[CANCER RESEARCH 46, 347-354, January 1986]

Duct-like Morphogenesis of Longnecker Pancreatic Acinar Carcinoma CellsMaintained in Vitro on Seminiferous Tubular Basement Membranes1

J. K. Reddy,2 Y. S. Kanwar,3 M. S. Rao, T. K. Watanabe, M. K. Reddy, I. Parsa, D. S. Longnecker, and S. Tafurí

Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611 [J. K. R., Y. S. K., M. S. P., T. K. W., M. K. Ft., S. T.¡;Department of Pathology,Downstate Medical Center, Brooklyn, New York 11203 [I. P.]; and Department of Pathology, Dartmouth Medical School, Hanover, New Hampshire 03756 [D. S. L]

ABSTRACT

We have investigated the behavior of dissociated cells of amoderately differentiated Longnecker transplantable pancreaticacinar carcinoma (Longnecker ef al., Cancer Lett., 7: 197-202,

1979) maintained in vitro on acellular seminiferous tubular basement membranes of rat testis. The tumor cells, which grow assolid masses in vivo with little organization, undergo organogénesis in vitro into distinct duct-like structures with lumina in thepresence of basement membrane scaffoldings. These duct-like

structures were formed by flattened epithelial cells, which exhibited poorly differentiated acinar cell characteristics with few orno zymogen (secretory) granules. The cells lining the duct-like

structures retained the pancreatic acinar cell specific antigen asdetermined by indirect ¡mrnunofluorescence. DNA synthesis didnot accompany duct-like organization; however, all of the cellslining these structures continued to incorporate [3H]leucine for

up to 4-5 days of culture. They continued to synthesize and

secrete amylase, a marker protein of pancreatic acinar cells, intothe medium. These results demonstrate that neoplastic epithelialcells of Longnecker pancreatic tumor differentiate into duct-like

structures when they come into contact with a basement membrane scaffolding and do not accumulate well-formed secretory

granules. This is in marked contrast to the previously reportedin vitro differentiation of cells derived from another transplantablerat pancreatic acinar cell carcinoma where the neoplastic cellswere fully cytodifferentiated on seminiferous tubular basementmembrane without forming duct-like structures but accumulatedabundant well-developed zymogen granules (Watanabe et al.,Cancer Res., 44: 5361-5366, 1984). Although the basal lamina

promotes differentiation of cells of two different pancreatic carcinomas in vitro, we conclude that the in vitro expression ofmorphogenetic and cytodifferentiation patterns is dependentupon the intrinsic properties of cells of these two transplantablepancreatic tumors.

INTRODUCTION

ECM4 appears to regulate various developmental processes

such as cell migration, proliferation, and differentiation (1-7).

Several recent studies have demonstrated that different types of

Received 6/18/85; revised 10/1/85; accepted 10/3/85.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This investigation was supported by NIH Grant CA 23055.2To whom requests for reprints should be addressed, at the Department of

Pathology, Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611.

3 Recipient of Research Career Development Award from the NIH (AM 01018).•Theabbreviations used are: ECM, extracellular matrix; COM, chemically de

fined medium; STBM, seminiferous tubular basement membrane; SDS, sodiumdodecyl sulfate.

normal epithelial cells grown in and on collagen matrices in vitroexhibit organization into three-dimensional tissue-like structures(7-16). Similarly capillary endothelial cells embedded in collagengels showed the capacity to form capillary-like networks in vitro

(17). Thus it appears that collagen and other ECM componentsincluding the basal lamina exert an inductive role on the expression of morphogenetic capabilities in vitro by normal epithelialand endothelial cells similar to that occurring in vivo (3, 5, 7,12,17). However, very little information exists regarding the role ofbasement membrane or ECM in the regulation of growth, differentiation, and morphogenesis of "neoplastic" cells in vitro, de

spite the notion that failure to maintain a basement membranemay be involved in the neoplastic disorganization of epithelialtissue architecture in vivo (18, 19). The present studies wereundertaken to examine the influence of basement membranescaffolding on in vitro growth and rearrangement of neoplasticacinar cells. We now report that dissociated cells from a moderately differentiated transplantable pancreatic acinar carcinoma(20), maintained in vitro on acellular rat testicular STBMs, organize into distinct duct-like structures, without evidence of abun

dant secretory granules unlike the previously reported observations of complete cytodifferentiation with well-developed numer

ous zymogen granules in neoplastic pancreatic acinar cells ofanother transplantable pancreatic tumor (21), when similarlymaintained in vitro on STBM (22). This model system of STBMand its components laminin and type IV collagen may be usefulfor delineating the mechanisms involved in differential regulationof differentiation and organization of cells.

