AmericanJournal ofPatholog, Vol. 137, No. 3, September 1990 Copyright © American Association ofPathologists

Differentiation Patterns in Two- and Threedimensional Culture Systems of Human Squamous Carcinoma Cell Lines Ruth Knuechel,*t Peter Keng,* Ferdinand Hofstaedter,t Virginia Langmuir, Robert M. Sutherland,t and David P. Penney* From the University ofRochester Cancer Center,* Rochester, New York; the Department ofPathology,t Universitjy ofRegensburg, Regensburg, Federal Republic of Germany; and the Life Sciences Division,* SRI International, Menlo Park, California

Relative quantification of the pattern of differentiation of two squamous carcinoma cell lines of the female genital tract, A431 and CaSki, was studied in various experimental tissue culture states that are frequently used to evaluate drug and radiation effects on human tumors. Two- and three-dimensional in vitro cultures, ie, monolayers and multicellular tumor spheroids (MCTS), and nude micexenograft tumors as in vivo tumor models were compared. In addition, epidermal growth factor (EGF) was used comparatively in the in vitro studies. Morphologic signs of epithelial differentiation could be recognized in both cell lines gradually increasingfrom monolayers to MCTS to xenograft tumors. Cytokeratin (CK) expression is described as stable in A431 cells. Using immunohistochemistry, however, partial masking of CK antigens was found when applying the antibody 8.12 on monolayer cells and could be quantified byflow cytometric measurements. Fundamental cellular changes were found in a CaSki xenograft tumor, which showed newly establishedfeatures ofa keratinizing carcinoma after late onset of tumor growth. Epidermal growth factor caused reduction of both intercellular contacts and later onset of necrosis in MCTS, leading to an increased viability of the spheroids. Significant differences in differentiation of the tumor model systems indicates that the characterization of differentiation with immunohistochemistry andflow cytometry is necessary to assist interpretation of data obtained with these different tumor models. (Am JPathol 1990, 13 7:725- 736)

Tumor growth and differentiation depends on autoregulative tumor cell interaction as well as on tissue and host interactions. Using cloned tumor cell lines in vitro avoids the high complexity of tumor heterogeneity in vivo. However many epigenetic variations of tumor cells still can be seen that might be biologically significant. Cloned tumor cell lines are most commonly used as monolayer cultures, thereby minimizing cell-cell interactions and effects of cell location and cell shape that may occur in three-dimensional culture, as well as host influences on tumor cells. The advantage of a controlled environment in vitro is maintained when cells are grown as three-dimensional tumors or multicellular tumor spheroids (MCTS). Three-dimensional tumor cell interactions with consequent gradients of cell-cycle heterogeneity, proliferation, and metabolism provide a more complex, and more in vivo-like, tissue culture model.1' 2 In contrast, when tumor cells are grown in athymic nude mice, cells are exposed to stromal cells and some host immune cells. Cellular interactions in this in vivo system are more complex than in the in vitro systems described. Two squamous carcinoma cell lines of the female genital tract were grown in the different experimental systems. These cell lines, as well as many in vivo squamous carcinomas of the female genital tract, have high amounts of epidermal growth factor (EGF) receptors3-4 and this feature is suggested to be an important component of their malignant phenotype. In the present study, the effect of EGF on differentiation in the in vitro cultures also was examined. A panel of CKs, intermediate filaments of epithelial cells, has been identified and antibodies against CKs can be used as markers for differentiation. Pairs of CK subunits are specific for a given epithelial phenotype5 and the CK pattern of normal cells, as well as that of tumor cells, is known to be stable and helpful for characterizing the differentiation of epithelial tissue.6

Supported by NIH Grant CA37618-07, Boehringer Ingelheim Fund for Basic Research in Medicine, and NCI Grant CA-37618. Accepted for publication April 25, 1990. Address reprint requests to Ruth Knuechel, Institute of Pathology, Universitaets Strasse 31, G-8400 Regensburg, FRG.

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For the quantification of anti-CK antibodies, immunohistochemistry and flow cytometry were applied. These are techniques that gain increasing importance for complementary tumor diagnosis with a broad spectrum of markers, especially monoclonal antibodies.

