Recapitulates Neural Crest Development - NCBI

American Journal of Pathology, Vol. 128, No. 3, September 1987 Copyright © American Association of Pathologists

Differentiation of Human Neuroblastoma Recapitulates Neural Crest Development Study of Morphology, Neurotransmitter Enzymes, and Extracellular Matrix Proteins

MARIA TSOKOS, MD, SUSANNA SCARPA, PhD, ROBERT A. ROSS, PhD, and TIMOTHY J. TRICHE, MD, PhD

From the Laboratory ofPathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, and

Department ofBiological Sciences, Fordham University, Bronx,

New York

Differentiation of human neuroblastoma (NB) was studied in vitro with five NB cell lines treated with dibutyryl cyclic adenosinemonophosphate and retinoic acid. Although the above agents induced different responses in the various cell lines, three overall morphoa neuronal, characterized logiccellphenotypes emerged: and neurosecretory granules, a flat by processes cell without pigment, which displayed basal lamina to Schwann cells, and a flat pigmented cell pertinent which exhibited melanosomes, similarly to melanocytes. The activity of the Schwann cell enzyme cyclic

nucleotidyl phosphohydrolase increased considerably condition, after induction of a predominantly flat cell phenotype. All studied NB cell lines were capable of synthesizing and expressing the extracellular matrix proteins laminin (LM), fibronectin (FN), and Type IV collagen; but a specific pattern of expression emerged after differentiation, which was proportional to normal tissue equivalents: neuronal-none; melanocytic-FN only; and Schwann cell-large amounts of FN, LM, and Type IV collagen. (Am J Pathol 1987, 128:484-496)

NEUROBLASTOMA (NB) is a neural crest-derived childhood tumor that undergoes spontaneous differentiation in vivo more frequently' and to a greater

from a rat peripheral neurotumor induced by injection of ethylnitrosourea in the area of the sciatic

extent (malignant NB to benign ganglioneuroma) than any other human cancer.2-5 Both ganglion cells and Schwann cells have been identified in these benign, differentiated tumors by electron microscopy (EM).5-8 Since both cell types are of neural crest origin,9-11 it has been presumed that NB cells in these

tilmorc have undergone both neuronal and Schwannian differentiation.12 Experimental proof of this, however, has been lacking. Instead, experimental evidence to date has documented only neuronal differentiation.13-19 The origin of Schwann cells in NB has been controversial, and conflicting theories, alternatively supporting NB cell differentiation12 or entrapment of neighboring Schwann cells,20 have been proposed within the past 5 years. More recently, dual neuronal and glial properties have been attributed to neural stem cells in two other biologic systems: a retinoblastoma cell line21'22 and a cell line derived

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in one

nerve.23

It is well known that the neural crest also gives rise melanocytes.9-'1,24 Further, certain presumed neural crest tumors routinely undergo melanocytic differentiation (ie, pigmented neuroectodermal tumor of infancy, melanotic Schwannoma).25'26 Electron microscopy of a melanotic medulloblastoma also revealed a neural crest type of melanin.27 Even to

Presented in part at The Third Advances in Neuroblastoma Research Conference, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, May 3-5, 1984, and published in the corresponding book Advances in Neuroblastora Research edited by A. E. Evans, G. J. D'Angio, and R. C. Seeger, and published by Alan R. Liss, Inc., New York (1985, pp 55-68). Accepted for publication April 29, 1987. Address reprint requests to Dr. Maria Tsokos, Laboratory of Pathology, National Institutes of Health, Building 10, Room 2A-10, Bethesda, MD 20892.

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examples of neuroblastoma have been reported undergo melanogenesis of neural crest type in

rare

to

vitro28 and

to show increased levels of the enzyme which is necessary for tyrosine oxidation tyrosinase, in the pathway of cutaneous melanin formation.29 Thus, the potential for melanocytic differentiation of NB based on known neural crest tissue derivatives and neoplastic counterparts has been corroborated. In the present study, we have attempted to demonstrate experimentally the potential for undifferentiated NB cells to undergo the three major pathways of differentiation mentioned above, and which are normally followed by differentiating neural crest cells, on the assumption that NB cells are the neoplastic analogs of normal neural crest cells. We have attempted to demonstrate this differentiation by conventional methods (morphology, enzymology) and less apparent approaches. In particular, we have analyzed the ability of NB cells to synthesize an extracellular matrix (ECM) protein, chiefly fibronectin (FN), laminin (LM), and Type IV collagen, since patterns of ECM synthesis are known to reflect tissue phenotype.30 Specifically, neuroblasts synthesize little if any ECM proteins,9,30,31 but extend neurites, an essential step in normal neurogenesis, on LM-, FN-, or Type IV collagen-coated substrates.32-34 Moreover, primitive neuroblasts of the neural tube and neural crest migrate on or through FN-coated pathways during embryogenesis.9'10'31'35 These substrata are possibly provided by Schwann cells in vivo, since only Schwann cells elaborate all three components in vitro.36-38 In contrast to both neuronal cells and Schwann cells, melanoma cells synthesize only FN.39 Thus, each of the three major neural crest-derived cell types expresses a distinctive pattern of ECM protein. We present evidence here, derived from morphologic, enzymologic, and ECM biochemical studies, which supports the concept that NB cells can give rise to neurones, Schwann cells, and melanocytes, or even mixtures thereof.

