ARTICLE |
Correspondence to: Per M. Almqvist, Dept. of Neurosurgery, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: per.almqvist@ks.se
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Summary |
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Nestin is an intermediate filament protein (IFP) expressed in undifferentiated cells during CNS development and in CNS tumors. Previous studies have arrived at different conclusions in terms of which types of CNS tumors express nestin. In this report we establish an immunohistochemical protocol using antigen retrieval, which significantly enhances staining with two polyclonal anti-nestin antisera, #130 and #4350. The staining pattern was identical for the two nestin antisera and very similar to that of vimentin, while glial fibrillary acidic protein (GFAP), immunoreactivity was absent from 9.5-week-old forebrain. The current study of 20 primary CNS tumors from pediatric patients included seven ependymomas, seven primitive neuroectodermal tumors (PNETs), five pilocytic astrocytomas, and one glioblastoma multiforme (GBM). All these tumors expressed nestin to various extents, in contrast to five brain metastases tested. Strong nestin immunoreactivity was found in malignant primary CNS tumors, whereas benign pilocytic astrocytomas showed low but consistent nestin expression. In all tumors nestin immunoreactivity was confined to the cytoplasm of tumor cells and was co-expressed with astrocyte markers vimentin, GFAP, and S-100. Vascular endothelial cells of all neoplasms also showed marked immunoreactivity for nestin and vimentin, whereas they were negative for GFAP and S-100. In conclusion, antiserum #4350 detected nestin in formalin-fixed, paraffin-embedded tissue sections by heat-induced antigen retrieval immunohistochemistry. Nestin was expressed in both highly malignant and low malignant gliomas, indicating the potential use of nestin as a diagnostic tumor marker in surgical pathology. (J Histochem Cytochem 50:147158, 2002)
Key Words: first trimester, telencephalon, nestin, vimentin, S-100, GFAP, immunohistochemistry, antigen retrieval, PNET, ependymoma, glioma
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Introduction |
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THE ADVENT of immunohistochemical (IHC) techniques to identify the expression of specific proteins has significantly advanced the typing of CNS tumors. These techniques have been of particular value in identification of intermediate filament proteins (IFPs) (
Nestin is an interesting IFP in this regard (
Nestin and vimentin are co-expressed in proliferating neuroepithelial cells in fetal rodent and human brain (-internexin, first detected at 15 weeks PCA in the developing hippocampus (
Nestin can be re-expressed in astrocytes of the adult CNS in response to cellular stress, including blunt focal skull trauma (
This justifies a re-evaluation of nestin expression in tumor specimens processed by formalin fixation (
The masking of antigens induced by fixation of tissues in formaldehyde-containing fixatives is a well-recognized problem. Several different methods have been used in an attempt to recover immunoreactivity, including trypsinization (
The present study confirms the value of heat-induced antigen retrieval for the localization of nestin in formalin-fixed tissue, and we emphasize the importance of proper tissue processing for optimal antigen detection using the #4350 nestin antiserum in IHC. We found that tumor cells of malignant gliomas expressed high levels of nestin, whereas pilocytic astrocytomas showed low but consistent nestin expression by tumor cells in contrast to normal brain tissue.
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Materials and Methods |
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Tissue Processing
Tumor specimens derived from surgical biopsies and autopsies were selected from the files of British Columbia's Children's Hospital. Surgical biopsies were from 1995 to 1998. One glioblastoma specimen was used as a nestin-positive control sample and five brain metastases, including three adenocarcinomas and two low-differentiated carcinomas, were used as nestin-negative controls. All tumors were processed according to routine protocols for histopathological diagnosis and were graded according the WHO grading system (
Nestin Antibodies
The anti-nestin antisera #130 and #4350 were produced in rabbits immunized with a bacterially produced fusion protein containing the 1300 carboxy-terminal amino acid residues of the rat nestin protein (#130) (
Immunohistochemistry
The formalin-fixed, paraffin-embedded tissue specimens were sectioned at 5 µm. Sections were mounted on silanized slides, deparaffinized in xylene, blocked in 3% H2O2 in absolute methanol, and processed for antigen retrieval by MW heating according to
For localization of IFPs and S-100 protein in tissue sections, an indirect IHC staining technique was used (Level 2 Ultra Streptavidin Detection System; Signet Laboratories, Dedham, MA). Both nestin rabbit antisera #130 and #4350 and rabbit antiserum against S-100 (Innovations Foundation; Toronto, ONT, Canada) were diluted 1:2000. Mouse MAbs to human GFAP and vimentin (Dako; Glostrup, Denmark) were used diluted 1:1000 and 1:100, respectively. Antibodies bound were visualized with 3-amino-9-ethyl-carbazole (AEC) (Sigma; St Louis, MO) as substrate chromogen. Slides were counterstained with Carazzi's hematoxylin and coverslipped using Entellan (Merck; Darmstadt, Germany). Brightfield microscopy with conventional photography was performed using a Leica DMRB microscope and a Nikon FII camera with Fuji T64 film.
