establishment and characterization of two new squamous ...scraping and selective trypsinization...

[CANCER RESEARCH 48, 2858-2866, May 15, 1988]

Establishment and Characterization of Two New Squamous Cell Carcinoma CellLines Derived from Tumors of the Head and Neck1

Peter G. Sacks,2 Steven M. ParnÃ©s,Gary E. Gallick, Zahra Mansouri, Rosemarie Lichtner,3 K. L. Satya-Prakash,4

Sen Pathak, and Donald F. ParsonsDepartments of Tumor Biology [P. G. S., G. E. G., Z. M., R. L.J, Head and Neck Surgery [P. G. SJ, and Genetics [K. L. S-P., S. P.], The University of Texas M.D.Anderson Hospital and Tumor Institute, Houston, Texas 77030; Division ofOtolaryngology fS. M. P.], Albany Medical Center, Albany, New York 12208; and WadsworthCenter for Laboratories and Research [D. F. P.], New York School Department of Health, Albany, New York 12201

ABSTRACT

Two human cell lines were established from untreated squamous cellcarcinomas of the head and neck. Line 183 was derived from a head andneck squamous cell carcinoma of the tonsil and 1483 from a head andneck squamous cell carcinoma of the retromolar trigone. Both lines growin a cobblestone pattern demonstrating their epithelial heritage. Immu-nofluorescence studies and one-dimensional polyacrylamide gel electro-phoresis indicated that both lines contain cytokeratins. Line 1483 is moreaggressive in nude mice, has a higher efficiency for anchorage-independent growth, expresses p21"" (product of the ras oncogene) at a higher

level, and is more aneuploid than 183. 1483 also grows as a multicellulartumor spheroid. Line 1483, which was established from the primarytumor of a patient with nodal metastasis, thus displays more progressedcharacteristics than line 183, which was established from a patient withno clinically positive nodes.

INTRODUCTION

Clinical survival rates have plateaued for patients with cancers of the head and neck, reflecting few significant advancesin treatment modalities and in understanding the biology of thedisease. Since the tumor represents only 5% of the total cancersin the U.S. (1), basic research has been largely neglected,although this type of tumor presents a formidable clinicalchallenge. We believe that tumors of the upper aerodigestivesystem have several features that suggest their potential usefulness as a model for HNSCC5 as well as for squamous cell

carcinoma in general: (a) most human tumors are carcinomas(1,2) and 90% of head and neck tumors also are carcinomas(2); (b) several premalignant lesions of the upper aerodigestivesystem are clinically recognized (3); (c) lesions of the upperaerodigestive system are usually superficial and can be clinicallymonitored; (</)epidemiolÃ³gica! data are available which correlate site and stage of the disease with progression to local ordistant mÃ©tastases(4); (e) patients with tumors of the upperaerodigestive system are at an increased risk of recurrence atthe primary site and for developing secondary neoplasms (5);(/) as a result of current therapeutic strategies, untreated clinical specimens are generally available.

Although the majority of human cancers are carcinomas,

Received 7/13/87; revised 12/22/87; accepted 2/22/88.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 work was supported by NIH grants CA29255 and CA38903 from the

National Cancer Institute.2To whom requests for reprints should be addressed, at Department of Tumor

Biology, P. O. Box 108, University of Texas System Cancer Center, M. D.Anderson Hospital and Tumor Institute, ISIS Holcombe Boulevard, Houston,TX 77030.

3 Present address: Institute for Immunology and Genetics, German CancerResearch Center, Heidelberg, Germany.

' Present address: Department of Pathology, Medical College of Georgia,

Augusta, GA 30912.5The abbreviations used are: HNSCC, head and neck squamous cell carcinoma;

MEM, minimal essential medium; FCS, fetal calf serum; DMEM, Dulbecco'sminimal essential medium; DPBS, Dulbecco's phosphate buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

experimental research has historically stressed mesenchymallyderived cells owing in large part to their adaptability to in vitroculture techniques. In this report we describe two new squamouscell carcinoma cell lines which were derived from untreatedprimary tumors of the head and neck. These cells have uniquebiological properties which may reflect important differencesin the tumors from which they were derived.