Materials and Methods Cell Cultures: Differentiation Experiments Five continuous NB cell lines were studied: SMSSAN, IMR-32, CHP-126, SK-N-SH, and SH-SY5Y. The SMS-SAN40 is purely adrenergic; the IMR-32,41 CHP- 126,42 and SK-N-SH43 cell lines exhibit a mixed pattern of neurotransmitter enzymes in vitro; however, IMR-32 is predominantly adrenergic, while the other two are predominantly cholinergic. The SH-SY5Y is a neuroblastic (adrenergic) clone of the SK-N-SH cell line, which shows spontaneous trans-

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formation to flat cells.44 All NB cell lines originated from adrenal tumors, except for SK-N-SH, which was established from a mediastinal tumor with chest wall extension. The CHP-126 cell line originated presumably from an adrenal NB; however, the tumor presented as a large retroperitoneal mass, and its exact site of origin is difficult to know with certainty. The SMS-SAN cell line was kindly provided by C. P. Reynolds. The SK-N-SH and SH-SY5Y cell lines were a generous gift of Dr. J. Biedler. CHP-126 and IMR-32 were purchased from the American Type Culture Collection (Rockville, Md). All cell lines were grown in Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum. For differentiation experiments, cells were plated in 60-mm tissue culture dishes (Falcon Labwear, Oxnard, Calif) at a density of 3 X 104 cells with 5 ml of medium. The differentiating agents, ie, N6, 0'2-dibu-

tyryl-adenosine 3':5-cyclic monophosphate (dibutyryl cyclic AMP [dbc-AMP], and all-trans-retinoic acid (retinoic acid [RA]) (Sigma, St. Louis, Mo), were added at 3-day intervals beginning on the third day after plating (three total doses) at final concentrations of 2 X 10-3 M dbc-AMP and 2.5 X 10-7 M RA. A concentration of 5 X 10-7 M RA was found optimal

in the SMS-SAN cell line. RA was dissolved in absolute alcohol at a 2.5 X 10-5 M concentration and dbc-AMP in sterile water at a 100 X 10-3 M concentration. Both agents were kept at -20 C as stock solutions. The final concentrations of the differentiating agents had been selected on the basis of dose-related morphologic differentiation and cellular toxicity curves (data not shown). The cells were examined daily with a contrast microscope for assessment of

morphologic changes.

Electron Microscopy Treated and untreated cells, after approximately 12 days in culture, were fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.4) at room temperature for 1-24 hours, postfixed in OSO4, and embedded in Maraglas 655 (Ladd Research Industries, Burlington, Vt). Sections were stained with uranyl acetate-lead citrate and examined in a Philips 400 electron microscope.

Neurotransmitter Enzyme Analysis Cells at the stationary phase from the CHP- 126 cell line before and after differentiation with dbc-AMP and RA were washed twice with phosphate-buffered saline (PBS; pH 7.4), and cell pellets were kept frozen

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(-80 C). The neurotransmitter enzyme analysis was performed on cell homogenates according to the method of Fonnum45 for the choline acetyltransferase (CAT) and the method of Glastris and Pfeiffer46 for 2',3'-cyclic nucleotide-3;-phosphohydrolase (CNP). Sodium Dodecyl Sulfate-Polyacrylamide Gel

Electrophoresis (SDS-PAGE) The synthesis of the ECM proteins was documented by metabolic radiolabeling of the cell cultures with 3H-leucine and 3H-proline (100-190 Ci/mmol) (Amersham, Arlington Heights, Ill) for 6 hours at 37 C in the presence of ascorbate (50 mg/100 ml), P,-amino-proprionitrile fumarate (10 mg/100 ml), and dextrose (300 mg/100 ml). SDS-PAGE was per-

formed on the media of these radiolabeled cell cultures by the method of Laemmli47 in the presence or absence ofdithiothreitol (final concentration 50 mM) with a 4% separating gel. Fluorograms were obtained by impregnating the methanol/acetic acid-fixed gels on Gel Bond (Marine Colloids, FMC Corporation, Rockland, Mass) with Enlightening (New England Nuclear, Boston, Mass), oven drying at 80 C, and exposure at -70 to Kodak XR-5 film. Molecular weights were determined by comparison with internal tritiated standards (Bethesda Research Laboratories, Gaithersburg, Md). The electrophoretic mobility of LM, FN, and Type IV collagen from the five investigated NB cell lines was compared with that ofimmunoprecipitated LM, FN, and Type IV collagen from cell lines known to produce the above proteins.

Quantitative Changes of the ECM Proteins Each one of the synthesized ECM proteins by undifferentiated and differentiated CHP-126 cells, as well as the total protein synthesis, were expressed as radioactivity per 100 cells for comparisons. The calculations were performed as follows: 1) Total radioactivity from cell layers and corresponding media was counted with a beta counter (Beckman Instruments, Irvine, CA) and expressed as

counts per minute. Cells were counted with a Coulter

Counter. 2) The fluorogram was evaluated by scanning densitometry (Gilford Spectrophotometer, Gilford Instrument Lab, Oberlin, Ohio) and plotted with a linear chart recorder attached to the spectrophotometer. The areas obtained as a result of the density of the lanes which corresponded to each one of the studied ECM proteins were traced with a Hewlett-Packard digitizer and expressed as a percentage of the total area of the fluorogram; the latter represented the total

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amount of synthesized protein. Since total radioactivity and cell numbers were known, all proteins were finally expressed as radioactivity (cpm) per 100 cells. Values thus obtained were used as reference points to compare changes in the synthesis of these

proteins in relation

to themselves before and after

treatment with dbc-AMP and RA and in

relation to

total protein synthesis. The percentage increase or decrease in the original value of each one of the ECM proteins per 100 cells was evaluated from the formula 1-b/a, where a equals radioactivity before treatment and b equals radioactivity of the same protein after treatment with dbc-AMP or RA.

Antisera Antisera against FN and Type IV collagen were raised by injection of purified human cellular FN (Bethesda Research Laboratories) and human Type IV collagen (Calbiochem-Behring, La Jolla, Calif) (500 ,ug in Freund's complete adjuvant) into New Zealand rabbits. Three or four injections were performed, and sera were collected 1 week after each injection. The antiserum against LM from mouse ES tumor was purchased from Bethesda Research Laboratories. Antisera to Type IV collagen, LM, and FN were purified by affinity column chromatography, and their specificity and titer assayed by enzyme-linked immunosorbent assay.48 All antisera against the ECM proteins were used at a concentration of 1: 100. The antiserum against the S-100 protein (polyclonal, made in rabbit) was purchased from Accurate (Westbury, NY) and used at a concentration of 1:300. The secondary antisera, ie, swine anti-rabbit IgG linked to fluorescein isothiocyanate (FITC), and normal rabbit serum were also purchased from Accurate and used at a concentration of 1:20.

