507
0 1991 The Japanese Society of
Pathology
Morphological and Cytogenetic Characterization and N-myc Oncogene Analysis of a Newly Established Neuroblastoma Cell Line
Ta kayuki Nojima', Syuiti Abe2, Yasushi Furutal, Kazuo Nagashima', AFM Saiful Alam3, Naoyuki Takada3, Fumiaki Sasaki3, and Yoshinobu Hata3 A permanent cell line established from a xenograft of neuroblastoma which occurred i n a 5-year-old girl was investigated for its morphological and biological characteristics. The cultured cells were tumorigenic in nude mice. Microscopically, each tumor consisted of small round to polygonal cells with irregular nuclei and prominent nucleoli, corresponding t o the features of the primary and xenografted tumor cells. Electron microscopic examination revealed that both the transplanted tumor cells and the cultured cells contained scanty microtubules and densecore neurosecretory granules. Chromosome analysis of this cell line showed monosomy for chromosomes 1,10,19 and X, and structural rearrangements involving chromosomes8,17 and 20, i n addition t o numerous double minutes. The N-myc oncogene was found to be amplified 40to 80-fold in the transplanted and cultured tumor cells, as well as in the primary tumor cells. In situ hybridization with a digoxigenin-labeled uridine-triphosphate N-myc RNA probe detected abundant mRNA i n the tumor cells. This neuroblastoma line may become a valuable in vitro experimental model system for studies aimed at better characterization of neuroblastoma. Acta Pathol Jpn 41 : 507-515, 1991.
Key words: Neuroblastoma cell line, Nude mouse, Karyotyping, N-myc, In situ hybridization
prognosis of this disease is very poor, but early diagnosis at an age of less than one year may sometimes ensure a favorable prognosis and/or a spontaneous remission. Although the prognostic factors of neuroblastoma are very complicated, S himada's clinico pat ho logic program (l), N-myc amplification (21, chromosome number (3) and serum ferritin level (4) have recently been found to be significant prognostic indicators. Various human neuroblastoma cell lines have been reported since the work of Murray and Stout (5). Continuous establishment of well characterized human neuroblastoma cell lines is necessary in order to compare the heterogeneity of this tumor among patients. Investigations of the biological characteristics of tumor cells are most important because of the biological complexity of neuroblastoma. Recently we established a new neuroblastoma cell line, designated HNB-18. The present paper describes the morphologic, immunohistochemical and chromosome characteristics of the tumor cells. We also present the results of Southern blot analysis of the N-myc oncogene copy number and in situ hybridization analysis using an antisense digoxigenin-labeled N-myc RNA probe for detection of mRNA in tumor cells.
INTRODUCTION
MATERIALS AND METHODS
Neuroblastoma is the third most common childhood cancer after leukemia and brain tumors. In general, the
Case report
Received November 8, 1990. Accepted for publication March 29, 1991. 'Department of Pathology and 3Department of Surgery, Hokkaido University School of Medicine, Sapporo. ZChromosome Research Unit, Faculty of Science, Hokkaido University, Sapporo. Mailing address : Takayuki Nojima, M.D., Department of Pathology, Hokkaido University School of Medicine, Kita 14 Nishi 5, Kita-ku, Sapporo 060, Japan.
A 5 year-old girl presented with a mass in the left supraclavicular fossa and complained of abdominal pain. She was admitted to the Hokkaido University Hospital in April 1986, and enlargement of the left adrenal gland was found. The rate of urinary excretion of vanillymandelic acid (VMA) was 7.2 mg/day, that of homovanillic acid (HMA) 13.3 mg/day, and that of dopamine 2,580 pg/day. The level of serum ferritin was 420ng/ml.
5 08
Neuroblastoma Cell Line (Nojima et a/.)
