establishment two clin pathol: molpathol 1996;49:m33-m39 establishment andcharacterisation oftwo...

_r Clin Pathol: Mol Pathol 1 996;49:M33-M39

Establishment and characterisation of two celllines derived from a primary adenocarcinoma ofthe duodenum

M Golding, G W H Stamp, T Oates, E-N Lalani

AbstractAims-To establish two cell lines from a

primary duodenal adenocarcinoma; todescribe the morphological, growth,ploidy, and immunophenotypic char-acteristics of these cell lines.Methods-The cell lines, designated DAC/S and DAC/E, were characterised usingboth in vitro and in vivo cell culture tech-niques, light and electron microscopy, im-munocytochemistry, and FACS analyses.Results-Both cell lines have an epithelialorigin, are aneuploid and display char-acteristics of transformed cells. The celllines differ from each other in morphology,doubling time and serum requirements.These cell lines are anchorage dependentand do not grow in nude mice.Conclusions-DACIS and DAC/E cell linesare derived from neoplastic epitheliumand could provide in vitro model systemsfor future investigations of the cell andmolecular biology of duodenal neoplasia.(J Clitn Pathol: Mol Pathol 1996;49:M33-M39)

Keywords: duodenum, adenocarcinoma, cell lines,integrins, intermediate filaments.

Department ofHistopathology, RoyalPostgraduate MedicalSchool, Du CaneRoad, LondonW12ONNM GoldingG W H StampT OatesE-N Lalani

Correspondence to:Dr E-N Lalani.

Accepted for publication9 November 1995

Development of primary adenomas and car-

cinomas in the small intestine of humans is a

rare event." The low frequency of neoplasiain this part of the gastrointestinal tract has beenconsidered surprising given the high incidenceof tumorigenesis in the oesophagus, stomachand large bowel. The scarcity of small bowelcancers has consequently resulted in limitedinformation regarding the clinical, pathologicaland biological characteristics of such malig-nancies.The establishment of continuous cell lines

from human cancers has provided the potentialto study aspects of the biology of various tu-mours, and to identify specific genetic al-terations present in the neoplastic cells whichare not confounded by the stromal elements.However, as many tumours exhibit con-

siderable heterogeneity, it is necessary for stud-ies to be carried out on a number of differentcell lines, in order to obtain a panel of datawhich may illustrate the mechanisms whichunderlie the disruption in growth control anddifferentiation which characterise malignantneoplasms. For this reason there is a perpetualrequirement for novel cell lines for ex-

perimental purposes, in the hope that new datamay be generated which contribute further to

our understanding of the causes and behaviourof cancers.

Immortalisation, which sometimes occursduring cellular transformation in vitro, is aproperty which is a prerequisite for the es-tablishment of a continuous cell line from aprimary culture. Unfortunately, this only oc-curs spontaneously in less than 10% of tumourcell cultures.3 If, however, immortalisation doesoccur, then this provides an opportunity toestablish cell clones from the primary culturefor subsequent characterisation. Characteris-ation typically involves determination ofmorphology, nutritional requirements, growthrates, differentiation properties, immuno-phenotype, ploidy, karyotype, and DNA finger-printing.34

In this report we describe the establishmentof two continuous cell lines derived from aprimary adenocarcinoma of the duodenum.

MethodsINITIATION OF THE PRIMARY CULTUREA duodenal tumour was obtained from a 45year old man at the Hammersmith Hospital,London. A 1 cm3 sample of tumour was takenand placed in sterile, ice cold phosphatebuffered saline (PBS; pH 7-2), and thin slicesadjacent to the tumour were fixed in 10%formal saline (10% formaldehyde (v/v) in 0 9%sodium chloride), embedded in paraffin waxand processed routinely for histology. Tissuesections of the sample showed a moderatelydifferentiated duodenal adenocarcinoma in-vading the intestinal wall and through into thepancreas.The tumour sample was divided into four

portions, each placed into a 10cm Petri dish(Nunc, Roskilde, Denmark), washed thor-oughly in cold (4°C) PBS and finely choppedusing crossed scalpels. Five millilitres of warm(37°C) Dulbecco's Modified Eagle's Medium(DMEM) supplemented with 10% fetal calfserum (FCS); 10 tg/ml insulin (Sigma, Poole,Dorset, UK); 5 ,ug/ml hydrocortisone (Sigma);and 0-6 mg/ml glutamine was added to theminced tissue and pipetted vigorously to obtaina fine suspension. The suspension was seededinto 10 Petri dishes (five 1 x 5 and five1 x 10 cm) and incubated at 37°C with 10%CO2 in air. After 48 hours, the medium wasaspirated and all adherent cells were washedthoroughly with cold (4°C) PBS, growthmedium (as above) was added and the dishesincubated as normal for seven to 10 days, afterwhich discrete colonies of cells had formed.