Immunofluorescence (IF) Indirect IF was performed in treated and untreated cells as previously described.21 Briefly, cells in tissue culture dishes were fixed in situ with cold (-20 C) methanol (3 minutes) and equal volumes of cold methanol/acetone (2 minutes). After being washed in phosphate-buffered saline (PBS), the cells were incubated at 4 C overnight either with primary antisera or with normal rabbit serum (negative control). On the next day, the primary antisera and the normal rabbit serum were washed off in PBS, and the secondary antiserum was applied at room temperature for 30 minutes. Excess secondary antiserum was also washed in PBS, and fluorescence was evaluated under a Zeiss standard microscope equipped with an epi-

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fluorescence illuminator and FITC narrow-band filter.

Results

Morphologic Studies The initial morphology of the studied NB cell lines was variable and appeared to relate to their enzymatic profile and to their adrenal versus extraadrenal origin. The purely or predominantly adrenergic (SMS-SAN and IMR-32) cell lines which were derived from classic adrenal childhood NB exhibited clusters of teardrop cells; the cell lines (SK-N-SH and CHP-126) which expressed a mixed, but predominately cholinergic neurotransmitter enzyme profile in vitro showed

487

a mixture of teardrop and polygonal cells, which were dispersed diffusely on the surface of the tissue culture disc. The SK-N-SH adrenergic clone (SH-SY5Y) originally possessed a teardrop morphology, but showed spontaneous conversion to a predominantly flat cell phenotype. After treatment with either agent, the former two cell lines differentiated into neuronal cells, which were arranged in cell balls with peripherally and radially extended neurites (Figure la). The latter three cell lines, on the other hand, differentiated into a mixture of neuronal and flat cells with dbc-AMP and predominantly (CHP-126), or exclusively (SK-NSH), flat cells with RA. The neuronal cells in the latter two cell lines were dispersed in small aggregates oftwo

Figure 1-Phase-contrast microscopy of differentiating NB cells in vitro, showing patterns of neuronal cell differentiation after treatment with dbc-AMP (a and b) and flat cell differentiation after treatment with RA (a and b) and flat cell differentiation after treatment with RA (c and d). a-The neuronal cells form tight clusters and exhibit long neuritic processes extending radially from the periphery of the cell clusters (SMS-SAN treated with dbc-AMP). (X60) b-The neuronal cells have round bodies and are arranged in single cells or small cell clusters with shorter neuritic processes (CHP-126 treated with dbc-AMP). (X100) c-Flat, substrate adherent cells (CHP-126 cell line treated with RA). (X100) d-Flat cells similar to the ones above, which, in addition, show black pigment granules (SK-N-SH cell line treated with RA). (X100)

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to three cells and communicated with shorter, ramifying neuritic processes (Figure lb); the flat cells were elongated, substrate adherent (Figure Ic), and occasionally contained cytoplasmic pigment granules (Figure ld). The SH-SY5Y neuroblastic clone, in spite ofits initial teardrop morphology and its known adrenergic profile, followed the pattern ofdifferentiation of the parental SK-N-SH cell line, instead of that of the other adrenergic cell lines. However, it should be noted that at the time of initiation of the experiments, the SH-SY5Y cell line had already shown spontaneous conversion to a predominantly flat cell phenotype. Minimal numbers of flat and neuronal cells were noted in the SMS-SAN and IMR-32 cell lines as well, before treatment with dbc-AMP and RA. The approximate percentages of neuronal versus flat cells in each cell line after treatment with dbcAMP and RA are shown in Table 1. By electron microscopy, the neuronal cells exhibited long processes with microtubules and neurosecretory granules (Figure 2). Most flat cells lacked specific ultrastructural features. Some of them, however, exhibited well-developed rough endoplasmic reticulum, which was occasionally dilated and filled with amorphous material, and well-developed intercellular attachments. Focal strands of basal lamina were seen on the outside surface of very few flat cells in the SH-SY5Y cell line (Figure 3a). In addition, some flat cells in the SK-N-SH and SH-SY5Y cell lines showed membrane-bound dark structures with the characteristic striated substructure of melanosomes (Figure 3b). Hybrid cells containing neurosecretory granules and melanosomes were observed as well. Cells exhibiting unequivocal morphologic differentiation into all three types, ie, neuronal, Schwannian, and melanocytic cells, were seen only in the cloned SH-SY5Y cell line.

Enzyme Analysis (The changes in the levels of activity of the neuronal enzyme CAT and the Schwannian enzyme CNP50

which were found in the CHP-126 cells before and

September 1987

after treatment with dbc-AMP and RA are shown diagrammatically in Figure 4. These findings further support the Schwannian character of these cells after treatment with RA, in spite of lack of obvious morphologic ultrastructural features, ie, basal lamina. The neuronal enzyme CAT was nondetectable, whereas the Schwannian enzyme CNP increased considerably after treatment with RA (15.9 nmol/hr/mg), when compared with the value (5.7 nmol/hr/mg) before treatment with RA. Less remarkable changes were noted when the same cells differentiated into a neuronal cell phenotype. ECM Proteins

Synthesis Detected by SDS-PAGE The fluorogram obtained from the media of all five NB cell lines under nonreducing and reducing conditions is shown in Figure 5. Four of five NB cell lines synthesized FN, detectable in their media as a single 480-kd band under nonreducing conditions and as a single 240-kd band under reducing conditions. These molecular weights corresponded to previously reported ones for cellular FN.39 LM was detected in the media of four cell lines and visualized best under nonreducing conditions as a single 1000-kd band.37 In general, the individual 200-kd and 400-kd subunits of LM were hardly evident under reducing conditions. Type IV collagen was detected in the media of only two NB cell lines, seen as a single 600-kd band under nonreducing conditions and as dimer below the 200kd molecular weight marker under reducing condi-

tions.36

Quantitative Changes in the ECM Proteins by the CHP-126 Cells The changes in the total protein and ECM protein synthesis by undifferentiated and differentiated CHP-126 cells are shown diagrammatically in Figure 6. Neuronal differentiation of the CHP- 126 cells was associated with a remarkable decrease in the synthesis of all three proteins (64% for FN, 20% for LM, 41 % for

Table 1-Quantitative Differentiation of NB Cells Into Neuronal and Flat Cells With dbcAMP or RA

dbc-AMP Cell lines SMS-SAN IMR-32 CHP-126 SK-N-SH SH-SY5Y

N 100% 100% 90% 50% 60%

N, neuronal; F, flat; U, undifferentiated.

