Induction of cell attachment and morphological differentiation in a pheochromocytoma cell line and embryonal sensory cells by the extracellular matrix

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BIOLOGY

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Induction of Cell Attachment and Morphological Differentiation in a Pheochromocytoma Cell Line and Embryonal Sensory Cells by the Extracellular Matrix I. VLODAVSKY,*~’ *Department

A. LEVI,?.”

I. LAX,~

Z. FUKS,*

AND

J. SCHLESSINGER~

of Rudiation & Clinical Oncology, Sharett Institute, Hadassah Univer.sity Hospitul, Jerusalem, and TDepartment of Chemical Iwnruuoloyy. Weizmann Institute of Sciencr, Rehowt, I.srael

Israel;

Received July 8, 19X1; accepted in revised form Mary 17, 19X1 Embryonic chicken sensory cells from dorsal root ganglia and a clonal line of pheochromocytoma cells (PC-12) extended neuronal-like processes within 24 hr of seeding on a naturally produced, basement membrane-like extracellular matrix (ECM) in the absence of nerve growth factor (NGF). Plating on ECM also induced a rapid cell attachment and flattening of these cells and supported the survival of embryonic sensory cells in primary cultures, Unlike the effect of NGF on PC-12 cells, the ECM-induced morphological differentiation was transient and led to disintegration and degeneration of processes bearing PC-12 cells. The ECM-induced morphological differentiation was not inhibited by anti-NGF antibodies, and the cells retained their ability to bind and internalize NGF in a manner similar to that observed on plastic. PC-12 cell attachment and flattening occurred on dishes coated with collagen type IV in a way similar to that observed on ECM, but precoating the dishes with tibronectin had no effect. Extension of cell processes was not induced by either substrate. Morphological differentiation but not the induction of cell adhesion and flattening was inhibited by either prefixation with glutaraldehyde, oxidation with periodate, or preexposure to concanavalin A of the ECM, suggesting that the ECM and in particular its sugar moieties play an active role in the induction of neurite outgrowth. It is suggested that close contact with the ECM provides chemical or mechanical cues that permit contactmediated elongation and directed growth of both embryonic and regenerating nerve fibers.

and orientation, to make it more responsive to physiologically occurring hormones and growth factors (Gospodarowicz and Tauber, 1980; Gospodarowicz et al., 1980b). Therefore, providing cells with a substrate most closely resembling their natural environment is essential if a meaningful extrapolation to the in ~ivo situation is to be drawn from in vitro studies. Primary cultures of embryonal sensory cells (LeviMontalcini and Angeletti, 1963,1968; Greene, 1977) and a clonal cell line PC-12, established from a transplantable rat pheochromocytoma (Greene and Tischler, 1976; Greene and Rein, 1977) exhibit both rapid and delayed responses to added nerve growth factor (NGF). Events occurring shortly after the addition of NGF include increased cell to substrate and cell to cell adhesion and changes in cell morphology (Connolly et al., 1979; Schubert and Whitlock, 1977; Schubert et al., 1978). Among the delayed responses observed are the extension of neuronal-like processes, development of electrical excitability, induction of specific enzymes, and long-term maintenance of cell viability (Levi-Montalcini and Angeletti, 1963, 1968; Greene, 1977; Greene and Tischler, 1976; Greene and Rein, 1977; Dichter et al., 1977; Schubert et al., 1978). Because basement membrane components may provide a chemical or mechanical cue to regenerating axons, so as to allow the precise reinner-

INTRODUCTION

The involvement of the extracellular matrix (ECM) in normal growth and differentiation in vivo has long been recognized by developmental biologists (Grobstein, 1967; Dodson, 1963; Hay and Meier, 1976; Wessels, 1964, 1977; Bernfield, 1978). Likewise, recent ix vitro studies have shown that cell interaction with a naturally produced, basement membrane like ECM induces dramatic effects on cell shape (Vlodavsky et al., 1980), growth characteristics (Gospodarowicz and Tauber; Gospodarowicz et ul.l980a,b) and response to naturally occurring hormones and growth factors (Gospodarowicz and Ill, 1980). The ECM has also been shown to modulate gene expression and cytodifferentiation and in some cases to shift the phenotypic expression of already differentiated cell types (Reddi and Anderson, 1976). The view put forth is that the ECM does not function merely as an inert structural support. Rather, it plays an active role in the control of cell growth and differentiation, possibly via modulation of cell shape ’ To whom all correspondence should be addressed: Department of Radiation & Clinical Oncology, Hadassah University Hospital, Kiryat Hadassah, 91 120 Jerusalem, Israel. ‘Permanent address: Laboratory of Cell Biology, Consiglio Nazionale delle Richerche, Roma, Italy. 285