MATERIALS AND METHODS

Materials purchased were as follows: L-[3,4,5-3H]Leucine (146 Ci/mmol) from Amersham Corp., Arlington Heights, IL; [mef/7y/-3H]thymidine

(1 Ci/mmol) from Research Products International Corp., Mt. Prospect,IL; DNase 1, soybean trypsin inhibitor, deoxycholate, and bovine serumalbumin (fraction V) from Sigma Chemical Co., St. Louis, MO; collagenase(class IV) from Worthington, Freehold, NJ; SDS, acrylamide, and relatedgel electrophoresis reagents from Bio-Rad, Richmond, CA; and ampho-line (pH 3.5-10) from LKB Instruments, Inc., Gaithersburg, MD. COM

was prepared as described by Parsa and Marsh (23); this was supplemented with vitamin C (10 mg/liter), hydrocortisone (4 mg/liter), insulin(8 mg/liter), and STI (0.2 mg/ml). The COM also contained the antibioticsgentamicin (50 mg/liter), penicillin (100 units/ml), streptomycin (100 /*g/ml), and Fungizone (0.25 M9/ml). Other chemicals were standard reagentgrade.

Preparation of Rat Testicular Seminiferous Tubular BasementMembrane. Acellular STBMs were prepared from adult F344 rats by amodification of the procedure (22) outlined by Meezan et al. (24) for theisolation of basement membranes from blood vessels and renal glomeruli.Testes were removed surgically under sterile conditions. After the tunicawas dissected out, the loose strands of seminiferous tubules were teasedapart in water containing 0.05% sodium azide. The blood vessels and

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interstitial capillary network were removed. Seminiferous tubules werechopped into ~5 mm segments with a razor blade. After these tubules

were washed in fresh sodium azide solution for 45 min at room temperature, the loose elements of germinal epithelium were removed byapplying gentle mechanical pressure with a rubber policeman; the seminiferous tubules were then washed out and treated with DNase 1 (100Kunitz units/ml) for 45 min at room temperature. Finally the seminiferoustubules were treated with 1% sodium deoxycholate for 45 min at roomtemperature. The germinal epithelium-denuded acellular STBMs (Fig. ~\A)

were collected and washed at least three times with 0.9% NaCI solutioncontaining 0.2% bovine serum albumin and once with COM.

Dissociation of Pancreatic Acinar Carcinoma Cells and in VitroMaintenance. Azaserine-induced pancreatic acinar carcinoma (CP-

33378) maintained as a transplantable tumor in Wistar/Lewis rats (20)was dissociated into single cells according to the procedures describedpreviously (25-27). Tumor cells (1 x 105 to 1 x 106 in 0.1 ml Krebs-

Ringer bicarbonate buffer) were mixed with supplemented COM containing -50-75 STBMs. The culture dishes were then placed on a Bélicorocker platform in an incubator at 37°C in an atmosphere of 5% CO2

and rocked gently for 4 to 6 h to facilitate contact and attachment oftumor cells to the floating STBMs. The unattached cells were removedwithin 12 h, and tumor cell-STBM aggregates maintained in culture for7-10 days.

