[CANCER RESEARCH 41, 1657-1663. May 1981]0008-5472/81/0041-0000$02.00

Tumorigenic Keratinocyte Lines Requiring Anchorage and FibroblastSupport Cultured from Human Squamous Cell Carcinomas1

James G. Rheinwald and Michael A. Beckett

Division of Cell Growth and Regulation. Sidney Farber Cancer Institute IJ. G. P., M. A. B.I. and Department of Physiology, Harvard Medical School ¡J.G. P.I.Boston, Massachusetts 02115

ABSTRACT

We have established cell lines from six human squamous cellcarcinomas (SCO of the epidermis and tongue, using culturemethods previously developed for clonal growth and serialcultivation of normal keratinocytes. The SCC lines all formrapidly growing, well-differentiated SCC's or progressively

growing squamous cysts in nude mice. In contrast to normalkeratinocytes, SCC cells form unstratified or very poorly stratifying colonies and do not require epidermal growth factor forsustained growth. The SCC lines vary in their requirement fora fibroblast feeder layer to support clonal growth, from complete independence to a dependence as absolute as normalkeratinocytes possess. Only one line forms large, progressivelygrowing colonies at high efficiency in semisolid medium; theother five lines exhibit only a small amount of abortive growthin semisolid medium, after which the cells appear to rapidlydegenerate. These results demonstrate that SCC's often grow

as established lines in culture, but they frequently possess invitro growth requirements similar to those of normal keratinocytes. Consequently, neither semisolid medium nor standardsurface culture media are appropriate for initiating primarySCC cultures or for selecting transformants out of carcinogen-

treated keratinocyte populations, because they do not provideconditions permissive for the growth of many malignant keratinocytes.

INTRODUCTION

Cell culture has long been perceived as an excellent systemin which to identify intrinsic differences between normal cellsand cancer cells and to determine how an alteration manifestedas abnormal behavior in culture gives rise to malignant behaviorin vivo. Much effort has been devoted to the analysis of experimentally induced variants of nontumorigenic cell lines in anattempt to correlate the acquisition of newly acquired pheno-

types in culture with the ability to form tumors in experimentalanimals. Beginning with studies of the growth properties ofvirus-transformed fibroblasts (12), much attention has beengiven to anchorage-independent growth ability as an important

marker of cancer cells. Studies of virally and chemically transformed rodent fibroblast lines (2, 22) have demonstrated astrong correlation between anchorage-independent growth in

culture and tumorigenicity in experimental animals. However,although cells from some types of naturally occurring humansolid tumors, such as melanoma (4), generally form colonies insemisolid medium, cells of many types of carcinoma (epithelialtissue-derived cancers) often do not (13, 24). Alternative

markers are therefore needed for identifying and selectingneoplastic variants of epithelial cell types in culture. Someestablished cell lines have been derived from virtually everytype of carcinoma, but the success rate has been extremelylow (5, 9), suggesting that only unusual forms of these tumorshave generally been studied in culture. Progress in understanding the biology of any type of carcinoma has been impeded bythe inability until recently to culture adequately either normalor malignant cells from most epithelial tissues.

The development of a cell culture system that satisfies tissue-specific growth requirements has permitted long-term serial

cultivation of the human keratinocyte (the cell of stratifiedsquamous epithelia) (19), which is able to divide only a fewtimes in standard culture media. Tissue size and architectureis normally controlled in stratified squamous epithelia by themaintenance of a balance between the rate at which cells in aproliferative compartment divide and the rate at which daughtercells leave this compartment and become committed to aprogram of terminal differentiation. Normal human keratinocytes retain this pattern of growth regulation in culture. Duringtheir finite culture lifetime, they express a specific requirementfor growth-promoting factors elaborated by connective tissue

fibroblasts (15, 19) and an inexorable tendency to lose divisioncapacity and become committed to terminal differentiation viaa mechanism subject to influence by EGF2 (14, 20, 27).