Four courses of cyclophosphamide, vincristine, adriamycin, and cisplatin were administered preoperatively. In September, 1986, the tumor mass and the left kidney were resected, but the paraaortic and mesenteric lymph nodes were incompletely resected due to adhesion to the aorta. The tumor mass excised at surgery measured 9 x 5 cm and was relatively firm and whitish gray t o tan in color. There were indications of invasion or metastasis to the left kidney, and paraaortic and mesenteric lymph nodes. Microscopic examination revealed sheets of small round to polygonal cells with scanty cytoplasm and poorly defined cell borders. The nuclei of the tumor cells were round with prominent nucleoli. Formation of rosettes or maturation toward ganglion cells was not found. The pathologic diagnosis was neuroblastoma, round cell type, stage IVa. The patient died of multiple pulmonary metastases in November, 1987. Heterotransplantation and cell culture The tissue from the primary tumor removed by surgical resection was minced with scissors into small fragments and inoculated into the subcutaneous space on the back of BALB/cAJc-nu nude mice (Nihon Clea, Tokyo, Japan). Serial tumor heterotransplantation was successful and the derived transplants at sixth passage were used for cell culture. Finely minced tumor tissues were suspended in 2 ml of 1.0% collagenase in RPMI-1640 medium at 37°C for 2 0 min. The supernatant was removed after centrifugation for 5 min at 1,500 rpm. Cells were seeded into plastic culture flasks (25 cmz) with RPMI- 1 6 4 0 medium supplemented with 10% heatinactivated fetal calf serum and incubated at 37°C in a humidified atmosphere of 5% CO, in air. The cells have since been subcultured more than 5 0 times in 2 4 months and can therefore be designated a permanent cell line, HNB-18. Doubling time of the cultured cell population was determined for 1 x l o 6cells seeded per 25-cmZ collagencoated Petri dish. Numbers of cells from three dishes were counted and averaged at 24-h intervals (24, 48, 72, 96, 1 2 0 h). To investigate the tumorigenicity of the HNB-18 cell line in nude mice, a suspension of 2 x lo7cells in 0.5 ml of potassium phosphate buffer (pH 7.4) was injected into the subcutaneous space on the back of BALB/c AJc-nu. Pathology studies Transplanted tumor specimens were fixed in neutral 10% formalin and stained with hematoxylin and eosin. Cultured cells were observed with a phase-contrast
microscope. Both the transplanted tumor in nude mice and cultured cells were processed for electron microscopic examination. The tumor specimens were fixed in 2% glutaraldehyde, post-fixed in 1% osmium tetroxide, and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Hitachi H-800 electron microscope. For immunohistochemical studies, we used the standard t hree-st ep indirect av idin- biot in- peroxida se met hod (6) on paraffin sections of the primary tumor, the transplanted tumor, and cultured cells. The antibodies used were those against neuron-specific enolase (NSE, DAKO, Copenhagen, Denmark, at dilution of 1 : 200), ferritin (DAKO, 1 : 800), neurofilament (DAKO, 2 0 0 kDa, prediluted), vimentin (DAKO, 1 : 100) and N-myc product (Nichirei, Tokyo, Japan, 62 kDa, 1 : 10). Catecholamine and ferritin assays One million cultured cells at 4 0 t h passage were seeded into Petri dishes and the culture medium was collected after 3 d a y s of incubation. Specimens of the tumor transplanted into nude mice were homogenized in 5 m M potassium phosphate buffer (pH 7.4). Cell-free supernatants, as well as culture medium, were stored at -85°C before transport to the Otsuka Assay Laboratories (Tokushima, Japan) for analysis of norepinephrine, epinephrine, dopamine and ferritin. Chromosome study Cultured cells were exposed to colcemid (0.2 pg/ml) for one hour at 37°C before harvest. Chromosome slides were prepared by air-drying after hypotonic treatment and fixation in methanol-acetic acid. Karyotype analysis was performed by G-banding. Chromosome abnormalities were classified according to the International System for Human Cytogenetic Nomenclature (1985) (7). Analysis of N-myc oncogene amplification and in situ hybridization High-molecular-weight DNA was extracted from the primary tumor, its xenograft and cultured tumor cells at 17th and 3 8 t h passage. Ten micrograms of DNA was digested with EcoRI, electrophoresed through 0.7% agarose gel, and transferred to a nylon membrane(8). The membrane was hybridized with a 32P-labeledN-myc probe (Oncor, Gaithersburg, USA). The relative copy number of the N-myc oncogene was estimated by densitometric comparison with a single-copy intensity of 10 pg of normal lymphocytes.
