[CANCER RESEARCH 44, 2595-2599, June 1984] Immunoradiometric and Immunohistochemical Demonstration of Neuron-specific Enolase in Experimental Rat Gliomas1 Stanley A. Vinores,2 Paul J. Marangos, Jose M. Bonnin,3 and Lucien J. Rubinstein Division of Neuropathology, Department ol Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908 [S. A. V., J, M. B., L J. R.], and Biological Psychiatry Branch, National Institute of Mental Health, NIH, Bethesda, Maryland 20205 [P. J. M.J ABSTRACT demonstration of NSE in the tumors obtained following their i.e. inoculation. A number of ENU-induced neural tumors in rats were A number of neural and nonneural tumor cell lines of rat and also examined immunohistochemically for the presence of NSE. human origin were assayed for neuron-specific enolase (NSE) by radioimmunoassay. Most neural tumor cell lines had appreciably MATERIALS AND METHODS higher levels of NSE than did the nonneural tumor cell lines, the highest levels being found in two anaplastic rat glioma lines (F98 Cell Lines. A number of neural and extraneural tumor cell lines of rat and T24). These two lines contained more than twice the amount and human origin were obtained (see Tables 1 and 2) for the determi of NSE found in a rat pheochromocytoma line (PC12) and in nation of their NSE content. The tumors from which the rat lines were neuroblastoma lines derived from rats (B35 and B50) or humans derived included anaplastic gliomas, malignant schwannomas, spongio- (IMR-32 and SHSY-5Y). Several of the rat glioma and schwan- blastoma, astrocytomas, mixed gliomas, neuroblastomas, pheochromo noma lines were inoculated intracerebrally into syngeneic rats. cytoma, hepatoma, and mammary adenocarcinoma. The human lines In the resulting tumors, NSE was demonstrable by immunohis- were derived from 2 neuroblastomas and one mammary adenocarci noma. Lines C6 and IMR-32 were obtained from the American Type tochemistry only in those from the F98 and T24 cell lines. A Culture Collection (Rockville, MD); line SHSY-5Y from Dr. J. Biedler (New number of ethylnitrosourea-induced rat tumors were also ex York, NY); clone PC12 from Dr. R. Goodman (Philadelphia, PA); clones amined immunohistochemically for NSE: NSE was demonstrated F98 and D74 from Dr. W. Wechsler (Dusseldorf, Germany); lines B35 in three anaplastic gliomas; three astrocytomas; and two mixed and 850 from Dr. D. Schubert (San Diego, CA); lines T9, T13, TU, T22, gliomas. Reactive astrocytes were also positive. Fibroadenomas T24, Y1196, Y2116, Y2121, 78-3853, 78-3864, and 78-4836 from Dr. of apocrine and mammary glands in rats were weakly positive, A. Koestner (East Lansing, Ml); line Q7 from Dr. J. Chou (Bethesda, MD); but other extraneural tumors tested were negative. and lines 64/24 and MCF7 from Dr. C. Eil (Bethesda, MD). Since normal neuronal elements, axonal swellings, and amine Inoculations i.e. of Tumor Cell Lines. Nine of the glioma lines and 2 precursor uptake and decarboxylation cells are strongly positive of the schwannoma lines (Table 1) were inoculated i.e. into the brains of syngeneic male Fischer rats weighing 90 to 110 g (Charles River, for NSE, whereas glia and most other normal cells are negative, Portage, Ml) as described previously (35). PC12 cells were inoculated in we hypothesize that the elevated metabolic demands imposed the same manner into New England Deaconess rats (Deaconess Hospi on neoplastic and reactive glial cells and on some extraneural tal, Boston, MA). Some of these rats were inoculated s.c. with PC12 tumors necessitate the opening up of metabolic pathways that cells for comparison. The tumor-bearing brains and, when present, the are normally operative only in neurons and neuroendocrine cells, visceral métastases, as well as s.c. PC12 pheochromocytomas, were therefore resulting in the synthesis of the more stable neuron- fixed by immersion in 10% buffered formalin after necropsy. specific form of enolase. Induction of Tumors by ENU. Tumors were induced transplacentally by a single injection of 20-mg/kg body weight of ENU in citrate buffer, pH 4.5, via the sciatic vein of pregnant Fischer rats on the 18th day of INTRODUCTION gestation or of ENU, 50 mg/kg, via the tail vein on the 16th day of NSE4 has been reported to be uniquely contained in all classes gestation. The tumors that developed in the offspring, usually between 150 and 500 days after birth, were fixed as described above after the of neurons (13, 21, 26) and in normal and neoplastic cells of the rats were sacrificed or had died from their tumors. APUD system (4, 5, 8,19, 25, 27, 30, 40, 41). Its appearance in Perfused Normal Rat Tissues. Normal rat tissues were fixed for 48 normal conditions correlates directly with structural and func hr at room temperature in 10% buffered formalin after intraventricular tional neurogenesis (15,25,37), and