The most important malignant variant of the keratinocyte isSCC. The expression of cell type-specific growth-regulatory

mechanisms by normal keratinocytes in culture suggested tous the feasibility of culturing SCC's to determine the necessary

conditions for their growth and to ascertain whether they consistently are "mutants" in any aspect of growth regulation that

might be causally related to their malignant growth ability. Wereport here the isolation of cell lines representing the tumori-genie populations of 6 human SCC's and a comparison of their

growth requirements in culture with those of normal keratinocytes. Because the culture environment was fully permissivefor the growth of normal keratinocytes, some of the tumor cellsthat we were able to culture were more similar to normal cellsin certain respects than SCC lines that had previously beenisolated; some of the lines retained a strong dependence uponfibroblast feeder support, and most were unable to grow progressively in semisolid medium.

MATERIALS AND METHODS

Tumors. Biopsies of SCC's of the tongue and pharynx were

obtained from the Head and Neck Tumor Clinic of the SidneyFarber Cancer Institute. Portions of surgically excised SCC's

1This investigation was supported by the Ellison Foundation, by Massachu

setts Division of the American Cancer Society Grant 1492-C. and by NationalCancer Institute Grants CA-26656 and CA-19589.

Received August 22. 1980; accepted January 16, 1981.

2 The abbreviations used are: EGF. epidermal growth factor; SCC. squamous

cell carcinoma; PBS. phosphate-buffered saline (0.137 M NaCI, 0.003 M KCI,0.016 M Na2HPO.,, and 0.002 M KH?PO.,).

MAY 1981 1657

J. G. Rheinwald and M. A. Beckett

of the epidermis were obtained from Peter Bent Brigham Hospital, Boston, Mass. SCC-4, SCC-9, SCC-15, and SCC-25were derived from SCO's of the tongue. SCC-4 was from a 55-

year-old male who had had radiation and methotrexate treat

ment for the tumor for 16 months before the biopsy was takenfor culture. SCC-9, SCC-15, and SCC-25 were from a 25-year-old, a 55-year-old, and a 70-year-old male, respectively. These

tumors were first detected less than 3 months before the biopsywas taken for culture, and no treatment had been given to thepatients prior to biopsy.

SCC-12 and SCC-13 were cultured from SCC's of the facial

epidermis. SCC-12 was from a 60-year-old male kidney trans

plant recipient who had been treated with immunosuppressivedrugs for the previous 7 years. SCC-13 was from a 56-year-

old female who had received a series of radiation treatmentsfor the tumor for several years before its surgical removal.

Preparation and Propagation of SCC Cultures. Cultureconditions and procedures were essentially those publishedpreviously for preparing keratinocyte cultures from normal skin(15, 18,19). Biopsies and portions of surgically excised tumorswere placed aseptically into culture medium for transport to thelaboratory, where processing was begun within 2 hr of removal.Samples were rinsed thoroughly with serum-free medium containing Mycostatin or Fungizone (both from Grand Island Biological Co., Grand Island, N. Y.) and then were cut into piecesapproximately 2 mm in diameter. Several pieces were fixed,sectioned, and stained with hematoxylin and eosin in order toconfirm that the sample indeed contained SSC. The remainingfragments were minced with scissors into pieces less than 1mm in diameter and then either were transferred to a stir flaskat 37°with 0.2% trypsin plus 0.2% collagenase to disaggre

gate into single cells or were distributed directly to culturedishes to initiate expiant outgrowth cultures. In either case, thetumor material was cultured with a mitomycin C-treated feeder

layer of the Swiss mouse embryonic fibroblast line 3T3 (31 ).Growth medium was Dulbecco's modified Eagle's medium plus

20% fetal calf serum plus hydrocortisone, 0.4 jug/ml. Serumlots were selected for optimal growth promotion of normalkeratinocytes.