Acta Pathologica Japonica 41 (7) : 1991
A genomic N-myc DNA clone (pSPT-N-myc, insert 2,143 bp, provided by Dr. F. Alt (9)) obtained from the Japanese Cancer Research Resources Bank was transcribed with SP6 RNA polymerase using digoxigeninlabeled uridine triphosphate as a substrate (RNA Labeling Kit, Boehringer Mannheim, Mannheim, Germany). The digoxigenin-labeled antisense RNA probe was synthesized in vitro (10). Frozen sections of the transplanted tumor 8 p m thick were fixed in 4% paraformaldehyde for 2 0 min at 4°C and washed in potassium phosphate buffer (pH 7.4). The slides were treated with 0.2 M HCI for 2 0 min at room temperature and then treated with 1 ,ug/ml proteinase K (Boehringer Mannheim). Hybridization was carried out a t 42°C overnight. Hybridization signals were detected by enzyme-linked immunoassay using an anti-digoxigenin alkaline phosphatase conjugate (Nucleic Acid Detection Kit, Boehringer Mannheim). Digoxigenin-labeled sense RNA homologous with the coding region of the N-myc oncogene was also synthesized using T7 RNA polymerase (Boehringer Mannheim), which was used as a negative control.
RESULTS Morphology of t h e transplanted t u m o r in nude mice Tumor heterotransplantation into nude mice has been performed more than 25 times, with 100% tumor "take" at each passage. The doubling time of the tumor volume was 1.7 days and the mean interval of transplantation was 32 days. Histologically, the xenografts were similar to the parent tumor. The tumor was composed of small polygonal cells with dense nuclei (Fig. 1).
5 09
There was massive necrosis in the tumors, but neither rosette formation nor fine fibrillar material was seen. Electron microscopy showed that the cells were closely packed, and that the scant intercellular spaces contained a small amount of collagen fibrils. A few intercellular junctions were recognized between the cells. The cytoplasm contained numerous free ribosomes, mitochondria, sparse rough endoplasmic reticulum, a moderately developed Golgi apparatus and small quantities of intermediate filaments. Arrays of microtubules and dense-cored vesicles were infrequently found in the cytoplasmic processes. These vesicles, approximately 100- 180 nm in diameter, were neurosecretory granules and had t rila minar membrane structures with cent ra I electrondense cores (Figs. 2a, b). Growth characteristics and morphology of the cultured cells Fibroblast-like cells became attached to the bottom of plastic flasks after initiation of culture. Within two or three days, most of these cells became detached and were seen floating in the medium. These suspended cells began to proliferate and aggregate slowly. Thereafter, confluence was achieved and serial passages were carried out every three weeks. These cells began to attach to the bottom of the flasks when the suspension culture reached 25th passage in 1 7 months. Most of the cells grew in a loosely adherent monolayer or formed cell clumps. A small proportion of cells were suspended in the medium. Microscopically, the adherent cells were tear drop-shaped, or polyhedral with short cytoplasmic processes (Fig. 3). The population-doubling time obtained at 4 7 t h passage was approximately 31.1 h. Electron microscopically, most of the cells had numerous microvillus-like cytoplasmic processes and the nuclei were large, round to oval with one prominent nucleolus, and consisted of diffuse chromatic material. The cytoplasm of the tumor cells contained abundant free ribosomes, mitochondria, Golgi apparatus, arrays of rough endoplasmic reticulum and a few microtubules. In some cells, small, dense-cored neurosecretory granules measuring 100-200 nm in diameter were seen throughout the cytoplasm including the cytoplasmic processes, but these granules were scant compared with those in the transplanted tumor cells. A few primitive intercellular junctions were observed between adjacent cells (Figs. 4a, b). Tumorigenicity in nude mice
~-
Figure 1. Histologic appearance of the transplanted tumor. Small polygonal cells with indistinct cytoplasm are evident. HE
Cultured cells inoculated into the subcutaneous space in the back of the nude mice formed tumor masses,
510
Neuroblastoma Cell Line (Nojima e t a/.)
Figure 2. Electron micrographs of the transplanted tumor in nude mice. a : Tumor cells are closely packed and contain free ribosomes, mitochondria, and rough endoplasmic reticulum in the cytoplasm. Neurosecretory granules (arrows) are identifiable ( x 11,000). b : Intermediate filaments (F) are present in the cytoplasm. N : Nucleus ( x 25,000).
reaching 15 mm in diameter within two months. Histologically, each tumor consisted of tightly packed small cells with irregular dense nuclei showing numerous mitotic figures. These cells were similar to the cells of the primary tumor and its xenografts.