the enzyme levels detected gravity perfusion, first briefly with phosphate-buffered saline (0.01 M have been used as an index for neuronal differentiation (11, 19, phosphate buffer, pH 7.6, containing 0.85% NaCI) and then with 10% 25, 39). It is absent from normal glia (21, 24). The purpose of buffered formalin for about 10 min, both at 37°C. this study was to determine by immunoradiometric assay the RIA. Cultured cells from the various cell lines tested (Tables 1 and 2) were washed with Ca2+-, Mg2+-free Dulbecco's phosphate-buffered sa levels of NSE in a variety of rat glioma and schwannoma cell line (0.2 g KCI:0.2 g KH2PO«:8g NaCI:2.16 g Na2HPO4-7H2O/liter) and lines and to correlate these levels with the immunohistochemical immediately frozen on dry ice in 107 cell aliquots. These were homoge ' Supported by Research Grant CA 31271 of the National Cancer Institute and nized and assayed for NSE using a double-antibody, solid-phase RIA by Neuropathology Research Training Grant T32 NSO7236 of the National Institute procedure as described previously (14, 20, 39). Assays were done in of Neurological and Communicable Diseases and Stroke, United States Department triplicate for each cell line. Cells from rat tumor lines were assayed with of Health and Human Services. 2To whom requests for reprints should be addressed. anti-rat NSE antiserum, and those from human lines were assayed with 3 Recipient of Clinical Fellowship 5732 of the American Cancer Society. anti-human NSE antiserum. Antisera were produced as described pre 4 The abbreviations used are: NSE, neuron-specific enolase; APUD, amine viously (17). precursor uptake and decarboxylation; GFA, glial tibnllary acidic; RIA. radioimmu Immunohistochemistry. All tissues from the i.e. implants and ENU- noassay; ENU, ethylnitrosourea; TBS:NGS, 0.05 M Tris buffer, pH 7.6, containing 0.9% NaCI solution and 1% normal goat serum; i.e., intracerebral(ly). induced tumors, as well as normal rat tissues, were embedded in paraffin, Received November 22, 1983; accepted February 24, 1984. sectioned, and stained for NSE by the peroxidase:antiperoxidase tech- JUNE 1984 2595 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1984 American Association for Cancer Research. S. A. Vinares et al. Tabtel with fresh buffered saline (except after the last centrifugation). Diluted Detection of NSE in rat neural tumors by RIA and peroxidase:antiperoxidase anti-NSE serum was added to the pellet in a ratio of 1 ml of 1:500 Cultured cells were assayed by RIA for NSE In aliquots of 107 cells. The standard antiserum/100 mg of washed liver powder (dry weight). This was incu curve for NSE detection ranged from 100 pg to 5 ng (20). The minimal detectable bated for 30 min at 37°and centrifuged as above. The supernatant was level was 100 pg/ml of solution. The resulting tumors were assayed by peroxi- dase:antiperoxidase following I.e. inoculation of these cells. Paraffin-embedded added to 50 mg of washed liver powder (dry weight)/ml, incubated, and sections were incubated with a 1:500 dilution of rabbit anti-rat NSE serum. centrifuged as before. The supernatant was then applied to deparaffin- ized sections as described above. of NSE by from which the NSE levels in out- peroxidase: cell tured cells (ng/mg antiperoxidase in CelllineF98T24T9Y211678-3864T13Y1196T22Y2121C6T1478-385378-4836D74PC12TumorderivedAnaplastialine was implants1407.1protein) i.e. RESULTS gliomaAnaplastic ±1330.0 glioma0Anaplastia Radioimmunoassay ±751 gliomaAnaplastic .3±61 gliomaMalignant 2.2±271.1 The quantitative values for NSE obtained from several cultured schwannomaMalignant ±754.9 lines of neural tumors (Tables 1 and 2) ranged from a low of schwannomaMalignant ±373.4 schwannomaSpongioblastomaAstrocytomaAstrocytomaMixed±741 267.7 ±75.6 (S.D.) ng/mg protein in the C6 astrocytoma to a .7±470.1 high of 1407.1 ± 102.8 ng/mg protein in the F98 anaplastic ±267.7 glioma. Most neural tumor lines had NSE levels that were appre ±1041.7 gliomaMixed ±421 ciably higher than those of the 3 nonneural tumors examined. gliomaMixed .6±289.8 However, the cells from one rat malignant schwannoma, 2 rat gliomaDifferentiated ±820.4 gliomas (including the C6 astrocytoma), and one human neuro gliomaPheochromocytomaDetection±649.1 ±102.8a58.817.96.14.214.222.626.221.375.65.428.12.335.244.9+"+————NT———NT——NT-blastoma had low levels of NSE that were within the range of ' Mean ±S.D. that of the rat hepatoma. " +, positive; -, negative; NT, not tested. c The T24 line was derived from a tumor which was originally diagnosed as an Immunohistochemistry astrocytoma, but the absence of GFA protein in the implants grown after numerous in vitro passages has prompted us to call the tumor an anapìasticglioma. Controls. No staining was seen in any tissue when normal Table 2 rabbit serum was substituted for NSE antiserum.