0012.lSOS/S2/100285-16$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

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vation of denervated muscle (Marshal et al., 1977) and because cells plated on ECM undergo rapid morphological changes (Vlodavsky et al., 1980) and no longer require the addition of mitogenic factors in order to proliferate and differentiate (Gospodarowicz and Tauber, 1980; Gospodarowicz et al., 1980b; Salomon et al., 1981), we have tested whether a mere interaction of either the PC-12 or primary sensory cells with the ECM can mimic the effect of NGF on cell attachment and flattening, morphological differentiation and survival. MATERIALS

AND METHODS

Cell culture conditions. PC-12 cells were grown on Falcon tissue culture dishes in a medium consisting of 85% RPM1 1640,10% heat-inactivated horse serum, 5% fetal calf serum, 50 pg/ml streptomycin, and 50 units/ ml penicillin. Cells were dissociated for replating by vigorous trituration and growth medium was replaced twice a week (Greene and Tischler, 1976; Connolly et al., 1979). Dorsal root ganglia were dissected from 8-day chicken embryos, collected, and cultured in RPM1 1640 medium consisting of 1% heat-inactivated horse serum, 2 mM glutamine, 50 units/ml penicillin, and 50 pg/ml streptomycin (Levi et al., 1980). A single-cell suspension was prepared by trypsinization (10 min, 37”C, 0.1% trypsin, Difco Inc., 1:250) and repeated suction with a Pasteur pipet (Greene 1977; Levi et al., 1980). Cells were then seeded in RPM1 medium, containing 1% horse serum, into tissue culture dishes coated with either ECM or poly-L-lysine (5 pg/35-mm plate) as described (Vlodavsky et al., 1980; Mazia et al., 1975). All cultures were maintained at 37°C in a water-saturated atmosphere containing 5% COZ. Cultures of bovine cornea1 endothelial cells were established from steer eyes as described (Gospodarowicz et al., 1977). Stock cultures were maintained in Dulbecco’s modified Eagle’s medium (DMEM, H-16), which was supplemented with 1OYo calf serum, 5% fetal calf serum, gentamycin (50 pg/ml), and Fungizone (2.5 pug/ ml). Fibroblast growth factor (FGF, 100 rig/ml) was added every other day until the cells were nearly confluent. Bovine brain FGF was purified as described (Gospodarowicz et al., 1977). Preparation of dishes coa.ted ‘with>ECM. Cornea1 endothelial cells dissociated with 0.05% trypsin/O.O2% EDTA solution (STV) were plated at an initial density of 4 X lo4 cells per 35mm dish (Falcon) and maintained in the presence of DMEM containing 10% calf serum, 5% fetal calf serum, 5% Dextran T-40, 50 pg/ml gentamycin, and 2.5 pg/ml Fungizone. FGF was added every other day at a concentration of 100 rig/ml until the cells reached confluence and the cultures were further

VOLUME 93, 1982

incubated for 6 days. The cultures were then washed once with phosphate-buffered saline (PBS) and exposed (30 min of gentle shaking at room temperature) to 0.5% Triton X-100 in PBS (v/v). The cell layer was dissolved, leaving the underlying ECM intact and firmly attached to the entire tissue culture dish (Gospodarowicz et al., 1980a; Vlodavsky et al., 1980). Remaining nuclei and cytoskeletons were removed by a 2- to 3-min exposure to 0.025 N NH,OH followed by four washes in PBS. ECM-coated dishes containing PBS could be stored before use at 4°C for up to 3 months. Coating of culture dishes with fibronectin and various types ofcollagen. Bovine plasma fibronectin was purified by affinity chromatography on a gelatin-Sepharose column (Engvall and Ruoslanti, 1977) followed by chromatography on a Sephadex G-200 column (Vlodavsky et al., 1980). The purified preparation (l-3 mg/ml) in 4 Murea was diluted in PBS and used at a concentration of 30 pg/ml to coat 35-mm tissue culture dishes by incubation (1 ml per dish) for 1 hr at 22°C followed by three washes in PBS as described (Orly and Sato, 1979). Collagen preparations (1 mg/ml), dissolved in 0.5 M acetic acid, were added to culture dishes containing 1 ml water at a final concentration of 25 pg/ml and allowed to dry exposed to air at room temperature as described (Murray et al., 1979). Collagen types I (rat tail), III (human fetal skin), and IV (human placenta) were kindly provided by Dr. S. Tseng (University of California at San Francisco). Attachment assay. PC-12 cells cultured on ECM were dissociated with STV into a single-cell suspension, suspended in growth medium, and seeded (2.5 X lo5 cells/ 35-mm dish) into either regular tissue culture dishes or dishes coated with various substrates. At different times after seeding, unattached cells were removed by a gentle trituration and rinsing of the dishes (twice) with PBS. The remaining firmly attached cells were dissociated with STV and duplicate cultures were counted electronically using a Coulter counter (Vlodavsky et al., 1980; Vlodavsky and Gospodarowicz, 1981). The variation in duplicate determination was less than 10%. PC-12 cells cultured on plastic did not yield a single-cell suspension but rather cell aggregates upon trypsinization or vigorous pipetting, and therefore could not be used as stock culture for the attachment assay. Scanning electrrm microscopy. PC-12 cells cultured on ECM were washed twice with PBS at 37°C and fixed (30 min at 37°C followed by 24 hr at 22°C) with 2.5% glutaraldehyde in PBS. Incubation with 2% guanidine hydrochloride and 2% tannic acid, postfixation with 2% osmium tetraoxide, dehydration, and critical point drying were performed as described (Murakami et al., 1977). Dried specimens were sputter coated with a thin

