Human fetal brain antigen expression common to tumors of neuroectodermal tissue origin : Gliomas, neuroblastomas, and melanomas

The antigenic relationship between human tumors of neuroectodermal origin and fetal brain were further investigated by characterization of two hybridoma antibodies derived from a fusion of P3-NS1/1-Ag 4-1 (NSI) myeloma cells and splenocytes hyperimmunized to second trimester human fetal brain homogenate. Monoclonal antibodies (MAs) 1H8cl 2 and 1H8cl 3 were analyzed by cel; surface radioimmunoassay (CS-RIA), quantitative absorption, indirect immunofluorescence, and peroxidase-antiperoxidase (PAP) immunohistology. MA 1H8cl 3 is the more broadly reactive, binding to 9/14 glioblastoma (GBM), 2/3 neuroblastoma, 1/2 melanoma, and 1 medulloblastoma cell line(s) by CS-RIA analysis, and to 12/15 GMB, fetal brain, spleen, and liver, and adult spleen by PAP analysis. MA 1H8cl 2 is more restricted, binding to 7/14 GBM, 2/3 neuroblastoma, 1 medulloblstoma, and 2/3 fetal skin fibroblast cell line(s) by CS-RIA, and to 9/15 GBM and fetal brain and spleen by PAP analysis. Control non-central nervous system tumors and normal adult tissue including brain, thymus, lymph node, liver, kidney, lung, skin, and pancreas, were unreactive with both 1H8cl 2 and 1H8cl 3 by CS-RIA, PAP, and potential interest in terms of cell lineage evolution and investigations of cell-cell interactions from immunologic, developmental, and neuronal communication viiewpoints. The most critical observation remains, however, that the operational specificity of MAs 1H8cl 2 and 1H8cl 3 within the adult central nervous system makes them useful reagents for the purification and biochemical characterization of the family of human neuroectodermal tumor-associated antigens.     

Characterization of three restricted specificity monoclonal antibodies raised against the human glioma cell line D-54 MG

Monoclonal antibodies ( MCAs ) have been derived from a fusion of P3-NS1/1-Ag 4-1 (NS1) myeloma cells and splenocytes immunized to human glioma cell line D-54 MG. MCAs 2F3 , 4C7 , and 5B7 were analyzed by cell surface radioimmunoassay (CS-RIA), quantitative absorption, indirect immunofluorescence, and peroxidase-anti-peroxidase (PAP) immunohistology of unfixed tissue samples. MCA 2F3 exhibits the most highly restricted pattern of reactivity we have observed, reacting only with 5/12 glioblastoma cell lines and 1/4 fetal skin lines by CS-RIA, and to 9/11 glioblastoma tissue samples by PAP and absorption analysis; this MCA is totally nonreactive with melanomas, neuroblastomas, meningiomas, and control non-central nervous system tumors, and to adult and fetal tissues including brain, thymus, spleen, liver, lung, heart, gut, skin, and muscle by PAP analysis. MCAs 4C7 and 5B7 demonstrate neuroectodermal tumor cross-reactivity profiles, reacting with either melanomas ( 5B7 ) or melanomas and neuroblastomas ( 4C7 ); both are reactive with fetal skin, brain, and thymus of less than or equal to 16 weeks of gestational age. Other than this latter fetal antigen reactivity, these MCAs share the same negative reactivity profile described above for MCA 2F3 . Data from experiments using control or 0.02% EDTA-treated confluent cell monolayers of D-54 MG as antibody absorbents showed that the antigens detected are present in the extracellular matrix material remaining following cell removal. The data presented here establish that these highly restrictive anti-human glioma cell line MCAs are expressed in primary human gliomas; that the markers defined are developmental in nature, in that they are expressed by human fetal tissue, but not by adult tissue; and that in conjunction with previously characterized specificities, these markers of antigenic heterogeneity will be valuable in model system studies of therapeutic response heterogeneity.     

