Cell culture studies on human nerve sheath tumors

Pathology (1994), 26, pp. 29-32

CELL CULTURE STUDIES ON HUMAN NERVE SHEATH TUMORS KUSUM K H A R B A N D A , AMIT K. DINDA, CHITRA S A R K A R , ASIS K. KARAK, MEERA

MATHUR

AND SUBIMAL ROY

Department o f Pathology, A l l India Institute o f Medical Sciences, Ansari Nagar, New Delhi, India

Summary The main controversy about nerve sheath tumors (NSTs) has been their histogenesis, A Schwann cell origin has been proposed by many investigators for both schwannomas and neurofibromas. However Erlandson and Woodruff 1observed that while schwannomas appeared to be composed predominantly of Schwann cells, neurofibromas consisted of mainly perineurial cells. In addition, variable numbers of fibroblast-like cells and intermediate cells also have been reported in the 2 lesions. Whether these represent distinct cell types or variants of Schwann cells is still debatable. In an attempt to solve this controversy, the present study was undertaken to observe the morphology and the behaviour of these tumors in culture. These studies showed that all nerve sheath tumors are basically of Schwann cell origin and that intermediate cells and fibroblasts are variants of Schwann cell. Tissue culture studies done chiefly on schwannomas showed that the morphological features of schwannomas are preserved in 'in vitro culture' condition and therefore the difference between neurofibroma and schwannoma appears to be due to inherent differentiating property of the Schwann cells along with some environmental stimulus. Key words: Nerve sheath tumor, in vitro culture, S-100 protein, bromodeoxyuridine, labelling index, immunohistochemistry, phagocytosis, Schwann cell, intermediate cell.

(FLC) cells seen in cultures of neurofibromas were derived from Schwann cells, is a finding that has been further affirmed by the studies of Conley et al. 16 and Pettonnen et al. lr Our previous work on 102 cases of benign nerve sheath tumors (NST), studied with a combined approach of routine light microscopy, immunohistochemistry and electron microscopy, has suggested that all nerve sheath tumors are basically of Schwann cell origin. 18 As an extension of our previous work, nerve sheath tumors were subjected to in vitro studies to observe changes in morphological appearance, growth kinetics and phagocytic properties of cells in culture to resolve the controversy regarding the histogenesis of these tumors.

MATERIALS AND METHODS A total of 46 cases of NST from the cranial and spinal locations were studied (Table 1). Material was collected from surgically removed tissue from the Department of Neurosurgery. The age, sex, duration of symptoms and site of the tumor were recorded in each case. The tumor tissue was collected at the time of surgery in TC-199 medium for in vitro studies. Several fragments of tumor tissue were also fixed in 10% buffered formalin for routine histological studies.

Histology Accepted 10 August, 1993

INTRODUCTION There has been a lot of controversy regarding the origin of nerve sheath tumors which histologically have been divided into 2 distinct categories viz. schwannomas and neurofibromas which differ both in morphological appearance and in their biological behaviour, z-4 Schwannomas have been shown to consist predominantly of Schwann cells; in contrast, neurofibromas have been shown to consist of heterogenous cell population, including Schwann cells, perineurial cells and fibroblasts, together with scattered mast cells and phagocytic cells. 1,3-s A Schwann cell origin has been proposed by many both for schwannomas and neurofibromas. 2-4'9-12 Nakamura et a l ) 4 also put forth the hypothesis that the various cellular variants of neurofibromas may be immature cells of Schwann cell lineage since at later stage of neurofibroma, Schwann cells shared the main part of tumor proliferation. Tissue culture studies also corroborated the possibility that the 2 morphologically distinct cells i.e. Schwann-like (SLC) and fibroblast-like

The pieces fixed in formalin were routinely processed and paraffin embedded. Five micron thick sections were cut and stained by haematoxylin and eosin method for histological typing of tumor using WHO classification. 19

TABLE 1 Total cases Age range M:F Location

Schwannomas 1. Cranial Acoustic Cerebellar 5th cranial

2. Spinal Cervical Dorsal Sacral Neurofibromas

1. Cranial Orbit 2.

Spinal Cervical

46 9-62 yrs t:0.6 38 33 1

4 6 2 3 1

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Pathology (1994), 26, January

al.

Fig. 1 Phase contrast micrograph of a case of Schwannoma in primary explant culture depicting the morphological appearance of cells at the end of 3 days in culture showing both Antoni type A (arrow) and type B (arrowheads) cells (ttaematoxylin and eosin × 250).