layer of gold-palladium and examined with a JEOL 35 SEM at an accelerating voltage of 25-35 kV. Locakixutiov of cutecholartiines. PC-12 cells cultured for 48 hr on ECM were dried overnight under vacuum in a desiccator containing silica gel. The culture dishes (35 mm) were then placed in a desiccator containing dry paraformaldehyde and heated at 80°C for 1 hr. Control dishes were heated for 1 hr without paraformaldehyde. Cultures were observed in a fluorescence microscope (Zeizz) and micrographs taken with Kodak TriX film. A greenish fluorescence after paraformaldehyde exposure is indicative of the presence of catecholamines (Jacobowitz and Greene, 1974). NGF receptor assay. NGF, 2.5 S, purified according to the method of Bocchini and Angeletti (1969) was a generous gift of Dr. P. Calissano (Consigilio Nazionale delle Richerche, Rome). Affinity purified antibodies against NGF (Stockel et al., 1976) were generously provided by C. Cozzari (Consigilio Nazionale delle Richerche, Rome). 12”1-NGF was prepared according to the method of Young et al. (1979). For NGF binding studies, the PC-12 cells were seeded at 0.5 X 10” cells per well on either ECM or polylysinecoated Costar multiwell trays. Twenty-four hours later the medium was replaced and the cells were incubated (60 min, 37°C) with 0.4 ml growth medium containing increasing concentrations of ‘““I-NGF in the absence or presence of large excess (10 pg/ml) unlabeled NGF. as deSpecific binding of ‘““I-NGF was determined scribed (Levi et al., 1980). Image-intensijied video Juorescence microscopy. PC12 and dorsal root ganglia cells cultured on ECM were incubated (2 hr, 37°C in growth medium) with rhodamine-conjugated NGF (R-NGF, 130 rig/ml), prepared as previously described (Levi et al., 1980). Cells were washed three times with medium and R-NGF was localized by means of an intensified target (ISIT) camera (RCA TC 1040/H) which can detect very low levels of light (Schlessinger et al., 1978; Levi et al., 1980). RESULTS

Cell Attachment

and Flattening

PC-12 cells seeded on plastic formed loosely attached aggregates composed of tightly packed spheroidal cells (Fig. 1A). A similar morphology was adopted by cells seeded on dishes coated with either fibronectin (Fig. 1D) or with poly-L-lysine and the plated cells could be removed by gentle pipetting, regardless of their time in culture. In contrast, seeding of the same cell aggregates into dishes coated either with ECM (Fig. 1B) or collagen type IV (Fig. 1C) was associated with a rapid cell spreading and migration leading, in less than an hour, to the formation of colonies composed of flat, nonover-

lapping cells. These cells could be dissociated from the tissue culture dish only by means of trypsinization. The time course of PC-12 cell attachment to various substrates was studied with a single-cell suspension prepared by trypsinization of a stock culture maintained on ECM. Less than 5% of the cells seeded either on plastic or fibronectin became firmly attached and no cell flattening was observed, even after 3 hr in culture (Fig. 2). In contrast, 60 to 70% of the cells were firmly attached and flattened within 30 min when plated into dishes coated with either ECM or collagen type IV (Fig. 2). In all experiments, the attachment to type IV collagen was greater than that observed to type I or III collagen (Fig. 2). Cell flattening but not attachment was slower (l-2 hr) in a medium containing bovine serum albumin as compared to FCS, and the addition of serum prior to cell flattening induced a rapid (lo- to 15-min) Aattening of the attached cells. Firm cell attachment and flattening were also induced by precoating the dishes with medium conditioned by vascular endothelial cells (not shown) containing, among other components, both fibronectin and laminin (Vlodavsky and Gospodarowicz, 1981). Precoating the culture dishes with poly-L-lysine induced cell attachment to about half the extent seen on ECM but with no induction of cell flattening. Extension

