Distinguishing characteristics of a new neuroblastoma cell line

Distinguishing Characteristics of a New Neuroblastoma Cell Line James T. Casper, Jeffrey M. Trent, Joseph Harb, Vicki Piaskowski, Marilyn Helmsworth, Jeanne Finlan, and Daniel D. Von Hoff

ABSTRACT: The characteristics of a new neuroblastoma cell line (MC~NB-1) established from the bone marrow of a 2-year-old male are described. Morphologically, the cells appear as flattened and epithelial-like or as small and spherical. Electron microscopy demonstrated microtubules and dense core secretory granules. The doubling time was approximately 35 hr. Isoenzyme patterns and catecholamine secretion indicated a human line of neuronal origin. The soft agar tumor colony forming system demonstrated drug resistance in vitro comparable to in vivo nonresponsiveness. The stemline karyotype of MC-NB-1 is 44,Y, del(1) (p22:), - 4, - 7, + del(7)(q22 :), - 16, + t( 7 ;16 )(16pter-*16q2 4 : : 7q22--~ 7q32 ), - 17. Additionally, double-minute bodies were observed. However, no evidence of homogeneous staining regions (HSRs) were detected.

INTRODUCTION Neuroblastoma is one of the most c o m m o n malignant tumors in children, e x c e e d e d only by l e u k e m i a / l y m p h o m a and brain neoplasms [1]. Unfortunately, the prognosis for this disease has not i m p r o v e d over the past 20 years [2]. Paradoxically, neuroblastoma has the highest rate of spontaneous remission of any h u m a n malignancy [3]. Studies to date have not clarified the biological c o m p l e x i t y that allows this tumor to have such a high rate of spontaneous remission in younger children and yet be such a devastating disease in older children. Reports of neuroblastoma lines have appeared in the literature [4-7]. However, the c o n t i n u e d establishment of well-characterized h u m a n neuroblastoma lines is necessary to compare the heterogeneity of this disease b e t w e e n patients and of equal importance to elicit specific biological characteristics (i.e., c h r o m o s o m a l abnormalities, e n z y m e content, metastatic potential, etc.). In this regard we describe the u n i q u e characteristics of a n e w neuroblastoma line, MC-NB-1, established in our laboratory.

From the Midwest Children's Cancer Center, Milwaukee Children's Hospital, Department of Pediatrics, The Medical College of Wisconsin, Milwaukee, Wisconsin (J. T. C., J. H., V. P., M. H, J. F.); Section of Hematology-Oncology,The Cancer Center, University of Arizona, Tucson, Arizona (J. M. T.) and the University of Texas Health Sciences Center, Department of Medicine,Division of Oncology, San Antonio, Texas (B. D. V. H.). Address requests for reprints to Dr. J. T. Casper, Department of Pediatrics, Medical College of Wisconsin. Milwaukee Children's Hospital, 1700 West Wisconsin Avenue, Milwaukee, WI 53233. Received November 17, 1982; accepted January 10, 1983.

177 © Elsevier Science Publishing Co., Inc., 1983 52 Vanderbilt Ave., New York, NY 10017

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CASE REPORT

MC-NB-1 was derived from a 2-year-old white male who presented with an abdominal mass. Initial evaluation demonstrated an adrenal p r i m a r y with liver and bone marrow metasteses. The bone scan and skeletal survey were negative for metastatic disease. The VMA spot test was negative. Therapy consisted of c y c l o p h o s p h a m i d e , adriamycin, vincristine, and d i m e t h y l t r i a z e n o i m i d a z o l e c a r b o x a m i d e (DTIC). Metastatic foci of neuroblasts were again detected in a bone marrow aspirate 51/2 months after initiation of chemotherapy. This marrow sample was further evaluated as outlined below. The patient died with progressive disease 3 weeks later. MATERIALS AND METHODS

Cell culture. The marrow sample with metastatic foci was seeded directly into 25cm 2 tissue culture flasks. Media consisted of RPMI-1640 containing 20% heat inactivated fetal bovine serum (FBS), penicillin, and streptomycin (1%) (GIBCO). On day 11, foci of attached cells were noted. These were fed once a week with fresh culture media. After 1 m o n t h the cells were subcultured using t r y p s i n - v e r s e n e [1 part 0.25% trypsin (Sigma) in RPMI-1640 and 3 parts 0.02% versene (Fisher) in RPMI-1640]. The cells were routinely subcultured as they became confluent, approximately every 2 weeks.