MATERIALS AND METHODS

Establishment of Cell Lines. Surgical specimens were obtained fromthe operating room suite and transported to the laboratory in Eagle's

MEM supplemented with 10% FCS and the antibiotics gentamicin (SOMg/ml) and fungizone (5 Mg/ml)- Tumor tissue was rinsed in MEMcontaining nystatin (50 /Â¿g/ml)instead of fungizone and, under sterileconditions minced with scalpels into small-sized pieces of 1-3 mm.Pieces were placed in 35- or 60-mm Petri dishes with enough mediumto wet the surface and were incubated at 37Â°Cin a 5% CO2 and water-

saturated incubator. Within 3-10 days epithelial and fibroblast cellsmigrated from expiants. Fibroblast overgrowth was controlled by cellscraping and selective trypsinization (0.05% trypsin-2 HIM EDTA).During selective dissociation the dishes were monitored under a phasecontrast microscope and treatment was stopped when fibroblasts butnot epithelial cells were detached. When epithelial cells in the dishesreached 75-100% confluency and were free of fibroblasts, they weresubcultured in conditioned medium with an initial split ratio of notmore than 1:2. Line 183 was considered "established" (able to be

routinely passaged) after about 4 months in culture and line 1483 after6 months. Lines were routinely tested for mycoplasma contaminationby Hoechst staining and were always negative. The characteristics ofpatients from whom these cell lines were established are given in Table1. Histopathology showed that tumor 1483 was more aggressive in thatit exhibited local intiltration of subjacent muscle, fascia, and glandulartissues. This tumor also had spread to one lymph node.

In Vitro Culture Techniques. Cell lines were originally subculturedin Eagle's MEM with 10% FCS and gentamicin as above. For the cell

characterization in this study, the lines were maintained in DMEM-F12 (1:1) with 10% FCS and gentamicin and used between passages 15and 30. These cell lines stratify in culture and contain cell-cell junctionsthat make them difficult to separate into pure single cells. When singlecells were needed, vigorous pipetting of dissociated cells plus a 10-minsedimentation at l g was performed, and the top one-half of cells wasused.

Growth rate studies used single cell suspensions of log phase cells.Cells were plated in duplicate at 20 x IO3cells/60-mm Petri dish. Cell

numbers were counted on days 0, 2,4,6,8,10, 12, and 14, and doublingtimes were derived from the exponential growth phase. For platingefficiency on plastic, single cell suspensions were plated in 60-mm Petridishes at concentrations of 50, 100, 500, 1000, 2500, 5000 cells (fourdishes/concentration). Colonies were allowed to form for 14-21 days,fixed in 3:1 methanohglacial acetic acid, and stained with 0.3% mÃ©thylÃ¨neblue. Anchorage-independent growth was assayed by suspendingknown numbers of log phase cells in 0.5 ml of 0.5% agarose (Seaplaque,FMC) over a 1% agarose base. Agarose was made up with DMEM-F12 plus 10% FCS and cell numbers varied from 50 x IO4to 4 x 10*

cells per dish. Following gelling an additional 1 ml of medium wasoverlaid on the agarose, and the plates were refed every 7-10 days.Colonies were grown for 21 days and dishes were stained with the vital

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TWO NEW CELL LINES FROM HEAD AND NECK TUMORS

Table 1 Patient-tumor characteristics

Patient1-83

14-83Age54 66SexM MStageT3NOMOT2N1MOTumor

histologyWell-differentiated

SCCof the tonsil

Well-differentiated SCCarising from retromo-lar trigone

stain iodonitrotetrazolium chloride before counting (6). A colony wasdefined as a cluster, at least 50 urn in diameter, of red stained cells.

Multiceli tumor spheroids were initiated by seeding log-phase cells(50 x IO4 to 4 x 10s) onto either 60-mm nonadherent Petri dishes(Lab-Tek) or onto Petri dishes (60 or 100 mm) which had beenprecoated with 1.25% agar in complete DMEM-F12 (7). Spheroidalaggregates formed within 1 day and were maintained either on non-adherent dishes or in suspension culture in spinner flasks.

