Its Origin and Morphological Pleomorphism

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St. Marianna Med. J. Original Article Vol. 32, pp. 227῎234, 2004

Gliosarcoma: Its Origin and Morphological Pleomorphism

Hiroo Kobayashi1, 2, Takeshi Sakiyama1, Akio Kazama1, Hirotaka Koizumi1, Masayuki Takagi1, and Mamoru Tadokoro1

ῌReceived for Publication: August 9, 2004῍

Abstract The histogenesis of gliosarcoma has been concerned in that the sarcomatous component is of vascular origin, or fibroblasts, pluripotent mesenchymal cells of the perivascular adventitia or perivascular spaces, or a suggestive common origin. Recent molecular-biological analysis, however, makes one to consider the possibility of this tumor originating from common precursor cells. To clarify the histogenesis of this tumor, an experimental study using nude mice with transplanted human glioblastoma cells was carried out. The grown tumor demonstrated fibrosarcomatous features, di#ering from those of originally cultured glioblastoma cells, except for some remaining cells with positive GFAP immunoreactivity and intracytoplasmic intermediate-sized glial filaments on electron microscopy. The presence of Alu sequence in the grown tumor proved that the tumors were composed of human originated tissue. In addition, DNA restriction fragment length polymorphism analysis showed the transplanted tumor consisted of human cells. These results indicated that the fibrosarcomatous lesion of glioblastoma exografts in nude mice was derived only from originally cultured human glioblastoma cells, but not from the recipient mouse. It may thus be assumed that human gliosarcoma showing fibrosarcomatous features might have originated from the same totipotential cells.

Key Words gliosarcoma, glial ﬁlament, nude mouse, Alu sequence, RFLP,

histogenesis of this tumor has been concerned in Introduction that the sarcomatous component is of vascular ori- Glioblastoma is a poor prognostic brain tu- gin, or fibroblasts, pluripotent mesenchymal cells of mor, occupying one-third of primary brain tumors. the perivascular adventitia or perivascular spaces, The average life expectancy after diagnosis is about or a suggestive common origin2῎6῍. Recent molecu- one year. Histopathologically, the tumor shows lar-biological analysis, however, makes one to con- various characters and features such as heteromor- sider the possibility of this tumor originating from phism, pleomorphism, and sometimes even forming common precursor cells7῎10῍. From this point of view, a sarcoma. Gliosarcoma among them is defined as a an experimental study using nude mice transplanted glioblastoma variant characterized by a biphasic human glioblastoma cells is scheduled to prove the tissue pattern with alternating areas displaying glial histogenesis of the tumor, and to establish the ani- and mesenchymal di#erentiation according to the mal model of human glioblastoma. new World Health Organization ῌWHO῍ classific- ation, and corresponding to WHO grade IV1῍.The