of Cell Processes

Stimulation of cellular adhesion and flattening has been suggested as the first step in NGF-initiated neurite outgrowth (Schubert et al., 1978). It has also been shown that growth cones of embryonic sensory neurons prefer to elongate on surfaces to which they adhere most firmly (Letourneau, 1979). We have, therefore, studied whether the induction of a firm cell attachment and flattening by the ECM is associated with subsequent morphological differentiation in both the PC-12 and the primary sensory cells. (u) Pheoch,rornocytol?La cells. Extension of cell processes in the absence of added NGF was observed within 24 hr after cell seeding on ECM (Fig. 3B). A maximal response in about 40-50s of the cells was observed 4860 hr after seeding (Fig. 3C, D). This was followed by degeneration and disintegration of the extended processes and to a lesser extent by cell death (about 40% of the processes bearing cells), within 3-5 days after seeding (Figs. 3E, F). Thus, in comparison with the effect of NGF (Greene and Tischler, 19’76), the ECM-induced morphological differentiation was fast and mostly transitory. PC-12 cells devoid of neuronal-like processes exhibited no signs of degeneration or cessation of cell proliferation and formed islets of closely apposed and flattened cells. Due to their higher ra.te of proliferation

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FIG. 2. Time course of firm attachment and flattening of PC-12 on various substrates. PC-12 cells cultured on ECM-coated dishes for 24 hr were dissociated by treatment with STV into a single-cell suspension. Cells, 2.5 X lo’, were seeded in growth medium into regular 35-mm tissue culture dishes (a) or dishes coated with either ECM (O), bovine plasma fibronectin (25 pg/dish) (m), or 25 ~g each of either collagen type IV (A) or collagen type I (0). At various times after seeding, unattached cells were removed by gentle pipetting and rinsing with PBS. The remaining firmly attached cells were dissociated with STV and counted, as described under Materials and Methods.

when plated on ECM (doubling time 32-40 hr) there was, during the phase of active cell growth, a two- to fourfold higher number of cells on ECM than on regular culture dishes. This effect was even greater at low concentrations of serum (1% FCS + 1% HS) where the cells on plastic hardly proliferated as compared to a doubling time of about 48 hr on ECM. PC-12 cells plated on ECM survived also in serum-free medium (doubling time lo-12 days) as compared to cell death within 3-4 days on uncoated dishes. The best results in terms of process extension were obtained with single cells seeded on ECM at a low density (2-5 X lo4 cells per 35-mm dish) whether in the absence or in the presence of serum, where up to 80% of the cells exhibited, within 24-48 hr, processes at least twice as long as the cell diameter. These processes, although somewhat shorter and flatter than those induced by NGF, contained growth cones (Figs. 3C, D, and 4C), varicosities (Figs. 3D, E, and 4D) and branching points (Figs. 3 and 4) as revealed by phase (Fig. 3) and scanning electron (Fig. 4) microscopy. Preexposure of dried cultures to paraformaldehyde (1 hr, 80°C) yielded a greenish fluorescence along varicose processes (Fig. 4B) indicative of the presence of catecholamines (Jacobowitz and Greene, 1974). When differentiated PC-12 cells were removed from ECM-coated dishes and replated on plastic, they did not reextend their fibers. On the other hand, re-

plating on ECM was associated with the reextension of processes as readily as within 6 hr. The ECM-induced extension of cell processes was not inhibited in the presence of anti-NGF antibodies (not shown). Regardless of the time in culture, there was no induction of neuronal-like processes in cells seeded on plastic dishes coated with either fibronectin, each of the collagen types (I, III, IV, V), or with medium conditioned by vascular endothelial cells. (b) Dwsal root ganglia. The induction of morphological differentiation in explanted and dissociated primary cell cultures of chicken embryo sensory ganglia is demonstrated in Figs. 5 and 6, respectively. Embryonic sensory ganglia explanted on ECM-coated dishes exhibited, in the absence of added NGF, extensive outgrowth of cell processes within 24 hr (Fig. 5C). Morphologically, the fibers resembled those induced by NGF in ganglia maintained on dishes coated with either collagen or polylysine (Fig. 5A). The presence of anti-NGF antibodies greatly inhibited the effect of NGF (Fig. 5B) but had no inhibitory effect on the ECM-induced outgrowth of neurites (Fig. 5D). Therefore, the morphological differentiation of sensory neurons plated on ECM is in all likelihood not mediated by an endogenous NGF. That NGF is not a component of the ECM was further suggested by showing that ““I-goat anti-rabbit IgG binds in equal amounts to ECM that was preex-

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FIG. 3. Morphological differentiation of PC-12 cells seeded on ECM -coated dishes. Stock cultures maintained on plastic were dissociated dishes in RPM1 medium containing 10% HS and 5% by vigorous trituration and the cells seeded on poly-L-lysine (A), or ElCM (B-F)-coated FCS. Phase-contrast micrographs (200X) were taken 24 hr (B), 48 hr (C, D), and 90 hr (E, F) after seeding. Cells plated on poly-l-lysinecoated dishes form aggregates composed of round to ovoid cells (A), whereas cells plated on ECM migrate out of the cell aggregates and exhibit neuronal-like processes which are long and branched. Note the presence of growth cones (C, D) and varicosities (D, E). Degeneration of these processes as well as cell death were observed to begin 72 hr iafter seeding the cells on ECM (E, F).