RA

F

U

N

F

U

0 0 10% 40% 30%

0 0 0 10% 10%

100% 50% 30% 0O 0O

0 0 70% 100% 100%

0 50% 0 0 0

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Figure 2-The neuronal cells, especially the ones shown in Figure a,

showed well-defined ultrastructural features, such as neurites with microtubules and neurosecretory granules (arrows). (Uranyl acetate and lead ci-

trate, X26,200)

Type IV collagen), in spite of an overall increase in the total protein synthesis (25%). On the contrary, Schwannian differentiation (RA) was associated with increased synthesis of all three ECM proteins (170% for FN, 120% for LM, and 82% for Type IV collagen), as well as increased total protein synthesis (130%). These results show a disparity in the synthesis of FN, LM, and Type IV collagen by cells with a neuronal versus Schwannian differentiation. Expression of the ECM Proteins In general, IF was more sensitive in detecting ECM proteins in the NB cell lines. Since only minute amounts of certain proteins were occasionally detected in some NB cell lines by IF, their absence from the fluorograms of the SDS-PAGE can be explained on the basis of small amounts of protein, insufficient for detection by SDS-PAGE. IF was also successful in showing expression of the above proteins by cells with specific morphologic phenotypes. FN, LM, and type

IV collagen were exclusively seen in undifferentiated cells, or more so in cells with Schwannian differentiation (Figure 7a-c) and not in neuronal cells (Figure 7d). Melanocytes, on the other hand, expressed only FN. The pattern of staining was circular punctate for LM and Type IV collagen and diffuse or fibrillar for FN. Type IV collagen was present to appreciably lower amounts than LM. No staining was noted when normal rabbit serum was used instead of the primary antisera against LM, FN, and Type IV collagen (data not shown).

Expression of S-100 Protein In general, S-100 protein was not expressed in undifferentiated NB cells, except for rare cells in the CHP- 126 cell line. After treatment with RA, the number of S-100 positive cells increased in the CHP-126 cell line (Figure 8a and b). Sparse S-100 positive cells

were also seen in the SH-SY-5Y and SK-N-SH cell lines after treatment with RA.

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NEUROTRANSMITTER ENZYMES IN CHP-126 Changes with differentiation nmol/hr/mg

40 30

20 10 0

I

a

wff

Contirol

....

VIIA

.....

JJ

Neuronal

(dbc-AMP)

D

JJ..J

Schwannian (R.A.)

Choline acetyl Tranaferase

j

Cyclic Nucleotidyl Phosphohydrolase Figure 4-Diagrammatic presentation of the changes in the activity of cho-

line acetyl transferase (CAT) and cyclic nucleotidyl phosphohydrolase (CNP) by the CHP-126 cells before and after neuronal and Schwann cell differentiation with dbc-AMP and RA, correspondingly. Both enzymes show easily detectable levels of activity in the undifferentiated cells. Neuronal differentiation is accompanied by a slight increase of CAT and decrease of CNP, whereas Schwannian differentiation is accompanied by a remarkable decrease of CAT and increase of CNP. This reverse patter of synthesis of the neuronal (CAT) and the Schwannian (CNP) enzymes by the flat RA-treated CHP-126 cells supports their Schwannian nature at the biochemical level.

Discussion

Figure 3a-Electron micrograph of a flat nonpigmented cell (SH-SY5Y cell clone treated with RA). The cytoplasm contains prominent, occasionally dilated rough endoplasmic reticulum. A discontinuous basal lamina is seen on the surface (arrows). A well-developed intercellular attachment is also present (arrowhead). All of the above are features of Schwann cells and are not seen in neuronal cells. (Uranyl acetate and lead citrate, X33,0UO) (From Neuronal, Schwannian and melanocytic differentiation of human neuroblastoma cells in vitro, Advances in Neuroblastoma Research. Edited by AE Evans, GJ D'Angio, RC Seeger. New York, Alan R. Liss, 1985, pp 55-68.) b-A hybrid cell (SK-N-SH cell line treated with RA) contains both melanosomes (arrows) and neurosecretory granules (circles). At a higher magnification some of the melanosomes show the characteristic striated substructure (inset). (Uranyl acetate and lead citrate, X16,000; inset, X72,000)

Neuronal, Melanocytic, and Schwann Cell Differentiation of NB Cells The potential neuronal character of primitive or undifferentiated NB cells has long been recognized in vivo and in vitro,2'5'7'8 where both spontaneous and agent-induced neural differentiation has been reported.14-16,18,19,44,49,51 In vitro, this differentiation has typically been manifest as the extension of neurites, often massively so, from otherwise undifferentiated, round, poorly substrate adherent cells. Recently, another cell type, markedly flat and substrate adherent, has been reported in human NB cultures treated with bromodeoxyuridine or retinoic acid.'7-19,49,5' The identity of these cells has been uncertain, but the appearance of tyrosinase activity has suggested a melanocytic character.29 Until the present report, then, only neuronal and presumed melanocytic differentiation has been documented in human NB. We have assembled a body of evidence derived from morphologic, enzymologic, immunologic, and biochemical studies which confirms the melanocytic character of the flat cells noted above. The acknowledged neuronal differentiation has been confirmed and extended to include extracellular matrix bio-

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Figure 5-The fluorogram was obtained from radiolabeled media of control and NB cell lines under unreducing (-) and reducing (+) conditions. The six lanes on the left show immunoprecipitated LM, FN, and collagen Type IV from cell lines known to produce the above proteins, which were used as positive controls. The 10 lanes on the right show migration of proteins detected in the media of the 5 studied NB cell lines which were labeled with tritiated leucine and proline. FN and LM were seen in the media of all but one NB cell lines and collagen Type IV was seen only in one NB cell line. Lack of detection of some of the proteins in certain NB cell lines does not necessarily mean absence, but rather low levels, of synthesis of these proteins, since all proteins were detected in all studied NB cell lines by immunofluorescence.