Tumorigenicity. To determine in vivo tu morigeri icity, IO7viable cells

were injected into the lateral thoracic wall of nude mice (live mice/cellline) obtained from HarÃanSprague-Dawley. Mice were maintained inlaminar flow hoods under pathogen-free conditions. When tumors werebetween 1 and 2 cm in diameter individual mice were killed and selectedorgans (described in "Results") were processed for histology and ex

amined for the presence of metastasis.Chromosome Analyses. A number of modifications of standard chro

mosome preparation techniques were attempted to obtain ample mitoses in which optimally condensed chromosomes could be obtained.For example, mitotic shake-off 2 h after treatment with colcemid (0.04Mg/nil) yielded highly condensed chromosomes. The following protocolyielded both satisfactory mitotic index and chromosome morphologyfor subsequent banding. Cells were grown in I ,-15 medium supplemented with 10% FCS. At about 60% confluency, cultures were treatedwith colcemid (0.03 Mg/ml) for 6 h at 37Â°C.Cells were dislodged with

a rubber policeman, collected in 15-ml tubes and centrifuged at 1700rpm for 5 min. The pellet was suspended in 10 ml of a 1:1 mixture of0.075 M KC1 and 1.0% sodium citrate for 20 min at room temperature.Following hypotonie treatment, the cells were centrifuged and fixed infresh methanol:glacial acetic acid (3:1 by volume) for 10 min at roomtemperature and washed three times with fresh fixative. The fixed cellsuspension was then processed. Six- to 7-day-old slides were treated forvarious banding techniques following routine procedures (8). Someslides were conventionally stained in Giemsa for chromosome countsand identification of double minutes (DM). A minimum of 50 meta-

phase spreads were counted for modal number and at least five G-banded karyotypes were made for each cell line.

Keratin Analyses. Keratin content was analyzed by indirect immu-nofluorescence and SDS-PAGE analysis. For immunofluorescence,cells were grown on coverslips, rinsed in DPBS at 37Â°Cand fixed inmethanol for 5 min at â€”20Â°C.Coverslips were rinsed and incubated

with the antikeratin monoclonal antibody PKK1 (Labsystems) for l hat 37Â°C.After being rinsed in DPBS, the coverslips were incubated

with an anti-mouse IgG conjugated with fluorescein (Sigma) for 60 minat 37Â°C.Then they were rinsed and mounted in 9:1 glycerol:PBS (v/v)

with cover cement (Pfaltz & Bauer). Slides were examined with anOrthoplan Zeiss photomicroscope with epifluorescence. SDS-PAGEwas performed on confluent monolayers. Cells were rinsed and scrapedin Ca2+ Mg2+-free PBS containing yV-a-/Mosyl-L-arginine methyl ester

(1 mg/ml) and phenylmethylsulfonyl fluoride (2 HIM).The cell pelletwas extracted in 0.6 M KC1 containing 1% Triton X-100 and DNase (1mg/ml; 10 mM Mg2+). The insoluble intermediate filament pellet wassolubilized in 8 M urea-0.5 M Tris, containing 4% NP-40 and 5% ÃŸ-mercaptoethanol (pH 7.2). SDS-PAGE was performed by a modification of the procedure of Laemmli (9) using a 3% stacking and 12%running gel. Gels were stained with Coomassie blue or transferred tonitrocellulose using standard immunoblotting procedures described byTowbin ft al. (10). Keratins were identified on the nitrocellulose blotsusing the AE1-AE3 antikeratin monoclonal antibodies (Hybritech) withimmunodetection with the Vectastain ABC kit.

Detection of p21"". The determination of p21"" levels was performedas described previously (11). Briefly, p21"" was immunoprecipitatedfrom monolayer cultures (-70% confluent) with v-H-ros monoclonalantibody Y13-259 (Oncogene Sciences). The immunoprecipitate wasrun on SDS-PAGE (10.5% acrylamide) and transferred to nitrocellulose. Nonspecific binding sites on the nitrocellulose were blocked with3% bovine serum albumin after which the blot was probed with theanti-p21"" antibody, washed, and then probed with '"l-labeled goatanti-rat IgG (Amersham). The nitrocellulose filters were dried andautoradiographed at â€”70Â°C.

RESULTS

Tumor tissue from patients 1-83 and 14-83 produced epithelial outgrowths that were freed of fibroblasts as discussed in"Materials and Methods." Lines 183 and 1483 are epithelial in

Fig. I. Phase contrast micrographs of cell lines 183 (left) and 1483 (right) (145 x; bar, 100 iitn).