Primary cultures prepared by either method were subcul-

tured after about 2 weeks, at a time when individual colonieshad attained a size of 103 to 10" cells and before neighboring

colonies had merged to make a confluent monolayer. Cultureswere first relieved of the 3T3 feeder cells and any living humanfibroblasts by a 30-sec incubation with 0.02% EDTA followed

by vigorous pipetting of the fluid against the culture dishsurface. This procedure left only epithelial cells adherent to thedish, and these were disaggregated to single cells by a 15- to30-min incubation with 0.05% trypsin plus 0.01% EDTA at37°. The cells were replated at densities ranging from 15 to

5000 cells/sq cm in dishes containing 3T3 feeder cells.Tumor cell populations were serially passaged at 7- to 10-

day intervals by subculturing preconfluent cultures that hadbeen initiated from 3 x 103 to 3 X 10" cells/60-mm dish with

feeder cells. Each passage was equivalent to about 7 to 10cell generations. Primary or secondary cultures of each tumorwere cryogenically preserved as were cell populations at approximately the 10th and 20th passages.

A normal newborn foreskin keratinocyte population (strainN) was cultured in the same way, but in the presence of EGF(20), and was used between the 30th and 80th cell generations

in culture as a control in these experiments.Tumorigenicity of Cultured Cells in Nude (Athymic) Mice.

Nude BALB/c mice from 1 to 2 months old were given s.c.injections of 3 x 106 SCC cells suspended in 0.1 ml of culture

medium. SCC-25 was tested at the third and fourth passagesin a total of 4 mice. All other lines were tested at the 3rd to 5thpassage and also at the 8th to 20th passage, in a total of atleast 7 mice each. Mice were killed from 3 weeks to 3 monthsafter injection, and the tumors were fixed, sectioned in paraffin,and stained with hematoxylin and eosin.

Semisolid Medium. Medium was made semisolid for cellsuspension experiments by the addition of methylcellulose(4000 centipoises; Fisher Scientific Co., Pittsburgh, Pa.) to afinal concentration of 1.3% as described elsewhere (15, 17).To assess anchorage-independent growth, cells were inoculated into 60-mm plastic bacteriological Petri dishes (nottreated to promote cell attachment) at a density of 5 x 103

cells/ml in 10 ml methylcellulose medium. Colony-forming

efficiency was estimated by counting the number of colonies in10 random fields as observed through the inverted microscopeand relating the area of a magnified visual field to the surfacearea of the dish.

Indirect Immunofluorescence. Cells grown on 12-mm-di-ameter glass coverslips were fixed with 10% formalin in PBSfor 10 min at room temperature and were treated with —20°

methanol for 5 min. Antiserum raised in rabbits against humanepidermal stratum corneum keratins (kindly provided by T-T.Sun) was diluted 1/50 in PBS and placed on the coverlips fora 30-min incubation at 37°.After two 10-min rinses in a large

volume of PBS, fluorescein- or rhodamine-conjugated goatanti-rabbit IgG (Miles Laboratories, Elkhart, Ind.) diluted 1/20in PBS was applied for 30 min at 37°. Coverslips were then

thoroughly rinsed in PBS, mounted in glycerin jelly (SigmaChemical Co., St. Louis, Mo.), and viewed through a Zeissepifluorescent microscope with x25 Neofluor and x63 Plan-

apochromat immersion objectives.

RESULTS

Establishment of Cell Lines from Tumor Biopsies. Of 22biopsies of SCC placed in culture, 7 were heavily contaminatedwith yeast and were discarded. Two others proved not to havecontained tumor cells by histological examination. Of the remaining 13 samples, 4 yielded only keratinocyte colonies ofnormal morphology, and these populations senesced after 3 or4 passages. Three of the 13 yielded a mixture of colonies withnormal and abnormal morphology in the primary culture, butabnormal colonies were not detected after the second passage,and these populations senesced after 3 or 4 passages as well.The other 6 tumor samples also yielded normal and abnormalcolonies in the primary culture, but in these cases the abnormalcolony-forming cells persisted in subsequent passages and

behaved like established cell lines. The proportion of tumorsthat grew in culture as established lines varied with the tissueof origin: 4 of 4 SCC's of the tongue; 2 of 5 SCC's of theepidermis; and none of 4 SCC's of the pharynx.

