
Neurosurgery, 1-1-50
Japan.
Fukushima,
Shimizu,
Osaka
M.D.,
Depart
University
Medical
Fukushima-ku,
Osaka
Using
Masanobu YAMADA, Keiji SHIMIZU, Kazuyoshi TAMURA, Shusuke MORIUCHI, Eiichiro MABUCHI, Kae Chang PARK, Yasuyoshi MIYAO and Toru HAYAKAWA Department
of Neurosurgery,
Osaka University Medical School, Osaka
Abstract
An experimental model of meningeal dissemination was developed by intracisternal inoculation of human medulloblastoma (ONS-76) cells into nude mice. All mice died within 65 days after inoculation of 1 × 107 tumor cells. The median survival time was 56 days. Clinical signs and histological findings were similar to those in medulloblastoma patients with meningeal dissemination. Immunohisto chemical studies showed that ONS-76 cells in the subarachnoid space expressed major histocom patibility complex (MHC) class I antigens until 20 days after inoculation. After 30 days, expression of MHC class I antigens decreased and cells began to proliferate rapidly. Expression of MHC class I antigens on tumor cells may result in effective recognition by the host immune system. Key words:
meningeal
tumor,
cell line,
medulloblastoma,
Introduction The 5-year survival among medulloblastoma patients has improved to 70% in the last 15 years. 7,9,13)This dramatic improvement is mainly due to advances in radiation therapy. 2,1,12)Medulloblastomas are cer tainly radiosensitive, but are not always radiocurable because dosages are limited by adverse effects on the whole neural axis.') Eventually, tumor regrowth oc curs and cerebrospinal fluid (CSF) metastasis often causes death in medulloblastoma patients. To further advance the treatment of medullo blastoma, we have developed a murine model of leptomeningeal dissemination using human medullo blastoma cells. Here, we describe our histological results and the biology of disseminated cells in our murine model. Materials
and
Methods
I.
Cell line and animals This study used a human medulloblastoma cell line (ONS-76). The establishment and biological characterization were described previously. 16,11)The Received 1991
January
23,
1991;
Accepted
April
11,
experimental
model
ONS-76 cells demonstrate neuron-like characteris tics, expressing neuron-specific enolase and neuro filament proteins (145,200 kd). Flow cytometry showed that most ONS-76 cells continuously express ed major histocompatibility complex (MHC) class I antigens, and MHC class II antigens could be in duced in vitro after coculture with recombinant hu man interferon-gamma (200 U/ml) for 48 hours. 16) ONS-76 cells were maintained in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum at 37°C in a humid incubator under 5% CO2, and passaged in vitro more than 100 times. Twenty-five female athymic nude mice (BALB/ c, nu/nu, 8-12 weeks old; Japan SLC Inc., Hamamatsu, Shizuoka) were used. II.
Leptomeningeal dissemination model ONS-76 cells were harvested and suspended in phosphate buffer saline (PBS) at concentrations of 1 x 10', 5 x 106, and 1 x 106 cells/ 100 ,ul. Each 100 ,ul of cell suspension or saline was percutaneously transplanted into the cisterna magna of nude mice (n = 5) under ether anesthesia. The general condition and neurological signs were checked daily, and the median survival time (MST) calculated. The brain and spinal cord with the skull and vertebral column intact from dead mice were
fixed in 10% formalin. After decalcification, cor onal, transverse, and longitudinal blocks were cut and embedded in paraffin. Histological sections were prepared from each block and stained with HE. In another experiment, five mice inoculated with 1 x 10' tumor cells were sacrificed at 7, 20, and 30 days after inoculation to investigate the expression of MHC antigens on disseminated medulloblastoma cells. Animals were transcardially perfused with 100 ml PBS and the brains removed. Cryostat sections (8-10µm) were cut and fixed with cold acetone for 10 minutes. MHC class I antigens on disseminated cells were evaluated using the indirect immunoperox idase method with mouse monoclonal antibodies against human leukocyte antigens (HLA)-A, B, C (Serotec, Oxford,
U.K.). Results
I.
Leptomeningeal dissemination model Animals demonstrated severe anorexia and weight loss, and gradually became less active 30 days after tumor inoculation. Pareses of the hind limbs and urinary and rectal incontinence then developed. In the terminal stage, animals were prone with the back hyperflexed and the hind limbs extended. All five mice died within 65 days of intracisternal inoculation of 1 x 10' ONS-76 cells (Fig. 1). MST was 56 days. Only one of the five mice receiving 5 X 106 cells had died 50 days after tumor inoculation.
Fig. 2
Macroscopic appearance of coronal section of the brain from experimental animals, which died of meningeal dissemination. The whole ventricular system including the fourth ventri cle (arrow) was severely dilated. There was a tumor mass at the cranial base (arrowheads).