QUANTITATIVE CHANGES IN THE SYNTHESIS AND ACCUMULATION OF THE ECM-PROTEIN8 BY THE CHP-126 CELLS Totel Protein

FN

LM

IV

,

,

zoo

150

100 50 0

-50 -100

-150 -200

N- Neuronal

8- Schwannian

Figure 6-Schematic presentation of total protein, FN, LM, and Type IV collagen synthesis by the CHP-126 cells before and after neuronal and Schwannian differentiation. Total protein synthesis increased after differentiation into neuronal and in particular Schwann cells. On the other hand, the synthesis of all studies ECM proteins increased dramatically after Schwannian differentiation (bars above the baseline) and decreased after neuronal differentiation (bars below the baseline). The latter finding was more dramatic in view of the parallel increase of total protein synthesis in the same

condition.

chemical data. Most importantly, we have derived several lines of evidence which in aggregate provide compelling evidence of Schwannian differentiation in human NB, often in conjunction with melanocytic differentiation. Demonstration of neuronal or melanocytic differentiation is a relatively straightforward proposition, since unique morphologic features (dense core granules within neurites and melanosomes, respectively) characterize each. Reliable evidence for Schwann cell differentiation is far more problematic. Schwann cells in culture in the absence of neuronal cells have no unique light- or electron-microscopic features.52 We felt that corroborative data from two fairly novel experimental methods (neurotransmitter enzyme and extracellular matrix synthesis analysis), in conjunction with more conventional morphologic and immunocytochemical studies, would provide strong evidence in support of our hypothesis that NB cells can undergo differentiation typical of Schwann cells in addition to neuronal and melanocytic differentiation. The evidence presented here substantiates our be-

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Figure 7-Staining of flat, Schwann-like cells by immunofluorescence. a-Staining with antiserum against LM resulted in a punctuate, circular pattern. (X750) b-A similar but slightly different pattem was obtained with antiserum against Type IV collagen; positive staining in the region corresponding to the Golgi apparatus was noted as well. (X390) A punctuate instead of linear patter of staining with antisera against LM and Type IV collagen can be explained by the fact that basal lamina was not assembled in its usual linear form at the periphery of the Schwann cells. c-A flat cell with Schwannian differentiation stains densely with antiserum against FN. Less differentiated adjacent cells are only slightly positive. (X300) d-Neuronal cells are negative for LM. (X250) Similarly, they were negative for FN and Type IV collagen.

liefthat NB cells are capable of Schwann cell differentiation. First, we found the Schwann cell-associated marker S-100 protein53 only in flat cells suspected of Schwannian and/or melanocytic differentiation. Secondly, we found the Schwann cell-associated enzyme CNP50 in appreciable levels only in cells transformed to a flat phenotype after treatment with RA. Thirdly, ultrastructural studies identified basal lamina associated with rare flat cells in vitro; this structure is uniquely associated with Schwann cells in the present context. Finally, increased amounts of the appropriate basal lamina ECM constituents, LM and Type IV collagen, were identified immunologically, and even more importantly, as de novo synthesized proteins, only in putative Schwann cells. In our opinion, these findings can only be reconciled if one accepts the hypothesis that some, if not all, flat cells are undergoing at least early Schwann cell differentiation.

Melanocytic differentiation in NB cells was substantiated by several observations. In addition to melanosomes seen by EM, we found that cells with melanocytic phenotype ("flat," plus melanosomes seen by EM) synthesized and expressed only FN; this par-

allels similar observations on melanoma cells in vitro, where no LM or Type IV collagen synthesis was noted.39 This pattern of ECM synthesis distinguishes melanocytes from Schwann cells, which express all three (vide supra). The melanogenic potential of the SK-N-SH cell line is furthermore supported by the previously reported markedly elevated tyrosinase levels (the required first enzyme in melanogenesis) in the "flat," or "epithelioid," clone of this line (SK-NSH-EP).29 We have found identical flat cells in the SK-N-SH parent clone used in this study; these cells in particular have expressed the melanocytic characteristics noted above.

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port the notion that melanogenesis is a potential feature of nonadrenal NB, or peripherally localized, neural crest-derived tissues. In that respect, the extraadrenal (peripheral) NB may be a more primitive and pluripotential type of NB than classic adrenal NB.

Enzymes and Differentiation It is well documented that neuronal differentiation in human NB is associated with adrenergic NB cell lines with elevated levels of adrenergic neurotransmitter enzymes.'6,56'57 In the present study, we have demonstrated that the nonneuronal, flat cell phenotype following differentiation with RA occurs preferentially in NB cell lines with a mixed adrenergic/cholinergic neurotransmitter enzyme profile. These cells express high levels of Schwann cell-associated enzyme CNP or alternatively contain melanosomes (to name only two of several findings), indicating a capacity for Schwann cell or melanocytic differentiation. It is thus particularly intriguing that of all previous reports ofRA-induced differentiation of human NB,17-19'49'51 only three lines have been noted to develop the flat cell phenotype; all three (SK-N-SH, CHP- 100, SK-N-MC) were derived from NB expressing cholinergic neurotransmitter enzymes only.4244 The clinical presentation was further atypical in that the primary site was nonadrenal, there was no catecholamine excretion in the urine, and the patients were adolescents. This profile is extraordinarily rare in classic neuroblastoma and strongly suggests that these features, including the propensity for nonneural differentiation in vitro, after treatment with RA, delineate another form of NB. These tumors are probably related to, if not identical with, the neural tumor described in the literature under several names, most

Figure 8a-Undifferentiated (X310) b-Flat cells after (X700)

NB cells negative for S-100 RA treatment positive for S-100

protein. protein.