posed (90 min, 37°C) to either affinity purified rabbit anti-NGF antibodies or to nonimmune rabbit IgG. The ECM was also shown to mimic the effect of NGF

on dissociated dorsal root ganglia in terms of both neurite extension and cell survival (Fig. 6). Dissociated cells maintained on dishes coated with either poly-L-lysine

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FIG. 4. Extension of neuronal-like processes from PC-12 cells seeded on ECM. PC-12 cells from stock cultures were seeded in complete medium into 35-mm dishes coated with ECM (lo” cells per dish). Forty-eight hours after seeding the cultures were processed for SEM (A, C, D) and localization of catecholamines (B) as described under Materials and Methods. Plating on ECM is associated with an extension of long processes (A, 870x) whose neuronal character is demonstrated by the presence of branching points (A-D), growth cones (C, 2400X), and varicosities (D, 3700x). Exposure to paraformaldehyde (60 min, 80°C) yielded a greenish fluorescence along varicose processes indicative of the presence of catecholamines (B).

or collagen in the absence of NGF failed to survive for even 48 hr after plating and underwent degeneration and complete disintegration (Fig. 6B). In contrast, cells plated on ECM (Fig. 6A) survived, like cells maintained on polylysine in the presence of NGF (Fig. 6D), for over 3 weeks in culture and exhibited an extensive outgrowth of neurites. This extension of processes was observed within 24-48 hr after seeding, was not inhibited by antiNGF antibodies, and was similar to that observed with

cells plated on polylysine-coated dishes in the presence of NGF (Fig. 6D). After 1 week in culture, both intact and dissociated ganglia were overgrown by fibroblastic cells. That the ECM-induced extension of neuronal-like processes was not mediated by these nonneuronal ganglionic cells was demonstrated in the following way. Intact or dissociated (Fig. 6) dorsal root ganglia were plated on ECMcoated dishes containing glass coverslips coated with

FIG. 5. Extension of neuronal-like processes out of embryonic dorsal root ganglia explanted in the presence or absence of NGF on dishes coated with either polylysine or ECM, respectively. Chicken sensory ganglia were explanted on poly-L-lysine(A, B) or ECM (C, D)-coated dishes with (A, B) or without (C, D) 50 rig/ml NGF and in the presence (B, D) or absence (A, C) of affinity purified anti-NGF antibodies. Micrographs were taken 48 hr after seeding (phase contrast, 160X). An extensive outgrowth of neuronal processes is seen with cells maintained on either poly-L-lysine plus NGF or on ECM alone. Anti-NGF antibodies greatly inhibited the formation of these processes when added to cells maintained on dishes coated with poly-L-lysine (B) but there was no inhibitory effect on ECM (D).

FIG. 6. Neuronal differentiation of dissociated single primary sensory cells seeded into dishes coated with ECM and containing glass coverslips placed on top of the ECM. Cells, 2 X IO5 from dorsal root ganglia dissociated with trypsin were seeded (RPM1 supplemented with 1% HS) in direct contact with the ECM (A, C) and on polylysine-coated coverslips that were first placed on top of the ECM (B, D). NGF (5 rig/ml) was added to some of the dishes (C, D) and phase-contrast micrographs (160X) were taken 48 hr after seeding. Cells plated on ECM in the absence (A) or presence (C) of NGF extended neuronal-like processes that were similar in size and morphological appearance to those induced by NGF in cells plated on the polylysine-coated coverslip (D). In contrast, cell death and disintegration were observed after similarly seeding the cells on top of such a coverslip but in the absence of NGF (B). Similar results were obtained with coverslips coated with collagen and placed into either uncoated or ECM-coated dishes.

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polylysine. Forty-eight hours later, the cells on ECM showed an extensive outgrowth of processes (Fig. 6A) while the cells on coverslips exhibited no such processes and mostly died (Fig. 6B). Control cultures incubated with NGF showed neuronal-like processes both when plated on ECM (Fig. 6C) and glass coverslips (Fig. 6D). Cell Interaction with Modiified ECM

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(10 min, 85°C) had little or no effect on cell attachment and spreading but clearly inhibited the induction of processes outgrowth. The extension of neuronal-like processes from explanted ganglia (Fig. 8) was less readily inhibited by the various modifications of the ECM as compared with the PC-12 cells. Thus, exposure of the ECM to higher concentrations of periodate (50100 mM) was required to inhibit the outgrowth of processes from explanted dorsal root ganglia (Fig. SC). Dissociated single primary sensory cells failed to survive and degenerated within 24 hr when plated on either prefixed or periodate-treated ECM. These modifications had no effect on the outgrowth and proliferation of nonneuronal cells present in intact (Fig. 8) or dissociated ganglia.