No mouse inoculated within 3 months.
with 1 X 106 ONS-76
cells died
II.
Histological findings Macroscopic examination found various degrees of hydrocephalus and leptomeningeal thickening at the cranial base in all models (Fig. 2). Microscopic ex amination revealed tumor layers and nodules within the subarachnoid or epidural spaces from the cerebral hemisphere to the cauda equina. In par ticular, thick tumor layers occurred at the skull base involving major cranial nerves, such as the optic
Fig. 1
Survival curves for murine model after in tracisternal inoculation of ONS-76 cells (n = 5). All mice inoculated with 1 x 10' cells (0) died within 65 days of inoculation. MST was 56 days. Only one mouse receiving 5 x 106 (•) or less (1 x 106, A) tumor cells died within 3 months. o : mice injected with saline.
(Figs. 3A and 4A) or trigeminal nerves (Fig. 3B). Heavy infiltration of tumor cells was observed especially in the cistern around the brainstem. The spinal root ganglions were also preferentially in volved (Fig. 4B). The tumor cells extended into the perivascular space of the penetrating vessels (Vir chow-Robin's space) (Fig. 4C). Many mitotic figures were observed in the infiltrating cells (Fig. 4D). These histological findings were similar to those in medulloblastoma patients.
III. MHC class I antigen expression on inoculated ONS-76 cells MHC class I antigens (HLA-A, B, C) were detected on transplanted cells until 20 days after tumor inoculation (Fig. 5A, B). About 30 days after inoculation, expression of MHC class I antigens became almost undetectable (Fig. 5C). Discussion Fig. 3
Coronal sections through the frontal lobe and optic nerves (A), and the midbrain and trigeminal nerves (B). A: The tumor cells in filtrated the skull base, and the bilateral optic nerves (arrows) were encased by many tumor cells. Severe hydrocephalus was also observed. HE stain, x 10. B: The tumor cells encased the midbrain and filled the surrounding cis terns. Bilateral trigeminal nerves (arrows) were surrounded by thick layers of tumor cells. HE stain, x 10.
Fig. 4
Tumor cell dissemination along the CSF pathways is nearly always fatal in medulloblastoma patients despite improved irradiation techniques. The development of an effective therapeutic strategy to prevent meningeal ma requires animal
dissemination of medulloblasto models to investigate the patho
physiology. However, few animal models for menin geal dissemination using human medulloblastoma cells have been developed because of the difficulties in establishing a medulloblastoma cell line. Present culture techniques can now culture human medullo blastoma cells in vitro, and several useful cell lines
Photomicrographs of tumor cells. A: Massive tumor cells encased the optic nerve. HE stain, x 100. B: The spinal meninges were infiltrated by tumor cells (arrowhead) and the dorsal nerve root ganglions were also involved (arrow). HE stain, x 100. C: Perivascular extension of tumor cells. Virchow-Robin's space was markedly distended. HE stain, x 250. D: Many mitotic figures (arrow) could be observed in the infiltrating cells. HE stain, x 500.
Fig. 5
have
been
Immunohistochemical study of MHC class I antigens on the transplanted cells at days 7 (A), 20 (B), and 30 (C) after tumor inoculation. The tumor cells in the subarachnoid space positively stained for class I antigens (HLA-A, B, C) at days 7 and 20, but only a few cells expressed the antigens at day 30. x 200.
reported.4,s.lo,ls)
Friedman
et al.',')
re
ported experimental models for human medullo blastoma using TE-671 cells, which, however, were demonstrated to be a subline of human rhabdo myosarcoma.15) In 1988, Friedman et al.4) reported the first animal models with meningeal involvement using human medulloblastoma cell line (D341 Med). We have further developed murine models for meningeal dissemination using human medulloblas toma (ONS-76) cells. The neurological signs and histological findings in our models were very similar to those in medulloblastoma patients. ONS-76 cells expressed MHC class I antigens on their cell surface, and also in the subarachnoid space. MHC class I antigens were present until 20 days after tumor inoculation, but expression marked ly decreased after 30 days. Around this time, animals began to demonstrate anorexia and weight loss. This suggests that the inoculated tumor cells proliferated rapidly when autologous MHC antigen expression was reduced. In contrast, ONS-76 cells developed with difficulty as subcutaneous xenografts in athymic nude mice. ONS-76 cells easily propagated in the subarachnoid space, which is thought to be an im munologically privileged site. These results suggest that expression of MHC class I antigens results in effective recognition of the disseminating tumor cells by the host immune system,' 1,8,14,17) although im munological reactions in the subarachnoid space may be different from systemic host reactions."
There
may
be a relationship
between
the tumor
cell
phenotype, such as MHC antigen expression, and oc currence of meningeal metastasis. This model should be useful in investigating the clinical outcome of human medulloblastoma with meningeal dissemina tion.