I mmunohistochemistry The results of immunohistochemical staining are listed in Table 1. Most of the tumor cells in vitro and in vivo, as well as the primary tumor cells, reacted positively with
Table 1. Results of lmmunostaining Antigen
Figure 3. Phase-contrast micrograph of the cultured cells. Flat, fibroblast-type cells with short neurite-like processes are evident.
Neuron-specific enolase Ferritin Neurofilament Vimentin N-myc product
Primary tumor
Transplanted tumor
Cultured cell
+ +
+ +
+
+
+ + + + +
+-
+-
511
Acta Pathologica Japonica 4 1 (7) : 1 9 9 1
Figure 4. Electron micrographs of the cultured cells. a : The cytoplasm contains mitochondria, Golgi apparatus and free ribosomes. Intercellular junction (arrow) is present between the cells ( ~ 7 , 0 0 0 ) . b : Dense-core, neurosecretory granules 100-200 nm in diameter are present in the cytoplasm. N : Nucleus ( x 33,000).
anti- NSE, anti-ferritin, anti-neurofilament, and anti-Nmyc product. However, vimentin was detected only in the cytoplasm of cultured cells. Catecholamine and ferritin assays Activities of dopamine, norepinephrine, epinephrine and ferritin are presented in Table2. The culture medium showed no detectable accumulation of catecholamine or ferritin. On the other hand, a high level of ferritin and lower levels of dopamine and norepinephrine were detected in the transplanted tumor tissue.
Chromosome analysis Chromosomes of tumor cells from primary culture were examined, but analysis was unsuccessful. Tumor cells from the 32nd passage were used for chromosome studies, and numerous double minutes of various sizes were present in every cell examined (Fig. 5a). The modal chromosome number was 43. The karyotype was interpreted as 43, X, -X, -1, -8, -8, -10, -19, -20, +der(8)t(8 ; ?)(p21 ; ?), +der(8)t(8 ; ?) (p12 ; ?), +i(17q), +der(20)t(20 ; ?)(q13 ; ?) (Fig. 5b). Detection of N-myc oncogene
Table 2.
Catecholamine and Ferritin Activities
0 1991 The Japanese Society of
Pathology
Morphological and Cytogenetic Characterization and N-myc Oncogene Analysis of a Newly Established Neuroblastoma Cell Line
Ta kayuki Nojima', Syuiti Abe2, Yasushi Furutal, Kazuo Nagashima', AFM Saiful Alam3, Naoyuki Takada3, Fumiaki Sasaki3, and Yoshinobu Hata3 A permanent cell line established from a xenograft of neuroblastoma which occurred i n a 5-year-old girl was investigated for its morphological and biological characteristics. The cultured cells were tumorigenic in nude mice. Microscopically, each tumor consisted of small round to polygonal cells with irregular nuclei and prominent nucleoli, corresponding t o the features of the primary and xenografted tumor cells. Electron microscopic examination revealed that both the transplanted tumor cells and the cultured cells contained scanty microtubules and densecore neurosecretory granules. Chromosome analysis of this cell line showed monosomy for chromosomes 1,10,19 and X, and structural rearrangements involving chromosomes8,17 and 20, i n addition t o numerous double minutes. The N-myc oncogene was found to be amplified 40to 80-fold in the transplanted and cultured tumor cells, as well as in the primary tumor cells. In situ hybridization with a digoxigenin-labeled uridine-triphosphate N-myc RNA probe detected abundant mRNA i n the tumor cells. This neuroblastoma line may become a valuable in vitro experimental model system for studies aimed at better characterization of neuroblastoma. Acta Pathol Jpn 41 : 507-515, 1991.
Key words: Neuroblastoma cell line, Nude mouse, Karyotyping, N-myc, In situ hybridization
prognosis of this disease is very poor, but early diagnosis at an age of less than one year may sometimes ensure a favorable prognosis and/or a spontaneous remission. Although the prognostic factors of neuroblastoma are very complicated, S himada's clinico pat ho logic program (l), N-myc amplification (21, chromosome number (3) and serum ferritin level (4) have recently been found to be significant prognostic indicators. Various human neuroblastoma cell lines have been reported since the work of Murray and Stout (5). Continuous establishment of well characterized human neuroblastoma cell lines is necessary in order to compare the heterogeneity of this tumor among patients. Investigations of the biological characteristics of tumor cells are most important because of the biological complexity of neuroblastoma. Recently we established a new neuroblastoma cell line, designated HNB-18. The present paper describes the morphologic, immunohistochemical and chromosome characteristics of the tumor cells. We also present the results of Southern blot analysis of the N-myc oncogene copy number and in situ hybridization analysis using an antisense digoxigenin-labeled N-myc RNA probe for detection of mRNA in tumor cells.