It is interesting to note that melanogenesis in this study was associated with a non-adrenal and less differentiated morphologically NB cell line and its clone. This exactly parallels the results of a study of the melanocytic metabolite 5-S-cysteinyldopa in NB.28 In that study, actual melanogenesis was found only in three extraadrenal NB in vitro. The potential of neural

crest-derived tissue to undergo melanogenesis has also been reported in normal sensory ganglia by Cowell and Weston54 and in cultures of peripheral nervous tissue by Nichols and Weston.55 The above sup-

notably peripheral neuroepithelioma (PN).58 A pattern thus emerges in which classic NB differentiates predominantly into neuronal cells in response to RA, while nonadrenal, cholinergic, primitive NB in adolescents (?PN) preferentially undergoes Schwann cell or melanocytic differentiation in vitro. However, while this is the case for RA, bromodeoxyuridine appears to cause flat cell transformation of presumable Schwann cell origin, even in classic adrenal NB in vitro51; and, therefore, such a possibility cannot be

excluded, especially in view of the presence of both

neuronal and Schwann cells in well-differentiated classic adrenal NB in vivo. The association between neurotransmitter enzyme content and ultimate differentiation in human NB (ie, adrenergic with neuronal, cholinergic with Schwannian/melanocytic) parallels that seen in the

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developing neural crest. Cohen and Konigsberg have noted that certain neural crest cell clones give rise only to neuronal (catecholamine positive) cells, others produce purely melanocytic (tyrosinase positive) cells, and some produce both.59 This suggests that different stages of differentiation are associated with different patterns of neurotransmitter enzyme content. If so, then pluripotentiality (as manifested by the propensity for melanocytic and Schwann cell differentiation in addition to neuronal differentiation) should be associated with cholinergic, not adrenergic or mixed, patterns, since cholinergic neurotransmitters appear first in the development of the neural crest9'60,6I and adrenergic enzymes appear with terminal neuronal differentiation.9'62 Such was clearly the case here, which suggests that the cholinergic, pluripotential lines are less differentiated (or at least less committed to a specific differentiation pathway) than the classic NB lines. Extracellular Matrix, Neuroblastoma, and the Neural Crest As demonstrated in this study, differentiated neuroblasts, like normal neurones, produce ECM proteins (LM, FN, Type IV collagen). The simultaneous synthesis of small amounts of each by undifferentiated neuroblasts presumably reflects an initial lack of commitment to a specific differentiation pathway by these tumor cells. In contrast, normal Schwann cells and the NB tumor cells undergoing Schwann cell differentiation in this study synthesize large amounts of all three components, eventuating in a basal lamina in normal nerves, and even focally in tumor cells in this study. All nerves in the peripheral nervous system are ensheathed by Schwann cells, and Schwann cellneurone interaction is a fundamental issue in neurobiology.63'64 It is reasonable to assume that ECM proteins play a role in this interaction. Experimental data support the hypothesis that ECM plays some role in neural development. Neurite extension by developing neuroblasts in vitro occurs preferentially on LM, FN, or Type IV collagen-coated substrata.3234 Moreover, ECM proteins play a role in the early development of the neural crest. FN surrounds and separates the closing neural tube from adjacent nonneural tissue.3' It is produced by cephalic and sacral, but only to a minimal extent by trunk neural crest cells,35 and it constitutes the early migratory pathways of neural crest cells. 10,3' LM and Type IV collagen constitute the physiologic barrier for the dorsally and ventrally migrating neural crest cells, including colonization of the epidermis by melanoblasts. 1',0" LM and FN influence the pathways of mi-

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gration by neural crest cells, since LM- and FN-coated beads are excluded from the ventral migration pathway.65'66 Finally, analogies have been drawn between the development of the neural crest'0 and the process of tumor invasion and metastasis67; ECM in general and basal lamina in particular are fundamental to the latter and therefore to neural crest development, if the analogy is valid.

Summary The results reported here provide compelling evidence for the potential of NB cells to undergo neuronal, melanocytic, and Schwann cell differentiation in response to environmental stimuli such as dbc-AMP and RA. Such agents alone fail to induce identical differentiation in all NB lines and ultimately serve to unmask inherent differences between two major families of neural tumors of childhood, ie, classic adrenal and nonadrenal (peripheral) NB. Although neuronal and Schwannian differentiation of classic adrenal NB can occur in vitro, as shown in the present and previous studies, the propensity for such, and even more pluripotent (neuronal, Schwannian and melanocytic), differentiation is higher in nonadrenal, cholinergic neuroblastic tumors, which suggests origination of the latter from a more primitive neural crest cell. Furthermore, all the patterns of induced differentiation of neuroblastic tumors in vitro recapitulate different stages of neural crest development, thus supporting origin of these tumors from neural crest.

References 1. Everson TC: Spontaneous regression of cancer. Ann NY Acad Sci 1964, 114:721-735 2. Adam A, Hochholzer L: Ganglioneuroblastoma of the posterior mediastinum: A clinicopathologic review of 80 cases. Cancer 1981, 47:373-381 3. Bolande RP: Spontaneous regression and cytodifferentiation of cancer in early life: The oncogenic grace period. Surv Synth Pathol Res 1985, 4:296-311 4. Bove KE, McAdams AJ: Composite ganglioneuroblastoma: An assessment of the significance of histologic maturation in neuroblastoma beyond infancy. Arch Pathol Lab Med 1981, 105:325-330 5. Triche TJ, Askin FB: Neuroblastoma and the differential diagnosis of small, round blue-cell tumors. Hum Pathol 1983 14:569-595 6. Gonzales-Angulo A, Reyes HA, Reyna AN: The ultrastructure of ganglioneuroblastomas: Observation on neoplastic ganglion cells. Neurology 1965, 15:242-252 7. Romansky SG, Crocker DW, Shaw KNF: Ultrastructural studies on neuroblastoma. Cancer 1978, 42:2392-2398 8. Taxy JB: Electron microscopy in the diagnosis of neuroblastoma. Arch Pathol Lab Med 1980, 104:355-360 9. Le Douarin NM: Migration and differentiation of neural crest cells. Curr Top Dev Biol 1980, 16:31-85 10. Le Douarin N: The neural crest. New York, Cambridge University Press. 1982 11. Weston JA: A radioautographic analysis of the migra-

Vol. 128 * No. 3

12.