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Results |
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Cytoarchitecture of Fetal Forebrain
Brain tissue from an aborted first-trimester fetus was used for the study of nestin expression at an early developmental stage, for evaluation of staining protocols, and for titration of the nestin antisera. The tissue specimen had been paraformaldehyde-fixed, paraffin-embedded, and processed for IHC without previous storage. Hematoxylin (Carazzi) staining of tissue sections showed the characteristic cytoarchitecture of the outer telencephalic wall at the time of transition from the embryonic to the fetal stage, including the germinal matrix of the ventricular layer (VL) with the highest density of cells, the intermediate layer (IL), the cortical plate (CP) and subplate zone (SPZ), and the subpial layer (SPL) (Fig 1). The cortical plate was present on the surface of the entire neopallium at this stage (Fig 1, inset), with the subpial layer half the width of the cortical plate. External to the ventricular layer is the IL, followed by the SPZ. The subplate was at least twice the thickness of the CP, based on an increase of fibers travelling perpendicular to the developing cerebral cortex (
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Nestin Expression in Fetal Forebrain
To perform a comparative analysis of nestin antisera #130 and #4350, tissue sections of formalin-fixed human fetal forebrain were processed for IHC using a standard protocol. Nestin #130 antiserum specifically stained the ependymal cell layer, scattered cells in the germinal matrix of the ventricular layer, and radial fibers projecting to the SPZ (Fig 2A), whereas nestin #4350 antiserum gave a very faint immunostaining (Fig 2B). After antigen retrieval by MW heating of tissue slides, nestin immunostaining was enhanced for both nestin antisera, giving equally strong immunostaining at 1:2000 dilution and overnight incubation with primary antiserum (Fig 2C and Fig 2D). In addition, the staining patterns were identical for the two nestin antisera, with strong nestin immunoreactivity detected in ependymal cells lining the ventricle, in scattered cells of the germinal matrix of the ventricular layer, in radial glial cells extending fibers from the ventricular layer, traversing the intermediate layer towards the SPZ (Fig 2C and Fig 2D). There was a steep gradient of nestin immunoreactivity, with very intense staining of the columnar epithelium lining the ventricle and the nestin expression diminishing as the cells were located away from the germinal matrix. No nestin-positive cell bodies were seen outside the ventricular layer.
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Similar to nestin, vimentin was expressed by ependymal cells, by cells in the germinal matrix, and by radial fibers as they traversed the ventricular and the intermediate layer. In addition, vascular endothelial cells were strongly positive for vimentin. No GFAP immunoreactivity could be detected before or after heat-induced antigen retrieval (data not shown).
Immunohistochemical Staining for IFPs in Formalin-fixed Tissue
Sectioned material from formalin-fixed, paraffin-embedded surgical specimens of 20 pediatric brain tumors was analyzed for nestin expression by IHC after heat-induced antigen retrieval. The specimens consisted of seven ependymomas, seven PNETs, five pilocytic astrocytomas, and one glioblastoma multiforme (GBM).
Ependymomas. All seven ependymomas examined arose within the posterior fossa of children 210 years old, with a median age at diagnosis of 4 years. Six of these tumors (numbers 16 in Table 1) were classic ependymomas characterized by moderate cellularity, formation of perivascular pseudorosettes, nuclear-free zones, and fibrillary cell processes extending to the endothelium of the vessels. One anaplastic ependymoma was analyzed and showed increased cellularity and mitoses compared to the classic ependymomas.
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By standard IHC techniques, the classic ependymomas showed no immunoperoxidase staining with any of the two nestin antisera. However, after antigen retrieval there was cytoplasmic staining of the tumor cells and endothelial cells in all tumor specimens tested. The most prominent staining was noted in the cell processes of the perivascular spaces. In these areas the intermediate filaments stained as fine fibrillary processes that extended from the cytoplasm of the ependymal cells to the endothelium lining the vascular spaces. The intensity of staining and the staining pattern were very similar with the two nestin antisera. More than 50% of tumor cells were nestin-positive in all specimens, and staining intensity varied slightly. The vascular endothelium of ependymomas stained for nestin with high intensity in all samples tested (Table 1).
Staining of an anaplastic ependymoma (number 7, Table 1) with nestin antiserum #130 without antigen retrieval showed both tumor cell cytoplasmic staining and endothelial cell staining (Fig 3A), whereas nestin #4350 antiserum failed to give any specific immunostaining (Fig 3C). The staining intensity increased after antigen retrieval (Table 1). Fig 3B and Fig 3D show strong immunoperoxidase staining of tumor cells and vascular endothelial with either #130 or #4350 antiserum.
