Neu,,'ose/en~ Letters, 34 (1982) 315-320 Else der Scientific Publishers Ireland Ltd.

315

lrl~JH~ CELL LINF_,S FROM A SPONTANEOUS MURINE ASTROCYTOMA SHOW Y ARIATION IN ASTROCYTIC DIFFERENTIATION

•

O E ~ Y J. PlLKINGTON, PETER L. LANTOS, JOHN L. DARLING and DAVID G.T, : 'HOMAS

D e ~ of Neuropathology, Institute of Psychi~rtry, London SE5 8AF and Gough-Cooper Depart- me nt of Neurological S u ~ : Institme of Neurology, London WCIN 3BG (U.K.)

(Re~ved Ot,-tober 10th, 1982; Revised eersion receiw,,d November 15th, 1982; Accepted November 16tn,

1982)

Key words: cell lines - differentiation - astrocytoma - immunofluorescence - ultrastructure

Three cell lines origil~aUy derived from a spontaneous, transplantable, murine astrocytoma have been maintained in vitr3, lmmunofluorescence confirmed their astrocytic nature while electron microscc ?y revealed differen¢~ in the number and ratio of 10 mn f'daments and 24 nm microtubules in the 3 lines; a feature related to the degree of astrocytic differentiation.

Wilson [t3] has suggested that an ideal animal model for human glioma should be spontaneously arising, of glial origin, capable of intraparenchym~! growth, uniformly fatal within a reasonable time period, correlate with the therapeutic sen- sitivities of httman gliomas, capable of in vitro growth and be transplantable in- tracraldally arid subcutaneously in syngeneic animals. Wilson and Bates [14] have also proposed that the ideal model for chemotherapy studies should be 'a serially transplantable glioblastoma in the mouse'. Spontaneous brain tumours in mice are, however, n:ure, but Fraser [4] described anaplastic astrocytomas in 1.6o:0 of mice from the inbred VM strain; these tumours were capable of serial intracranial transplantation into syngeneic mice [5]. This model system however had two disad- vantages. The tumour could not be transplanted extracranially and, as ~rain hom~at~!:W~e~:for~ ~ transplantation, the system could not be quan- .~~.~:~ ~~ihav¢ ~ recently overco, me by Scrano et al. [12] who ~:~~!!~ii~:~: ifmmlthis sPontaneousl murine astrocytoma (SMA). Line ~ p ~ : ~ ~ ' s h - ~ i l a ~ h o m o g e n i z a t i o n , t .r~psinizatio~ and subsequent ~ ! ~ f i ~ ~ ~ : ~ O i " an SMA-¢ontaining mo~e bra~n. Ceils from t ~ i i e ~ ~ - ~ / i a ~ : ~ ~ t 0 n e a l l y i n t o ~ 'ageneic animals at 30 day inter- ~ii~i~ i i ~ ~ d ~ i ~ ! 0 . ! 1 4 , " - - - - - - 0 . 2 5 and •0'33 brain equivalents (one brain ~ ~ ! ~ ~ : ~ i ~ : t i s s t ~ : from the cerebral hemispheres of the SMA-

~ ) ~ i : ~ r ~ ~ia 45.5e70 incidence of tumour nodules, subsequent culture of which led to ~he establishment of line VMDK P540. Cell suspensions from the i.p. tumour nodules were also injected s.c. into syngeneic mice and gave a 100%

0304.3940/82/0000-{RR~/$ {}2.75 © 1982 Elsevier Scientific Pub:ishers Ireland Ltd.

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tumour incidence. Culture of cells from these neoplasms gave rise to line VMDK P560. Intracerebral inoculation of lines P497, P540 and P560 into syngeneic mice gave rise to infiltrating astrocytomas in 80, 100 and 100¢/a of cases respectively [i2]. Since biochemical assays for various 'glial markers' employed by Serano et al. [12] faile6 to establish unequivocally the glial nature of the 3 lines, we carried out im- munofluorescent studies using two astrocyte-specific markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), and examined their ultrastructural characteristics with special reference to the incidence of 10 nm glial filaments, a reliable guide to the degree of astrocytic diffc.reatiation [7].

Cells were maintained in Ham's Fl0 medium contailfing 10070 foetal calf serum and buffered with 20 mM HEPES solution (complete medium), supplemented with penicillir (50 IU/ml) and streptomycin (50 mg//~l). For this study the cell lines P49"/, P540 and P560 were examined at in vitro passages 39-47, 19-29 and ~5-16 respec- tively. Cells grown on coverslips for immunofluorescence :;tudies were washed twice in Hunk's buffered saline solution (HBSS), fixed in acid alcohol for l0 min and washed in phosphate-buffered saline (PBS). Rabbit anti-ovine GS at a 1/100 dilu- tion, or rabbit anti-bovine GFAP at 1/500, was applied for 1 h at room temperature. "[he procedure for raising the GS antibody has been described elsewhere [11]. After washing off the primary antibody swine anti-rabbit immunoglobulin conjugated to rhodamine (TRITC) or fluorescein (FITC) was applied for a further 1 a. The coverslips were then washed in PBS, mounted in :;0°70 glycerol and examined with

Zeiss photomicroscope Ill, incorporating reflected light fluorescence excitation and barrier filters for TRITC and FITC.