INTRODUCTION
MATERIALS AND METHODS
Neuroblastoma is the third most common childhood cancer after leukemia and brain tumors. In general, the
Case report
Received November 8, 1990. Accepted for publication March 29, 1991. 'Department of Pathology and 3Department of Surgery, Hokkaido University School of Medicine, Sapporo. ZChromosome Research Unit, Faculty of Science, Hokkaido University, Sapporo. Mailing address : Takayuki Nojima, M.D., Department of Pathology, Hokkaido University School of Medicine, Kita 14 Nishi 5, Kita-ku, Sapporo 060, Japan.
A 5 year-old girl presented with a mass in the left supraclavicular fossa and complained of abdominal pain. She was admitted to the Hokkaido University Hospital in April 1986, and enlargement of the left adrenal gland was found. The rate of urinary excretion of vanillymandelic acid (VMA) was 7.2 mg/day, that of homovanillic acid (HMA) 13.3 mg/day, and that of dopamine 2,580 pg/day. The level of serum ferritin was 420ng/ml.
5 08
Neuroblastoma Cell Line (Nojima et a/.)
Four courses of cyclophosphamide, vincristine, adriamycin, and cisplatin were administered preoperatively. In September, 1986, the tumor mass and the left kidney were resected, but the paraaortic and mesenteric lymph nodes were incompletely resected due to adhesion to the aorta. The tumor mass excised at surgery measured 9 x 5 cm and was relatively firm and whitish gray t o tan in color. There were indications of invasion or metastasis to the left kidney, and paraaortic and mesenteric lymph nodes. Microscopic examination revealed sheets of small round to polygonal cells with scanty cytoplasm and poorly defined cell borders. The nuclei of the tumor cells were round with prominent nucleoli. Formation of rosettes or maturation toward ganglion cells was not found. The pathologic diagnosis was neuroblastoma, round cell type, stage IVa. The patient died of multiple pulmonary metastases in November, 1987. Heterotransplantation and cell culture The tissue from the primary tumor removed by surgical resection was minced with scissors into small fragments and inoculated into the subcutaneous space on the back of BALB/cAJc-nu nude mice (Nihon Clea, Tokyo, Japan). Serial tumor heterotransplantation was successful and the derived transplants at sixth passage were used for cell culture. Finely minced tumor tissues were suspended in 2 ml of 1.0% collagenase in RPMI-1640 medium at 37°C for 2 0 min. The supernatant was removed after centrifugation for 5 min at 1,500 rpm. Cells were seeded into plastic culture flasks (25 cmz) with RPMI- 1 6 4 0 medium supplemented with 10% heatinactivated fetal calf serum and incubated at 37°C in a humidified atmosphere of 5% CO, in air. The cells have since been subcultured more than 5 0 times in 2 4 months and can therefore be designated a permanent cell line, HNB-18. Doubling time of the cultured cell population was determined for 1 x l o 6cells seeded per 25-cmZ collagencoated Petri dish. Numbers of cells from three dishes were counted and averaged at 24-h intervals (24, 48, 72, 96, 1 2 0 h). To investigate the tumorigenicity of the HNB-18 cell line in nude mice, a suspension of 2 x lo7cells in 0.5 ml of potassium phosphate buffer (pH 7.4) was injected into the subcutaneous space on the back of BALB/c AJc-nu. Pathology studies Transplanted tumor specimens were fixed in neutral 10% formalin and stained with hematoxylin and eosin. Cultured cells were observed with a phase-contrast