1 Department of Pathology, St Marianna University School of Medicine 2 Hospital of Ishioka Neurosurgery

9 228 Kobayashi H Sakiyama T et al

tions were stained with hematoxylin-eosinῌHE῍ and Materials and Methods reticulin silver impregnation. Immunohistochemis- Animal model try on para$n section was performed with a strep- Male nude mice ῌBalbῒc: nuῒnu῍ at 5 weeks of toavidin-biotin staining kit ῌDAKO, Glostrup, age were purchased from Japan Clea Laboratory. Denmark῍ using diaminobenzidin ῌDAB῍ as the The mice were fed with autoclaved water and steril- chromogen. The sections were treated with anti- ized feed by irradiation ῌCE-2, Japan Clea Lab. GFAP antibody ῌDAKO, Glostrup, Denmark῍ at Tokyo, Japan῍ and kept in box fixed by autoclaved 1 : 4000 working dilution, and visualized by DAB. chips of wood in the filter conditioned Isolac ῌSanki Samples for transmission electron microscopy ῌH- Kagaku Inc., Kanagawa, Japan῍. Sixteen mice were 7500, Hitachi Ltd., Tokyo, Japan῍ were fixed with xonografted. Additional 6 mice were xenografted 2.5ῑ glutaraldehyde and osmium acid, embedded for short term e#ects of transplantation. Mice were in Epon 812 ῌNakarai, Kyoto, Japan῍, and ultrathin sacrificed under ether anesthesia, approved by the sections were made. institution[s guide for the care and use of laboratory animals. Molecular analysis and in situ hybridization The tumor was frozen under liquid nitrogen, Cell culture of glioblastoma multiform and xeno- and BIOFOOD identification kit ῌBIOTOOLS B& grafted glioblastoma M Labs, S.A., Spain῍ was applied to distinguish Glioblastoma cell line ῌU-118 MG῍ was pur- human DNA from mouse DNA. The process was chased from American Type Culture Collection. done following the manufacture[s protocol. Human The cells were cultured in MEM ῌminimum essen- control DNA was freshly isolated from leukocytes tial medium, Invitrogen Corp., CA, USA῍ contain- of a healthy donor and mouse DNA from liver of ing 10ῑ fetal calf serum in the CO2 incubator under an experimental animal. DNAs of the transplanted the condition of 5ῑ concentration. The cells were tumors were obtained from formalin-fixed and par- cultured in 75 cm2 flasks and on slide chambers at a$n-embedded sections through needle dissection the same time. The cells in flasks were collected by ῌLaser Microdissection CRI 337, Cell Robotics, rubber scratch and divided for xenograft and for Inc., NM, USA῍. A 359-bp DNA product was am- electron microscopic findings. The cultured glio- plified by PCR program using special restriction blastoma cells on slide chamber, on the 1st,3rd,4th, enzyme ῌEnzyme 3῍, resulted in the di#erent gel and 7th day were stained immunohistochemically pattern between human DNA and mouse DNA in a with GFAP. The xenografted lesions were exam- species-specific manner. The formalin-fixed, par- ined on the 3rd and 7th day after subcutaneous injec- a$n embedded sections were used for in situ hy- tion of 1῎107 cells in 100 ml of PBS into the back. bridization for detecting Alu sequence in the nuclei Xenografted human glioblastoma tumors were es- of transplanted tumor cells. The slides were micro- tablished in nude mice by subcutaneous injection of waved in citrate bu#er for 5 min, prehibridized with 2῎106 cells in 100 ml of PBS, into the back of 16 salmon sperm DNA, and add probe ῌAlu DNA, individual mice11῍. The grown tumors were meas- ISH probe, PR-1001ΐ01ΐ2601, Inno Genex, CA, ured on the 25th day and following every week up to USA῍. Heat the slide to 85ῌC for 10 min and incu- on the 25th week after transplantation, and meas- bate in a moist chember at 37ῌC for overnight. Post- ured tumor size at 25 days, 18 weeks, and 25 weeks hybridization wash and detection according to the after transplantation. Grown tumors on the 18th manufacture[s manual ῌInno Genex, CA, USA῍ week after transplantation were examined for were done and a kit ῌDAKO, Glostrup, Denmark῍ pathological findings and molecular analysis. The was used with minor changes. purchased U-118 MG cells ῌpassage number 446῍ Results were used at passage 446ΐ450. Tumor growth and size Pathology and Immunohistochemistry The transplanted glioblastoma cells prolifer- The xenografted lesion on the 3rd day and the ated to form a solitary tumor in the subcutaneous grown tumor on the 18th week after transplantation region on the back of 14 in 16 mice, measuring 2῎ were fixed in 10ῑ bu#ered formalin ῌpH 7.0῍ and 2 mm up to 5x5mm ῌ2.9ῐ0.9῎3.4ῐ0.9 mm; n῏ embedded in para$n. The para$n-embedded sec- 14῍,4῎4mmupto18῎22 mm ῌ12.5ῐ4.0῎16.5ῐ

10 Gliosarcoma, origin and pleomorphism 229

Fig. 2. Electron microscopically, cultured glioblastoma Fig. 1. Cultured glioblastoma cells show positive cells show intermediate-sized filament: glial immunostaining with GFAP ῌb, x200῍, but are filament. Inserted white square line under low not immuno-reactive to normal human fibro- magnification ῌa῍ was magnified to identify the blasts ῌa, x200῍. glial filament at high magnification ῌb῍.