References 1)
Alon Y, Hammerling GJ, Segal S, Bar EM: Associa tion in the expression of Kirsten-ras oncogene and the major histocompatibility complex class I antigens in fibrosarcoma tumor cell variants exhibiting different metastatic capabilities. Cancer Res 47: 2553-2557, 1987
2)
Dewit L, Van-Dam J, Rijnders A, Van-de-Velde G, Ang KK, Van-der-Schueren E: A modified radio therapy technique in the treatment of medulloblas toma. Int J Radiat Oncol Biol Phys 10: 231-241, 1984
3)
Friedman HS, Bigner SH, McComb RD, Schold SC Jr, Pasternak JF, Groothuis DR, Bigner DD: A model for human medulloblastoma. J Neuropathol Exp Neurol 42: 485-503, 1983 Friedman HS, Burger PC, Bigner SH, Trojanowski JQ, Brodeur GM, He XM, Wikstrand CJ, Kurtzberg J, Berens ME, Halperin EC, Bigner DD: Phenotypic and genotypic analysis of a human medulloblastoma cell line and transplantable xenograft (D341 Med) demonstrating amplification of c-myc. Am J Pathol 130: 472-484, 1988 Friedman HS, Burger PC, Bigner SH, Trojanowski
4)
5)
6)
7)
JQ, Wikstrand CJ, Halperin EC, Bigner DD: Establishment and characterization of the human medulloblastoma cell line and transplantable xenograft D283 medulloblastoma. J Neuropathol Exp Neurol 44: 592-605, 1985 Friedman HS, Schold SC Jr, Varia M, Bigner DD: Chemotherapy and radiation therapy of human medulloblastoma in athymic nude mice. Cancer Res 43: 3088-3093, 1983 Inoya H, Takakura K, Shitara N, Manaka S: Treat ment of medulloblastoma. Prog Exp Tumor Res 30: 91-99, 1987
8) Karre K, Ljunggren HG, Piontek G, Kiessling R: Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defense strategy. Nature 319: 675-678, 1986 9)
10)
11)
12)
13)
Levin VA, Vestnys PS, Edwards MS, Wara WM, Fulton D, Barger G, Seager M, Wilson CB: Improve ment in survival produced by sequential therapies in the treatment of recurrent medulloblastoma. Cancer 51: 1364-1370, 1983 McAllister RM, Isaacs H, Rongey R, Peer M, Au W, Soukup SW, Gardner MB: Establishment of a human medulloblastoma cell line. Int J Cancer 20: 206-212, 1977 Nicholas MK, Arnason BGW: Immunologic con siderations in transplantation to the central nervous system, in Seil FJ (ed): Neural Regeneration and Transplantation. New York, Alan R Liss, 1989, pp 239-284 Norris DG, Bruce DA, Byrd RL, Schut L, Littman P, Bilaniuk LT, Zimmerman RA, Capp R: Improved relapse-free survival in medulloblastoma utilizing modern techniques. Neurosurgery 9: 661-664, 1981 Park TS, Hoffman HJ, Hendrick EB, Humphreys PR, Becker LE: Medulloblastoma: Clinical presenta
tion
and management.
Experience
Sick Children, Toronto, 543-552, 1983
at the Hospital
1950-1980.
J Neurosurg
for 58:
14)
Plaksin D, Gelber C, Feldman M, Eisenbach L: Reversal of the metastatic phenotype in Lewis lung carcinoma cells after transfection with syngeneic H - 2Kb gene. Proc Natl Acad Sci USA 85: 4463-4467, 1988 15) Stratton MR, Darling J, Pilkington GJ, Lantos PL, Reeves BR, Cooper CS: Characterization of the human cell line TE671. Carcinogenesis 10: 899-905, 1989
16) Tamura K, Shimizu K, Yamada M, Okamoto Y, Matsui Y, Park KC, Mabuchi E, Moriuchi S, Mogami H: Expression of major histocompatibility complex on human medulloblastoma cells with neu ronal differentiation. Cancer Res 49: 5380-5384, 1989 17)
Wallich R, Bulbuc N, Hammerling GJ, Katzav S, Segal S, Feldman M: Abrogation of metastatic prop erties of tumor cells by de nove expression of H-2K antigens following H-2 gene transfection. Nature 315: 301-305, 1985 18) Yamada M, Shimizu K, Tamura K, Okamoto Y, Matsui Y, Moriuchi S, Park KC, Mabuchi E, Yamamoto K, Hayakawa T, Mogami H: Establish ment and biological characterization of human medulloblastoma cell lines. No To Shinkei 41: 695 -702, 1989 (in Japanese)
Address
reprint
ment School, 553,
requests
of
to:
K.
Neurosurgery, 1-1-50
Japan.
Fukushima,
Shimizu,
Osaka
M.D.,
Depart
University
Medical
Fukushima-ku,
Osaka