13. 14. 15.

16. 17.

18.

19. 20. 21.

22. 23. 24. 25.

26.

27. 28. 29.

30. 31.

DIFFERENTIATION OF NEUROBLASTOMA IN VITRO

tion and localization of trunk neural crest cells in the chick. Dev Biol 1963, 6:279-3 10 Bolande RR: Neurofibromatosis: The quintessential neurocristopathy: pathogenetic concepts and relationships, Advances in Neurology: Neurofibromatosis (Von Recklinhausen's Disease). Vol 29. Edited by VM Riccardi, and JJ Mulvihill. New York, Raven Press, 1981 Goldstein MN, Burdman JA, Journey U: Long-term tissue culture of neuroblastomas: II. Morphological evidence for differentiation and maturation. J Natl Cancer Inst 1964, 32:165-174 Murray MR, Stout AP: Distinctive characteristics of the sympathicoblastoma cultivated in vitro. Am J Pathol 1947, 23:429-441 Prasad KN: Differentiation of neuroblastoma cells in culture. Biol Rev 1975, 50:129-265 Prasad KN, Kumar S: Role of cyclic AMP in differentiation of human neuroblastoma cells in culture. Cancer 1975, 36:1338-1343 Sidell N: Retinoic acid-induced growth inhibition and morphologic differentiation of human neuroblastoma cells in vitro. J Natl Cancer Inst 1982, 68:589-593 Sidell N, Altman A, Haussler MR, Seeger RC: Effects of retinoic acid (RA) on the growth and phenotypic expression of several human neuroblastoma cell lines. Exp Cell Res 1983, 148:21-30 Tsokos M, Scarpa S, Thorgeirsson UP, Liotta LA, Triche TJ: Differentiation, matrix protein synthesis and in vitro invasiveness of human neuroblastoma (Abstr). Fed Proc 1983, 42:525A Sidman RL, O'Gorman SV: Cellular interactions in Schwann cell development,12 1981 Kyritsis AP, Tsokos M, Triche TJ, Chader GJ: Retinoblastoma-Origin from a primitive neuroectodermal cell? Nature 1984, 307:471-473 Tsokos M, Kyritsis AP, Chader GJ, Triche TJ: Differentiation of human retinoblastoma in vitro into cell types with characteristics observed in embryonal or mature retina. Am J Pathol 1986, 123:542-552 Sueoka N, Droms, K: On neuronal and glial differentiation of a pluripotent stem cell line, RT4-AC: A branch determination. Curr Top Dev Biol 1986, pp 211-221 Rawles ME: Origin of pigment cells from neural crest in the mouse embryo. Physiol Zool 1947, 20:248-266 Dehner LP. Sibley RK, Sauk JJ, Vickers RA, Nesbit ME, Leonard AS, Waite DE, Neeley JE, Ophoven J: Malignant melanotic neuroectodermal tumor of infancy: A clinical, pathologic, ultrastructural and tissue culture study. Cancer 1979, 43:1389-1410. Mennermeyer RP, Hammar SP, Tytus JS, Hallman RO, Raisis JE, Bockus D: Melanotic Schwannoma: Clinical and ultrastructural study of three cases with evidence of intracellular melanin synthesis. Am J Surg Pathol 1979, 1:3-10 Boesel CP, Suhan JP, Sayers MP: Melanotic medulloblastoma: Report of a case with ultrastructural findings. J Neuropathol Exp Neurol 1978, 37:531-543 Aubert C, Janiaud P, Hansson C, Rorsman H, Rosengren E: Melanogenesis in cultured human neuroblastomas. Ann Clin Res 1980, 12:288-294 Ross RA, Biedler JL: Presence and regulation of tyrosinase activity in human neuroblastoma cell variants in vitro. Cancer Res 1983, 45:1628-1632 Alitalo K, Keski-Oja, J, Vaheri, A: Extracellular matrix proteins characterize human tumor cell lines. Int J Cancer 1981, 27:755-761 Newgreen DF, Thiery JP: Fibronectin in early avian embryo: Synthesis and distribution along the migratory pathways of neural crest cells. Cell Tissue Res 1980, 211:269-291