GD3 expression by cultured human tumor cells of neuroectodermal origin

Summary Seven monoclonal antibodies (mAbs) reactive with ganglioside II³(NeuAc)₂-LacCer (GD3) were generated; four of these mAbs (DMAb-21, DMAb-22, DMAb-23, and DMAb-24) by immunizing mice with GD3 adsorbed to Salmonella minnesota and the remaining three (DMAb-7, DMAb-8, and DMAb-17) with melanoma line SK-MEL 28, which contains 1.4 nmol sialic acid of GD3 per mg protein. The specificities of the mAbs were defined by high-performance thin-layer chromatography (HPTLC) immunostain and solid-phase radioimmunoassay (SP-RIA) with a panel of purified gangliosides. DMAb-7 and DMAb-8 reacted with GD3, IV³(NeuAc)₂nLcOse₄Cer(3,8-LD1), and very weakly with IV³(NeuAc)₂II³NeuAc-GgOse₄Cer (GTla), but not with II³NeuAc-LacCer (GM3), II³NeuAcGgOse₃Cer(GM2), II³NeuAc-GgOse₄Cer(GM1), II³NeuAc, IV³NeuAcGgOse₄Cer (GD1a), II³(NeuAc)₂GgOse₃(GD2), II³(NeuAc)₂GgOse₄Cer (GD1b), IV³NeuAcII³(NeuAc)₂, GgOse₄Cer(GT1b), suggesting the binding epitope to be a terminal tetrasaccharide NeuAc2-8NeuAc2-3Gal1-4(Glc or GlcNAc). DMAb-7 and DMAb-8 were used to investigate the expression of GD3 on cultured human tumor cells of neuroectodermal origin. Thirteen of 19 gliomas, 3 of 5 medulloblastomas, 5 of 5 neuroblastomas, 2 of 2 melanomas, and 1 of 3 teratomas were shown to react with DMAb-8 and/or DMAb-7 by cell surface-RIA (CS-RIA) and immunofluorescence (IF) assays. HPTLC and densitometric analysis confirmed these results, as positive immunostains in the GD3 region were obtained with oligoganglioside fractions from 9 glioma, 1 medulloblastoma, 2 neuroblastoma, 1 melanoma, and 1 teratoma cell line. Glioma cell line U-105 MG and medulloblastoma cell line Daoy contain GD3 as shown by HPTLC immunostain analysis of extracts, although GD3 was undetectable on the cell surface as determined by CS-RIA and IF. There was no detectable GD3 found in gangliosides isolated from cell lines U-373 MG, D-54 MG, TE-671, and PA-1, which were negative for both DMAb-7 and DMAb-8 by CS-RIA and IF assay. Our results provide evidence that GD3 is expressed extensively with significant quantitative heterogeneity on cultured human neuroectodermal tumor cells including glioma, medulloblastoma, neuroblastoma, and melanoma.Supported by NIH grants R37 CA11898, NS 20023, and CA32672 and by grants from the Swedish Medical Research Council (project no. 03X-627), Swedish Cancer Society (project no. 2260-B88-01X) and the National Swedish Board for Technical Development (project no. 84-4667)     

Five novel cell surface antigens of CNS neoplasms

Optimal monoclonal antibody-mediated immunotherapy requires the identification of tumor-restricted cell surface antigens. We have identified and partially characterized 5 new monoclonal antibodies generated against malignant astrocytoma, medulloblastoma, neuroblastoma and melanoma which were used to define 5 neuroectodermal tumor antigenic systems. CNT/1 identifies a 57-kDa, heat-stable, trypsin-sensitive neuroblastoma surface antigen, which is expressed intracellularly in many malignant gliomas, medulloblastomas, ependymomas, breast and ovarian carcinomas. CNT/2 reacts with a 130-kDa, heat-labile, trypsin- and neuraminidase-resistant antigen restricted to low-grade astrocytomas and malignant gliomas. CNT/11 reacts with a 70-kDa, heat-labile, trypsin-sensitive antigen coded for by a gene on chromosome 12, and is restricted to astrocytomas, neuroblastomas and sarcomas. CNT/8 identifies a heat-labile, trypsin-sensitive antigen whose gene has been localized to chromosome 15 and is expressed by neuroectodermal and mesodermally derived tumors and few epithelial cancers. The B2.6 antigen is identified only in terms of serologic reactivity with a subset of cultured astrocytomas and melanomas. Neuroectodermal tumor-associated antigens may be categorized as lineage-consistent, lineage-independent and putatively tumor-restricted in their expression. These restricted antibodies may be potentially useful reagents to consider for monoclonal antibody-mediated immunotherapy of CNS neoplasms.     