In an attempt to distinguish between the ECM-mediated cell adhesion and flattening and its induction of neuronal-like processes, the ECM was subjected to various modifications and tested for its ability to induce cell attachment and morphological differentiation. It was found that extension of processes in PC-12 cells (Figs. 7B, D, F) and to a lesser extent in the primary sensory cells (Fig. 8) was inhibited by pretreating the Interaction with NGF ECM with either sodium periodate (10 m&f, 30 min, Cells plated on ECM were exposed to either rhoda22”(J), concanavalin A (50 pg/ml, 30 min, 22”C), or glumine-conjugated NGF (R-NGF) (Levi et al., 1980) (Fig. taraldehyde (O.l%, 5 min, 22°C) (Figs. 7,8). In contrast, these treatments had a very small or no effect on the 9) or to lz51-NGF to study whether they retain the abilinduction of initial cell attachment and flattening (Figs. ity to bind and internalize NGF in a manner similar 7A, C & E). The involvement of the ECM sugar moieties to that of cells maintained on polylysine-coated dishes. in the induction of neurite extension is suggested by its The distribution of R-NGF (added at a concentration sensitivity to oxidation with sodium periodate and to of 130 rig/ml for 2 hr at 37°C) on differentiated PC-12 preincubation with concanavalin A. PC-12 cells seeded (Fig. 9B) and primary sensory cells (Figs. 9D, F), was on periodate-treated ECM showed, during the first 3- detected by means of an image-intensified video camera, 4 hr, firm cell attachment and flattening in a manner 48 hr after seeding the cells on ECM. As with cells similar to that observed with cells plated on intact ECM maintained on polylysine (Levi et al., 1980), R-NGF was (Fig. 7C). Upon further incubation, cell retraction and distributed both in the PC-12 and in the primary senaggregation occurred and, within 24 hr after seeding, sory cells along the extended processes (Fig. 9F) and the culture adopted the morphological appearance of mostly as large clusters in proximity to the nucleus in cells plated on plastic and gave no sign of cell processes the main cell body (Figs. 9B, D). These large fluorescent (Fig. 7D). There was little or no effect when the ECM clusters could not be removed by treatment with trypsin was concomitantly exposed to equal amounts of per- (0.25% trypsin, 15 min, 37”C), indicating that they had undergone internalization and subsequent segregation, iodate and sodium borohydride and a partial reversion by adding the borohydride subsequent to the periodate mostly toward the main body. Nonneuronal cells present in primary cultures of chicken sensory cells exhibtreatment. Cell retraction and reaggregation following the initial attachment and flattening was less evident ited a diffuse nonspecific fluorescence over the entire on either prefixed (0.1% glutaraldehyde, 5 min, 22°C) cell surface. Experiments with lz51-NGF have demonstrated that (Fig. 7B) or concanavalin-treated (Fig. 7F) ECM as compared with periodate-modified ECM (Fig. 7D). Nev- PC-12 cells seeded on either ECM or polylysine-coated ertheless, the subsequent induction of morphological dishes bind NGF in a similar way in terms of binding differentiation was in each case greatly inhibited and capacity (6 X lo4 NGF molecules/cell), saturability, and the proportion of processes bearing cells did not exceed rate of release of cell-associated ‘251-NGF. The latter 10% of the entire cell population. Similarly, heating property was tested in cells washed free of unbound FIG. 7. Flattening and morphological differentiation of PC-12 cells seeded on native vs modified ECM. Dishes coated with ECM were preexposed to either glutaraldehyde (0.1% in PBS for 5 min at room temperature) followed by 5-min exposure to 0.1 M glycine (A, B), sodium periodate (10 mM in PBS for 30 min at room temperature) (C, D), or concanavalin A (50 fig/ml for 30 min at room temperature) (E, F). The modified ECM-coated dishes were then washed four times with PBS, and tested for the induction of cell flattening (A, C, E) and neurite extension (B, D, F) in PC-12 cells. Phase-contrast micrographs (320X) were taken 1 hr (cell flattening) and 48 hr (neurite extension) after seeding. All treatments greatly inhibited the ECM-induced extension of neuronal-like processes but had a very small or no inhibitory effect on the initial induction of cell attachment and falttening. Cells seeded on periodate-treated ECM exhibited rapid attachment and flattening but later underwent retraction to adopt, 24 hr after seeding, the morphology of cells maintained on plastic.

FIG. 8. Neurite outgrowth from dorsal root ganglia seeded on native vs modified ECM. Dishes coated with ECM were pretreated with either 0.1% glutaraldehyde (B), 50 mM sodium periodate (C), or 100 rg/ml concanavalin A (D) as described in the legend to Fig. 7. Embryonic dorsal root ganglia were then plated on untreated (A) ECM, and each of the modified dishes, and 24 hr later tested for outgrowth of neuronal-like processes. The various modifications of the ECM greatly inhibited neurite extension, whereas the outgrowth of fibroblasts was not inhibited.