Light and electron microscopy. Cells were obtained for light m i c r o s c o p y following trypsinization as described above. Aliquots were w a s h e d and cytocentrifuge slides prepared with a S h a n d o n cytospin and stained with W r i g h t - G i e m s a . Cells were prepared for electron m i c r o s c o p y by fixation in glutaraldehyde in cacodylate buffer, followed by postfixation in 1% o s m i u m tetroxide in cacodylate buffer. Ultrathin sections were stained with uranyl acetate and lead citrate and e x a m i n e d using a JEOL 100S electron microscope. Isoenzyme analysis. These studies were k i n d l y performed by Dr. Robert Sparkes, UCLA. Lysates of cultured neuroblastoma cells were analyzed electrophoretically by standard techniques for the enzymes: adenylate kinase [8], 6-phosphategluconate dehydrogenase [8], adenosine deaminase [9], p h o s p h o g l u c o m a t a s e I [8], acid phosphatase [10], glucose 6-phosphate dehydrogenase [11], and esterase D [12]. Population-doubling time. Cells were grown in 25-cm 2 flasks until confluent. For doubling time determinations 1 x 106 cells were seeded into 25-cm 2 flasks. Cells from two flasks were removed as above, counted, and averaged at 30, 45, 56, and 96 hr (Exp. I) or 48, 72, and 96 hr (Exp. II).

Tumor-colony-forming assay. Cells were cultured in the bilayer agar system as previously described [13, 14]. In vitro drug sensitivity studies were performed by incubating the cells with and without drugs for 1 hr at 37°C in Hank's balanced salt solution [15]. All cultures were incubated at 37°C in 7% CO2 in humified air. All assays were performed in triplicate. The drugs tested i n c l u d e d chlorambucil, 0.2 ~g/ml; adriamycin, 0.4 ~g/ml; and vincristine, 0.1 ~g/ml. Chlorambucil was chosen as the alkylating agent because c y c l o p h o s p h a m i d e requires metabolic activation. A 70% decrease in tumor-colony-forming units was arbitrarily chosen as a definition for in vitro responsiveness [15]. Catecholamine secretion. Culture m e d i a were decanted from tissue culture flasks 5 days after subculturing. In a d d i t i o n to MC-NB-1, established neuroblastoma lines

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SK-N-SH, SK-N-MC, LAN-2, and B-lymphoblastoid line Raji (American Type Culture Collection) were used for comparison. Cell-free supernatants were stored at -20°C before shipment to the Upjohn Company for analysis of norepinephrine, epinephrine, and dopamine content according to the method of Peuler and Johnson [16].

Cytogenetics. Cultures were harvested for cytogenetic analysis using the procedure described by Trent et el. [17]. Briefly, monolayers were incubated at 37°C for 2 hr in 2.5 ml of culture medium containing 0.1 M colchicine. Cells were removed, washed, and resuspended in 5 ml of 0.075 M KC1 prewarmed to 37°C for 10 min. The cells were fixed (3:1 absolute methanol to glacial acetic acid) and stored at -9°C. Slides were prepared by standard air-dry techniques and cells G-banded [18] or C-banded [19].