Antigenic heterogeneity of human anaplastic gliomas and glioma-derived cell lines defined by monoclonal antibodies

The antigenic heterogeneity of human neuroectodermal tumors defined by both murine and human monoclonal antibodies (MAs) is reported; no patterns of reactivity defining degree of anaplasia, in vitro morphology, or immunogen used were apparent. We investigated the reactivity of 20 distinct murine MAs defining markers of glioma-associated or predominantly lymphoid distribution for 13 human glioma-derived (HGL) cell lines and frozen sections of 19 human glioblastoma multiforme (GBM) and six astrocytomas (AST). Methods included radioimmunoassay, immunofluorescence, immunohistochemistry, and absorption analysis. Two markers, HLA-A,B and human Thy-1, exhibited no deviation; all HGL cell lines tested bound high levels of specific MA. Individual HGL cell line reactivity with the MA panel ranged from 30 to 70%. HGL cell lines (7/13) which reacted with greater than or equal to 50% of the antiglioma MAs had the highest (30-70%) positive reactivity rates with the anti-lymphoid marker MA panel; complex antigenicity in one system correlated with multiple antigens in the other. Within the anti-lymphoid marker MA panel, subpopulations of 4/13 HGL cell lines were clearly positive for the HLA-DR (Ia) antigens; another 3/13 HGL cell lines were strongly positive for common acute lymphocytic leukemia antigen (CALLA). With the exception of Thymocyte 1 antigen (Thy-1), reactivity for early and mature T-cell markers was infrequent and sporadic. Lymphoid marker expression by HGL cell lines is highly heterogeneous, ranging from few (Thy-1 and HLA-A,B) to complex expression of Ia, T-cell, and lymphoid tumor markers. GBM and AST tissues were antigenically less complex; for each of 6/8 anti-glioma MA, 70-100% of GBM and 66-100% of AST were positive. Two MAs were highly reactive (7/10, 8/9) with GBM sections and minimally so (1/6) with AST. Antigenic expression in gliomas is complex and heterogeneous; however, clear differences in lymphoid marker expression, the identification of widely and rarely expressed glioma-associated antigens, and the potential of immunologic differentiation between GBM and AST by large panels of MAs will serve to reduce the complexity and may be of potential diagnostic or prognostic significance.     

Nervous system antigen-5, an antigenic cell surface component of neuroectodermal origin.

The antigenic cell surface component NS-5 (nervous system antigen-5) is recognized by antiserum raised in C3H.SW/Sn mice against cerebellum of 4-day-old C57BL/6J mice. When analyzed in the cytotoxicity test the antiserum detects a cell surface antigen or set of antigens present not only an cerebellum but also other parts of the central nervous system, including retina, as well as on mature spermatozoa and to a lesser degree on kidney. All other non-neural tissues tested, liver, splee, thymocytes, muscle, testis, adrenal gland and epidermis do not express detectable amounts of the antigen. Among seven murine tumors of the nervous system, medulloepithelioma shows high levels of NS-5 expression, whereas neuroblastoma Cl300, glioma G26, glioblastome, ependymoblastoma, ependymoblastoma EPA and glioblastoma G26l do not carry detectable NS-5. All mouse strains tested (C57BL/6J, C3H.SW/Sn, C3H/HeDiSn, A/J, AKR/J, BALB/cJ and DBA/2) express similar levels of NS-5. The antigen is demonstrable not only on postnatal day 4 neural tissue, but also in lower amounts on adult nervous system. On embryonic day 9, the earliest stage tested, and at all subsequent stages during embryonic development, NS-K is already present in brain and spinal cord, but not in gut.     