microscope. Both the transplanted tumor in nude mice and cultured cells were processed for electron microscopic examination. The tumor specimens were fixed in 2% glutaraldehyde, post-fixed in 1% osmium tetroxide, and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Hitachi H-800 electron microscope. For immunohistochemical studies, we used the standard t hree-st ep indirect av idin- biot in- peroxida se met hod (6) on paraffin sections of the primary tumor, the transplanted tumor, and cultured cells. The antibodies used were those against neuron-specific enolase (NSE, DAKO, Copenhagen, Denmark, at dilution of 1 : 200), ferritin (DAKO, 1 : 800), neurofilament (DAKO, 2 0 0 kDa, prediluted), vimentin (DAKO, 1 : 100) and N-myc product (Nichirei, Tokyo, Japan, 62 kDa, 1 : 10). Catecholamine and ferritin assays One million cultured cells at 4 0 t h passage were seeded into Petri dishes and the culture medium was collected after 3 d a y s of incubation. Specimens of the tumor transplanted into nude mice were homogenized in 5 m M potassium phosphate buffer (pH 7.4). Cell-free supernatants, as well as culture medium, were stored at -85°C before transport to the Otsuka Assay Laboratories (Tokushima, Japan) for analysis of norepinephrine, epinephrine, dopamine and ferritin. Chromosome study Cultured cells were exposed to colcemid (0.2 pg/ml) for one hour at 37°C before harvest. Chromosome slides were prepared by air-drying after hypotonic treatment and fixation in methanol-acetic acid. Karyotype analysis was performed by G-banding. Chromosome abnormalities were classified according to the International System for Human Cytogenetic Nomenclature (1985) (7). Analysis of N-myc oncogene amplification and in situ hybridization High-molecular-weight DNA was extracted from the primary tumor, its xenograft and cultured tumor cells at 17th and 3 8 t h passage. Ten micrograms of DNA was digested with EcoRI, electrophoresed through 0.7% agarose gel, and transferred to a nylon membrane(8). The membrane was hybridized with a 32P-labeledN-myc probe (Oncor, Gaithersburg, USA). The relative copy number of the N-myc oncogene was estimated by densitometric comparison with a single-copy intensity of 10 pg of normal lymphocytes.
Acta Pathologica Japonica 41 (7) : 1991
A genomic N-myc DNA clone (pSPT-N-myc, insert 2,143 bp, provided by Dr. F. Alt (9)) obtained from the Japanese Cancer Research Resources Bank was transcribed with SP6 RNA polymerase using digoxigeninlabeled uridine triphosphate as a substrate (RNA Labeling Kit, Boehringer Mannheim, Mannheim, Germany). The digoxigenin-labeled antisense RNA probe was synthesized in vitro (10). Frozen sections of the transplanted tumor 8 p m thick were fixed in 4% paraformaldehyde for 2 0 min at 4°C and washed in potassium phosphate buffer (pH 7.4). The slides were treated with 0.2 M HCI for 2 0 min at room temperature and then treated with 1 ,ug/ml proteinase K (Boehringer Mannheim). Hybridization was carried out a t 42°C overnight. Hybridization signals were detected by enzyme-linked immunoassay using an anti-digoxigenin alkaline phosphatase conjugate (Nucleic Acid Detection Kit, Boehringer Mannheim). Digoxigenin-labeled sense RNA homologous with the coding region of the N-myc oncogene was also synthesized using T7 RNA polymerase (Boehringer Mannheim), which was used as a negative control.
RESULTS Morphology of t h e transplanted t u m o r in nude mice Tumor heterotransplantation into nude mice has been performed more than 25 times, with 100% tumor "take" at each passage. The doubling time of the tumor volume was 1.7 days and the mean interval of transplantation was 32 days. Histologically, the xenografts were similar to the parent tumor. The tumor was composed of small polygonal cells with dense nuclei (Fig. 1).