RESULTS Growth in Culture By light microscopy, cells with two different morphologic features were observed. One type was epithelial-like and grew in a diffuse monolayer; the other morphologic population was sperical and grew on top of the epithelial-like population. However, when the spherical cells were subcultured they readily attached and the two populations were still present. The doubling time for MC-NB-1 in the first experiment was 38 hr, whereas that for the second experiment was approximately 33 hr. Wright-Giemsa-stained preparations demonstrated cells slightly larger than lymphocytes with a large nucleus and scant cytoplasm. These cells resembled the metastatic foci observed in the patient's bone marrow. Electron Microscopy Electron microscopy revealed small round cells which were loosely aggregated to form small nests of three to several closely apposed cells. The nuclei were large, ovoid to rounded, with prominent nucleoli and diffuse chromatic material. The nuclear margins were regular, with occasional slight indentations. The cytoplasm was scant, consisting of numerous polyribosomes, few small mitochondria, few short profiles of rough endoplasmic reticulum, and occasional small lipid inclusions. Cytoplasmic processes extended from the cell periphery into open spaces between cells. Long, slender, microvillus-like processes contained microfilaments, whereas the broader, short processes representing neuronal extensions contained mitochondria and other organelles, including neurotubules and small, dense-core neurosecretory granules which measured 85-100 nm in diameter (Fig. 1). No desmosomes were observed, but occasional desmosome-like structures were encountered between adjacent cell processes (Fig. 1, inset).

Isoenzyme Analysis and Catecholamine Secretion The esterase D pattern present in MC-NB-1 differed when compared to three other neuroblastoma lines. Furthermore, none of the lines had isoenzyme patterns characteristic of HeLa cells. Measurement of catecholamine secreted into culture media was determined in four neuroblastoma lines and a B-cell line as a control. Dopamine was detected in the media of MC-NB-1 and SK-N-SH but not in SK-N-MC (Table 1). LAN-2 was the only line to secrete detectable amounts of norepinepherine.

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F i g u r e 1 Details of cytoplasm and cellular processes. Slender processes are filled with neurofilaments (F), whereas other process reveal mitochondria (M), polyribosomes (r), and densecore membrane bound granules (long arrows). Neurotubules (short arrows) are seen in the cytoplasm and within a process. N = nucleus; L : lipid inclusion, x 18,750. Inset: A desmosome-like structure between a neuroblastoma cell and a cellular process, x 32,625.

Tumor-Colony-FormingAssay A n a v e r a g e of 246 c o l o n i e s w e r e f o r m e d p e r 5 × 105 c e l l s p l a t e d for a p l a t i n g e f f i c i e n c y of 0.05%. Cells p r e i n c u b a t e d w i t h e i t h e r c h l o r a m b u c i l , a d r i a m y c i n , or v i n c r i s t i n e s h o w e d little or n o d e c r e a s e i n t h e n u m b e r of c o l o n i e s f o r m e d (adriam y c i n = 255 c o l o n i e s ; c h l o r a m b u c i l = 255 c o l o n i e s ; v i n c r i s t i n e = 146 c o l o n i e s ) .

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Table 1

Fractionated catecholamines from cell line culture m e d i a Cell line (pg/ml of medium)

Catecholamine

MC-NB-1

SK-N-MC

SK-N-SH

LAN-2

RAJI

Norepinephrine Epinephrine Dopamine

14 11 251

9 3 23

31 29 139

128 67 1

7 8 17

15

75

Efficiency of recovery (%) 60

76

80

As p r e d i c t e d by this assay, the patient's neuroblasts did not r e s p o n d to these drugs clinically. This finding is similar to an earlier observation on neuroblastoma colonies in short-term agar culture [15].