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FIG. 9. Distribution of R-NGF on differentiated PC-12 and chicken sensory cells seeded on ECM. PC-12 (A, B) and chicken sensory cells (C-F) maintained on ECM for 24 hr, and exhibiting neuronal-like processes, were incubated with R-NGF (130 rig/ml) for 2 hr at 37°C. RNGF was localized (image-intensified fluorescence) along the neurites (F), and in the main cell body (B, D). (Phase contrast (A, C, E) and fluorescence (B, D, F) micrographs 2000X).

lz51-NGF and further incubated at 37°C for up to 60 min. It was found that up to 50% of the radioactivity associated with cells seeded on either polylysine or ECM was released to the medium within 60 min of incubation at 3’7°C and with similar kinetics. It was also found that preplating of PC-12 cells on ECM did not “down regulate” their NGF receptor sites. In these experiments, cells were preincubated in close contact with the ECM for different lengths of time (up to 8 hr) and subsequently incubated with lz51-NGF (10 rig/ml, 30 min, 37°C) and tested for specific cell-associated radioactivity. As time proceeded, there was at most a 10% reduction in ‘251-NGF binding capacity, as was the case with control cells seeded on polylysine-coated dishes for the same periods of time.

DISCUSSION

Studies with nonneuronal cell types maintained on a naturally produced basement membrane-like extracellular matrix have shown that the cells adhere rapidly and firmly to this substrate and subsequently adopt a morphological appearance, growth characteristics and biological responses that are not expressed when the same cells are maintained on either the plastic surfaces of regular tissue culture dishes or on dishes coated with either fibronectin, laminin, or each of the collagen types (Vlodavsky et al., 1980; Vlodavsky and Gospodarowicz, 1981; Gospodarowicz et al., 1980b). In the present study, we report that the same phenomenon is observed with pheochromocytoma (PC-12) and primary sensory cells

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that express their differentiation phenotype when maintained in close contact with the ECM but not on plastic, whether coated with either collagen or fibronectin or uncoated. Increased cell to substrate adhesion has been shown to be both necessary and sufficient to induce neurite formation in C-1300 neuroblastoma (Patrick et al., 1978). Similarly, a stimulation of cellular adhesion was suggested as the first step required for NGF-initiated neurite outgrowth by PC-12 and sympathetic nerve cells maintained in vitro (Schubert and Whitlock, 197’7; Schubert et al., 1978). That this may in fact be the case was demonstrated by showing that plating on ECM induced rapid cell attachment and flattening followed by extension of neuronal-like processes in both the PC-12 and the primary chicken sensory cells, and in the absence of added NGF. Although cell attachment and flattening may represent an essential stage in the processes of cell preparation for neurite outgrowth, it was in itself not sufficient to induce morphological differentiation. First, cell attachment and flattening were induced by cell plating on dishes coated with collagen type IV to the same extent as on dishes coated with ECM, but this was not associated with subsequent extension of processes in both the PC-12 and the primary sensory cells. Second, various modifications of the ECM had only a small effect or none at all on the induction of cell attachment and flattening but greatly inhibited the formation of neuronal-like processes. Third, whereas essentially all the PC-12 cells seeded on ECM underwent a firm attachment and flattening within lo-15 min, a rather prolonged period of time (24-48 hr) was required for the appearance of neuronal-like extensions and only 50-60% of the total PC-12 cell population formed such extensions. That increased adhesion to substrate is not sufficient to promote neurite outgrowth was also concluded by Chandler and Hershman (1980). Whereas the basement membrane collagen (type IV) may be the component responsible for the rapid attachment and flattening of PC-12 cells, the sugar moieties of the ECM seem to be involved in the subsequent induction of morphological differentiation. This was demonstrated by the inhibition of cell processes outgrowth on ECM that was pretreated with either periodate or concanavalin A. Neurite extension was also greatly inhibited by a mild prefixation of the ECM, suggesting that the ECM does not function merely as an inert physical support for the seeded cells, but, rather, is actively involved in the induction of the observed biological responses. Studies with nonneuronal cell types have demonstrated that periodate oxidation of the ECM stimulated its induction of vascular endothelial cell proliferation but inhibited the attachment and subsequent flattening of human colon carcinoma cells (Vlodavsky and Fuks, manuscript