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Fig. 2. Electron micrographie overviews of confluent monolayers of lines 183 and 1483. Three layers of stratification are evident in (lop) line 183 (4400 x; bar, 5;im); Hirer, higher magnification at arrow (41,400 x; bar, 240 nm) and (bottom) line 1483 (4400 x; bar, 5 /mi).

Table 2 Ce// /mÂ«growth parameters

Colony formation(%)183

1483Doubling

time~36h

-36 hPlastic

(J?,range)~3%(0.5-7.0)

~6%( 1.6-8.4)Agarose

(X,range)~1.4%(0.4-2.7)

~4.0%(2.0-5.7)Tumorigenicity"

(nudemice)4/55/5

" Growing tumors formed within 4 weeks.

nature and grow in typical epithelial cobblestone patterns (Fig.1). The lines are morphologically distinct with 1483 cells beingmore compact and individual cell boundaries easily recognizable. Cells of 183 are more spread and grow with recognizablecell piling occurring in nests of all sizes (Fig. 1, left). Both celllines grow as individual epithelial nests that enlarge and coalesce in time. In the center of individual nests, phase contrastmicroscopy showed a piling of cells. Electron microscopy indicated that the piling is epithelial stratification with cells beingelongated and joined by desmosomes. The qualitative impression was that 183 cells have more desmosomal junctions than1483 cells, but normal squamous differentiation was not found(Fig. 2). The uppermost layer of cells are not terminally differentiated, i.e., squames are not formed. The major ultrastructuraldifferences found between lines 1483 and 183 were that 1483

cells contain more mitochondria and their nuclei have moreprominent heterochromatin. In addition, 183 cells spread moreas is evident from the thickness of equivalent cell layers (Fig.2). Necrotic sloughed cells were found in 1483 cultures; at highmagnification these appeared to have both a thickened cellmembrane (cornified envelope) and an abundance of filamentous material (data not shown). This suggested that differentiation, although it is atypical, occurs to a limited extent.

Cell Growth. Growth related parameters are shown in Table2. Cell line 1483 plated on plastic and grew in 0.5% agarosewith a higher efficiency than line 183. Both lines had similargrowth rates. Although clonal growth can be obtained, lowersplit ratios of 1:3 to 1:5 maintained healthier appearing cellsfor routine passaging, probably because of their epithelial natureand nest-like growth pattern with intercellular cooperative effects.

Chromosome Analysis. As shown in Fig. 3, cell line 183 isclose to triploid (range, 60-80) with a bimodal distribution at69 and 73 chromosomes. Cell line 1483 is close to tetraploid(range, 60->100) with a modal chromosome number of 74. Avariety of harvesting techniques were attempted to obtain met-aphase spreads suitable for banding since the standard procedure produced supercondensed chromosome morphology.

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80 62 64 66 6B 70 72 74 76 7Â» Â»0

CHROMOSOME NUMBER

24 -

22 -

20 -

16 -

16 -

14 -

12 -

10 -

6 -

* -

4 -

2

O I M.I60 62 64 66 66 70 72 74 76 7Â« 60 >

CHROMOSOME NUMBER

Fig. 3. Chromosome distributions for lines 183 (tup) and 1483 (bottom).

Giemsa-banded karyotypes of cell lines 183 and 1483 are shown

in Fig. 4. The two cell lines share three marker chromosomes:Ml = del I(p21), M2 = del 6(q22), and M3 = (13q;13). Eachcell line has several unique markers. Cell line 183 has four suchmarkers: del(7q), del (lip), t(2p;?), and t(20p;?) (Fig. 4, toparrows). Cell line 1483 has six unique marker chromosomes:del (3p), t(5p;?), t(10p;6p), t(15p;17q), t(18q;?), andt(14p;ABR) (Fig. 4, bottom). These anomalies were clonal forboth cell lines. Other alterations were nonclonal and could notbe identified in some cases. Giemsa-banding patterns confirmedthat both cell lines were of human origin.

Keratin Analyses. Both cell lines were examined for thepresence of cytokeratins by indirect immunofluorescence techniques using an anticytokeratin monoclonal antibody, PKK1.As shown in Fig. 5, both lines contain an extensive cytokeratinnetwork that reflects their epithelial heritage. The keratin content was further analyzed by one-dimensional SDS-PAGE.Since keratin polypeptide content is known to vary with differ-entiative state ( 12, 13), confluent monolayers were used for thekeratin extractions for more precise comparison of the two celllines. An example of an SDS electrophoretic gel in which 20Mgof protein/lane were analyzed is shown in Fig. 6. Inumino-

blotting with the broad spectrum anticytokeratin monoclonalantibodies AE1-AE3 confirmed that these bands in the M,40,000 to 70,000 range were keratins (data not shown).