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Golding, Stamp, Oates, Lalani

CELL CLONING AND PROPAGATIONEach colony of cells exhibiting a distinctivemorphology was selected using the cloning ringtechnique3 and expanded by seeding initiallyinto 24 well plates and subsequently into sixwell plates. The resultant cell suspension wasthen seeded into 25 cm3 culture flasks (Nunc)standing vertically. Once confluent, the cellswere trypsinised (0 25% trypsin in versene),the flask laid horizontally and incubation wascontinued until confluent. Using this method,five clones were isolated and subsequently ex-panded. Only two of the original five cloneswere viable after one month in culture and thesewere named according to their morphology,DAC/S (duodenal adenocarcinoma/spindle)and DAC/E (duodenal adenocarcinoma/epi-thelioid).

MINIMAL GROWTH REQUIREMENTExperiments were set up as follows: DAC/Sand DAC/E cells (1 x 105) were suspended inserum free DMEM medium and seeded intoeach well of a 24 well plate and incubated for24 hours. The medium was then aspiratedand replaced with one of the following media:DMEM alone; DIMEM + 10% FCS; DMEM+10% FCS and insulin (I), or glutamine (G),or hydrocortisone (H), or all three (I,G,H (allconcentrations as above)). The effect on cellgrowth (quadruplicate samples) was as-

certained over 10 days by viewing at dailyintervals. As a result of this analysis, DAC/Scells were subsequently grown in DMEM+10% FCS and DAC/E in DMEM+ 10% FCS+ I, G and H.

CALCULATION OF DOUBLING TIMES

DAC/E and DAC/S cells (3 x 104) were seededinto each well of a 24 well plate and incubatedin 1 ml medium (as above). Cells from threewells were trypsinised at 24 hour intervals,triplicate counts were made and averaged foreach cell line at a given time point. This was

repeated until the plateau phase was reached,and from the data the lag time, populationdoubling time, and plateau density were de-termined.3

FACS ANALYSIS FOR PLOIDY STATUS

Cells ofboth clones were grown until confluentin 75 cm3 culture flasks (Falcon, Oxford, UK),trypsinised and a single cell suspension pro-duced by syringing the cells three times through

Table 1 Antibodies used for immunocytochemistry

Antibody Source Specificity Dilution

CAM 5-2 ICRF, London, UK Cytokeratin 1:2 (sn)V9 Sigma, Poole, Dorset, UK Vimentin 1:1000D33 Dako, High Wycombe, UK Desmin 1:1001A4 Sigma, Poole, Dorset, UK Smooth muscle actin 1:300PR-3B10 ICRF, London, UK CEA Neat (sn)HAS-3 F Watt, ICRF, London, UK a2 integrin 1:50DH12 B de Stroper, Amsterdam, The ,B1 integrin 1:500

Netherlands4F10 M Pignatelli, ICRF, London, UK x6 integrin Neat (sn)HECD-1 M Takechi, Kyoto, Japan E-cadherin Neat (sn)

sn = hybridoma supematant.

a 25 gauge needle. Cell suspensions containingapproximately 105 cells of each clone werecentrifuged at 1000 x g, the supematant fluidaspirated and the cell pellet resuspended in2 ml cold PBS and gently vortexed to removeany excess medium. The resulting cell sus-pension was then centrifuged at 1000 x g andthe cell pellet was fixed by resuspending in icecold 70% ethanol with periodic vortexing for30 minutes. This was followed by washing forfive minutes in 100 mM Tris buffer (pH 7 5)and staining with 0 01% propidium iodide.The nuclear DNA content was analysed usinga fluorescence activated cell sorter (FACS IV,Becton Dickinson, Gosport, UK). The normallymphoblastic cell line RPET 001 served as acontrol.