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Three ependymomas (numbers 57 in Table 1) were analyzed for vimentin, GFAP, and S-100 expression. Clearly, all three of these glial markers were co-expressed with nestin in tumor cells (Fig 4). Endothelial cells expressed nestin (Fig 3) and vimentin (Fig 4B), whereas they were devoid of GFAP and S-100 (Fig 4C and Fig 4D).
Primitive Neuroectodermal Tumors. All PNETs examined arose within the posterior fossa of children 2.516 years old. Median age at diagnosis was 7.5 years. These tumors were characterized by dense cellularity and were composed of sheets of cells with hyperchromatic, angulated nuclei and minimal amounts of cytoplasm. True rosettes were identified in some areas of the tumors. None of the tumor samples showed further differentiation along glial or neuronal cell lines. Mitoses and karryhorectic cells were present in the tumor.
Neither of the nestin antisera showed distinct immunostaining of tumor cells before antigen retrieval, although weak staining of endothelial cells by the #130 antiserum was observed in some cases. After antigen retrieval, 5/7 specimens showed coarse tumor cell cytoplasmic staining by the #130 nestin antiserum. More than 50% of cells were positive in all samples and intensity of staining was intermediate (Table 1). Antiserum #4350 stained tumor cells in all PNETs tested. Staining pattern and intensity varied extensively among tumor specimens, while these parameters correlated well in 5/7 tumors comparing #130 and #4350 antisera (Fig 5A and Fig 5B) Vascular endothelial cells showed very high staining intensity for both nestin antisera in all PNETs tested (Table 1).
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GFAP and S-100 expression were detected in a minority of tumor cells after antigen retrieval. The tumor cells showed cytoplasmic staining with a very high intensity in two of the samples tested. Endothelial cells were devoid of GFAP and S-100 immunoreactivity but stained positive for vimentin. In contrast, vimentin could not be detected in tumor cells of the two PNETs tested.
Glioblastoma Multiforme. Tumor tissue of a GBM located in the suprasellar region of a 14-year-old boy was used as a positive tumor control sample for the analyses of IFP expression. The histological picture was characterized by increased cellularity, mitoses, and endothelial proliferation. The tumor cells were plump, with abundant eosinophilic cytoplasm, and were devoid of astrocytic morphology. Areas of necrosis were present.
Nestin antiserum #130 stained the majority of tumor cells, whereas no staining was obtained with the #4350 antiserum before antigen retrieval. After antigen retrieval, the cell cytoplasm of most tumor cells, if not all, stained intensely with both nestin antisera (Fig 5C and Fig 5D). In addition, these cells showed strong staining for vimentin, GFAP, and S-100. Endothelial cells within the tumor tissue were stained for nestin and vimentin but not for GFAP or S-100 (Table 1).
Pilocytic Astrocytomas. Pilocytic astrocytomas were from children 712 years of age, of whom none was diagnosed with neurofibromatosis 1 (NF1). Four of five tumors originated from the cerebellum and one was located in the thalamus. Histologically, these astrocytomas were characterized by a biphasic pattern with microcysts filled with mucin, bordered by round astrocytes, and alternating with dense fibrillary areas composed of astrocytes with hair-like cell processes. One of the astrocytomas showed many Rosenthal fibers.
Nestin immunoreactivity of tumor cells was negative before antigen retrieval, whereas nestin #130 antiserum showed occasional staining of endothelial cells only. After antigen retrieval, both nestin antisera stained the vast majority of tumor cells at a low but consistent intensity (Fig 5E and Fig 5F). Tumor cells showed a delicate nestin immunoperoxidase staining of the cytoplasm with its hair-like cell processes, and staining pattern and intensity correlated well between the two nestin antisera (Table 1; Fig 5E and Fig 5F). The glial markers vimentin, GFAP, and S-100 were all expressed by tumor cells, whereas only nestin and vimentin were found in endothelial cells (Table 1).
Negative Tumor Controls. Five brain metastases of non-neuroepithelial origin were used as tumor control samples. All five were negative for nestin (Table 1). However, some reactive astrocytes surrounding two of these metastases were weakly nestin-immunoreactive, as were some endothelial cells in the metastatic tumors (not shown).
Nestin Immunostaining of Fresh Frozen Tissue
Freshly frozen tumor specimens were available for eight of the 20 tumors studied (Table 2). These samples were cryostat-sectioned, acetone-fixed, and processed for nestin IHC as described in the Materials and Methods. In all tissue sections, nestin antiserum #4350 stained tumor cells and endothelial cells without preparatory MW heating. Antigen retrieval further increased the intensity of nestin staining in most tissue sections studied, whereas MW heating had the opposite effect on antiserum #130 immunostaining. This antiserum gave weak immunoperoxidase staining of cryostat sections and failed to detect nestin in more than half of the tissue sections tested (Table 2).