Tumorigenicity. To determine whether these cell lines weretumorigenic, IO7 viable cells were injected into nude mice (five

mice/cell line). As is shown in Table 2, both lines producedtumors. Histologically, line 183 produced more highly keratin-

ized tumors (Fig. 7, top) with many areas of pearl formation(pathological diagnosis of well differentiated SCC). Tumorsfrom animals injected with line 1483 were histologically morediverse with the majority of the tumor being more undifferen-tiated (Fig. 7, bottom) but individual cell keratinization andareas of overt keratinization could also be found (pathologicaldiagnosis of moderately differentiated SCC). Line 1483 displayed a higher malignant potential in that two mice developedrapidly growing tumors and died within 7 weeks. We used theanterior lateral thoracic wall as the site of tumor inoculationsince this site was reported to enhance spontaneous metastasis(14). Mice were maintained up to 7 months and then killed.Autopsies were performed and selected organs (lung, liver,axillary lymph nodes, kidney, ovary, spleen) were examinedhistologically for mÃ©tastases.In one mouse injected with cellsof line 1483, a metastatic deposit was found in a vessel adjacentto a lymph node.

Spheroid Formation. Cell lines were tested for their ability toaggregate, a property that has been correlated with the transformed state (15). When plated on nonadherent surfaces, bothlines aggregate and form tightly compact spheroidal masses.These were similar in morphology to aggregates produced bysuch tumor spheroid-forming lines as V-79 and EMT6. Weattempted therefore to culture these aggregates both in stationary and suspension cultures to test whether they had spheroidforming capacities. Line 183 aggregates could not be propagated as spheroidal masses and died after several days. Line1483 aggregates remained viable on either nonadherent surfacesor in spinner flasks and grew as multicellular tumor spheroids.

p21"" Determinations. Since the elevation of p21"" gene prod

ucts in some types of tumors may correlate with the stage ofmalignancy, we determined levels of p21, the protein productsof the c-ras oncogene, as described in " Materials and Methods."The antibody Y13-259 (16) recognizes all p21"" proteins of thec-ras genes. In Fig. 8, p2\ras levels of lines 183 and 1483 werecompared to LSI80, a colon carcinoma elevated in p21ras. Line1483 showed an increased p2l"" level compared with that of

line 183. Densitometric scanning showed the increase to beapproximately 25-fold.

DISCUSSION

Historically, some of the oldest tumor cell lines are from thehead and neck. In the 1950s, KB from the floor of the mouth(17) and Hep 2 and 3 (18) from larynx and neck nodes (buccalmucosa primary), respectively, were developed. Now however,these lines are considered to be Hela contaminated and this,coupled to their 30 years in culture raises questions about theirsuitability as models for studying tumors of the head and neck.Interest and success in developing carcinoma cell lines hasrecently been renewed. In 1978, Krause and colleagues at AnnArbor, MI, presented their initial work of establishing HNSCCcell lines using expiant outgrowth techniques with primary orxenografted primary tumors. They produced three cell linesfrom 194 patients with short term epithelial culture of anadditional nine patients still in progress (19). They continuedthis work and now have perhaps the largest collection ofHNSCC cell lines (20). The establishment of carcinoma celllines is considered difficult and low success rates have beenreported. For example, three esophageal carcinoma lines wereestablished by one group using 100 specimens (21), and recentlyanother group reported the establishment of two head and necklines from more than 100 specimens (22). In 1981 two teamsreported the development of HNSCC cell lines. With expiant

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Fig. 4. G-banded karyotypes. Unique chromosomes (arrows) and three shared markersMl, M2, M3 are described in text. Top, line183; bottom, line 1483.

ir<irjx Ã¼%Ml o ^ 4 5

n B:IMII Â«Â«<

M3 14 15

Â«rIH Ittt

19 20 21 22

)f I Ir*V V

1 Ml 3 \

78 9 / 10 11 12

III iM3 14 f

H10 17 ^ 18

19 20 21 22 x x

k* /*~* mu

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Fig. 5. Immunofluorescent micrographs of lines 183 (left) and 1483 (right) stained for cytokeratins. (1100 X; bar,