Fcr eIectron microscopy cell monolayers, when confluent, were removed from thei, culture flasks by washing twice in HBSS and treatment with 0.25070 w/v trypsin (1/300). Cells were resuspended in comple',e medium and then centrifuged at 100 r.p.m, for 5 min. Surplus medium was removed by resuspension in HBSS and fur- ther centrifugation prior to fixation in one-half strength Karnovsky fixative [6] for 20 rain. Secondary fixation in l~0 osmium tetroxide in 0.1 M cacodylate buffer at pH 7.4 for 15 rain was carried out after washing in buffer. A final wash in buffer

: ~ . ~ " ; . . . . I . - . ! ! . [

' I

• T ' : , . . ' ~, ~ . . .. • . .

~ - ~,_. , : !

.I !

I !

O ,¢ . ~ _

Fig. !. Po.qiti~ e immunofluorescence staining in the cytoplasm of cells from line VMDK P540. a: rabbit anti-GFAP serum (FITC) x 450. b: rabbit ~,nti-GS serum (TRITC) x 450.

317

was followed by processing through ascending grades of alcohol (50°7o, 75°70, 80V0, 90°70 and 2 x 100070 - 10 rain each), absolute alcohol/Spurr resin mixture (1 h) and fresh Spurt resin (3 h). The cells were centrifuged at each stage and resuspended in the appropriate solution. Polymerization was carried out at 60°C for 8 h. Sections for electron microscopy were cut and stained with uranyl acetate and lead citrate before examination in an H600 electron microscope (Hitachi).

Immunocytochemical studies revealed a positive cytoplasmic fluorescence of all 3 VMDK cell lines with both GFAP (Fig. la) and GS (Fig. lb) while nuclei remained negative.

Each line showed an homogeneous population of cells upon fine structurai ex- amination (Fig. 2). All cells shared certain features, including a aigh nuclear-cytoplasmic ratio and irregular ~uclei wi:h evenly distributed chromatin and ch:mped peripheral heterochromatin. Their cytoplasm contained numerous

Fig. 2. Ce!ls from line VMDK P560 are an homogei~eous population with irregular nuclei, x 4800.

318

mitochondria, polyribosomes, lipid droplets, occasional lysosomes and coated ve;icles at the cell membrane. Golgi bodies and rough endoplasmic reticulum cister- nae were present and often well developed in lines P540 and P560. Short irregular processes characterized the cell surface. While these common features were shared by the 3 cell lines, the most striking fine structural difference was in the number and distribution of 10 nm filaments ate5 24 nm microtubules. A count of 1000 cells from each line revealed the presence of filaments in 15%0 of P497, 18% of P540 and 98% of P560 cells (Fig. 3). However, while few cells in lines P497 and P540 contained filaments the cytoplasm displayed large numbers of microtubules (Fig. 4) which were only occasionally seen in line P560.

The cytosl:eletal protein GFAP and cytoplasmic enzyme GS are both astrocyte- specific markers in normal and neoplastic brain [1, 91. The positive identification of these two markers in all 3 VMDK cell lines therefore provides im- munocytochemical confirmation of their astrocytic nature. Fine structural examina- tior. of cells provides an invaluable approach to the characterization of normal and neoplastic glial cells in vitro. Comparison of the fine structure of normal and neoplastic astrocytes in vitro has shown only quantitatiue differences [2, 3, 8]. The present study shows that the 3 VMDK cell lines examined, P497, P540 and P560,

Fig. 3. Numerous 10 nm filaments (f) are present in a cell from line VMDK P560. N, Nucleus. × 67,500.

3!9

possess the fine structural features of astrocytes, particuhtrly the presence of 10 nm f'darlents. In addit 'on, all 3 neoplastic astrocytic cell lines contain microtubules, but the ratio of microtubules to filaments varied substantially between the lines, it has beer~ demonstrated in ethylnitrosourea-induced gliomas irt tats that poorly differen- tiated neoplastic astrocytes contain many microtubules but few filaments, while there is a reduction in the number of microtubules witl~L a concurrent increase of filaments in the more differentiated astrocytic cells [7]. Thus the ratio of these organelles seen in the VMDK cell lines may reflect the degree of differentiation, with P560 being the best differentiated. However, the possibility canno~ be excluded that this finding may be due to the lower in vitro passage level of the P560 cells examin- ed. l)iff~zrentiati,~n may be lost with in vitro passaging, therefo:'e cells c,f higher

Fig. 4. Several microtubules (arrows) are present in the cytoplasm of a cell from line VMDK P540. N,

Nucleus. × 7'I,000.