Cell culture The tumor tissue brought in the tissue culture medium was thoroughly washed with phosphate buffered saline (PBS pH 7.4) and after removal of blood and electrocoagulated portion, it was cut into small fragments of about 1 mm 3 size. They were explanted - - 6 explants per coverslip. Four coverslips were inserted into one 25 cm 2 Nunc tissue culture flask containing TC-199 medium supplemented with 10% fetal calf serum, penicillin (t00 U/mL), streptomycin (100/~g/mL) and fungizone (0.25 ~g/mL) and HEPES buffer (10 raM). For each tumor, a minimum of 4 flasks each containing 4 coverslips was put up. The flasks were then incubated at 37 °C in 5% COJalr. The cultures were fed regularly depending upon the requirement in each case. The medium was gently decanted and replaced at time intervals ranging from 4 to 7 days. The following studies were done on the cultured cells. 1. Morphology The morphological features were noted daily using phase contrast microscope. 2. S-100 immunohistochemistry In all cases, one coverslip at a time was randomly harvested at wkly intervals from 2 flasks - - at the end of first, second and third wk respectively. The cells were thoroughly washed in PBS and fixed in acid alcohol for one hr at 4 °C. Subsequently, the coverslips were then stained by the avidin biotin peroxidase complex (ABC) technique 2° using polyclonal anti-S-100 antibody at a dilution of 1:500 with overnight incubation. The primary antibody was obtained from Dakopatt A/S, Denmark and the ABC reagents from Vector Laboratories, Burlingame, USA. Diaminobenzidine (DAB) was used as the chromogen and counter staining was done with haematoxylin. 3. Bromodeoxyuridine labelling index (BrdU-LI) In all cases, sequential BrdU-LI estimation was done at wkly intervals. For this, one coverslip at a time was taken out fi'om each of 2 flasks at the end of first, second and third wk. After harvesting, the coverslips containing the cultured cells were immediately transferred to a petri dish and exposed to 10 uM BrdU (Sigma) in the medium for one hr at 37 °C. Then they were thoroughly washed in PBS and the cells were fixed in chilled 70% alcohol for one hr at 4 °C. Subsequently, the DNA in the cultured cells was denatured by treating the coverslips with IN HCL at 60°C for 8 rains as detailedearlier, zx The coverslips were then stained by the avidin biotin peroxidase complex (ABC) technique2° using monoclonal anti-BrdU antibody at a dilution of 1:30 (Becton-Dickinson, Mountain View, USA). DAB was used as the chromogen and counterstaining was done using light haematoxylin and eosin stain.

Fig. 2 S-100 positivity of polygonal cell rich area of a case of neurofibroma at the end of second week in culture. Avidin biotin peroxidase (ABC) complex counterstained with light haematoxylin x 250.

4. Analysis On each coverslip 400-500 cells were randomly analyzed for the average S-phase fraction = i.e. labelling index (LI) which was expressed as the percentage of BrdU labelled nuclei to the total number of cells counted. 5. Phagocytic studies At the end of 2 wks in culture, the coverslips containing the cultured cells were exposed to 0.008% India ink in the medium for one h. Subsequently the cultured cells were thoroughly washed with PBS, fixed for 10 rains with 10% buffered formalin and stained with haematoxylin and eosin. 6. Transmission electron microscopy This was done on the centrifuged pellet of the cultured cells in 9 cases of schwannomas and one case of neurofibroma. For this, the cell pellet was fixed in 2.5 % glutaraldehyde, post-fixed in 1% osmium tetroxide, routinely processed and embedded in Epon. Ultrathin sections were examined under Philips TEM Model 301,

RESULTS

Clinicopath ological features The clinical features and histopathological diagnosis according to the W H O classification 19 of all the cases is listed in Table 1. Growth pattern Schwannomas showed a strikingly uniform growth pattern which was vigorous and abundant with aggregat i o n o f b o t h A n t o n i t y p e A ( e l o n g a t e d cells, b i p o l a r , w h o s e n u c l e u s c a u s e d t h e cell b o d y t o b u l g e a t its m i d p o i n t ) a n d A n t o n i t y p e B ( f l a t t e n e d cells s h o w i n g anastomozing multiple processes). This appearance was observed as early as 3 days in vitro and persisted t h r o u g h o u t t h e p e r i o d o f s t u d y ( F i g . 1). T h e 2 c a s e s o f n e u r o f i b r o m a s i n a d d i t i o n t o A n t o n i t y p e A a n d B cells a l s o s h o w e d s o m e l a r g e p o l y g o n a l cells.