4.4 mm; n῏10῍, and 4῎4mmupto33῎28῎26 invasive character of glioblastoma multiform was mm ῌ14.3ῐ2.5῎16.5ῐ3.1 mm; n῏5῍ in size at 25 preserved although its morphological feature was days, 18 weeks, and 25 weeks after transplantation, fibrosarcomatous. In addition, electron micro- respectively. scopically, there were a few cells with a bundle of intracytoplasmic intermediate-sized glial filaments, Pathologic features of the tumor which was direct proof of glial origin, or partially The cultured glioblastoma cells ῌU118 MG῍ remaining elements of glial filament among the cells showed positive GFAP immunostaining on whole with hardly any remnant of glial filament ῌFig. 4῍ at experimental day ῌ1st to 7th cultured day῍, but the 18 weeks after transplantation, which indicated the cultured control human fibroblasts were negative remnant of the original character of oligoblastoma ῌFig. 1῍. Also, there were intracytoplasmic interme- even in fibrosarcomatous cell appearance. diate-sized filaments ῌglial filament῍ on electron microscopy ῌFig. 2῍. Molecular aspects of the tumor The xenografted lesion was hard to notice as a The results from the existence of Alu sequence tumor on the 3rd and 7th day. However, histopathol- in the formalin-fixed, para$n-embedded tissue ogically, the region transplanted after 3 days pre- specimen of grown tumor proved that the tumors served the features of glioblastoma keeping GFAP were composed of human originated tissue ῌFig. 5 positive staining and with focally appearing re- a῍. In order to identify whether the components ticulin fibers in the stroma. originated from only human glioblastoma or not, All established tumors were whitish pink and the result of restriction fragment length polymor- relatively firm with homogeneous fibrous tissue like phism ῌRFLP῍ helped to identify human and mouse sarcomatous appearance from 4 to 25 weeks after tissue using a 359-bp DNA product amplified by transplantation. They showed smooth and circum- PCR and digested to specify the species-specific scribed cut-surface, grossly, without massive adhe- restriction length pattern. The RFLP analysis sion and invasion to the host tissues, except for a showed di#erent pattern of digested DNA length few adhesive lesions. Histologically, the tumor from human and mouse ῌFig. 5b῍. The restriction demonstrated fibrosarcomatous features, losing length patterns of the transplanted tumors were positive GFAP immuno-reactivity resulted in hard identical with human DNA without mixture of any to find and demonstrated increased reticulin fibers mouse restricted DNA fragment. Therefore, there at 18 weeks after transplantation ῌFig. 3῍. However, might be no mixture or less detectable sensitivity of a few positive GFAP immuno-reactive cells were mouse tissue in the transplanted and formed tumor. found in the muscle layer, which indicated that the

11 230 Kobayashi H Sakiyama T et al

Fig. 3. The histological feature of xenografted lesion in the back of nude mouse at 3 days ῌa, b, c, x100῍ and grown tumor at 18 weeks ῌd, e, f, x100῍ after transplantation. The fibrosarcomatous features are shown by HE staining ῌa, d῍. The preserved feature of glioblastoma with positive GFAP staining was noticed ῌb: arrows denote typical positive cells῍, and also showed scattered reticulin fibers ῌc῍ on the 3rd day. However, minimal remaining original character of glioblastoma with positive GFAP immunoreactivity ῌe῍ is shown among the fibrosarcomatous pattern. On the contrary, characteristic fibrosarcomatous features were clearly demonstrated by silver stained reticulin fibers at 18 weeks after transplantation ῌf῍.

Fig. 4. Inserted arrow area on low magnification ῌa῍ was magnified to identify the glial filament at high magnification ῌb῍. Electron microscopically, a few fibroblastic cells showed bundles of intra-cytoplasmic intermediate-sized filaments: glial filament ῌb῍, which indicated that the cell originated from glial cell at 18 weeks after transplantation. Coincidently, among numerous tumors cells ῌc῍, partial glial components ῌarrow; magnified inset at the right῍ were hardly noticed.