495

32. Carbonetto S, Gruver MM, Turner DC: Nerve fiber growth in culture on fibronectin, collagen and glycosaminoglycan substrates. J Neurosci 1983, 3:2324-2335 33. Manthorpe M, Engvall E, Ruoslahti E, Longo FM, Davis GE, Varon S: Laminin promotes neuritic regeneration from cultured peripheral and central neurons. J Cell Biol 1983, 97:1882-1890 34. Rogers SL, Letourneau PC, Palm SL, McCarthy J, Furcht LT: Neurite extension by peripheral and central nervous system neurons in response to substratumbound fibronectin and laminin. Dev Biol 1983, 98:212-220 35. Duband JL, Thiery JP: Distribution of fibronectin in the early phase ofavian cephalic neural crest migration. Dev Biol 1982, 93:308-323 36. Carey DJ, Eldridge CF, Cornbrooks CJ, Timpl R, Bunge RP: Biosynthesis of type IV collagen by cultured rat Schwann cells. J Cell Biol 1983, 97:473-479 37. Cornbrooks CJ, Carey DJ, McDonald JA, Timpl R, Bunge RP: In vivo and in vitro observations on laminin production by Schwann cells. Proc Natl Acad Sci USA 1983, 80:3850-3854 38. Palm SL, Furcht LT: Production of laminin and fibronectin by Schwannoma cells: Cell-protein interactions in vitro and protein localization in peripheral nerve in vivo. J Cell Biol 1983, 96:1218-1226 39. Bumol TF, Resified RA: Biosynthesis and secretion of fibronectin in human melanoma cells. J Cell Biochem 1983, 21:129-140 40. Reynolds CP, Frenkel EP, Smith GR: Growth characteristics of neuroblastoma in vitro correlate with patient survival. Trans Assoc Am Physicians 1980, 93:203-211 41. Tumilowicz JJ, Nichols WW, Cholon JJ, Green AE: Definition of a continuous human cell line derived from neuroblastoma. Cancer Res 1970, 30:2110-2118 42. Schlesinger HR, Gerson JM, Moorhead PS, Maguire H, Hummeler K: Establishment and characterization of human neuroblastoma cell lines. Cancer Res 1976, 36:3094-3100 43. Biedler JL, Helson L, Spengler BA: Morphology and growth, tumorigenicity and cytogenetics of human neuroblastoma cells in continuous culture. Cancer Res 1973, 33:2643-2652 44. Ross RA, Joh TH, Reis DJ, Spegler BA, Biedler JL: Expression of neurotransmitter-synthesizing enzymes in human neuroblastoma cells: Relationship to morphological diversity, Advances in Neuroblastoma Research Edited by AE Evans. New York, Raven Press, 1980, pp 151-160 45. Fonnum F: Radiochemical micro-assays for the determination ofcholine acetyltransferase and acetylcholinesterase activities. Biochem J 1969, 115:465-471 46. Glastris B, Pfeiffer SE: Mammalian membrane marker enzymes: Sensitive assay for 5'-nucleotidase and assay for mammalian 2',3'-cyclic nucleotide-3'-phosphohydrolase. Methods Enzymol 1974, 32:124-131 47. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685 48. Engvall EP, Perlmann P: Enzyme linked immunosorbent assay (ELISA). Quantitative assay for immunoglobulin G. Immunochemistry 1971, 8:871-879 49. Haussler M, Sidell N, Kelly M, Donaldson C, Altman A, Mangelsdorf D: Specific high-affinity binding and biologic action of retinoic acid in human neuroblastoma cell lines. Proc Natl Acad Sci 1983, 80:5525-5529 50. Reddy B, Askanas V, King-Engle W: Demonstration of 2',3'-cyclic nucleotide 3'-phosphohydrolase in cultured human Schwann cells. J Neurochem 1982, 39:887-889 51. Reynolds CP, Maples J: Modulation of cell surface an-

496

52.

53. 54. 55.

56. 57.

58.

TSOKOS ET AL

tigens accompanies morphological differentiation of human neuroblastoma cell lines, Advances in Neuroblastoma Research. Edited by AE Evans, GJ D'Angio, RC Seeger. New York, Alan R. Liss, 1985, pp 13-37 Askansas V, King Engel W, Dalakas MC, Lawrence JV, Carter LS: Human Schwann cells in tissue culture: Histochemical and ultrastructural studies. Arch Neurol 1980, 37:329-337 Kahn HJ, Marks A, Thom H, Baumal R: Role ofantibody to S-100 protein in diagnostic pathology. Am J Clin Pathol 1983, 79:341-347 Cowell LA, Weston JA: An analysis of melanogenesis in cultured chick embryo spinal ganglia. Dev Biol 1970, 22:670-697 Nichols DH, Weston JA: Melanogenesis in cultures of peripheral nervous tissue: 1. The origin and prospective fate of cells giving rise of melanocytes. Dev Biol 1977, 60:217-225 Richelson E: Stimulation oftyrosine hydroxylase activity in an adrenergic clone of mouse neuroblastoma by dibutyryl cyclic AMP. Nature 1973, 242:175-177 Waymire JC, Weiner N, Prasad K: Regulation of tyrosine hydroxylase activity in cultured mouse neuroblastoma cells: Elevations induced by analogs ofadenosine 3',5'-cyclic monophosphate. Proc Natl Acad Sci 1972, 69:2241-2242 Tsokos M, Donner L, Reynolds CP, Triche TJ: Peripheral neuroepithelioma: An entity distinct from classic neuroblastoma (Abstr). Lab Invest 1985, 52:70A

AJP * September 1987

59. Cohen AM, Konigsberg IR: A clonal approach to the problem of neural crest determination. Dev Biol 1975, 46:262-280 60. Cochard P, Coltey P: Cholinergic traits in the neural crest: Acetylcholinesterase in crest cells of the chick embryo. Dev Biol 1983, 98:221-238 61. Smith J, Fauquet M, Ziller C, Le Douarin NM: Acetylcholine synthesis by mesencephalic neural crest cells in the process of migration in vivo. Nature 1979, 282:853-855 62. Cochard P, Goldstein M, Blank IB: Ontogenetic appearance and disappearance of tyrosine hydroxylase and catecholamines in the rat embryo. Proc Natl Acad Sci 1978, 75:2986-2990 63. Bunge MB, Williams AK, Wood PM: NeuronSchwann cell interaction in basal lamina formation. Dev Biol 1982, 92:449-460 64. Mudge AW: Schwann cells induce morphologic transformation of sensory neurons in vitro. Nature 1984, 309:367-369 65. Bronner-Fraser M: Distribution of latex beads and retinal pigment epithelial cells along the ventral neural crest pathway. Dev Biol 1982, 91:50-63 66. Bronner-Fraser M: Latex beads as probes of a neural crest pathway: Effects of laminin, collagen, and surface charge on bead translocation. J Cell Biol 1984, 98:1947-1960 67. Liotta LA, Rao CN, Barsky SH: Tumor Invasion and extracellular matrix. Lab Invest 1983, 49:636-649