Tissue Autoradiography. Incorporation of [3H]leucine (20 ¿iCi/ml)intoproteins and [3H]thymidine (120 MCi/ml) into DNA by tumor cells main

tained on STBMs was assessed by light and electron microscopicautoradiography as described previously (28). The cells on STBMs werelabeled either continuously for up to 96 h or for 1 h prior to harvesting

at 48, 72, and 96 h.Morphological Procedures. Unfixed STBM-tumor cell aggregates

were photographed using a Zeiss Ultraphot III microscope using eitherphase optics or with lowered condenser. For light microscopy, the STBM-

tumor cell complexes and fragments of transplantable tumor were fixedin Bouin's fluid and embedded in paraffin, and 3-4-/¿m-thick sections

were stained with hematoxylin and eosin. For transmission electronmicroscopy, samples were fixed with 2.5% glutaraldehyde in 0.1 Msodium cacodylate (pH 7.4) for 1 h, postfixed in 1% OsO4, and processed

as described previously (28).Immunofluorescence. For fluorescence microscopy, pancreatic aci

nar carcinoma cells maintained in vitro on STBM for 3 to 7 days, as wellas portions of transplanted tumor, were fixed in Bouin's fluid and

embedded in paraffin. Sections 3-4 urn thick were cut and processed

for the indirect immunofluorescence localization of pancreatic acinar cellspecific antigen using the monoclonal rat anti-mouse pancreatic acinarcell (ACR-1 ) antibodies as described previously (29).

[35S]Methionine Labeling. Pancreatic acinar carcinoma cells were

maintained on STBM for 4, 24, 48, and 72 h. They were labeled with[^SJmethionine (100 ¿iCi/mlmethionine-free medium) for 4 h before

harvesting at the time intervals indicated. At the end of the designatedincubation period, the STBM cell aggregates were pelleted (2 x 500rpm/10 min). The lysates of the cells were prepared and processed forone-dimensional electrophoresis as indicated below. Similarly the me

dium was centrifuged at 50,000 rpm for 45 min to sediment cell debrisand the supernatant was analyzed.

SDS-Polyacrylamide Gel Electrophoresis. One-dimensional electrophoresis of ["Sjmethionine-labeled tumor cell lysates and the culture

medium was performed on SDS-polyacrylamide slab gels according to

the method of Laemmli (30). The gels were dried and autoradiograms

were prepared.Immunoblot Analysis. The secretory proteins in the incubation me

dium of tumor cells maintained on STBM for 48-72 h were subjected toone-dimensional SDS-polyacrylamide gel electrophoresis and were trans

ferred electrophoretically to nitrocellulose sheets. The sheets weretreated with rabbit anti-rat amylase antiserum and then 125l-labeled

protein A and autoradiographed.

RESULTS

Morphological Studies. Azaserine-induced Longnecker trans

plantable pancreatic acinar carcinoma of the Wistar/Lewis ratwas dissociable into viable single cells by the method used forthe dissociation of nafenopin-induced pancreatic acinar carci

noma of the Fischer 344 rat established by Reddy and Rao (21).Dissociated tumor cells attached readily to the STBM within 1 to4 h (Fig. 1B). This adhesive property enabled the removal ofexcess or nonadherent tumor cells from the culture medium. Thecell density on STBM was adjustable either by the number oftumor cells added in relation to the quantity of STBM segmentsin culture dishes or by varying the length of contact time withSTBM prior to removal of free cells from the culture medium.The tumor cell-STBM aggregates floated freely in the medium

as individual segments or formed entangled aggregates. These,however, did not attach to the plastic surface.

The general morphological pattern of these tumor cell-STBM

aggregates was monitored with an Ultraphot III microscope.Within 8 to 12 h, the tumor cells attached to STBM began toflatten out somewhat and appeared to establish cell to cellcontact. By 18 to 24 h, small gland-like structures with retractile

cavities developed. Within 24 to 96 h, the entire surface of theSTBM segments was covered with numerous duct-like struc

tures (Fig. 1C). The size of these duct formations increasedprogressively with time for up to 5 days. By phase contrastmicroscopy, their lumina appeared to contain translucent fluid(Fig. 1C). These cavities excluded trypan blue, suggesting thatthey were in fact authenticated lumina surrounded by tumor cellsand that they do not represent the lumina or "holes" of the STBM

lined by the cells.The histological appearance of the Longnecker pancreatic

acinar carcinoma maintained in vivo as transplanted tumor andof duct-like formations by tumor cells maintained in vitro on