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Discussion |
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Nestin is a commonly used marker for undifferentiated cells in the developing CNS and for CNS tumors. In this report we establish an improved protocol for immunohistochemical detection of nestin, based on antigen retrieval. Using this protocol, we show that nestin is abundantly expressed by tumor cells and endothelial cells of ependymomas and PNETs, whereas pilocytic astrocytomas showed low but consistent nestin expression by tumor cells in contrast to normal brain tissue.
Effects of Microwave Heating
Encouraged by recent reports on improved immunoreactivity of IFPs without loss of specificity by heat-induced antigen retrieval, we have evaluated the use of antiserum #4350 to identify nestin in formalin-fixed CNS tissue and tumors. Nestin antiserum #4350 (
Microwave heating greatly increased the sensitivity of both nestin antisera. Although being weakly reactive before heating, antiserum #4350 gave strong immunostaining of formalin-fixed embryonic human telencephalon, identical to that of nestin antiserum #130, after microwave heating (Fig 2). Therefore, antigen retrieval did not affect the staining specificity but gave increased sensitivity with improved signal-tonoise ratio optimal at a 1:2000 dilution of either nestin antiserum.
Although the exact mechanism of antigen retrieval is not yet understood, it is believed that the crosslinks between proteins are broken, which makes the antigen accessible to the antibody. Improved detection of several intermediate filament proteins in formalin-fixed brain tissue has been reported, including phosphorylated and non-phosphorylated NF (
Nestin and Vimentin Expression During CNS Development
Previous studies have shown that vimentin and nestin are concurrently expressed during rodent CNS development, from neural tube closure to the end of gliogenesis (
Nestin Expression in Brain Tumors
Two previous studies report on nestin expression in CNS tumors. Interestingly, their results differ despite the use of nestin antiserum #129 in both studies (
The study by
In view of these differences, the aim of this study was to establish an optimal protocol for nestin IHC and to analyze expression in primary CNS tumors. To this end, we used the #130 antiserum previously used and the recently developed #4350 antiserum against human nestin. With the #130 antiserum, we could confirm the previous findings of apparently low nestin immunoreactivity in formalin-fixed tumor tissue samples using a standard staining protocol. After MW heating for 10 min in acetate buffer, nestin immunoreactivity increased significantly without loss of specificity. The morphological resolution and low background staining were maintained. These findings are in agreement with the work by
Although no immunoreactivity could be detected by #4350 antiserum before MW heating of tumor sections, #4350 antiserum gave a weak signal, as analyzed by standard IHC of human fetal forebrain. This is probably a result of different processing of fetal and tumor tissue specimens. Whereas the fetal tissue specimens were processed immediately after fixation, tumor samples had been stored, paraffin-embedded, for 1 to several years. Extensive paraformaldehyde fixation and/or prolonged storage was reported to have a negative effect on antigenic sites and required extended heating for antigen retrieval (
A GBM tissue sample was included as a control sample in the tumor group because it had been reported previously to be high in nestin (
Nestin expression in astrocytic tumors has previously been considered to be restricted to astrocytomas of WHO grade II-IV (
Consistent nestin expression was also found in vascular endothelial cells of normal CNS tissue and in tumor samples (Table 1). Immunostained consecutive tissue sections of an anaplastic ependymoma, as shown in Fig 3, clearly show that endothelial cells of tumor vessels co-express nestin and vimentin, whereas they are negative for GFAP and S-100. Previously, endothelial cells positive for nestin were reported in primary CNS tumors (
With special reference to surgical pathology, we wanted to study the immunoreactivity of nestin in fresh frozen tumor specimens that had been cryostat sectioned and simply fixed with cold acetone. On this substrate, both nestin antisera could detect nestin without prior antigen retrieval. However, antiserum #4350 was superior to antiserum #130 with respect to identification and intensity of immunostaining (Table 2). Antigen retrieval by MW heating, as described for formalin-fixed tissue sections, could further improve the immunoreactivity of nestin as detected by the #4350 antiserum, whereas it had no or a negative effect on immunostaining by the #130 antiserum. This finding indicates the potential use of nestin antiserum #4350 for immunostaining of tissue smears and cryostat-sectioned tissue samples from stereotactic biopsies and open surgery to identify neoplastic CNS cells.
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Acknowledgments |
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Supported by the Swedish Medical Research Council, grants B96-13X-11570, K96-17R-12045 and K97-17F-12023, the Foundations of the Karolinska Institute, the Magnus Bergvall Fund for Medical Research and the Foundations of Sven Jerring, of Tore Nilson, and of Barncancerfonden.
Received for publication March 12, 2001; accepted October 3, 2001.
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