Glioma-associated antigens defined by monoclonal antibodies against an avian sarcoma virus-induced rat astrocytoma

S69-c15 is a highly immunogenic cell line derived from an avian sarcoma virus (ASV)-induced astrocytoma in F-344 rats. Monoclonal antibody (Mab) production was attempted by fusing F-344 rat splenocytes and mouse P3 X 63/Ag8.653 myeloma cells after a syngeneic immunization protocol. 336 fusion clones were screened by cell surface radioimmunoassay (CS-RIA) against the immunizing line S69-c15, rat kidney fibroblast line S203-c11 and Walker rat carcinoma line. Mabs 7G4, 9F1, 10E3 and 10E7 which reacted only with S69-c15 were chosen. Further analysis demonstrated that these Mabs reacted only with rat (13/23 astrocytomas, 2/4 gliomas, 1/11 neurinomas) or mouse (2/10 astrocytomas) neurogenic tumor cells induced by both viral and chemical agents. Reciprocal competition assays suggested that 7G4, 9F1 and 10E3 recognized the same epitope and that 10E7 reacted with a spatially close determinant. Antigen activity could not be found in adult rat tissues (brain, heart, lung, liver, kidney, spleen, thymus, intestine, muscle and peripheral nerve) and fetal brain (8, 12, 20 days gestation) by either absorption analysis or tissue staining. Preliminary characterization indicated that the epitope may be polypeptide-associated. Further antigen purification and tumor localization can be attempted with these Mabs.     

Immunohistochemical study of placental form of glutathione S-transferase in human brain tumors and fetal brains

The activity of glutathione S-transferase placental form (GST-pi) was examined in 100 cases including various histologic subtypes and grading of human brain tumors and 10 cases of fetal brains by immunohistochemical studies. The 69% of cases with brain tumors were shown to be positive for GST-pi. This activity in neuroepithelial tumors tended to increase in order to tumor grading, however, medulloblastoma and primitive neuroectodermal tumor (PNET) were not immunoreactive with GST-pi. Embryonal carcinoma showed strong staining, although fetal brains were negative. The metastatic brain tumors showed the same reactivity with GST-pi as those of original carcinomas. Moreover, the difference of GST-pi activity was investigated on some brain tumors treated with or without antitumor drug, such as 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). The 85% of recurrent cases showed strong staining with GST-pi, and GST-pi activity seemed to be increased after treated with ACNU. The present study indicated that GST-pi might be a useful marker for human brain tumor, as the same conclusion was applicable to other neoplastic lesions examined previously. It is suggested that the increased GST-pi activity with malignancy of tumor may indicate the tendency to recurrence. The presence of such activity in tumor cells may also imply their acquired multidrug resistance. Our findings suggest that the evaluation of GST-pi activity in brain tumors will offer a predictive value for eventual behavior of the tumor.     

S-antigen and rod-opsin immunoreactions in midline brain neoplasms of transgenic mice: similarities to pineal cell tumors and certain medulloblastomas in man

Transgenic mice expressing the large T-antigen of the simian virus 40 (SV 40) under the control of 1) the enhancer of Moloney murine sarcoma virus (MSV) and 2) the SV 40 promoter develop undifferentiated neuroectodermal tumors located in the midline of the dorsal brain surface, abnormalities in lens fiber differentiation and retinal dysplasia. In this study the brain neoplasms of six adult animals and the brain of one 11-day old mouse were examined by conventional histology and immunocytochemical demonstration of S-antigen, rod-opsin, neuron-specific enolase, neurofilaments (160 and 200 kDa) and glial fibrillary acidic protein. According to histologic criteria the neoplasms were characterized as "primitive" neuroectodermal tumors composed mainly of small cells with scanty and ill-defined cytoplasm. Neoplastic cells displaying immunoreactive S-antigen were found in five brain tumors; three of these tumors also contained a limited number of rod-opsin immunoreactive neoplastic cells. Some tumor cells had neurite-like processes containing immunoreactive neurofilament (200 kDa). No pathologic lesions were found in the brain of the 11-day old animal. Tumors in transgenic mice may resemble pineal cell tumors and a special subtype of medulloblastoma in man. These neoplasms contain S-antigen immunoreactive and also rod-opsin immunoreactive tumors cells in certain cases. The findings suggest that transgenic mice expressing the large T-antigen of SV 40 may become a valuable animal model for analysing the origin, histogenesis and development of primitive neuroectodermal tumors with photoreceptor-like features (pineal cell tumors and certain medulloblastomas).     