STBM are shown in Fig. 2, A and B, respectively. Under in vivoconditions, this less-well-differentiated tumor displayed the ap

pearance of a solid mass of relatively small cells, without evidence of gland formation (Fig. 2A). By electron microscopy, thecells of this tumor showed usual arrays of rough endoplasmicreticulum and a few variable numbers of zymogen (secretory)granules in some tumor cells (not illustrated). In contrast, thehistology of dissociated cells of this tumor maintained in vitro onSTBMs showed duct-like morphogenetic organizations (Fig. 2ß).Duct-like structures consisted of a lumen surrounded by flattened

epithelial cells resting on the STBM.Examination of semithin sections of tumor cell-STBM aggre

gates demonstrated that the majority of pancreatic acinar cellsdelimited a lumen. Thin sections showed that the duct-like struc

tures consisted of three or more cells that resembled acinar cellswith their bases resting on the STBM (Fig. 3) and their apiceswith few microvilli toward the lumen (Fig. 4A). The lateral plasmamembranes of these cells had points of apposition resemblingtight junction formation (Fig. 46). These cells contained largenuclei and prominent strands of rough endoplasmic reticulum.The zymogen granules were sparse in the majority of cells liningthese duct-like structures. When present, these granules were

irregular in shape and smaller than typical zymogen granules(Figs. 3A and 4).

Pancreatic carcinoma cells plated in plastic dishes in theabsence of STBMs formed solid clumps or sheets in the culture

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medium and displayed no gland-like organizations. Deletion of

soybean trypsin inhibitor from the culture medium did not inhibitgland-like formations in cells maintained on STBM.

Immunofluorescence Studies. In order to determine whetherduct-like formations in vitro result in the persistence or loss ofexpression of acinar cell-specific ACR-1 antigen (29), we utilized

a monoclonal murine IgG which specifically stained normal andneoplastic rat pancreatic acinar cells. The ACR-1 antigen has

been tentatively identified as a M, 80,000 glycoprotein (29). Withthis monoclonal antibody, the plasma membranes of both in vivotumor transplants and the epithelium lining the duct-like struc

tures formed in vitro on STBM showed intense fluorescence(Fig. 2, C and D).

DNA and Protein Synthesis. DMA synthesis did not accompany the reorganization of pancreatic acinar cells on STBM intoduct-like structures. Although 10-15% of the freshly dissociatedneoplastic cells incorporated [3H]thymidine, none of the cellsmaintained on STBM for up to 4 days in the presence of [3H]-

thymidine showed any nuclear labeling by electron microscopicautoradiography (not illustrated). However, all these cells continued to incorporate [3H]leucine as evidenced by autoradiography

indicating their viability (Fig. 33).SDS-Polyacrylamide Gel Electrophoresis and Immunoblot

Studies. Autoradiograms of one-dimensional gel electrophoresis

of lysates of the neoplastic cells and media showed the consistent presence of an amylase band (M, -55,000) during the entire

period of labeling (Fig. 5). The identity of the band as amylasewas confirmed by the immunoblot analysis where a single bandcorresponding to the M, ~55,000 was seen (Fig. 5).

DISCUSSION

The present study demonstrates that dissociated neoplasticpancreatic cells derived from azaserine-induced transplantable

LYSATES MEDIUM IMMUNOBLOT(AMYLASE )

72 48 24 4 48 72

Fig. 5. Autoradiograms of one-dimensional SDS-polyacrylamide gel electrophoresis profiles of proteins of lysates of [^SJmethionine-labeled cells and media,

maintained on STBMs in culture for 4, 24, 48, and 72 h. At all time points, thereappears to be adequate synthesis and discharge of cellular proteins including thatof amylase (arrow). Right, immunoblot of the lysate of the cells maintained for 72 hon STBMs. The antibody in this case is directed against pancreatic amylase.