Cytogenetics Results of the cytogenetic assessment of the MC-NB-1 line were taken from subpassage number 62. Chromosome analysis for m o d a l n u m b e r revealed a near d i p l o i d profile with the m o d e at 44. The chromosome range was from 28-47 with greater than 97% of the 132 cells scored evidencing h y p o d i p l o i d y . Results of the detailed analysis of MC-NB-1 using both G- and C-binding analysis revealed a pattern of c h r o m o s o m e change consistant with previous reports on direct tumor specimens and neuroblastoma cell lines [20, 21]. G-banding revealed the major stem line as 44,XY,del(1)(p22 :), - 4, - 7, + del(7)(q22 :), - 16, + t(7;16)(16pter-~ 16q24::7q22-->7q32),-17 (Fig. 2). The finding of a simple deletion for the distal segment of the short arm of chromosome 1 was a c o m m o n feature on all cells examined. A d d i t i o n a l l y , all cells contained a recognizable c h r o m o s o m e 1 6 q + . Gb a n d i n g analysis demonstrated the origin of the extra long-arm material distal to band 16q24 as a translocated segment from the long arm of c h r o m o s o m e 7 (q22--~q32). This translocation resulted in a partial deletion in the long arm of one chromosome 7(q22:). It appears the terminal segment of the translocated chromosome 7(q33-~qter) is deleted, thus resulting in m o n o s o m y for a portion of the distal long arm of c h r o m o s o m e 7 (Fig. 3). In a d d i t i o n to the finding of clonal chromosomal abnormalities in this cell line, a p p r o x i m a t e l y 14% of tumor mitoses counted d i s p l a y e d m u l t i p l e copies of double minute bodies (DMs). The number of DMs per cell varied widely, from 10 to 200 DMs per cell (Fig. 4). The actual percentage of cells with h u n d r e d s of copies of DMs was very small (<2%). Also, in no instance were h o m o g e n e o u s l y staining regions (HSRs) observed in cultured cells from the MC-NB-1 line.

DISCUSSION We have characterized a neuroblastoma cell line derived from the bone marrow of a 2-year-old boy. The isoenzyme pattern, d o p a m i n e secretion, and m i c r o s c o p i c features of these cells are consistent with a h u m a n neuronal origin. The anchorage i n d e p e n d e n t growth of these cells in soft agar is a characteristic associated with malignant transformation [22]. The neuroblastoma nature of the cells is established by the presence of neuritic processes that contain dense-core granules, w h i c h probably represent catecholamine granules, and neurotubules. Some of the neuritic pro-

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1

2

3

6

7

8

13

14

19

i Ii 4

5

me 9

10

11

12

t5

16

17

18

20

21

22

X

Y

Figure 2 G-banded metaphase of MC-NB-l-passage 62 showing a variety of chromosome abnormalities. The stemline karyotype of this line is 44,XY,del(1)(p22:), - 4 , - 7 , +del(7)(q22:), -16, +t(7;16) (16pter-+16q24::7q22--~7q32),-17 (see Fig 3). Loss of chromosome 9 pictured in this cell was n o t a clonal alteration. cesses appear to form poorly d e v e l o p e d synaptic junctions with adjacent cells. These are characteristic features of neuroblastoma, and serve to distinguish this from other neoplasms such as reticulum ceil sarcoma, small cell osteogenic sarcoma, embryonal rhabdomyosarcoma, and eosinophilic granuloma, all of w h i c h lack these granules. These ceils also lack glycogen "lakes" w h i c h are characteristic of Ewing's sarcoma. Poorly d e v e l o p e d processes with few granules and occasional neurotubules and dense-core granules within the cell bady, and the absence of welld e v e l o p e d desmosomes, is indicative of a poorly differentiated neoplasm. The cytogenetic findings were also consistent with neuroblastoma. The findings of a n e a r - d i p l o i d chromosome n u m b e r in this cell line is in general agreement with earlier p u b l i s h e d reports [20, 21]. In a recent detailed report, Brodeur, et al. have reported on a total of ten p r i m a r y h u m a n neuroblastomas and established neuroblastoma cell lines [21]. T w e n t y were near-diploid, one was near-triploid, and three were near-tetraploid. Chromosomal banding analysis revealed the most consistent structural chromosome abnormality in these tumors was the partial deletion of the short arm of chromosome 1 ( l p - ) . A total of 14 of the 20 tumors studied had evidence of a partial or complete loss of one chromosome 1 short arm with breakpoints in the range p13--~p33, In all reported cases with deletion of l p (including this current report), deletion of all material distal to band p32 has been observed. The actual significance in relation to h u m a n cancer of a partial deletion involving only one of two