SOFT AGAR GROWTH ASSAYAnchorage independent growth was assessedas follows: a single cell suspension of 5 x 104cells from each clone was seeded in a 0 3%agar layer on top of a 0 5% agar base (bothlayers contained DMEM medium with 10%FCS) in 5 cm Petri dishes (Nunc). After fourdays of culture at 37°C in a humid atmosphereof 10% CO2 in air, the cultures were overlaidwith fresh DMEM medium with 10% FCS.These overlays were replaced every three daysand after six weeks on this regimen, any col-onies of more than 20 cells were counted induplicate dishes and compared with parallelcultures of the breast cancer cell line T47D,which served as a positive control.

COLLAGEN GEL CULTURESCells (5 x 105) of each clone were added to6 ml collagen Type I (Vitrogen 100), preparedaccording to manufacturer's instructions and1 ml of this mix was placed into each of six pre-warmed (37°C) organ culture dishes (Falcon).The gel was left to set in the incubator for90 minutes before adding culture medium (asabove). Cell growth was analysed daily by phasecontrast microscopy. Growth medium waschanged every three days, and at weekly in-tervals for a period of six weeks. One gel ofeach clone was fixed in formal saline for 24hours, processed and embedded in paraffin waxfor routine histology.

XENOGRAFTING INTO ATHYMIC NUDE MICE

Cells were grown to confluency in 75 cm3 cul-ture flasks, trypsinised as above and re-suspended in DMEM+ 15% FCS. Six NIMRNu/Nu athymic female mice (three for eachcell line) were injected subcutaneously in bothflanks with 107 cells suspended in 05 ml ofmedium. Progress of the xenografts was mon-itored by weekly inspection for up to sixmonths.

IMMUNOCYTOCHEMISTRYDAC/S, DAC/E and T47D (positive control)cells were grown on four well slides (CA Hend-ley Ltd, Loughton, Essex, UK) until 50%

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Establishment and characterisation of cell lines

Figure 1 A, Phase contrast image of DACIS cellsshowing fibroblastoid, spindle morphology; B, DACIE cellsdisplaying a regular polygonal morphology, typical ofepithelial cells. Note the nuclear pleomorphism andpresence of cytoplasmic vacuoles.

confluent. Slides were rinsed well in coldPBS, fixed in a 1:1 mix of methanol:acetone(-20°C) for 10 minutes and air dried. Slideswere stained by the standard indirect im-munoperoxidase method using mouse anti-human monoclonal primary antibodies (table1). The cells were incubated with the primaryantibody for 40 minutes at room temperatureand rinsed, followed by incubation with per-oxidase conjugated rabbit anti-mouse im-munoglobulins (Dako, High Wycombe, UK)at a 1 in 30 dilution for 40 minutes at room

temperature. The slides were rinsed in PBS,developed with 0-25 mg/ml diaminobenzidine(DAB) for three to five minutes, rinsed indistilled water, counterstained with haema-toxylin and mounted in aqueous Hydromount.Paraffin wax sections (5 jpm) of the originaltumour were immunostained using the stand-ard horseradish peroxidase conjugated Strept-avidin/biotin complex (Strept ABC) technique.

ELECTRON MICROSCOPYCells from each clone were grown to confluencyon plastic microslips (Thermanox ScientificLtd, Naperville, Illinois, USA), rinsed withPBS, fixed in 2% gluteraldehyde for one hour,washed with PBS, postfixed in osmium tetr-oxide for one hour, dehydrated and embeddedin TAAB epoxy resin. The cells were thenfreeze fractured in liquid nitrogen, 50-60 nmsections were cut, stained with uranyl acetateand lead citrate and examined using a PhilipsCM10 transmission electron microscope.

DC- .

Figure 2 A, Electron micrographs of DACIE cellsshowing suface microvilli and invagination of nuclei.Note the abundance of euchromatin and irregularlydistributed heterochromatin (scale bar= 2 um); B, DACIScells displaying interdigitation of microvilli of adjacentcells. Phagolysosomes and electron dense membrane boundvacuoles similar to zymogen granules are also present.Note the abnormal nucleolus with an electron dense coreand the flocculent material within mucus granules (scalebar= 1 plm).

ResultsTwo clones of cells from the original primarypopulation were propagated.