The abnormal colonies from all tumors were different fromnormal keratinocyte colonies in that stratification was absentor greatly reduced. The cells appeared to adhere less tightly ormore reversibly to one another, since they showed more heterogeneity in shape and packing density and tended to round

1658 CANCER RESEARCH VOL. 41

Cell Lines from Human SCO

up more above the level of the surrounding monolayer when Inmitosis (compare Fig. 1a and Fig. 1b). Indirect immunofluores-

cence with an antiserum specific for human stratum corneumkeratin proteins (28) showed that the cells of all 6 lines contained a dense cytoplasmic matrix of keratin filament bundles(Fig. 1c) consistent with their epithelial origin (7, 29, 30). Thecells growing in early-passage cultures of the different tumors

had distinguishing morphological characteristics (Fig. 2), suchas a tendency to stratify slightly and abnormally in large colonies or at confluence (SCC-9) and the rare (SCC-4) or frequent(SCC-25) presence of loosely adherent, rounded cells. Thelines also displayed differences in their 2-dimensional packingdensities in growing colonies (i.e., how flattened the cellsappeared). These characters were preserved by the respectivecell populations indefinitely through many subcultures, suggesting that the long-term cultured populations were descended without change from the major colony-forming cells inthe primary cultures.

Colony-forming efficiency in low-density platings with the

feeder layer was 15 to 40% in the secondary and subsequentcultures, much higher than that displayed by normal keratino-cytes (19, 20). Cultures grew with population-doubling timesof about 24 hr. In contrast to normal diploid keratinocytes (20),EGF was not required by any of the SCC lines to permitcontinued exponential growth to confluence or to effect a highcolony-forming efficiency at subculture. All of the lines grew

progressively and at only slightly slower rates when the serumsupplement was reduced to 5%. The lines also grew satisfactorily with the majority of fetal calf serum lots tested, in contrastto the fastidious requirement of normal keratinocytes for a 20%supplement of only certain lots.

Each line was found to have a unique aneuploid karyotypewith model chromosome numbers ranging from 50 to 80.3 Five

of the 6 lines have undergone at least 200 and the mostrecently isolated line, SCC-25, has undergone about 60 cellgenerations in culture to date without significant alterations inthe properties described below.

Tumor Formation in Nude (Athymic) Mice. When 3 x 106

cells were injected s.c. into nude mice, all 6 SCC lines produced rapidly growing tumors in every animal inoculated.Growth was visually apparent within 10 days, and the tumordiameters increased to 5 to 10 mm by 3 to 6 weeks. SCC-4,SCC-9, and SCC-15 formed well-differentiated SCC's with

characteristic small nests of differentiated cells surrounded byseveral layers of dividing cells, intermixed with connectivetissue (compare Fig. 1d and Fig. 1e). SCC-13 tended to formlarge differentiating cysts with a more organized structure aswell as regions of histologically typical SCC. SCC-12 regularlyformed one or 2 large well-organized differentiating cysts (Fig.1f), growing to a maximum diameter of 5 to 10 mm, accumulating fluid in the center, and remaining for the life of the mouse.SCC-25 formed a single large differentiating cyst similar to thatof SCC-12 but without fluid accumulation. None of the SCClines grew invasively beyond the dermis but remained encapsulated by a thin sphere of mouse dermal connective tissue. Incontrast, diploid keratinocytes cultured from normal epidermisformed only a small 1- to 2-mm-diameter lump that regressedand disappeared after 2 to 3 weeks. Tumors formed from 3 ofthe lines (SCC-4, SCC-9, and SCC-13) were placed back inculture. Cells with the distinctive morphology and karyotype of