93-

66-

45-

31-

21-

14-1

Fig. 6. One-dimensional SDS-PAGE analysis of cytokeratin content. Lane 1,(standards) phosphorylase B, M, 92,500; bovine serum albumin, M, 66,200;ovalbumin, M, 45,000; carbonic anhydrase, M, 31,000; soybean trypsin inhibitor,M, 21,500; lysozyme, M, 14,400. Lane 2, line 1483. Lane 3, line 183.

outgrowth techniques similar to the methods we have used, aseries of cell lines designated HN1-HN10 were derived from

patients who had received radiotherapy, with or without chemotherapy (23). The second group used feeder layer techniques toestablish two carcinoma cell lines of the epidermis and four ofthe tongue (24). One patient with tongue carcinoma (SCC-4)

had received prior radiation and chemotherapy and three hadnot been treated (SCC-9, -15, and -25). These lines, developed

by using inactivated 3T3 mouse cell feeder layers, displayed afibroblast growth dependence ranging from very strong dependence (SCC-9) to total independence (SCC-25), and one of theepidermal carcinoma lines exhibited total dependence. Anotherresearch group recently established four new HNSCC cell linesusing outgrowth techniques on tumors from patients who hadreceived radiotherapy (25). Thus, while the number of availableHNSCC cell lines is expanding, there are still a limited numberin existence and fewer which are useful as models for development and progression of this important type of tumor.

Tumors are well known to be heterogeneous in a number ofparameters (26). Radiotherapy and chemotherapy, by eliminating sensitive cell populations, may alter a tumor's in vivo

progression and cell heterogeneity (27). The two new HNSCCcell lines we described here were established from untreatedprimary tumors and we believe that such cell lines are morelikely to represent the initial tumor than lines derived fromtherapeutically treated patients. When nine HNSCC cell lineswere karyotyped, those from untreated patients had lowermodal numbers of chromosomes than those derived fromtreated patients (28). Karyotypic analyses of our cell linesdemonstrated that they are bona fide lines and are neither cross-contaminated with each other nor contaminated with Hela.

As was shown in Table 1, line 1483 was developed from aprimary tumor that was more advanced and histopathologicallymore aggressive than the tumor of line 183. This behavior wasmaintained by the 1483 cells in culture. The cell line was moreaggressive in nude mice in that two animals developed rapidlygrowing tumors and died within 7 weeks; a 1483 spontaneousmetastasis was found. In addition, line 1483 plated more efficiently on plastic and in soft agarose. Chromosome analysesshowed 1483 to be more aneuploid and to contain more markerchromosomes. The level of p21, product of the ras oncogene,was higher and we have also reported that the level of epidermalgrowth factor receptor is also increased in 1483 as comparedto 183 (29). Lastly, line 1483 grows as a multiceli tumorspheroid and 183 does not.

Multiceli tumor spheroids are believed to be of an interme-

2863




Fig. 7. HistolÃ³gica!sections of nude mousetumors. Top, representative section of 183 tumor showing several areas of terminal differentiation (pearl formation); bottom, section of1483 tumor showing major histolÃ³gica! pattern of undifferentiated HNSCC. As discussedin "Results," areas of terminal differentiation

can be found (not shown) (220 x; har. 50 ion).

diate complexity to monolayer cultures and in vivo tumors (30).Although both normal and transformed human cells may aggregate, only certain tumor cells will grow as spheroids. In astudy of 20 surgical specimens 16 formed spheroids (31), when22 xenografts were tested only five were capable of spheroidgrowth (32) and in another test of 27 lines, only 16 formedspheroids (33). Normal cells may aggregate but they do notnormally grow with one exception being cells of lymphoidorigin (33). Of our two lines, both aggregate but only 1483 willgrow. To our knowledge, this is the first report of humansquamous cell carcinoma cells growing as tumor spheroids.Normal keratinocytes have an anchorage dependence forgrowth and the possibility that squamous cell carcinomas alsohave this need may account for poor growth of cell lines and invivo tumors in semisolid medium (34, 35). The ability of line1483 to grow as a tumor spheroid is probably mechanisticallyrelated to its increased ability to grow in anchorage-independent

conditions. Another recently developed HNSCC cell line alsoshows spheroid forming capacity and this line also grows in

anchorage independence with an efficiency of about 5%.6 Inaddition, both lines 1483 and 183 grow in anchorage-independ

ent conditions at higher rates than that reported for otherHNSCC cell lines (24, 25). In anchorage-dependent studies,

however, colony formation was significantly lower than thatreported by one group (25) but probably equivalent to a seriesof tongue carcinomas in which three out of four cell lines werehighly dependent on fibroblast conditioning for clonal growth(24).