Material and Methods The cell line CaSki, derived from a human epidermoid carcinoma of the cervix,7 and the cell line A431, derived from a human epidermoid carcinoma of the vulva,8 were used throughout these studies. The cell lines were obtained for the Cancer Center Laboratory in Rochester from the American Type Tissue Collection in 1983 (A431) and 1986 (CaSki) and were used over a restricted range of 10 passages to minimize cell changes resulting from longterm culture. CaSki cells were maintained in Rosewell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum, 5 mmol/l (millimolar) L-glutamine, and 100 lU/ml streptomycin/penicillin. A431 cells were maintained in Dulbecco's modified Eagle's medium (Gibco) with the same supplements as were added to RPMI medium. These media are called 'regular medium' in the following text. Monolayers were grown in T150 flasks (Corning, Corning, NY) and incubated at 37°C humidified atmosphere of 3% CO2 and 97% air (CaSki) or 5% CO2 and 95% air (A431). Monolayer cells were kept in exponential growth and split once or twice a week at a ratio of 1:3 to 1:5. Single-cell suspensions were obtained from monolayers by detaching cells with a mixture of 0.01% trypsin (Worthington Biochemical Corp., Freehold, NJ) and 0.016% ethylene diamine tetra acetic acid (EDTA) (Sigma Chemical Co., St. Louis, MO).

In Vitro Cultures Monolayers of CaSki and A431 cells were initiated using 2 x 106 exponentially growing cells per Ti 50 flask in 20 ml of regular medium. Cells were grown for 2 or 10 days, respectively, with replacement of medium every other day. Multicellular tumor spheroids were initiated from single-cell suspensions of exponentially growing cells (8 x 106 for A431 and 6 x 106 for CaSki) in 10-cm petri dishes (Nunc, Naperville, IL) on a layer of 2% Bacto Agar (Difco, Detroit, Ml) in 10 ml of regular medium or medium containing 10 ng/ml EGF (culture grade, Collaborative Research, Lexington, MA). After incubating those cells for 4 days at 370C in a humidified incubator, small MCTSs were formed and were separated from single cells by sedimentation in 50-ml centrifuge tubes (Corning) for 10 minutes. The separated MCTSs were transferred into suspension

spinner flasks (Bellco, Vineland, NJ) and stirred continuously on magnetic stirrer plates (Bellco) at a speed of 1 10 rpm. Large-volume flasks containing 250 to 350 MCTSs in 300 ml medium were used to maximize equilibration of the medium with the gaseous phase over it. Medium with and without additional EGF was replaced every other day, and flasks were gassed with 5% CO2 in air for 2 minutes before continuing incubation in a warm room at 37°C. Multicellular tumor spheroids were grown during a period of 14 days in regular medium and up to 30 days in medium supplemented with EGF. At days 14, 21, and 30, MCTS were harvested from the flasks, washed twice in phosphate-buffered saline (PBS) solution, and handled for further processing.

In Vivo Xenografts Athymic female nude mice (NCR-nu, Madison, WI) were used for injection of tumor cells at an age from 3 weeks to 2 months. A431 tumor cells were injected bilaterally as 1 X 107 cells in 0.1 ml of PBS subcutaneously into the lower flanks. CaSki cells were injected at a concentration range of from 1 to 3 X 107 cells in 0.1 ml of PBS subcutaneously into the lower flanks, axilla region, or hind legs.

Tissue Processing and Analytical Methodology Histochemistry and Transmission Electron Microscopy Monolayers and MCTSs of CaSki and A431 cells were prepared in the same way before fixation for light microscopy and transmission electron microscopy (TEM). Fresh specimens were rinsed three times in 0.1 mol/l (molar) cacodylate buffer, pH 7.2, to remove excess media. Specimens for light microscopy were fixed for 24 hours in 2.5% glutaraldehyde in cacodylate buffer, pH 7.2, at 4°C. Then specimens were rinsed 2 times in buffer, dehydrated in increasing concentrations of ethanol, and infiltrated with and embedded in Immunobed (Polysciences, Warrington, PA). Embedded specimens were allowed to polymerize at room temperature for 1 to 8 hours. Sections measuring 1.5- to 5-^,m thick were stained with methylene blue-azure 11 and observed with a light microscope. For TEM, fixation in 2.5% glutaraldehyde was for 1 hour, followed by rinsing overnight in buffer. Specimens were postfixed for 1.5 hours in 1% Os04 in cacodylate buffer, dehydrated in increasing concentrations of ethanol, and embedded in Spurr epoxy resin. Thick sections (1.0 ,um) were stained with methylene blue-azure 11 for histologic

Differentiation Patterns in 2D and 3D Culture Systems AJP September 1990, Vol.

Table 1.