12 Gliosarcoma, origin and pleomorphism 231

Fig. 5. Alu sequence is found as small dots ῌarrow῍ in the nucleus of transplanted tumor cells by in situ hybridization ῌa, x200῍. RFLPs analysis of transplanted tumors. Enzyme 3 in a BIOFOOD kit digested a 359-bp PCR product in a species-specific manner; 198῏161 and 315῏44-bp fragments in humanῌarrows῍ and mouseῌarrow head῍, respectively. Two representative tumors ῌT1, T2῍ obviously showed human specific RFLP profile ῌb῍.

region17῍. Both hypotheses for the sarcomatous com- Discussion ponents have been concerned with the mixture of Glioblastoma, the most frequent malignant neuro-originated ectodermal glioblast and meso- brain tumor shows various characters and features dermal sarcoma. However, recent molecular and such as anaplasia and dedi#erentiation and some- biological analysis, such as common mutation of p times even form a sarcoma. Recently, glioblastoma 53 in both components and resemblance to chromo- has been classified into two types, primary glioblas- somal abnormality, makes one to reinforce the con- toma and secondary glioblastoma, lead by the di#e- sideration for the possibility of this tumor to origi- rent genetic pathways with little overlapping1῍. Glio- nate from common precursor cells5ῐ9῍. Further ge- sarcoma is comprised in approximately 2 to 8῎ of netic analysis of both primary gliosarcoma and a total glioblastomas1, 10, 12, 13῍. It is morphologically de- secondary one strongly supports the concept of a fined as a glioblastoma variant, corresponding to monoclonal origin of gliosarcomas by identical ge- WHO grade IV1῍. It was described originally in 1895 netic alterations in both gliomatous and sarcoma- by Strobe et al and defined as a glioblastoma sub- tous components10῍. From this point of view, the type by Feigin et al10, 14῍. Gliosarcomas are morpho- previous cases in humans can be classified morphologically characterized by a biphasic tissue pattern, logically into three di#erent phenotypes, such as showing a glial di#erentiation pattern and a mesen- anaplastic sarcomatous cells proliferating around chymal one1῍. It has been hitherto widely accepted vessels mixed with gliomatous cells, existence of a that the histogenesis of this tumor has been con- fibro-sarcomatous tumor between glioblastoma and cerned in that the sarcomatous component is of meninx, and mingled and mixed fibro-sarcomatous vascular endothelial cell and pericytic origin, or of components of glioblastoma and sarcoma17ῐ19῍. the potential cell origin from the mesenchymal tis- Our experimental study using nude mice trans- sue component of the meninx close to the planted with human glioblastoma cells may give a glioblastoma2ῐ6, 14, 15῍. The former hypothesis was sup- direct proof to solve the histogenesis of the gliosar- ported by experimental work of transplantation of coma. The experimentally established tumor, origi- glioblastoma multiform fragments obtaining the nating from human glioblastoma multiform, formation of endothelial sarcoma16῍. The latter hy- showed the third type feature of gliosarcoma; fibr- pothesis was supported by the study of the expres- osarcoma, which could not be discriminated from a sion of a-smooth muscle actin in the sarcomatous glioblastoma component and a fibrosarcoma one.