NUTRITIONAL REQUIREMENTS ANDMORPHOLOGICAL CHARACTERISTICSDAC/S and DAC/E cells grew as monolayersand showed contact inhibition. Cell growthwas optimal under the influence of insulin,hydrocortisone and glutamine; when sub-confluent, both cell lines were well spread andrandomly orientated on the plastic substrate.They had multipolar, fibroblastic type mor-phologies, though this was much more apparentfor DAC/S at confluency under phase contrastillumination with the cells arranging themselvesinto parallel arrays and whorls (fig 1A). Bycontrast, DAC/E, when confluent, exhibitedregularly orientated cells with polygonal, epi-thelial-like characteristics, with the cells havingwell defined borders (fig 1B). After a week atconfluency, some cells developed numerouscytoplasmic vacuoles and seemed to haveruffled borders.DAC/S, unlike DAC/E, grew with a limited

capacity in serum free DMEM medium alone

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G3(olduigl, Stallip. O(acs, I,.ll.a ,l

A RPET001 Normal lymphoblastic cell line

o1

B DAC/E 1

0

10 101401 C DAC/S

102 103

102 103

AI

0 4-A

Figuitre 3 FACS traces for (A) the normial cdiploid conitr-olcell luite RPETOO1, (B) DACIE and (C) DAC/S. BothduiodeIIal cell linles s.cent to be pentaploid. (Sec text fo)explanatiOn.)

and proliferated for four passages withoutadded serum.

Ultrastructural analysis revealed long micro-

villi; on higher magnification, interdigitationof these microvillous processes of adjacent cellswas observed but no junctional complexes were

seen (fig 2B). Both DAC/S and DAC/E hadlarge nuclei with a high proportion of irregularly

distributed euchromatin and large nucleoli withan unusual organisation which secmed to con-tain variable densities of RNA (fig 2A). Theabnormal mucus filled vacuoles seen in fig 2Bmay represent dilatated endoplasmic reticulumor an altered mucus content, both of wshichare characteristic of tumour cells of glandularorigin.

GROWTH RATESBoth DAC/S and DAC E cells attained sat-uration density at about eight days after seed-ing, and cell counts were terminated at dav 1O.From the growth curves (data not shown), thelag time for both cell lines is approximately 24hours, followed by a log phase of about fourdays. The calculated cell population doublingtimes are 24 and 42 hours for DAC S andDAC/E, respectively.

FACS ANALYSISFigures 3A-3C represent typical FACS plots,with relative cell number on the vertical axisand relative fluorescence (DNA content) onthe horizontal axis; both arc in arbitrarv units.Figure 3A shows a tvpical tracc for a normaldiploid population with the two peaks rep-resenting cells in GI and G2 phases of the cellcycle, in sharp contrast to fig 3C (representingDAC/S) revealing an ill defined populationwhich is less dramatic for DAC E (fig 3B).These results indicate that both cell lines areclearly aneuploid, possessing a DNA contentof approximately 5N.

GROWTH IN AGAR

After six weeks in culture, ncither cell lineformed colonies in agar. Howcver, a few cvto-plasmic processes werc seen cmanating frommany of the DAC/S cells within the first weekin culture, but these regressed after 10 days.In contrast, DAC'E showed no growth in agarand all of the cells had senesced within aboutthree weeks of seeding.

GROW'TH IN COLLAGEN GEL

DAC/S cells displayed extensive growth in thegels, extending numerous cytoplasmic pro-cesses into the collagen matrix within 12 hoursof seeding, although no complex structuresdeveloped. In contrast, DAC E cells did notgrow, remaining spherical and showed no ob-vious interaction with the collagen. Ex-amination of a haematoxvlin and eosin stainedpreparation of DAC/E revealed cells with cos-inophilic cytoplasm and pyknotic nuclei in-dicative of cell death (data not shown).

GROWTH IN NUDE MICE

Six months after inoculation of cells, tissuefrom the area of injection was taken from twoof the animals, which, after staining withhaematoxylin and eosin, revealed no tumourgrowth and only fibrotic scar tissue remaining(data not shown).

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IMMUNOCYTOCHEMISTRYTable 2 summarises the immunocytochemicalprofiles of both cell lines. Figure 4A showsthe moderately well differentiated adeno-carcinoma, from which DAC/S and DAC/Ewere derived, invading deep into the stb-mucosa and muscularis propria of the duo-denum.Both cell lines express cytokeratin and vi-

mentin (figs 4B and 4C, respectively) illus-trating the characteristic filamentous natureof these proteins. Figure 4D shows the mem-brane specific localisation of 31 integrin on the

C

E

Table 2 Immunocytochemistry results

Antigen T47D DACIS DACIE

Cytokeratin + + + + + + + +Vimentin + + + + + + + +Desmin - - -Smooth muscle actinCEA +/- + + + + + +t2 integrin + + + + ++ + + +t6 integrin + + +1 integrin + + + +++ + + +E-cadherin + + +

Staining intensity: -= negative; -/ + = equivocal; + = weak;+ + =moderate; + + + = strong.

