In Vitro Cell. Dev. Biol. 30A:813-816, December 1994 © 1994 Society for In Vitro Biology 1071-2690/94 $01.50+0.00

Let ter to the Ed i tor PRIMITIVE NEUROECTODERMAL TUMOR CELL LINES DERIVED

FROM A METASTATIC PEDIATRIC TUMOR

Dear Editor: We have established two cell lines in serum-free culture from

different stages of a metastatic primitive neuroectodermal tumor (PNET) isolated from a 2-year-old patient. Among human brain tumors, PNETs are rare malignant multipotent pediatric neoplasia capable of exhibiting all stages of neurogenic maturation (5,15). This tumor, diagnosed as progressive PNET, was first found in a retroperitoneal mass; it recurred in the perirectal space and metas- tasized as a perianal lesion. Two specimens of the tumor were re- trieved from the perirectal space and the perianal lesion. A cell line derived from a perirectal lesion was designated RS-1, and the sec- ond cell line derived from a perianal mass was designated RS-2. Tumor specimens were cut, minced, and seeded in 60 mm plastic dishes (Falcon, Oxnard, CA). The cells were maintained in a humidi- fied atmosphere of 5% CO2 at 37 ° C in RD medium supplemented with 6 factors (6F) (17). 6 factors consisted of 10 #g/ml bovine insulin, 5 #g/ml human transferrin, 50/. tg/ml fatty acid-free bo- vine serum albumin, 10 #M 2-mercaptoethanol, 10 #M 2-amino- ethanol, and 10 nM sodium selenite (all from Sigma, St. Louis, Mo.). Each factor was made as a sterile 100)< concentrate and stored at 4 ° C. RD medium was a 1:1 mixture (by volume) of RPMI 1640 medium (Kyokuto, Tokyo, Japan) and Dulbecco's modified Eagle's medium (Kyokuto), to which was added 0.01% sodium pyruvate (Sigma), 2.2 g/liter sodium bicarbonate (Wako Chemicals Tokyo, Japan), 15 mM HEPES (Dojin, Tokyo, Japan), streptomycin and kanamycin solution (GIBCO Laboratories, Grand Island, NY). There are reports in the literature of PNET cells being cultured for short periods of time to determine chromosome aberrations or to examine nerve growth factor (NGF) receptor expression (1,6,7,10, 11,19,22,24,25), but long-term culture of such cells has not been described. Among several culture conditions tested, RD medium supplemented with 6 factors gave the greatest degree of cell pro- liferation. The doubling time of RS-1 was 48 h, and the doubling time of RS-2 was 24 h (Fig. 1).

Ewing's sarcoma is closely related to PNET histogenically (8,12-14,20), and it is sometimes difficult to distinguish PNET from Ewing's sarcoma. Under the light microscope RS-1 and RS-2 cells appeared small in size with short neurites. Immunohistocbemi- cal examination revealed that RS-1 cells expressed neuron-specific enolase protein (NSE) (Fig. 2 A), neurofilament (NF) (Fig. 2 B), S-100 protein (S-100) (Fig. 2 C), and vimentin (Fig. 2 D). Expres- sion of glial fibillary acidic protein (GFAP) was not found. Accord- ing to an old classification system, NSE expression is significant in making a diagnosis of PNET. According to a newly proposed classifi- cation system, expression of two different neural markers can lead to a diagnosis of PNET (5,15). Our results showed that RS-1 cells had a typical PNET phenotype. Although RS-2 cells expressed vi- mentin (Fig. 2 H) and S-100 (Fig. 2 G), which is a neural marker,

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NF was barely detectable in RS-2 (Fig. 2 F). Neither NSE (Fig. 2 E) nor GFAP were detected in RS-2 cells. In Ewing's sarcoma, no neural marker such as S-100, NSE, NF, or GFAP can be identified (8). Thus, we concluded that RS-2 cells had an undifferentiated phenotype similar to Ewing's sarcoma. We examined the karyotype of both cell lines at the 40th passage. The stemline karyotype of RS-1 was 50×X in 32% of the cells, and it was characterized as +12, +15, +19, +21, +i(lq), - 1 1 , - 1 4 , t(l l ,14) (p15;q(11,2)) (data not shown). The stemline karyotype of RS-2 was 50)<)< in 20% and 48)<>( in 22% of the cells, and it was characterized as 48)<)<, +1, +15, +21, +22, - 1 1 , - 1 4 , t ( l l ;14) (p15;q(l l ,2)) (data not shown). The most consistent cytogenetic alteration in PNET was reported to be i(17q) (2). It was reported that a t(11;22) was consistently found in two neuroepithelial malignancies, neuro- epithehoma and Ewing's sarcoma, arising outside the central net-

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FIG. 1. Growth curves of RS-1 and RS-2 cells. The RS-t (©) and RS-2 (O) ceils were seeded at 1 X 104 cells per ml in RD medium supplemented with 6 factors in 24-well plates (Falcon), and cell numbers were counted daily with a Coulter particle counter (Coulter Electronics, Hialeah, FL).

814 FURUE ET AL.

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RS-1 at 1 X 104 cells/well (O) and RS-2 at 5 × 103 cells/well (0) were seeded in a 24-well dish in RD medium supplemented with 6 factors. FGF-1 was added to each well at 0, 0.01, 0.1, 1.0, 5, and 50 ng/ml. The cells were counted after 5 d in culture.

vous system (CNS) (23). RS-1 and RS-2 cells differed in that they had translocations involving chromosomes 11 and 14.