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in preparation). It was also shown that the attachment and flattening of different cell types is mediated by different adhesive glycoproteins present in the ECM (Vlodavsky and Gospodarowicz, 1981). These results may indicate that different components of the ECM (collagen, glycosaminoglycans, and glycoproteins) may be involved in the modulation of cell shape, proliferative response, and expression of differentiated functions observed when the cells are plated on ECM. Similarly, various cell types may interact with different components of the ECM or respond differently to a given modification of the ECM. It is also likely that a combination of the various components present in the correct ratio and forming a tridimensional configuration similar to the extracellular scaffolding in viva is required to elicit the observed biological responses. Such a requirement can only be fulfilled by providing the cells with a naturally produced, basement membrane-like ECM (Vlodavsky et al., 1980; Gospodarowicz and Tauber, 1980). A number of laboratories have reported factors which, by binding to the appropriate substrate can influence the elongation and migration of growth cones (Johnston and Wessels, 1980). Among these are components of media conditioned over heart cells (Collins, 1978) and other mammalian and avian cell types (Adler et al., 1981). The presence of such factors associated with the ECM cannot be excluded, although there was no induction of cell processes by conditioned media from cultured bovine endothelial cells. Similarly, the possibility of a bovine NGF found in the ECM is unlikely because there was no binding of anti-mouse NGF to the ECM nor any effect on the outgrowth of cell processes. Cell plating on ECM had also no effect on their subsequent capacity to bind mNGF. These results do not exclude the possibility of a bovine NGF present in the ECM if such a molecule is not recognized by anti-mouse NGF or does not compete with mNGF on binding to cell surface receptor sites. Furthermore, the ECM-mediated differentiation of PC-12 cells was faster and mostly transient as compared to that induced by NGF and hence is most likely elicited via a different mechanism. Whereas the ECM may fulfill a permissive role in neuronal differentiation, NGF may be responsible for stabilization and long-term maintenance of the differentiated state. Similarly, the ECM has been shown to have a permissive effect on the proliferation of various nonneuronal types of cells (Gospodarowicz et al., 1980a). The observed retraction and disintegration of cell processes might be due to a destruction of active components in the ECM by degradative enzymes secreted by the PC-12 cells. Our study on the interaction of cells plated on ECM with either R-NGF or ‘%I-NGF has demonstrated that

the cells retain their ability to bind and internalize NGF in a manner similar to that observed with cells cultured on plastic. The localization of R-NGF along processes induced by cell plating on the ECM suggests that these processes are in all likelihood identical to those induced by NGF when added to neuronal cells maintained on plastic. It is also likely that in a mixed cell population such as the primary chicken sensory cells, the cells that respond to a close contact with the ECM are also those that bind, internalize, and respond to NGF. Neuronal outgrowth in such cultures can be mediated by nonneuronal cells via a stimulated production of a neurite promoting factor (Adler et a,l., 1981) or by the nonneuronal cells providing an adequate terrain for neurite elongation (Wessels et al., 1980). That both possibilities are unlikely was suggested by the following observations: First, there was no induction of neurite outgrowth from cells plated on top of a glass coverslip coated with either polylysine or collagen and placed next to cells of the same preparation plated on ECM. Second, neuronal differentiation occurred as early as 24 hr after seeding, when the number of nonneuronal cells was small and similar on surfaces coated with ECM or polylysine. Contacts with nonneuronal cells were particularly rare when dissociated ganglia were seeded at a low cell density on either substrate and the cultures observed within 24-48 hr. Third, there was little or no neuronal differentiation on a modified ECM despite a lack of a visible effect on the outgrowth of nonneuronal cells. In addition to the induction of cell adhesion and neuronal-like extensions, plating on ECM allowed the survival of embryonic sensory cells in primary cultures, as well as accelerated the proliferation of PC-12 cells and extended their life span in serum free medium. Similarly, it has been shown in other cell systems that cells plated on ECM no longer require an addition of a polypeptide growth factor in order to express differentiated properties, and to exhibit active cell proliferation and long term survival (Gospodarowicz et al., 1980a,b; Salomon et al., 1981). It is suggested that provision of a proper substrate can dictate an adequate cell shape and orientation which allows the cells to respond to naturally occurring hormones and growth factors to which they do not respond when maintained on artificial substrates. The structural complexity and specificity which exists in the nervous system may result, at least partially, from an ordered or directed growth of nerve fibers during embryogenesis. It has been suggested by the behavior of embryonic cells during morphogenesis that cell movements are controlled by alterations in the ECM (Wessels, 1977). Contact-mediated interactions may similarly restrict axon elongation to certain path-

ways, as well as guide and attract neurite outgrowth from distant parts of the nervous system. This was suggested by studies showing that growth cones preferentially elongate on surfaces to which they adhere more firmly (Letourneau, 1979; Wessels, 1980) and by the possible role of the ECM in allowing the precise reinnervation of original synaptic sites on denervated muscle cells (Marshall et al., 1977). This work was supported hy grants from the Israel Cancer Research Fund (IV.) and the Israel Academy of Sciences (I.V.) and by grants from the NIH (CA 30289 to I.V. and CA 25820 to J.S.) and the United States-Israel Binational Science Foundation (J.S. and I.V.). A.L. is a recipient of a long-term EMBO Fellowship. The authors wish to acknowledge the expert technical assistance of Mrs. Ruth Atzmon. We thank Drs. H. Gamliel and D. Gurfel for their help with the scanning electron microscopy. h’ote added in proqf: Similar with the ECM were recently Gospodarowicz (Neurosciwm~, tion).

results on the interaction of PC12 cells independently ohtained by Fujii and 1982, in press, personal communica-

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