B

D

F

G H .q

Figure 4 A, Haematoxylin and eosin stained section of the well differentiated duodenal adenocarcinoma, here seeninvading the intestinal muscularis. DACIE cells stained for (B) cytokeratin and (C) vimentin. D, T47D cellsimmunoreactive for the fll integrin subunit. DACIE and DACIS stained for the (E) a2 and (F) fl subunits. Intense,granular staining of (G) DACIE and (H) DACIS with PR 3B10; the majority of cells (>95%) were CEA positive.

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epithelial cell line T47D. Interestingly. suchmembranous expression is not reflected in theDAC/S or DAC/E cells where the integrinsubunits ct2 and 11 seem to be located pre-dominantly within cytoplasmic granules (figs4E and 4F). Both cell lines are intensely positivefor carcinoembryonic antigen (CEA) (figs 4Gand 4H), which is also distinctly located withingranules widely distributed throughout thecytoplasm of the majority of the cells.

In summary, we propose that DAC/S arerapidly growing 5N cells with a spindle mor-phology capable of serum free growth. Theyseem to be anchorage dependent, are nottumorigenic in nude mice but display extensive,poorly organised growth in Type I collagen.Immunophenotypic analysis suggests an epi-thelial (cytokeratin positive), de-differentiated(CEA positive) phenotype. DAC/E cells havesimilar properties, except that they are epi-thelioid, are slow growing and senesce whencultured in collagen.

DiscussionPrimary adenocarcinoma of the duodenum isa very rare lesion, constituting only about 03%of all gastrointestinal cancers,' 2 and con-sequently data concerning the cellular and mo-lecular properties associated with such tumoursare very limited. We have established two celllines, DAC/S and DAC/E, from a primaryduodenal adenocarcinoma, which have sub-sequently been characterised to determine theirorigin and tumorigenic potential.DAC/E cells have a regular, polygonal mor-

phology and grow as monolayers with each cellhaving a well defined boundary with adjacentcells when confluent (fig 1 B). This morphologyis characteristic of epithelial cells, in sharp con-trast to DAC/S which are spindle shaped andform parallel arrays and whorls on confluency(fig lA).

Ultrastructurally, both cell lines have an un-usual complement of morphological char-acteristics with long, extensive but irregularmicrovilli a prominent feature. Microvilli areusually uniform in size and regularly arranged,although they may be drastically deformed insome pathological conditions, particularlyadenocarcinomas.5 Cells containing large ab-normal mucus vacuoles were observed, sug-gesting that these cells have a secretoryfunction, as would be expected of cells derivedfrom glandular tissue. The mucus of normalcells is usually of a more electron dense naturefrom that seen here, and the presence of se-verely distorted vacuoles is abnormal.5 Thenuclear characteristics of DAC/S and DAC/Ecells are also unusual. Nuclei with large, coarseclumps of heterochromatin with invaginationof the nucleus (fig 2B) is a common feature ofneoplastic cells.5 Enlarged, distorted nucleoli,characteristic of adenocarcinoma cells, are alsoevident.Both cell lines also varied in their growth

requirements, which was exemplified by theability ofDAC/S to grow in serum free medium.This indicates that the cells are capable ofsecreting essential growth factors which could

act in an autocrine/paracrine manner, whichfurther reflects the less well differentiated DAC/S phenotype. Such properties indicate a de-creased ability to respond to regulatory en-vironmental cues, a common feature ofneoplastic cells, and it would be interesting todetermine the putative growth factor(s) in-volved in this autocrine/paracrine mechanism.In contrast, DAC/E required supplementationwith FCS, insulin, glutamine and hydro-cortisone for optimal growth. This is indicativeof a better differentiated phenotype, in thatthese cells will only respond to appropriategrowth stimuli, similar to the situation ex-perienced under normal in vivo conditions.The population doubling time (PDT) of