5 09
There was massive necrosis in the tumors, but neither rosette formation nor fine fibrillar material was seen. Electron microscopy showed that the cells were closely packed, and that the scant intercellular spaces contained a small amount of collagen fibrils. A few intercellular junctions were recognized between the cells. The cytoplasm contained numerous free ribosomes, mitochondria, sparse rough endoplasmic reticulum, a moderately developed Golgi apparatus and small quantities of intermediate filaments. Arrays of microtubules and dense-cored vesicles were infrequently found in the cytoplasmic processes. These vesicles, approximately 100- 180 nm in diameter, were neurosecretory granules and had t rila minar membrane structures with cent ra I electrondense cores (Figs. 2a, b). Growth characteristics and morphology of the cultured cells Fibroblast-like cells became attached to the bottom of plastic flasks after initiation of culture. Within two or three days, most of these cells became detached and were seen floating in the medium. These suspended cells began to proliferate and aggregate slowly. Thereafter, confluence was achieved and serial passages were carried out every three weeks. These cells began to attach to the bottom of the flasks when the suspension culture reached 25th passage in 1 7 months. Most of the cells grew in a loosely adherent monolayer or formed cell clumps. A small proportion of cells were suspended in the medium. Microscopically, the adherent cells were tear drop-shaped, or polyhedral with short cytoplasmic processes (Fig. 3). The population-doubling time obtained at 4 7 t h passage was approximately 31.1 h. Electron microscopically, most of the cells had numerous microvillus-like cytoplasmic processes and the nuclei were large, round to oval with one prominent nucleolus, and consisted of diffuse chromatic material. The cytoplasm of the tumor cells contained abundant free ribosomes, mitochondria, Golgi apparatus, arrays of rough endoplasmic reticulum and a few microtubules. In some cells, small, dense-cored neurosecretory granules measuring 100-200 nm in diameter were seen throughout the cytoplasm including the cytoplasmic processes, but these granules were scant compared with those in the transplanted tumor cells. A few primitive intercellular junctions were observed between adjacent cells (Figs. 4a, b). Tumorigenicity in nude mice
~-
Figure 1. Histologic appearance of the transplanted tumor. Small polygonal cells with indistinct cytoplasm are evident. HE
Cultured cells inoculated into the subcutaneous space in the back of the nude mice formed tumor masses,
510
Neuroblastoma Cell Line (Nojima e t a/.)
Figure 2. Electron micrographs of the transplanted tumor in nude mice. a : Tumor cells are closely packed and contain free ribosomes, mitochondria, and rough endoplasmic reticulum in the cytoplasm. Neurosecretory granules (arrows) are identifiable ( x 11,000). b : Intermediate filaments (F) are present in the cytoplasm. N : Nucleus ( x 25,000).
reaching 15 mm in diameter within two months. Histologically, each tumor consisted of tightly packed small cells with irregular dense nuclei showing numerous mitotic figures. These cells were similar to the cells of the primary tumor and its xenografts.
I mmunohistochemistry The results of immunohistochemical staining are listed in Table 1. Most of the tumor cells in vitro and in vivo, as well as the primary tumor cells, reacted positively with
Table 1. Results of lmmunostaining Antigen
Figure 3. Phase-contrast micrograph of the cultured cells. Flat, fibroblast-type cells with short neurite-like processes are evident.
Neuron-specific enolase Ferritin Neurofilament Vimentin N-myc product
Primary tumor
Transplanted tumor
Cultured cell
+ +
+ +
+
+
+ + + + +
+-
+-
511
Acta Pathologica Japonica 4 1 (7) : 1 9 9 1
Figure 4. Electron micrographs of the cultured cells. a : The cytoplasm contains mitochondria, Golgi apparatus and free ribosomes. Intercellular junction (arrow) is present between the cells ( ~ 7 , 0 0 0 ) . b : Dense-core, neurosecretory granules 100-200 nm in diameter are present in the cytoplasm. N : Nucleus ( x 33,000).
anti- NSE, anti-ferritin, anti-neurofilament, and anti-Nmyc product. However, vimentin was detected only in the cytoplasm of cultured cells. Catecholamine and ferritin assays Activities of dopamine, norepinephrine, epinephrine and ferritin are presented in Table2. The culture medium showed no detectable accumulation of catecholamine or ferritin. On the other hand, a high level of ferritin and lower levels of dopamine and norepinephrine were detected in the transplanted tumor tissue.
Chromosome analysis Chromosomes of tumor cells from primary culture were examined, but analysis was unsuccessful. Tumor cells from the 32nd passage were used for chromosome studies, and numerous double minutes of various sizes were present in every cell examined (Fig. 5a). The modal chromosome number was 43. The karyotype was interpreted as 43, X, -X, -1, -8, -8, -10, -19, -20, +der(8)t(8 ; ?)(p21 ; ?), +der(8)t(8 ; ?) (p12 ; ?), +i(17q), +der(20)t(20 ; ?)(q13 ; ?) (Fig. 5b). Detection of N-myc oncogene
Table 2.
Catecholamine and Ferritin Activities