Mouse fetal brain specific protein "GP68" is expressed in human tumor cells]

It is well known that some fetal antigens are expressed in malignant tumor cells. Likewise, brain tumors, especially histologically malignant cases, may have any antigenic relationships with fetal brain. So, we investigated the relationship by immunohistochemical technique, utilizing a polyclonal antibody to mouse fetal stage-specific polypeptide "GP68". We prepared GP68 from homogenate of head part of embryos at the 14th day of gestation mice by RCA-1 agarose column chromatography. And immunized it to Japanese white rabbits and the titer was measured by enzyme-linked immunosorbent assay. We analyzed operatively resected brain tumors and autopsy brain tissues. Frozen tissues were fixed in cold acetone and immunostained with anti-GP68 serum according to biotin-streptavidin peroxidase method. Remained tissues were homogenized in Laemmli's sample buffer and electrophoresed. The proteins were transferred to nitrocellulose membrane and immunostained with anti-GP68. Normal brain tissues were not positively stained, except for capillary endothelium which showed a weak staining. On the other hand, brain tumors of neuroectodermal origin were positively stained in varying degrees, and other tumors were negative. It is especially noteworthy that, in astrocytoma cases, there exists a definite correlation between the intensity of stain and the degree of histological malignancy. Immunoblot studies demonstrated a very weak band at 68 KD in normal brain and meningioma. In contrast, very strong band at the same position was seen in malignant astrocytomas. These results suggested that in brain tumors, especially those of neuroectodermal origin, GP68 antigen is expressed and the degree of expression is related to their histological malignancy. So this fetal antigen may be useful for evaluation of biological malignancy of gliomas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigenic phenotypes of cultured malignant astrocytomas: identification of lineage-consistent, lineage-independent and putative tumor-restricted antigenic expression

The treatment of CNS neoplasms with monoclonal antibody-mediated immunotherapy optimally requires the identification of tumor restricted cell surface antigens. However, little is known regarding the antigenic phenotype(s) of malignant astrocytomas. The interrelated expression of four neuroectodermal tumor antigens, CNT/11, AJ8, A010 and CNT/2, has been studied in cultured malignant gliomas and correlated with anchorage independent growth, morphology, glial fibrillary acidic protein, and the surface expression of other antigens. Many of these latter antigens have been reported to be expressed by specific fetal and differentiated adult cell lineages or tissues, as well as certain classes of malignant tumors. The tumor-associated expression of these antigens may be broadly classified as lineage-consistent, lineage-independent or putatively tumor-restricted. Malignant glioma tumor antigenic heterogeneity represents the expression of neuroectodermal and non-neuroectodermal cell surface markers. The importance of this observation is 2-fold. Lineage-independent antigen expression may be an indication of altered genome regulatory processes within tumor cells, and thus reflect the degree of anaplasia. The identification of lineage-consistent and lineage-independent tumor associated antigens may contribute to the selection of "target" antigens and the prediction of toxicity for monoclonal antibody mediated immunotherapy.     