DAC/S is approximately 24 hours, which iscomparable with normal epithelial cell lines.3The relatively slower PDT of DAC/E is un-remarkable in that tumour cell lines derivedfrom other regions of the gastrointestinal tracthave been shown to vary greatly in their growthrates, from 12 to 273 hours.6

Ploidy analysis was performed to determinethe DNA content of both cell lines. Naturally,all normal cells within the gastrointestinal tractare diploid, but both DAC/S and DAC/E havea predominant ploidy of 5N. These cell linescould be further analysed by karyotyping andgenetic fingerprinting. However, 65% of colo-rectal cancers are aneuploid, with many celllines derived from such lesions having variablekaryotypes.7

Collagen gel can provide a suitable matrixfor the morphogenesis of primitive epithelialstructures such as glands and, indeed, in vitrostudies of this nature have been successful ininducing glandular differentiation.8'0 DAC/Sproliferated and grew rapidly in collagen, yetthis growth seemed to occur in a random fash-ion with no evidence ofany attempt at glandulardifferentiation even when the cells were closelyassociated in clumps. In contrast, DAC/E cellsshowed no obvious interaction with the col-lagen, possibly because of their requirementfor a more complex support matrix such asMatrigel, which may be more effective at in-ducing differentiation.The assessment of anchorage independent

growth in agar showed both DAC/S and DAC/E to be non-viable in such a system. Generally,anchorage independent growth shows a highcorrelation with tumorigenicity," though only avery small proportion (<1 %) oftumour derivedcells are clonogenic in semi-solid media. Thisis because agar seems to enable only the mosthighly malignant cells to clone.'2Both cell lines were non-tumorigenic when

xenografted into nude mice, which was dis-appointing but not surprising considering thatthe frequency of "takes" (that is, growth oftransplanted tumour in the host) is, on average,only 30%.'" However, there are many examplesof cell lines derived from poorly differentiatedtumours that readily form colonies in soft agar,but are not tumorigenic in nude mice,'4 andthe technique and site of xenografting as wellas the strain of host animal may have a bearingon the "take" rate."3' We are currently at-tempting to define the conditions required to

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promote the growth of these cell lines in vivo.The original tumour stained positively for

cytokeratin and was negative for vimentin (datanot shown), yet both cell lines derived fromthis tumour expressed both in tissue culture.This is not unusual as cells which rapidly pro-liferate in vitro are known to be able to alternatetheir expression of intermediate filaments, ex-pressing high levels of cytokeratin or vimentin,or both.'6 The absence of desmin or smoothmuscle actin indicates that neither cell lineoriginated from muscle or myofibroblasts.Normal differentiation antigens, such as se-

cretory components, can be lost in transformedcells, whereas oncodevelopmental antigens areincreased,'7 reflecting the ability of neoplasticcells to de-differentiate to their primitive an-cestral phenotype. Carcinoembryonic antigenis found predominantly in tumours of the gas-trointestinal tract,6 and qualitatively both celllines express abundant CEA (figs 4G and 4H),indicating that these cells express at least oneoncofetal protein. However, CEA expression isnot conclusive evidence of malignancy, as thisprotein can be expressed, albeit rarely, in non-malignant conditions."'The pattern of integrin expression by cul-

tured cells may be different from that observedin tissues in vivo, which reflects the regulation ofintegrins by the complex extracellular matrix.'8The cells of many tissues express x2(31 anda6pl, which bind collagen and laminin, re-spectively. Both cell lines were negative for theat6 integrin subunit, but were positive for the(1 and at2 integrin components, yet did notdisplay the membrane specific localisation thatis characteristic of integrins (fig 4D). Thismight be because of mutational loss or ab-normal expression of certain integrins whichfrequently occurs in colorectal adeno-carcinomas and is associated with a more ag-gressive, poorly differentiated and henceinvasive phenotype in melanomas and breastcancer.'8 Indeed, many examples exist where achange in integrin expression is associated witha change in cells from a normal to a malignantphenotype.'8'9 Moreover, the ,B1 and at2 inte-grin subunits are clearly confined to cy-toplasmic granules in DAC/S and DAC/E cells,suggesting a defective intracellular receptortranslocation mechanism. In addition, neitherof the cell lines expressed the cell adhesionmolecule E-cadherin, a protein normally as-sociated with all normal epithelia, but which isfrequently lost in carcinomas. The absence ofE-cadherin immunoreactivity in DAC/E andDAC/S could be a result of the loss of theHECD-1 epitope through mutation or de-letion. It may be connected with the inabilityto form colonies in soft agar because of thelack of intercellular cooperation.

