Its Origin and Morphological Pleomorphism

227 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῍.

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