acinar carcinoma (20), when maintained on STBMs, undergoorganization into duct-like structures. This peculiar morphoge-

netic change, perhaps, represents one of the solitary exampleswhere basal lamina as a whole or one of its constituents iscapable of influencing the dedifferentiated neoplastic epithelialcells in vitro into the formation of organized ductules, structureswhich are very much reminiscent of the histological architectureof normal pancreas. On the other hand, in vivo these neoplasticcells lack epithelial organization, cell orientation, or gland formation (20). The tumor cells of another transplantable pancreaticacinar carcinoma established in a F344 rat by Reddy and Rao(21) fail to organize in vivo into discernible acinar patterns butexhibit polarization of secretory granules especially in perivas-

cular areas (31). Thus it appears that STBMs play a significantregulatory role in the inductive assemblage of singly dispersedcells into ductules. Conversely it can be safely stated that theinability to produce or maintain complete and structural basallamina in vivo may be responsible for the failure of neoplasticcells to undergo morphogenetic or histogenetic differentiation.Indeed Ingber et al. (18) reported the absence or abnormallyscanty distribution of certain components of basal lamina, i.e.,type IV collagen and laminin, in another transplantable pancreatictumor established in F344 rats by Reddy and Rao (21). Theseobservations do suggest a role of basal lamina, whether derivedfrom STBMs or other sources, in influencing the morphogenesisof neoplastic cells.

Available evidence strongly supports the role of mesenchymeand ECM components including basement membrane as informative or inductive molecules in growth regulation and morphogenetic organization of normal epithelial tissues under in vitroand in vivo conditions (2-6, 32-35). Several examples of the

inductive effect of collagen overlays and matrices on the establishment of tissue-like structures by normal epithelial cells in

culture have been reported. These include cells from mammarygland (8, 11, 16), submandibular gland (36), and thyroid (9), aswell as established cell lines derived from canine kidney (12) andendothelial cells of bovine adrenal cortex (17). In most of theseinvestigations, the behavior of either normal cells or cultured celllines was studied. Our results provide, for the first time, anunequivocal example of the in vitro reorganization of neoplasticpancreatic acinar cells in the presence of a basement membranescaffolding, although these cells are derived from a solid tumorwith disorganized architecture and maintained in vivo for severaltransplant generations. However, they retain the capacity to formdistinct morphogenetic patterns in vitro if they are provided withappropriate inductive environment. The striking difference in thecytodifferentiation potential between F344 rat pancreatic acinarcarcinoma cells and Longnecker pancreatic acinar carcinomacells when maintained in vitro on STBMs suggests that basallamina or some component of basal lamina is sufficient to exertdifferentiate stimulus, but we think that the ability to expressmorphogenetic patterns and to accumulate secretory granulesis due to intrinsic differences between these two pancreaticacinar tumors. It appears that Longnecker pancreatic acinarcarcinoma cells rapidly discharge secretory proteins into themedium and possess very little intrinsic capability for storage.

The interaction of pancreatic carcinoma cells with the STBMappears sufficient to induce the characteristic duct-like histolog

ical architecture in the absence of DNA synthesis and cell replication. A specific mesenchymal factor (33,34) appears to control

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cell proliferation and cytodifferentiation during embryonic development and may be required for the proliferation of these cellsin vitro. The present studies, however, suggest that this factormay not be essential for epithelial polarization and cell-cell inter

action. The structures formed under the in vitro experimentalcondition described in the present study depict the histologicalappearance of ductular structures in normal rat pancreas andthe pancreatic pseudoductules induced under certain experimental conditions (37-41). However, the duct-like cells lining thevacuoles described in the present study retain acinar cell-specific

antigen and secrete amylase in spite of the absence of characteristic cytodifferentiation features of acinar cells. This is particularly relevant in relation to our earlier study in which neoplastiapancreatic acinar cells derived from another transplantable tumoraccumulated numerous secretory granules and exhibited characteristic acinar cytodifferentiation when maintained in vitro onSTBM (22). However, those cells failed to undergo histogeneticor morphogenetic differentiation such as the one observed in thepresent study. It would be of particular interest to ascertainwhether the cells lining the duct-like structures formed under in

vitro conditions can be induced to express alkaline phosphataseand carbonic anhydrase similar to that observed in the 5-bro-modeoxyuridine-treated embryonic pancreas (42).