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1

183

t

t = i

=t=. I Ip-

7q- 7 16q+ 16

Figure 3 Composite of clonal karyotypic abnormalities from three G-banded cells. The normal chromosome 1, 7, and 16 are compared to their aberrent homologues. (See text for further description.)

homologues is still indeterminate. However, the obvious effect of such a deletion is to produce " p s e u d o d o m i n a n c e " at the now singly represented loci on the nondeleted homologue. The effect of such pseudodominance could effect levels of enzyme expression from involved loci, and could provide a background for expression of the "two-mutation model" for cancer origin proposed by Knudson [23]. The remaining structural chromosome abnormalities in this cell line also appear to fit previously described patterns of chromosome change. Specifically, in the MC-NB-1 line, a 16q+ chromosome was identified as having risen form a translocation between chromosomes 7 and 16. In reports by both Biedler and Spengler [24] and Brodeur et al. [20] on the established cell line IMR-32, a chromosome 16q+ also was observed. Interestingly, in the IMR-32 karyotype published by Brodeur et al. [20], there appears to be a deletion of 7q(q22 : :?) similar to that observed in the MCNB-1 line. However, the karyotyped cell shown in the original report of this line by Biedler and Spengler [24] does not appear to have a deletion of 7q. The differences which make MC-NB-1 unique from IMR-32 reside in the presence of a chromosome l p - in all cells (with no additional chromosome 1 alterations), and the consistent presence of a Y chromosome. Although two morphologically unique cell types appeared in culture, no evidence of karyotypically unique subpopulations was demonstrated. Finally, multiple copies of DMs were observed in 14% of cells from this line. However, the functional significance of DMs in this line is currently unclear. The aforementioned chromosomal changes, especially deletion of the short arm of chromosome 1, is strong supporting e v i d e n c e for the origin of this culture f r o m a h u m a n neuroblastoma. Continued analysis of patients' tumor cells should enhance our understanding

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A

B

Figure 4

(A) Multiple copies of double minute bodies (DMs) MC-NB-1 (arrows) in mitotic cells taken from passage 62 of the MCN-1 line. Note the heterogeneity in DM size. {Chromosomes were intentionally overdeveloped in printing to allow easier identification of DMs.) (B) Several hundred copies of DMs within a single mitotic cell. Cells with >100 copies of DMs were infrequently observed (see text).

of their biological complexity. Though fresh, viable tumor cells would be best suited for enzyme, surface antigen, and cytogenetic analysis, the investigator is limited by the n u m b e r of cells obtained. The establishment of cell lines allows the accumulation of significant n u m b e r s of cells for preparative work. It may also ensure that contaminating normal cells (present in fresh specimens) w o u l d be elimi-

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nated in cell lines. Furthermore, cell lines such as MC-NB-1 can be shared readily b et ween investigators. A problem to date with establishment of cell lines has been that only those neuroblastomas w h i c h are clinically aggressive have been adapted to long-term culture. Neuroblastoma cell lines from patients w i t h low-grade disease have only recently been established [7]. Establishment of well-characterized cell lines should en h an ce our knowledge of the antigenic characteristics of these cells and allow for production of m o n o c l o n a l antisera to these antigens [25]. Knowledge of the classes of antigens present on neuroblasts--i.e., differentiation vs n e u r o b l a s t o m a - - s p e c i f i c - - i s n e e d e d in order to establish a rational basis for i m m u n o t h e r a p y for patients w h o are u n r e s p o n s i v e to conventional therapy [26]. This research was supported by Grants CA 26226, CA 29476, and CA 17094, and the Midwest Athletes Against Childhood Cancer Fund. We wish to thank Dr. Robert Sparkes, UCLA School of Medicine, for performing the isoenzyme assays and Bettie Lyles for preparation of the manuscript. We would also like to acknowledge the excellent technical work of Ms. Sharon Olson. This line is available to other investigators upon request.

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