In conclusion, two continuous cell lines havebeen established from biopsy material obtainedfrom a primary duodenal adenocarcinoma.Both cell lines, namely DAC/S and DAC/E,are immortal, transformed and it is likely thatthey represent cell lineages which have ori-ginated from an epithelial component of theoriginal tumour. Both cell lines have verysimilar immunophenotypes, but different mor-phologies and growth properties. The tumor-igenic potential of both cell lines has not yetbeen firmly established, but further ex-periments are under way to evaluate this.

Virtually all human cell lines are char-acterised only after years of intensive work andadditional, extensive studies are required tofully characterise these unique cell lines. Thismay then provide an opportunity to elucidatethe molecular mechanisms operating in DAC/S and DAC/E, which will ultimately contributeto our understanding of gastrointestinal car-cinogenesis.This work is supported by the Medical Research Council.

1 Spira IA, Ghazi A, Wolff WI. Primary adenocarcinoma ofthe duodenum. Cancer 1977;39:1721-6.

2 Lien GS, Mori M, Enjoji M. Primary carcinoma of the smallintestine. A clinicopathologic and immunohistochemicalstudy. Cancer 1988;61:316-23.

3 Freshney RI. Culture of animal cells. A manual of basic tech-niques. 3rd edn. New York: Wiley-Liss, 1994.

4 Bartek J, Bartokova J, Lalani E-N, Brezina V, Taylor Pa-pamitriou J. Selective immortalisation of phenotypicallydistinct epithelial cell types by microinjection of SV40DNA into cultured human milk cells. Int J Cancer 1990;45:1105-12.

5 Ghadially NF. Ultrastructural pathology of the cell and matrix.2nd edn. London: Butterworths, 1982.

6 Rutzky LP. In vitro model for human colon cancer: Bio-logical properties. In: In vitro models for cancer research. Vol1. 1990:159-82.

7 McGee JO'D, Isaacson PG, Wright NA (eds). Pathology ofsystems. In: Oxford textbook of pathology. Vol 2A. 1992:1266.

8 Del Buono R, Pignatelli M, Hall PA. Control of differ-entiation in a rectal adenocarcinoma cell line: the role ofdiffusable and cell-associated factors. J Pathol 1991;164:59-66.

9 Pignatelli M, Bodmer WF. Genetics and biochemistry ofcollagen binding-triggered glandular differentiation in ahuman colon carcinoma cell line. Proc Natl Acad Sci USA1988;85:5561-5.

10 Kirkland SC. Polarity and differentiation of human rectaladenocarcinoma cells in suspension and collagen gel cul-tures. J Cell Sci 1988;91:615-21.

11 Hamburger AW, Salmon SF. Primary bioassay of humantumour stem cells. Science 1977;97:461-3.

12 Neugut AI, Weinstein IB. Use of agarose in the de-termination of anchorage-independent growth. In Vitro1979;15:351.

13 Giovanella BC, Fogh J. The nude mouse in cancer research.Adv Cancer Res 1985;44:69-120.

14 Whelan R, Gibby E, Sheer D, Povey S, Hill BT. Char-acterisation of a continuous cell line in culture establishedfrom a Krukenberg tumour of the ovary arising from aprimary gastric adenocarcinoma. Eur J Cancer Clin Oncol1988;24: 1397-408.

15 Stamp GWH, Quaba A, Braithwaite A, Wright NAW. Basalcell carcinoma xenografts in nude mice: Studies on epi-thelial differentiation and stromal relationships. J Pathol1988;156:213-25.

16 Connel ND, Rheinwald JG. Regulation of the cytoskeletonin mesothelial cells: Reversible loss of keratin and increasein vimentin during rapid growth in culture. Cell 1983;34:245-53.

17 McGee JO'D, Isaacson PG, Wright NA (eds). Principles ofpathology. In: Oxford textbook of pathology. Vol 1. 1992:575.

18 Pignatelli M, Stamp GWH. Integrins in tumour de-velopment and spread. Cancer Surv 1995;24: 113-27.

19 Belrew J. The role of cell adhesion molecules in cancerinvasion and metastasis. Breast Cancer Res Treat 1993;24:175-84.

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