The mechanism by which collagen and other ECM componentsinduce organogénesis and morphogenesis is not well understood. The formation of polarized epithelium, cell-cell interaction,and tissue-specific architectural imprint may be intrinsic properties of normal and in certain instances of neoplastia cells as well,but these may be influenced by specific components of thebasement membranes. Among the basement membrane components which could modulate the cytodifferentiation events arevarious species of sulfated and nonsulfated proteoglycans. Thenonsulfated proteoglycans which have been shown to influencethe morphogenesis include hyaluronic acid (2, 4) while the sulfated one is chondroitin sulfate (7, 43). In addition, the heparansulfate-proteoglycan has been implicated in the regulation ofembryonic-morphogenetic developmental events (44, 45). Ad

mittedly the role of proteoglycans in this investigation is notproven but would be one of the important issues which needsto be addressed in future investigations. Finally it seems that ourrelatively simple model system will greatly facilitate the investigation of the events involved in the morphogenesis and reorganization of neoplastia epithelial cells.

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43. Conn, R. H., Banjerjee, S. D., and Bemfield, M. R. Basal lamina of embryonicsalivary epithelia. Nature of glycosaminoglycan and organization of extracellular materials. J. Cell Biol., 75: 464-478,1977.

44. Reeves, W. H., Kanwar, Y. S., and Farquhar, M. G. Assembly of the glomerularfiltration surface. Differentiation of anionic sites in glomerular capillaries ofnewborn rat kidney. J. Cell Biol., 85: 735-753,1980.

45. Kanwar, Y. S., Jakubowski, M. L., Rosenzweig, L. J., and Gibbons, J. T. Denovo cellular synthesis of sulfated proteoglycans of developing renal gtomer-ulus in vivo. Proc. Nati. Acad. Sci. USA, 87: 7108-7111,1984.

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Fig. 1. Ught microscopic appearance of isolated acellular STBM segment of rat testis (A) and pancreatic acinar carcinoma cells maintained in vitro on such STBM for4 h (ß)and 48 h (C). The STBM in A, stained with 0.01% toluidine blue, shows the hollow tubular configuration with arrows pointing to the lumen. In ß,pancreatic acinarcarcinoma cells attached to the STBM (bm) at 4 h of culture show marked tendency to aggregate into clumps of several cells. In C, pancreatic carcinoma cells maintainedon STBM for 48 h reveal duct-like structures with retractile lumina (d). A few zymogen granules (arrows) are seen in some cells. A, x 40; B, x 400; C, x 1,200.

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DUCT-LIKE MORPHOGENESIS OF A PANCREATIC CARCINOMA

Fig. 2. Histological (A and B) and immunofluorescent (C and 0) micrographs of Bouin's-fixed and paraffin-embedded Longnecker pancreatic acinar carcinoma

maintained in vivo as a transplantable tumor (A and C) and pancreatic carcinoma cells maintained in vitro on STBMs for 72 h (ßand D). Sections in C and D are stainedfor pancreatic acinar cell specific ACR-1 antigen with monoclonal antibodies. The tumor cells cultured on STBM (bm) reveal duct-like organizations with lumina (d). S,stroma. A, H & E, x 180; B, H & E, x 400; C, x 540; D, x 220.

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Fig. 3. Low power electron micrographs of pancreatic acinar tumor cells cultured on STBM for 72 h {A and B). Pancreatic tumor cells are organized around circularto oval lumina (LUMEN) to form duct-tike structures. These structures are surrounded by STBM (bm). A few cells lining these duct-like structures contain focal collectionsof small secretory granules in the cytoplasm facing the luminal surface. B, autoradiograms showing (3H]leucme incorporation in cells lining the duct-like structures. N.

nucleus; Er, endoplasmi reticulum. A, x 1,900; B, x 2,800.

353

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DUCT-LIKE MORPHOGENESIS OF A PANCREATIC CARCINOMA

Fig. 4. Electron micrographs of ductules indicating the detailed structure of the cells. The cells contain plentiful rough endoplasmic reticulum and some zymogengranules (gr). The junctions are readily seen between the adjoiningcells (a/rows).B, higher magnificationof the inset of A to better illustrate the presenceof granules (gr)and cell junctions (arrows).A, x 8,000; B, x 30,000.

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1986;46:347-354. Cancer Res   J. K. Reddy, Y. S. Kanwar, M. S. Rao, et al.   Basement Membranes

on Seminiferous Tubularin VitroCarcinoma Cells Maintained Duct-like Morphogenesis of Longnecker Pancreatic Acinar

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