Table 1

Clonal growth requirements of SCC lines for fibroblast support and anchorage

Cultures of the 6 SCC lines and the normal newborn foreskin keratinocytestrain N. growing exponentially with 3T3 feeder cells, were subcultured at 103 or3 X 103 cells/60-mm dish in the presence or absence of 3T3 feeder cells invarious media for surface growth and at 5 x 103 cells/ml in medium made

semisolid with methylcellulose for anchorage-independent growth. Dulbecco smodification of Eagle's medium and Medium 199 were supplemented with 20%

fetal calf serum and hydrocortisone, 0.4 jig/ml. Cultures grown with feeder cellswere fed with Dulbecco's modification of Eagle's medium. 3T3 CMa was made

with Medium 199 plus fetal calf serum plus hydrocortisone. Surface cultureswere grown until the 3T3 feeder layer cultures were nearly confluent (usuallyabout 12 days), and then all dishes were fixed and stained. Cultures were rated+ + + for growth of a large proportion of colonies to £2000 cells/colony, + +for £200 cells/colony, + for 360 cells/colony, ±for very slow and abortivegrowth, and —for failure to undergo more than several divisions. Semisolidmedium was regular medium or 3T3 CM containing either the normal Ca2*

concentration of 1.8 mM or the reduced level of 0.2 mw plus 1.3% methylcellulose. Five x 10* cells were dispersed in 10 ml of this medium and were observedfor 3 weeks. Better suspension growth generally occurred with the lower Ca2*

level. Growth response under the best condition for suspension culture is givenfor each strain. Numbers given are the averaged results of three experimentsusing cells ranging from the 5th to 20th passage.

CelllineStrain

NSCC-4SCC-9SCC-12SCC-13SCC-15SCC-25Surface

culture Semisolidmedium3T3

Colony-feeder Medium Maximum forming effi-layer 199 3T3 CMa colony size ciency(%)+

+ +" ± 1<0.0001+

+ + + + + + 100 1+ + + ± ++ 4-1 5C 0.1+ + + ++" + + +d 40 0.02+ + + ± 15-40C 0.02+ + + -I- ++ 4-15° 0.2

a CM, conditioned medium.b Medium supplemented with EGF. 20 ng/ml.0 Varied in different experiments.

' E. Eberling, R. Kitchin. and J. Rheinwald, unpublished observations

Varied in different experiments.d Less than one-fifth as many colonies as in cultures grown with a 3T3 feeder

layer.

the injected cells grew out in each case.Dependence of SCC Cells on Fibroblast Feeder Support.

Each cell line was initiated from the tumor and serially passagedwith a 3T3 feeder layer in order to avoid selecting against cellspossessing the normal keratinocyte requirement for fibroblastsupport (19). At approximately the 5th, 10th, and 15th passage, each line was subcultured into various media at clonaldensities (i.e., 50 to 150 cells/sq cm) in the absence of 3T3feeder cells to test dependence upon fibroblast feeder functions for clonal growth (Table 1). SCC-4, SCC-12, SCC-15,and SCC-25 could form progressively growing colonies from

single cells in regular medium, but the growth rates of all butSCC-25 were increased substantially if the medium had beenconditioned by 3T3 cells. SCC-9 grew very poorly in regular

medium but grew much better in medium conditioned by 3T3cells than did the normal keratinocyte strain N. SCC-13 dis

played as stringent a fibroblast feeder requirement as did strainN. Thus, SCC lines varied from having as complete a dependence on fibroblast support for clonal growth as have normaldiploid keratinocytes to having completely lost this requirement.