13 232 Kobayashi H Sakiyama T et al

In the experimentally established gliosarcoma, or not. Various cytokines are expressed in brain however, it was necessary to solve the question tumor, particularly in gliomas. They may be in- whether the fibrosarcoma component originating volved in tumor proliferation, angiogenesis, and from human only or penetrated and mingled with immune surveillance evasion23῍. Further investiga- fibroblasts originating from mouse to form a sar- tion including various cytokines and growth fac- coma. From the previous report on human tumor tors, using this established animal model of human xenograft, it was uncertain whether the tumor was glioblastoma, may clarify the mechanism to form only originating from human or mixture with fibr- fibrosarcoma among the tumor lesion of glioblas- oblasts of mice. They were established by direct toma. transplantation from human tumor itself, or once Acknowledgements cultured cells transplanted into nude mice and its produced tumor were cut into small pieces ῌ2ῐ2῏3 We are grateful to Ms S. Ohnuma and Ms C. ῐ3mm῍ and inoculated in nude mice again20, 21῍. Hashimoto for their technical assistant, and to visit- Also, when the grafted tumor in nude mice was ing Professor Dr Eisei Ishikawa for his valuable introduced into the culture system, it was known suggestion. that human tumor cells were overwhelmed by the References fibroblasts originated from mice22῍. Our results indicated that the fibrosarcomatous 1῎ Ohgaki H, Biernat W, Reis R, Hegi M and component did not originate from the recipient Kleihues P. Gliosarcoma. In: Kleihues P and mouse, but from only human. The molecular study Cavenee WK ῌeds.῍, Pathology and genetic of for the discrimination from human and mouse tis- tumors of the nervous system, 2nd ed., IARC sue gave the solution. Before the di#erentiation to Press, Lyon, 2000: 42ῒ44 form a fibrosarcoma in the nude mouse, the cul- 2῎ Schi#er D, Dutto A, Cavalla P, Mauro A and tured glioblastoma cells maintained the character of Migheli A. GFAP, FVIIIῑRag, laminin, and glial cells, proven by positive GFAP staining and fibronectin in gliosarcomas: an immunohisto- keeping glial filaments electron microscopically. chemical study. Acta Neuropathol ῌBerl῍ 1984; However, after transplantation the proliferating tu- 63: 108ῒ116 mor cells lost almost all characters of glial cells. 3῎ Kochi N and Budka H. Contribution of histio- Only a few cells kept their original character, deter- cytic cells to sarcomatous development of the mined by a few positive GFAP cells and character- gliosarcoma. An immunohistochemical study. istic electron microscopical finding of glial cell. Acta Neuropathol ῌBerl῍ 1987; 73: 124ῒ130 Most cells lost the characteristic glial filaments or 4῎ Grant JW, Steart PV, Aguzzi A, Jones DB and partially retained them losing their original charac- Gallagher PJ. Gliosarcoma: an immunohisto- ter. From our results, however, xenografted grown chemical study. Acta Neuropathol ῌBerl῍ 1989; tumors were of human origin. And the result 79: 305ῒ309 proved to establish the animal model of human 5῎ Meis JM, Ho KL and Nelson JS. Gliosarcoma: glioblastoma for further investigation. In addition, a histologic and immunohistochemical rea$r- it may be assumed that gliosarcoma showing fibr- mation. Mod Pathol 1990; 13: 19ῒ24S. osarcomatous features in human might have origi- 6῎ Jones H, Steart PV and Weller RO. Spindle- nated from the same totipotential cells. At present, cell glioblastoma or gliosarcoma? Neuropathol there are three hypotheses; vascular pericyte origin, Appl Neurobiol 1991; 17: 177ῒ187 mesenchymal cell origin, and precursor cell origin. 7῎ Albercht JH, Connell JM and Brunner JM, The possibility of several growth factors surround- Distribution of p53 protein expression in glio- ing the glioblastoma cells might a#ect these three sarcoma: an immunohistochemiocal study. di#erent phenotypic expression, in that when close Acta NeuropatholῌBerl῍ 1993; 85: 222ῒ226 to the blood vessels they might be a#ected to lead to 8῎ Biernat W, Aguzzi A, Sure U, Grant JW, Klei- endothelial proliferation and when close to the hues P and Hegi ME. Identical mutation of the meninx they might be a#ected to lead to mesenchy- p53 tumor suppressor gene in the gliomatous mal proliferation between the glioblastoma and and the sarcomatous components of gliosarco- meninx, and fibroblastic di#erentiation. It is still mas suggest a common origin from glial cells. J unclear whether these hypotheses exist individually Neuropathol Exp Neurol 1995; 54: 651ῒ656

14 Gliosarcoma, origin and pleomorphism 233

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