Microsatellite instability and expression of DNA mismatch repair genes in malignant astrocytic tumors from adult and pediatric patients

Microsatellite instability (MSI) is used as a molecular marker for defective DNA mismatch repair (MMR) genes. We report here alterations of MSI in 15 malignant astrocytomas (WHO grade III) and glioblastomas (GBM; WHO grade IV) of pediatric patients (2 - 21 years) and 12 GBM from adults (44 - 68 years) by comparative analysis of BAT25/BAT26 loci and 10 other microsatellite markers. High-level microsatellite instability (MSI-H) occurred in 4 of the 15 pediatric cases (26.7%) and in 1 of the 12 adult GBM cases (8.3%). Low-level microsatellite instability (MSI-L) was observed in 6 pediatric cases (40%) and 8 adult GBM (66.7%). Unstable BAT-25 locus was found in 1 of the MSI-H pediatric cases. Thus, 2 unstable cases showed no instability of this marker. For BAT-26, such a discordance was even more profound: in 1 of MSI-H cases, we obtained no PCR product and the remaining 3 showed no alterations of this marker. MSH2 (Human MutS, Homologue2) protein was detected in all but 3 pediatric cases (1 highly unstable and 2 low-level unstable) and in all adult cases. MLH1 (Human MutL, Homologue 1) protein was detected in all but 2 pediatric cases (1 highly unstable and 1 low-level unstable). Thus, 2 highly unstable pediatric cases showed no detectable MLH1/MSH2 proteins. Our data support earlier observations that MSI occurs predominantly in malignant astrocytic tumors of young patients, which lends support to the hypothesis of different molecular mechanisms of pediatric brain tumors. Surprisingly, we found no significant correlation between the status of 10 microsatellite markers and that of either BAT25 or BAT26 loci or with the expression of MMR genes.     

Immunohistochemical expression of inducible nitric oxide synthase (iNOS) in human brain tumors: relationships of iNOS to superoxide dismutase (SOD) proteins (SOD1 and SOD2), Ki-67 antigen (MIB-1) and p53 protein

In this study, inducible nitric oxide synthase (iNOS) expression in a series of 158 human primary brain tumors was analyzed. To gain some insight into the biological significance of iNOS expression in tumor cells, comparative immunohistochemical analyses were employed to characterize the expression of iNOS, superoxide dismutase (SOD) proteins (SOD1 and SOD2), Ki-67 antigen (MIB-1) and p53 protein in these cells. Sixteen (39.0%) of the 41 glioblastoma multiforme (GBM) specimens showed iNOS immunoreactivity. Positive immunoreactions with iNOS were also detected in 2/8 anaplastic astrocytomas, 1/17 astrocytomas, 1/14 medulloblastomas and 1/11 primitive neuroectodermal tumors, but no positive reactions were observed in oligodendrogliomas (0/11), ependymomas (0/5), schwannomas (0/21), meningiomas (0/23) or pituitary adenomas (0/7). The MIB-1 labeling index of GBMs that expressed iNOS was significantly higher than that of GBMs that did not (0.025< P <0.05, Wilcoxon rank-sum test). Unlike iNOS-negative tumors, all iNOS-positive tumors coexpressed SOD1 or SOD2. In particular, there was a significant correlation between iNOS induction and SOD1 expression (P =1.65x10(-10), Fisher's exact test) in GBM specimens. There was no significant relationship between iNOS and p53 protein in any type of primary brain tumor (P >0.05, Fisher's exact test). No significant immunohistochemical reactions with iNOS, MIB-1 or p53 protein were observed in normal brain tissue sections. We conclude that primary brain tumors express iNOS, and that iNOS expression in brain tumor cells may depend, in part, on cellular proliferation potential. Based on the fact that SOD1 scavenges oxidative-stress species originating from large amounts of nitric oxide (NO) produced by iNOS, iNOS-expressing brain tumor cells may protect themselves against NO cytotoxicity by overinducing SOD1.     