Paniel ofAntibodiesAgainst Cytokeratins Used

Jbr Immniniiohistocbenistrjy ancd Flouw CJtometry Clone 8.60 8.12 CK5 4.62 KL1 *

Cytokeratin number*

Dilution in PBS

(1),10,11 13,16 18 19 6,10,11,12

1:10 1:10 1:10 1:5 1:5

Company Bioyedat Bioyedat Bioyedat Bioyedat AMACt

Numbers according to the catalogue of cytokeratins by Moll et al.9

t Bioyeda, Israel, distributed through Accurate Chemicals and Scien-

tific Inc., Westbury, NY. t AMAC, Inc., Westbrook, ME.

observation or orientation. Thin sections (40 to 80 nm) were stained with lead citrate and uranyl acetate and observed and photographed in a Zeiss 10A transmission electron microscope.

Immunohistochemistry For immunohistochemical staining of monolayers, cells were seeded as 2000 cells/chamber (8-chamber Lab-Tec slides, Nunc) and grown for 2 and 10 days. Monolayers subsequently were washed twice in PBS and used directly for staining or stored at -70°C. Multicellular tumor spheroids also were washed twice in PBS, mounted in a drop of Tissue Tec (Miles Inc., Elkhart, IA), and frozen in cold isopentane immersed in liquid nitrogen. After excision of nude mice tumors, fresh tumor material was frozen in the same way as the MCTSs. Frozen sections of both types of tumor tissue were transferred to Poly-L-lysine-coated slides and stored overnight at 40C before staining. Fresh or frozen monolayers or sections of frozen tissue, stored at 4°C overnight, were fixed in acetone (-20°C) and then stained with an indirect immunofluorescence technique. After rehydration of tissue in PBS following fixation, a panel of antibodies to different CKs (Table 1) was applied. Antibodies diluted in PBS were used at 50 ,ul/section, and sections were incubated for 45 minutes. After two rinses in PBS, incubation with the second antibody (FITC-conjugated rabbit anti-mouse IgG, Dakopatts) at a concentration of 1:10 in PBS followed for 30 minutes. Slides were washed twice in PBS and mounted in a mixture of glycerol:water, 9:1, containing para-phenylenediamine to avoid fading. Finally sections were sealed with nail polish surrounding glass cover slips.

Staining for Flow Cytometry Immunofluorescence staining and DNA staining for flow cytometry required single-cell suspensions. Monolayer cells were detached with a mixture of 0.01% trypsin and 0.016% EDTA, and enzyme action was stopped with medium containing 10% serum. Cells were spun down for 10 minutes at 400g and then washed twice in PBS.

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Multicellular tumor spheroids were dissociated in a mixture of 0.05% trypsin and 0.08% EDTA in PBS by placing them in donut dishes (Nunc) that were mechanically agitated for 10 minutes at 370C. Later MCTSs were broken up with a syringe to obtain a homogeneous single-cell suspension. Then trypsin action was stopped with medium containing 10% serum. Cells were spun down for 10 minutes at 400g and washed twice in PBS. Nude mice tumors were dissociated according to the method of Allalunis-Turner.10 Fresh tumors were minced thoroughly with surgical scissors on a watch glass and then transferred to 20 ml of the following enzyme cocktail: 0.025% collagenase (Sigma), 0.05% pronase (Calbiochem, Behring, La Jolla, CA), 0.4% DNAse in Hanks Buffered Salt Solution (Gibco). Incubation in this cocktail was carried out at 370C for 1 hour. Following incubation, the tumor suspensions were passed through 200-gauge metal meshes to remove tumor clumps. The cell suspensions were washed twice in PBS. Viable cells of all three types of tumor sources were counted with trypan blue exclusion using a hemocytometer. For further staining with anti-CK antibodies, cells were fixed in -20°C cold ethanol (70%) at a concentration of 1 X 107 cells in 3 ml ethanol and stored at 4°C for a minimum of 24 hours before staining. An indirect immunofluorescence method was used to stain single-cell suspensions for flow cytometry. Cell samples in ethanol were spun down for 10 minutes at 400g and consequently resuspended twice in PBS. Anti-CK antibodies were applied for 60 minutes in the same concentration as described for staining of sections (Table 1). Cells were washed twice in PBS containing 2% fetal calf serum (FCS) (PBS/FCS) and then incubated with the second antibody (FITC-conjugated rabbit anti-mouse IgG, Dakopatts) for 30 minutes at a concentration of 1:10 for all first antibodies except for KL1, which required a dilution of 1:5 for comparable fluorescence levels. Cells were washed twice in PBS/FCS and then incubated in 1 mg/ ml RNAse (1 ml/106 cells, Sigma) for 30 minutes at room temperature as the first step for DNA staining. Cells were spun down again and resuspended in 10 ,g/ml propidium iodide (1 mI/i 06 cells, Sigma). Before measurement on the flow cytometer, the cell suspensions were filtered through a 37-,um nylon mesh to exclude cell clumps.