Growth in Semisolid Medium. The SCC cells were suspended in regular or 3T3-conditioned medium containing methylcellulose either with the usual Ca2+ concentration of 1.8 mw

or with the reduced concentration of 0.22 mM. With an efficiency of about 1%, SCC-4 cells formed colonies that grewprogressively over a 2- to 3-week period to a maximum size ofabout 100 cells (Table 1). SCC-9, SCC-15, and SCC-25 pro

duced only microcolonies, never greater than a size of 15 cells,that underwent no further growth after the first 4 to 7 days and

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J. G. Rheinwald and M. A. Beckett

began degenerating at this time. In some experiments, nogrowth was observed from plating 5 x 104 cells of SCC-9 or

SCC-15. SCC-12 and SCC-13 produced colonies with an effi

ciency of about 0.02% that grew to a maximum size of about40 cells, although most colonies stopped growing at a smallersize. Normal diploid epidermal keratinocytes did not divide atall in the same methylcellulose medium, while HeLa cellsformed colonies larger than 100 cells with an efficiency ofabout 2%.

DISCUSSION

Behavior of Many Cultured SCC's as Established Lines. Alarge proportion of human SCC's of the epidermis and of theoral cavity and pharynx (the "head and neck" clinical category)

could be cultured as established cell lines that expressed theirtumorigenicity in nude mice. Grown in primary culture with a3T3 feeder layer, the carcinoma cells possessed a characteristic abnormal morphology in colonies initiated from single cellsor from explanted tumor fragments. They were therefore easilydistinguished from the well-organized stratified colonies formed

by any normal keratinocytes that were present in the tumorbiopsy. Similar morphological differences between normal andneoplastic keratinocytes have been documented in previousstudies of SCC's of a number of tissue types in primary culture

(1, 25). Each of the 6 SCC lines described here retained itsunique aneuploid karyotype and particular distinctive morphological characteristics indefinitely from the first several passages through subsequent serial cultivation. Although this factdoes not constitute conclusive evidence, it is consistent withthe notion that these lines represent the major stem populationsof their respective tumors instead of being founded by rarevariants that arise in culture and are selected for during serialcultivation. Thus, the 3T3 feeder layer, a permissive culturesystem for normal keratinocytes, also appears to support thegrowth of the malignant stem cells of SCC without imposingselection pressure to favor fitter variants having propertiesdifferent from those of the original tumor cell population.

Nevertheless, our success rate for culturing SCC's as estab

lished lines varied substantially among the 3 anatomical locations studied (epidermis, tongue, and pharynx), in spite of thefact that keratinocyte colonies of normal morphology grew inthe primary cultures of all tumors. We are continuing to placenew tumors in culture to determine whether this pattern issustained. We do not know why some of the biopsies thatcontained tumor tissue yielded no cells of abnormal appearance in culture. The tumor stem population may have been toosmall in those cases, and the physical association with a largenumber of normal keratinocytes in culture may have inhibitedthe growth or suppressed the abnormal morphology of the SCCcells. Abnormal SCC-like colonies were detected in the primary

cultures of 2 of the 4 pharyngeal tumors and one of the 3epidermal tumors that still senesced after several passages.The abnormal colonies disappeared from these populationsduring serial cultivation at least as rapidly as did the colonieswith normal morphology. Limited culture life span appears tobe the rule rather than the exception for most human solidtumor types (5, 9, 11). The 7 of 13 tumors in our study that didnot yield established lines senesced before a sufficient numberof cells could be obtained to permit comparison of their properties with those of the 6 immortal tumor cell populations

described here.Tumorigenicity in Nude Mice. When injected s.c. into nude

mice, 3 of the lines formed progressively growing tumors withhistology characteristic of well-differentiated SCC (i.e., small

nests of cells with central cell enlargement and differentiationintermixed with a network of host connective tissue). The other3 lines tended to form only one or 2 large, progressivelygrowing, differentiating squamous cysts. There were no apparent correlations between the histological organization of thenude mouse tumor formed by an SCC line and either thedegree of squamous differentiation in the original biopsy fromwhich the line was derived or the growth requirements of theline in culture. It is possible that the SCC lines vary in theirability to induce a stromal proliferative response in the host tothe extent that some fail to become well infiltrated with dermalconnective tissue.