S-I O0 immunohistochemistry S-100 positivity was shown both by Antoni type A and B cells. T h e s t a i n i n g w a s h o w e v e r m o r e i n t e n s e i n A t y p e cells. T h e l a r g e p o l y g o n a l cells i n n e u r o f i b r o m a s a l s o s h o w e d i n t e n s e S - 1 0 0 p o s i t i v i t y (Fig. 2). T h e p o s i t i v i t y

HUMANNERVESHEATHTUMORSIN CULTURE 31

Fig. 3 Photomicrograph depicting intense phagocytosis of India ink by both Antoni type A and B cells, x 250. persisted till the end of 3 wks in culture after which the cultures revealed degenerative change. A few cases where the culture persisted till the end of 4th wk in vitro still showed S-100 positivity.

Phagocytosis Both Antoni type A and B cells showed definite property of phagocytosis (Fig. 3). The polygonal cells in neurofibromas also showed evidence of phagocytosis.

BrdU-LI Akhough vigorous growth of cells was evident, the kinetic studies using BrdU showed very low labelling index (LI) ranging f r o m 0-0.5°7o at the end of first wk (Fig. 4). Only one case of NST (spinal sacral) showed a very high first wk BrdU-LI of 23.4°/0 (Fig. 5). The cells showed fibroblastic m o r p h o l o g y and an LI of 20.833% at the end of 2nd wk. In all other cultures, BrdU-LI was 0O7ofrom 2nd wk onwards. The cases of neurofibromas with the population of polygonal cells also showed very low labelling.

Transmission electron microscopy (TEM) studies Cultured cells showed dense bodies (lysosomes) in the t u m o r cells. Continuous basal lamina could be demonstrated in some of them. In addition, in one case of n e u r o f i b r o m a few cells showed discontinuous basal lamina while other cells resembled fibroblasts without any basal lamina. It was interesting to note that all cases except one (a typical case with high Brdu-LI) showed no regrowth/ repopulation on subculturing.

DISCUSSION The work was designed to resolve the controversy in the histogenesis of nerve sheath tumors. All the NST in culture showed admixture of Antoni type A and B cells. In addition, neurofibromas showed polygonal cells (fibroblast ?/intermediate ?) which showed intense S-100 positivity. These cells also showed evidence of phagocytosis. Ultrastructurally they appeared to be intermediate cells. Sharma et al. 18 have also observed intermediate cells in neurofibromas. In the present study, the S-100

Fig. 4 Photomicrograph of bromodeoxyuridinelabelled cultured cells of NST. The arrow indicates the positively stained nuclei. Note the low BrdU-LI at the end of first week in culture. ABC counterstained with light haematoxylin and eosin x 250. positivity of these intermediate cells in culture as well as their phagocytic property and ultrastructural features clearly indicate that these cells are variants of Sct-oatann cells. The cell kinetic studies in culture of NST revealed a BrdU-LI of less than 1 o70 which is in concordance with the reports of Lee et al. 22 on the in vivo infusion studies in intracranial neurilemmomas and coincides with the slow growth of these tumors. No significant difference of proliferative rate among the different morphological types of cells (Antoni type A and B and intermediate cells) was seen in in vitro culture. Thus the in vitro BrdU-LI may reflect the growth potential of individual tumors and clearly the one NST with very high LI needs to be closely followed up. To conclude, this study suggests that all NST are basically of Schwann cell origin and that intermediate cell and fibroblast are the derivatives of Schwann cell. Also, the in vitro BrdU-LI m a y reflect the biological behaviour of the tumor. In in vitro culture the morphological features of schwannoma and neurofibroma were relatively preserved along with the distribution of their constituent cell types. The property of morphological differentiation of the

Fig. 5 Photomicrograph of a case of spinal Schwannoma showing high BrdU-LI at the end of the first week of culture. ABC counterstained with light haematoxylin and eosin x 250.

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Pathology (1994), 26, January

Schwann cell into different cell types does not appear to alter in artificial culture condition. Therefore, it is possible that morphological difference between neurofibroma and schwannoma mainly depends upon the inherent differentiating property of the Schwann cells. However, the possibility of an added environmental stimulus cannot be completely excluded because Schwann cells have been shown to change their morphological forms depending on their association with a x o n s , za

11. Murray M, Stout A. Schwann cell versus fibroblasts as the origin of the specific nerve sheath tumours. Observations upon normal nerve sheath and neurilemmoma in vitro. Amer J Path 1940; 16: 41-60.

This stud), was funded by a grant from the Council of Scientific and Industrial Research, New Delhi, India.

14. Nakamura T, Mitsus H, Kasuga T. Early lesion of N-Nitroso-Nethylurea-induced hamster neurofibromatosis model. Can Res 1991; 51: 434-8.

ACKNOWLEDGEMENT

Address for correspondence: C.S., Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India.

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