Epidermal growth factor (EGF), fibroblast growth factor-1 (FGF- 1), or FGF-2 are regarded as trophic factors for neural-derived cells such as glial cells, neuroblastomas, PC12 cells, or astrocytomas (3,4,9,16,26). Schweigerer et al. (21) reported that FGF-2 inhib- ited the proliferation of Ewing's sarcoma cells, and it was reported that FGF-2 mRNA transcripts were found in Ewing's sarcoma (18). We examined the effects of EGF, FGF, or heparin on RS-1 and RS-2 cell proliferation in serum-free medium. RS-1 and RS-2 cells in R D + 6 factor medium were incubated in 24-well plates for 5 d with EGF (Sigma), FGF-2 (Sigma), or FGF-1 (Kyokuto) at 0 - 5 0 ng/ml, or heparin (Sigma) at 0 - 2 5 0 gg/ml . Although previous studies reported that EGF stimulated proliferation of neuroblas- tomas, glial cells, and astrocytes (4,16), neither RS-1 nor RS-2 were growth stimulated by exogenous EGF (results not shown).

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PNET CELL LINES IN SERUM FREE MEDIUM 815

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FIc. 4. Effect of heparin on the proliferation of RS-] and RS-2 cells. Heparin was added to concentrations of 0, 0.05, 0.5, 5, 50, and 250 #g/ml to cultures of RS-1 (C)) and RS-2 ceils (0) as described in the legend to Fig. 3. The cells were counted after 5 d in culture.

Similarly, proliferation of RS-1 and RS-2 cells was neither stimu- lated nor inhibited by FGF-2 (results not shown). In contrast, both PNET cell lines responded to FGF-I: FGF-1 promoted the prolifer- ation of RS-1 cells and inhibited the proliferation of RS-2 cells (Fig. 3). Heparin alone stimulated the proliferation of RS-1 cells, but it marginally inhibited the proliferation of RS-2 cells (Fig. 4). The parallel effects of FGF-1 and heparin suggested that exogenous heparin may have activated endogenous FGF-1 in the extracellular matrices of these cells. Previous studies reported that FGF induced proliferation and differentiation of neuroectodermal cells, astroglial cells, oligodendrocytes, and PC12 cells (12,13). However, RS-1 and RS-2 cells exhibited different responses to FGF-1 even though the cells were derived from a single patient.

Because FGF-1 and heparin stimulated the proliferation of RS-1 cells but inhibited the proliferation of RS-2 cells, RS-2 cells were thought to be more malignant and less well differentiated than RS-1 cells. Consistent with this idea the growth rate of RS-2 was higher than that of RS-1, and immunohistochemically RS-2 cells exhibited an undifferentiated phenotype. These properties suggested that the difference in responses of these ceils to FGF-1 and heparin might

FtG. 2. Immunohistochemical analyses of RS-1 and RS-2 cells. Expression in RS-1 ceils of (A) NSE; (B) NF; (C) S-IO0; and (D) vimentin. Expression in RS-2 cells of (E) NSE; (F) NF; (G) S-IO0; and (H) vimentin. The cells were seeded in 4 chamber glass dishes coated with fibronectin (UBI, Lake Placid, NY) and cultured for 2 d. The cells were fixed with 70% alcohol at the temperature of 0 ° C for 15 min and blocked background staining by 1% porcine serum albumin (Dako, Carpinteria, CA) for 30 min. The streptavidin-biotin peroxidase complex method (SBC method) (Nichirei Co., Tokyo, Japan) was used to investigate expression of NSE (Dako), NF (Dako), S-100 protein (Dako, Tokyo, Japan), GFAP (Dako), vimentin (Dako), and cytokeratin (Becton Dickinson, Oxnard, CA) in PNET cells. The cells were incubated for 1 h with each antibody at room temperature. Then, the cells were washed with phosphate buffered saline, and incubated with biotinylated rabbit anti-mouse immunoglobulin or goat anti-rabbit immunoglobulin for 1 h and with streptavidin for 30 rain (Nichirei). As a negative control, normal mouse serum or rabbit serum was substituted for antibody. Peroxidase activity was detected by incubation with 0.5 mg/ml diaminobenzidine in 0.01% H202/Tris-HCI buffer. The slides were dehydrated and mounted. RS-1 expressed NSE (A), NF (B), S-100 protein (C), and vimentin (D); GFAP and keratin were absent from RS-I. RS-2 expressed vimentin (H), and S-IO0 (G) but did not express NSE (E), NF (F), GFAP, or keratin.

816 FURUE ET AL.

be related to their extent of differentiation and degree of malig- nancy. These two cell lines may be useful in further studies of mechanisms of tumor recurrence or metastasis.

ACKNOWLEDGMENTS

This research was supported by the Japanese Ministry of Education, Science, and Culture and by grant 3276 from the Council for Tobacco Research-USA, Inc.

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20. Schmidt, D.; Hen'mann, C.; Jurgens, H., et at. Malignant peripheral neuroectodermal tumor and its necessary distinction from Ewing's sarcoma. Cancer 68:2251-2259; 1991.

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Miho Furue 1 Yukiehi Tanaka Tetsuji Okamoto Yoshirou Sasaki Masakazu lkeda Kouichi Nishihira

J. Denry Sato

Department of Biochemistry, Kanagawa Dental College, Yokosuka City, 238 Japan (M.F.); Department of Oral and Maxillofacial

Surgery I, Hiroshima University School of Dentistry, Hiroshima City, 734 Japan (T. O.), Departments of Dentistry (M. I.),

Pathology (Y. T. and Y. S.), and Oncology (K. N.), Kanagawa Children Medical Center, Yokohama City, 232 Japan;

W. Alton Jones Cell Science Center, Lake Placid, New York 12946 U.S.A. (J. D. S.)

(Received 18 April 1994)

I To whom correspondence should be addressed at 1:82 Inaoka-cho, Yokosura City, Kanagawa, Japan 238