Growth Requirements of SCC Cells in Culture. Cell lineshave been isolated without fibroblast feeder layer support fromhuman (9) and mouse (32) SCC's and after in vivo transfor

mation of primary mouse epidermal cultures (3, 6, 8, 23). Thus,malignant transformation of the keratinocyte can be accompanied by loss of a stringent requirement for fibroblast support.However, the culture systems used in the previous studiescould not have supported the growth of a class of carcinomacells that retained normal keratinocyte growth requirements.Not all established mouse epidermal lines selected for independence from fibroblasts are tumorigenic (3), and many thatare give rise to anaplastic tumors rather than differentiatingSCC's (3, 23). This indicates that loss of the fibroblast feeder

requirement is not sufficient to permit malignant growth by akeratinocyte and that selection for this phenotype may yieldatypical neoplastic variants.

By initially culturing carcinoma cell populations under non-selective conditions, we isolated SCC lines that proved to varygreatly in their requirements for fibroblast feeder support fromcomplete dependence (e.g., SCC-13) to complete independence (e.g., SCC-25). SCC-13 and SCC-9 would probably not

have been isolated under standard culture conditions. Evensome lines that undoubtedly could have been isolated in theabsence of fibroblast feeder support (e.g., SCC-4 and SCC-15) nevertheless are stimulated substantially by 3T3 conditioned medium or a 3T3 feeder layer (Table 1). It is likely thatserial cultivation of such lines in the absence of fibroblastsupport would have caused these populations to become enriched in more rapidly growing variants, and the lines wouldhave eventually evolved indifference to fibroblast feeding.Change in this direction does not seem to have occurred inthese lines over the course of at least 200 cell generations ofcultivation with a 3T3 feeder layer. SV40-infected human diploid keratinocyte populations are transformed into longer-livedlines that gradually evolve to independence from fibroblastsupport (26), but the culture conditions used may have selectedfor rare calls with that property.

It is important to note also that serial cultivation of SCC cellswith the 3T3 feeder layer did not seem to impose selectionpressure in the direction of increased fibroblast dependence;early-passage SCC-4 and SCC-25 cells did not grow better

when plated in the absence of feeder cells than they did aftermore than 200 cell generations of continuous culture with thefeeder layer. It is therefore unlikely that feeder-independent

cells were initially important constituents of the tumors that

1660 CANCER RESEARCH VOL. 41

Cell Lines from Human SCC

gave rise to SCC-13 and SCC-9 but that they were selected

against in culture in favor of cells that were more dependent onthe feeder layer.

Of the 6 SCC lines studied here, only one (SCC-4) formed

large progressively growing colonies in semisolid medium. Theother lines were able to undergo at most 2 to 5 divisions at avery low frequency. Because normal diploid keratinocytes areunable to divide even once in suspension, the ability of all theSCC lines to undergo at least some abortive growth is asignificant deviation from normal keratinocyte behavior, implying a somewhat reduced requirement for anchorage. However,the low frequency and small size of the colonies produced bymost of the SCC lines and their tendency to degenerate quicklyprobably would have precluded the use of semisolid mediumto select for the malignant stem cells in primary cultures of theoriginal tumors. A similar conclusion was reached by Rupniakand Hill (21), who recently reported that none of 17 primarylesions of SCC of the head and neck yielded colonies fromsingle cells in primary soft-agar suspension cultures. However,the presence in the disaggregated biopsies of viable tumorcells with colony-forming ability in surface cultures was not

determined in that study.As reported elsewhere (16), the SCC lines other than SCC-

4 retain the keratinocyte property of being triggered to terminally differentiate by anchorage deprivation (10, 20), althoughthey become committed to differentiate (with the concomitantloss of division potential) at a slower rate than do normalkeratinocytes. This suggests that SCC cells may have a somewhat reduced requirement for anchorage but that suspensionof the cells in semisolid medium is an inadequate method fordetecting this attribute, because the cells differentiate terminally before much, if any, growth can occur.

