Establishment and characterization of nine human brain tumor cell lines

Dear Editor: The most common type of primary brain tumor is glioma, which includes astrocytomas, oligodendrogliomas, ependymomas, anaplastic astrocytomas, and glioblastomas (GBLs). GBL is the most frequent and malignant brain tumor and accounts for 12-15% of all primary brain tumors (Zulch, 1986). Oligodendroglioma accounts for 4.2% of all primary brain tumors (Mork et al., 1985). GBL has been cultured in vitro, and several human GBL cell lines have been reported recently (Manuelidis, 1965; Collins, 1983; Bakir et al., 1998; Perzelova et al., 1998). However, a very limited number of oligodendroglioma cell lines have been established and characterized. The abundance of established brain tumor cell lines and the diversity among them are of great advantage to cancer research. Well-characterized human malignant brain tumor cell lines can contribute significantly to the understanding of the biology of human brain tumors in many fields. Herein, we describe the characteristics of newly-established human brain tumor cell lines including six GBL (SNU-201, -444, -466, -489, -626, and -1105), two oligodendroglioma (SNU-738 and -791), and one primitive neuroectodermal tumor (PNET) (SNU-1118) cell lines. The cell lines were established from pathologically proven brain tumors (Table 1). Solid tumors were finely minced with scissors and dissociated into small aggregates by pipetting. Appropriate amounts of fine neoplastic tissue fragments were seeded into 25-cm 2 flasks. Tumor cells were initially cultured in ACL-4 medium supplemented with 5% heat-inactivated fetal bovine serum (AR5). The AR5 medium has proved useful in the establishment of human cancer cell lines (Park et al., 1995; Ku et al., 1997). The composition of AR5 medium has been described previously (Park et al., 1995). Once established, the cultures were maintained in Roswell Park Memorial Institute 1640 medium (GIBCO/BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum. Cell cultures were maintained in a humidified incubator at 37 ~ C in an atmosphere containing 5% CO2. We began to establish human brain cancer cell lines in 1989 and have obtained a culture success rate of 26% (9 in 34 trials). All tumor lines grew as monolayers and showed high viability (85-95%) with varying doubling times (48-92 h) and absence of mycoplasma and bacteria contamination. Deoxyribonucleic acid (DNA) fingerprinting using two highly polymorphic microsatellite markers showed that the newly-established brain tumor cell lines were unique and unrelated (Fig. 1). These data excluded any possibility of cross-contamination between the cell lines. Clinical and morphologic characteristics are summarized in Table 1. The tumor cells from GBLs showed nuclear augulation, hyperchromatism, and pleomorphism. Mitoses were frequently noted, and vascular proliferation was prominent. Necroses were extensive in all tumors. In the original tumors of SNU-201, -466, -489, and -1105, pseudopalisading necrosis was noted. In immunohistochemical studies, the tumor cells expressed glial fibrillary acidic protein (GFAP), the marker for astrocytic cells, in the cytoplasm. The original tumor of SNU-444 was a recurrent GBL (Fig. 2A). Six months before initiation of the cell line, the patient had undergone craniotomy followed by radiation therapy. The initial tumor was diagnosed as GBL by histologic examination. The other patients in this study received no prior therapy. The original tumors of anaplastic oligodendrogliomas consisted of rather uniform cells with round, delicate nuclei and perinuclear halos (Fig. 2C). The tumors showed hypercellularity, nuclear hyperchromasia, conspicuous mitotic activity, and endothelial proliferation. Necrosis was present in the original tumor of SNU-738. In immunohistochemical studies, most of the tumor cells did not express GFAP. GFAP was focally expressed in the cytoplasm of minigemistocytes. The SNU-738 patient received no prior therapy. Five years before initiation of the cell line, the SNU-791 patient had undergone craniotomy, and the initial tumor was diagnosed as oligodendroglioma by histologic examination. The patient had received postoperative radiation therapy with 5550 rad. The cell line of SNU-1118 was derived from a PNET (Fig. 2E). One year before initiation of the cell line, the patient was diagnosed by biopsy as having a PNET. The extracranial component of the tumor was located in the left infratemporal fossa with the intracranial tumor in the left temporal lobe. The patient received 13 cycles of chemotherapy followed by craniotomy. The intracranial component of the tumor consisted of hypercellular primitive cells with extensive necrosis. The tumor cells had hyperchromatic nuclei and scanty cytoplasm (Fig. 2E). The tumor focally showed nodular arrangement. In immunohistochemical studies, the tumor cells focally expressed GFAP, S100 protein, neuron-specific enolase, vimentin, and desmin. The histological features were consistent with those of a PNET with glial, neuronal, and mesenchymal differentiation. The extracranial component of the tumor showed the histological features of a melanotic neuroectodennal tumor. The tumor was diagnosed as a malignant melanotic neuroectodermal tumor with glial, neuronal, and mesenchymal differentiation. The molecular mechanisms that underlie the development of brain tumor have not been elucidated. However, the inactivation of tumor suppressor genes has been increasingly recognized as a major mechanism in tumor development. This study demonstrated the inactivation of various tumor suppressor genes such as p53, p16, p15, PTEN, FHIT, and DCC in the nine new human brain cancer cell lines (Table 2). For the p53 gene, we examined exons 4-9, in which about 98% of the mutations have been detected (Nigro et al., 1989). Mutation of the p53 gene was found in five cell lines. The SNU201 cell line showed a point mutation of cagTAC to caaTAC at a splice acceptor of intron 4 (Fig. 3A); the SNU-444 line had a missense mutation of TGT (Cys) to TAT (Tyr) at codon 275; the SNU-