Immunofluorescence and one-dimensional SDS-PAGEanalysis demonstrated that both cell lines contained cytokera-tins. Keratin analysis did not detect high molecular weightkeratins (M, 65,000-67,000) which are characteristic of termi

nal squamous differentiation (12, 13). This agrees with themorphological results from electron microscopic analysis (Fig.2) in which normal squamous differentiation was not found tooccur in cell culture. In line 183 some superbasal cells containincreased amounts of tonofilaments as shown in Fig. 21!, but

" Unpublished observations.

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variety of biochemical and biological systems, as has been donewith the HN series of cell lines (46). These two cell lines ( 183and 1483) show good tumorigenic potential in nude mice, andthis, coupled to their increased ability to grow under anchorage-independent conditions, suggest their potential usefulness intherapeutic based studies. The ability of one of these lines togrow as a multicellular tumor spheroid also opens this modeland its numerous applications (47) to studies aimed at a betterunderstanding of squamous cell carcinomas of the head andneck.

ACKNOWLEDGMENTS

We thank Brooks Myers for electron microscopy, Eleanor FelonÃaand Susan Lyman for help in preparing this manuscript, and TaniaBusch for photographic work.

Fig. 8. Immunoblot of immunoprecipitated p21"". Lane I, LSI80, a coloncarcinoma elevated in p21""; Lane 2, line 183; Lane 3, line 1483. LC, light chain

IgG.

most superficial cells were similar in appearance to the moreundifferentiated cells of Fig. Iti. Whether cells with increasedtonofilaments are indicative of partial keratinization or solelyrepresent tumor heterogeneity is at present unknown.

Using a broad spectrum antibody which recognizes all c-rasproteins (16), cell line 1483 was found to have approximatelya 25-fold increase in p21rai. It is recognized that both over-expression of the normal c-ras protooncogene or activation ofthe protooncogene may lead to tumorigenesis (36). The biological significance of the increase in p21 in line 1483 is unknown,and ras levels have not been looked for in other HNSCC celllines. In one of the few studies in which head and neck cancerswere examined for oncogenes, increased transcriptional levelsof Ha-ras and Ki-ras were found (37). The ras oncogenes havebeen correlated with such biological functions as anchorageindependence and growth factor secretion; line 1483, which hasthe higher level, is biologically more progressed. In addition,we have found that epidermal growth factor receptor, analogousto the c-erb-B oncogene, is also elevated in line 1483 (29),similar to the elevation of epidermal growth factor receptorreported in some HNSCC cell lines (38, 39). This overexpres-sion was related to increased RNA whereas DNA from theselines showed neither gene amplification nor rearrangement(40).

In Giemsa-band analyses we found several unique markersfor each line and three shared markers. Although as discussedabove, several groups have established HNSCC cell lines, detailed chromosome analyses have not been reported for most.A study of six of the HN series (28), showed abnormalities onchromosome 1 to be common to all. Our two cell lines alsohave a deletion in chromosome 1 and whether aberrations onchromosome 1 will be important for the development ofHNSCCs is at present unknown. Aberrations on chromosome1 have been correlated with heÃ±Ãalological malignancies (41),and several oncogenes, Nras (42), the human homologue ofchicken \-SK (43), B-Lym (44), and src (45) have been mappedto chromosome 1.

The two HNSCC cell lines described in this report differ ina number of biological and biochemical parameters. We viewcell lines as tools with which studies aimed at understandingsquamous cell carcinoma biology can be undertaken using a

2865

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1988;48:2858-2866. Cancer Res Peter G. Sacks, Steven M. Parnes, Gary E. Gallick, et al. NeckCarcinoma Cell Lines Derived from Tumors of the Head and Establishment and Characterization of Two New Squamous Cell

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