Expression of p67 (Munc-18) in adult human brain and neuroectodermal tumors of human central nervous system

p67 (Munc-18), is a neuron-specific protein of 67 kDa, known for its ability to bind with syntaxin and also to copurify with neuronal cdc2-like kinase. Earlier, in situ hybridization and immunocytochemical analysis of rat trigeminal ganglion and hippocampal cells demonstrated the specific localization of p67 in nerve cells and its rich distribution in axons. In the present study, we have looked for p67 expression in normal human brain and various neuroectodermal tumors. Immunohistochemical and Western immunoblot analysis of normal human brain tissue using antibodies against the N- and C-termini of p67 demonstrated the specific localization of this protein in postmitotic neurons but not in glia. Among neuroectodermal tumors, expression of p67 was observed in 100% of the tumors of neuronal origin studied, especially in the mature neuronal cell population of these tumors. Western immunoblot analysis of non-neuronal neuroectodermal tumors failed to reveal the expression of this protein in majority of cases. However, in gliomas and meningiomas, mild cytoplasmic immunohistochemical staining of neoplastic cells was noted in 64.7% and 25% of cases, respectively. Observed mild immunohistochemical staining of these tumors could be due to immunoreactivity to low molecular weight degraded products of p67, as seen on Western blot. The findings suggest that p67, by virtue of its ability to be expressed in postmitotic neurons of adult human brain and in tumors of neuronal origin, may serve as a molecular tool to understand the growth and differentiation of the nervous system in general. Received: 8 September 1998 / Revised: 23 December 1998, 1 March 1999 / Accepted: 12 April 1999     

Monoclonal antibodies against human astrocytomas and their reactivity pattern

The establishment of hybridomas after fusion of X63-Ag8.653 mouse myeloma cells and splenocytes from BALB/c mice hyperimmunized against human astrocytomas is presented. The animals were primed with 5 × 10⁶ chemically modified uncultured or cultured glioma cells. Six weeks after the last immunization step an intrasplenal booster injection was administrated and 3 days later the spleen cells were prepared for fusion experiments. According to the specificity analysis of the generated antibodies 7 hybridoma products (MUC 7-22, MUC 8-22, MUC 10-22, MUC 11-22, MUC 14-22, MUC 15-22 and MUC 2-63) react with gliomas, neuroblastomas and melanomas as well as with embryonic and fetal cells but do not recognize non-neurogenic tumors. The selected monoclonal antibodies (McAbs) of IgG₁ and IgG_2a isotypes are not extensively characterized but these antibodies have been demonstrated to be reactive with a panel of glioma cell lines with varying patterns of antigen distribution. Using the McAbs described above and a series of cryosections of glioma biopsies and paraffin sections of the same material as well as glioma cultures established from these, variable antigenic profiles among glioma cell populations could be demonstrated. From these results it is evident that there is not only a distinct degree of antigenic heterogeneity among and within brain tumors, but also that the pattern of antigenic expression can change continuously. Some of the glioma associated antigens recognized by the selected antibodies persist after fixation with methanol/acetone and Karnovsky's fixative and probably are oncoembryonic/oncofetal antigen(s). The data suggest that the use of McAbs recognizing tumor associated oncofetal antigens in immunohistochemistry facilitates objective typing of intracranial malignancies and precise analysis of fine needle brain/tumor biopsies in a sensitive and reproducible manner.     

Midline brain tumors in MSV-SV 40-transgenic mice originate from the pineal organ

Summary Adult transgenic mice expressing the large T-antigen of the Simian virus 40 (SV 40) under the control of the Moloney murine sarcoma virus (MSV) enhancer and the SV 40 promoter develop inheritable uniform midline brain neoplasms showing features of primitive neuroectodermal tumors. The origin and histogenesis of these tumors were investigated in the present study. The brain and pineal organ of fetal and young transgenic mice less than 3 months old displayed normal macroscopic and microscopic features. In 3.5-month-old animals, the pineal organ was considerably enlarged due to hyperplasia, finally leading to tumor formation. Immunocytochemical demonstration of large T-antigen showed that this oncoprotein was already expressed in the nuclei of certain cells in the pineal organ of fetuses (16 and 18 days old) and newborn animals, but was absent from all other parts of the brain. The immunocytochemical demonstration of S-antigen (arrestin), a highly characteristic marker for pinealocytes, was used for further characterization of the large T-antigenimmunoreactive cells. The fetal pineal organ did not contain immunoreactive S-antigen. This first occurred in certain pinealocytes of newborn mice. Double immunostaining revealed that in newborn and older transgenic mice the immunoreactive large T-antigen was exclusively found in nuclei of cells containing S-antigen immunoreaction in their cytoplasm. Thus, transformed pinealocytes appear as stem cells of the experimental tumors. The results of this study suggest that primitive neuroectodermal tumors and the normal tissue from which they originate share certain molecular and immunocytochemical features.Supported by grants from the Deutsche Forschungsgemeinschaft to H-W K (Ko 758/3-2)     