32~

passage number show less differentiated features. A l ~ e l y , the ,~,.'ffere~a:es seen in the degree of cellular m a m ~ i o n may be the result of the different methods of derivation of the 3 cell lines.

This fme structural and Lmmunocytocbemical study has therefore revealed an astrocytic nature of the 3 VMDK cell lines examined. The f'flament to-microtulmle ratio reflects the degree of differentiation in vitro and therefore is a useful indk~___m_ of malignancy of tumours in vivo, produced by intracerebral injection of these cell lines into syngeneic mice. Although the cells display distinctive astrocytic features by morphological and immunofluorescent examination, a comprehen.qive cell biolo- gical and immunocytochemical survey is now in progress in order to characterize fully the celh from the 3 astrocytoma lines both in vivc and in vitro.

The authocs would like to thank Dr. D.D. Bigner, who kindly supplied the ~ cell lines for this study, and Dr. B. Anderton and Dr. J. Kalm for the gift of the GFAP antiserum. "l*his work was supported by grants from the Bethlem Royal Hospital- Maudsley Hospital Research Fund (G.J.P. and P.L.L.) and the Brain Research T¢ust and the Cancer Re,.~arch Campaign (LL.D. and D.G.T.T.).

! Bignami, A., En8, L.F.. Dahl, D. and Uyeda, C.f. , Loc~tza,~km of the gl~d flbrillm3t acidic protein in astrocyles by immunofluore~m~, Braip g a . , 4J (1972) 4,~:9-4J5.

2 Colliiis, V.P., Brunk, U.T., Fredriktson, B.A. and Wc~,rmttk, B., The fine structure of growing humar glib. and glioma cells. "~i, ole cell preparations, Acta path. microbiol. ~ . , ~'/A (1979) 29-36.

3 Collins, V.P., Forsby, N. Brunk, U.T. E ~ n , J.L.E. and We~crmark, B., Ultrastructural features of cuhurea hur:)an g~fa and glL~ma cells, Acta path. mlo'oblol. Icand., 87A (19"/9) 19-28.

4 Fraser, H., Astrocytomas in an inbred mouse grain, J. Path. 103 (1971) 266-290. 5 Fraser, H., Spontaneol. s a~tro ~ o m u in inbred mice: serial tramunl~ion studies with intact cells. In

St. Kornyey, S. Tariska and ¢3. Gosztonyi (IF.&.), Proc. Vllth Int. Coast. of Neuropmh., Vol. 1, Budapest, Excerpta M~dica, Amsterdam, 1974. pp. 491-494.

6 Karnovsky, M.J. A formald~'lyde-81utaraldehyd¢ fumtive of high om~b~ity for use in electron rr'.!croscopy, J. Cell Biol., 27 ~196~5) 137A-IJEA.

7 lantos, P.L., The distribution and role of micr~ubules and fdannamts in the neoplastic mtlocytes of experimental gliomas, Neuropath. appl. Neurobiol., 3 (197"/) 281-296.

8 l~4acintyre, E.H., Pont~n, J. and Vatter, A.E., The ultrmtmctur¢ of hmnan and routine mtrocytes and of human fibrobl~sts in culture, Acta path. microb/oi, stand., 80A 09"]2) 267-283.

9 Notenberg, M.D., Th<- distribution of glutamine syntheta~ in the rat central nervom system, J. Histcehem. Cytochem. 27 (19',~) 756-762.

10 Peters, A., Palay, S.L. and Webster, H. de F. (Eds), The Fine Structure of the Nerv~r~ System. The Neuroglial Cells, Saunders, Philadelphia, 19"/6, pp. 231-26~.

11 Pilkington, G.J. and Lantos, P.L., The role of 81utamin¢ s]mtheta~ in the diagnom of cerebral tumours, Neuropath appi. Neurobiol., 8 (1982) 22/-236.

12 Serano, R.D., Pegram, C.N. and Bigner, D.D., Tumorigenic cell ¢~ure lines from a spontaneous VM/DK murine astrocytorna tSMA), A-'~a neuropath., 51 (1980) 53---64.

13 Wiisen, C.B., in Biology of Brain Tumors, UICC Technical Rel~rt $¢ri~ Vol. 30, International Union Against Cancer, Geneva, 1978, pp. ~8~-199.

14 Wilson, C.B. and Bates, I:.A., Transplantable brain tumors, in W.M. Kirsch, E. Grossi-Paolettl and P. Paoletti (Eds), The Experimental Biology of Braiin Tumors, Thomas, Springfield, IL, 1972, pp. 19-56.