Our data indicate that many human SCC's behave like estab

lished lines in culture and express their tumorigenic potentialin nude mice. However, they can also retain the normal keratinocyte requirements for factors produced by fibroblasts andfor anchorage in order to grow in culture. Therefore, the in vitrogrowth and selection conditions that have been demonstratedto be useful for some human tumor types and for experimentalfibroblast transformation are not appropriate for human SCC.

ACKNOWLEDGMENTS

We are grateful to Drs. T. Ervin. D. Miller, and M. Meshad of the Sidney FarberCancer Institute and to Drs. J. Corson, J. Mulliken, and J. Murray of Peter BentBrigham Hospital for supplying tumor specimens. We thank Dr. T-T. Sun forproviding anti-keratin antiserum and for advice on the technique of indirect¡mmunofluorescence. We acknowledge the skillful technical assistance of E.Eberling and M. Wilde and thank L. Dillon for preparing the manuscript.

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J. G. Rheinwald and M. A. Beckett

Fig. 1. Human squamous cell carcinoma in culture and in vivo, a, colony of normal human epidermal keratinocyte strain N growing with a 3T3 feeder layer andEGF. All the cells are tightly adherent to one another and to the culture dish. The colony is stratified with an upper layer of very flattened cells the boundaries of whichare in a different focal plane and are therefore indistinct. Normal oral epithelial keratinocytes form colonies of identical morphology. Phase-contrast, x 160. b, colonyof human SCC line SCC-15 growing with a 3T3 feeder layer. Cells are less tightly adherent, often adopt a rounded morphology, and do not stratify. Occasionally largeend-stage cells are formed. Phase-contrast, x 160. c, fluorescence of a 3-cell colony of the SCC-4 line stained with rabbit anti-keratin antiserum and rhodamine-conjugated goat anti-rabbit IgG. The cells contain a network of keratin filament bundles in a pattern similar to that found in normal keratinocytes. Filament bundlesinsert perpendicularly into the desmosome attachment plaques that form at regions of cell contact. Apparent differences in keratin content among cells may be anillusion caused by differences in the degree of cell spreading, x 630. d. section of a portion of the human tumor biopsy from which SCC-9 was derived. This is atypical well-differentiated SCC, with nests of carcinoma cells becoming enlarged and showing signs of nuclear degeneration toward the centers of the nests. Theclusters of carcinoma cells are interspersed with lighter-staining connective tissue. H&E, x 80. e, section of the tumor formed in a nude mouse by cultured SCC-9cells. SCC-9 cells were serially cultured for about 70 cell generations and then injected s.c. into a nude mouse. A large well-differentiated SCC had formed 26 dayslater. H&E, x 80. f, section of the tumor formed in a nude mouse by cultured SCC-12 cells. SCC-12 cells were serially cultured for about 50 cell generations and theninjected s.c. into a nude mouse. The tumor consisted of 2 well-organized cysts with central differentiation in the form of multiple layers of flattened cells 37 days later.The centers of the cysts contained fluid. H&E, x 80.

1662 CANCER RESEARCH VOL. 41

Cell Lines from Human SCC

dFig. 2. Colony morphology of human SCC lines, a, SCC-4, 25th passage, x 200; b, SCC-25, 16th passage, x 200; c, SCC-13, 6th passage, x 200; d, portion

of large colony of SCC-9, 35th passage, showing several abnormally stratifying regions x 200.

MAY 1981 1663

1981;41:1657-1663. Cancer Res   James G. Rheinwald and Michael A. Beckett  CarcinomasFibroblast Support Cultured from Human Squamous Cell Tumorigenic Keratinocyte Lines Requiring Anchorage and

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