Yes-associated protein 1 is widely expressed in human brain tumors and promotes glioblastoma growth

The hippo pathway and its downstream mediator yes-associated protein 1 (YAP1) regulate mammalian organ size in part through modulating progenitor cell numbers. YAP1 has also been implicated as an oncogene in multiple human cancers. Currently, little is known about the expression of YAP1 either in normal human brain tissue or in central nervous system neoplasms. We used immunohistochemistry to evaluate nuclear YAP1 expression in the fetal and normal adult human brains and in 264 brain tumors. YAP1 was expressed in fetal and adult brain regions known to harbor neural progenitor cells, but there was little YAP1 immunoreactivity in the adult cerebral cortex. YAP1 protein was also readily detected in the nuclei of human brain tumors. In medulloblastoma, the expression varied between histologic subtypes and was most prominent in nodular/desmoplastic tumors. In gliomas, it was frequently expressed in infiltrating astrocytomas and oligodendrogliomas but rarely in pilocytic astrocytomas. Using a loss-of-function approach, we show that YAP1 promoted growth of glioblastoma cell lines in vitro. High levels of YAP1 messenger RNA expression were associated with aggressive molecular subsets of glioblastoma and with a nonsignificant trend toward reduced mean survival in human astrocytoma patients. These findings suggest that YAP1 may play an important role in normal human brain development and that it could represent a new target in human brain tumors.     

CD9 of mouse brain is implicated in neurite outgrowth and cell migration in vitro and is associated with the α6/β1 integrin and the neural adhesion molecule L1

We describe here a novel monoclonal antibody (mab INTRODUCTION H6) which recognizes CD9, an integral cell surface constituent previously described in cells of the hematopoietic lineage and involved in the aggregation of platelets. Mab H6 was raised against membranes of immature mouse astrocytes and reacted with a protein of 25–27 kD in detergent extracts of adult mouse brain membranes. Sequence analysis of the N-terminal amino acids revealed an identity of 96% with CD9 from mouse kidney. CD9 was localized in the central and peripheral mouse nervous systems: in the spinal cord of 11-day-old mouse embryos, CD9 was strongly expressed in the floor and roof plates. In the adult mouse sciatic nerve, myelin sheaths were highly CD9immunoreactive. Mab H6 reacted with the cell surfaces of both glial cells and neurons in culture and inhibited migration of neuronal cell bodies, neurite fasciculation and outgrowth of astrocytic processes from cerebellar microexplants. Neurite outgrowth from isolated small cerebellar neurons was increased in the presence of mab H6 on substrate-coated laminin, but not on substrate-coated poly-L-lysine. Addition of mab H6 elicited an increase in intracellular Ca²⁺ concentration in these cells on substrate-coated laminin. Immunoprecipitates of CD9 from cultured mouse neuroblastoma N2A cells contained the α6/β1 integrin. Moreover, preparations of CD9 immunoaffinity-purified from adult mouse brain using a mab H6 column contained the neural adhesion molecule L1, but not other neural adhesion molecules. CD9 bound to L1, but not to NCAM or MAG. Both the α6/β1 integrin and L1 could be induced to coredistribute with CD9 on the surface of cultured neuroblastoma N2A cells. The combined observations suggest that CD9 can associate with L1 and α6/β1 integrin to influence neural cell interactions in vitro. © 1996 Wiley-Liss, Inc.