Controls As controls for immunohistochemistry and flow cytometry, anti-CK antibody clones that are known to be negative in the cell lines were used: clones 4.62 and 8.60 for A431 cells11 and clone 8.60 for monolayer CaSki cells (nonkeratinizing cervical carcinoma).

Flow Cytometry A Coulter profile flow cytometer (Coulter Electronics, Hialeah, FL) was used for all measurements. Fluores-

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AJP September 1990, Vol. 1-3 7, No).

Figure 1. Morphologic differences in 8-day-old CaSki MCTS groun int regular medium (RM), diameter approximateli' 0.3 mm, anld in EGF-conitaintinig mediium (EM), dianmeter approximately 0.4 mm. a: RM, light microscopy (LM), methylene bluie-azure 11 (MBA),

original magnificationi X65. b: IM1, TETI, original magnificationz X600. c: EM, LM, MBA, originlal maglificationl X65. d: EM, TEM, original magnification X 3-300.

cence distributions of up to 20,000 cells/sample were analyzed using a 1 5-mW argon laser at 488 nm wavelength. Green fluorescence from fluorescein-isothio-cyanate (FITC) was collected in logarithmic scale as a measurement of the percentage of positivity of the CKs in the cell population. Red fluorescence from propidium iodide was collected as a measure of DNA content. Filters used were a 550-nm dichroic, 530-nm short pass for green fluorescence, and 590-nm long pass for red fluorescence.

Data Analysis Data files were transferred to disks after measurement on the flow cytometer. These data were analyzed on an IBM computer using the Epics Cytologic software, version 2.0. (Coulter Electronics). Fluorescence histograms were normalized to 10,000 cells. Control histograms were subtracted from positive histograms to define the percentage of positivity. For each data point, two to four separate experiments were used, and the mean as well as the standard error of the mean were calculated. Bitmaps were used in two-dimensional histograms showing red (DNA) fluorescence and green (antibody) fluorescence of samples from xenograft tumors. Host cells (bitmap 1) were separated from A431 tumor cells (bitmap 2), and the green fluorescence of bitmap 1 and

bitmap 2 were displayed separately as one-parameter histograms for further analysis.

Photo Documentation

Stained tissue sections were photographed using a Zeiss Photomicroscope (Carl Zeiss, Oberkochen, West Germany). For fluorescence photography, a Tri-X-pan film (400 ASA, Kodak, Rochester, NY) was used, and photos for regular light microscopy were taken with a Technical Pan Film (50 ASA, Kodak).

Results

Light Microscopy and Transmission Electron Microscopy Epidermal Growth Factor Effects As shown previously in this laboratory,12 MCTSs grown in regular medium (RM) differ from those grown in EGF-containing medium (EM). No significant difference could be found in the growth of monolayers, except for a rounding of cells in both cell lines grown in EM. However

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AIP September 1990, Vol. 1 3 7, No. 3

MCTSs grown in EM reached a higher average diameter and showed an increased stability of the viable rim. Morphologically the earliest difference was a rounded surface of cells of early MCTSs (4 to 9 days old) of both cell lines grown in EM (Figures 1c and d), in comparison with a majority of flattened surface cells in MCTSs grown in RM (Figures 1a and b). Light microscopy of 8-day-old MCTSs showed irregular single-cell necrosis in MCTSs grown in RM (Figure 1 b), in contrast to lack of necrosis and more frequent mitosis in the center of MCTSs grown in EM (Figure 1 d). Multicellular tumor spheroids could be grown for a longer time in EM. In these older MCTSs, surface cells were flattened and an increasing number of single cells were found in the surrounding medium in the spinner flasks. In older A431 MCTSs in EM (30 days), attenuated desmosomes were found within the viable rim; Caski MCTS of this age showed many true desmosomes. In old spheroids of both cell lines grown in EM, greater amounts of intercellular debris were present and autophagic vacuoles were numerous. However these spheroids also showed cells rich in polyribosomes, with only few other organelles. Multicellular tumor spheroids of both cell lines, grown in RM, began to break apart (A431, average diameter more than 0.4 cm; CaSki, average diameter more than 0.3 cm), and within this period of growth, mature epithelial contacts could not be found.

Morphology of A431 Cells Monolayers of A431 cells were composed of a mixture of cuboidal and spindle-shaped cells, forming nests of dense, overlapping cells in confluent cultures. Ultrastructural features included a rich cytoplasmic component of polyribosomes and sparse intermediate filaments. Many microvilli and cellular interdigitations could be seen in confluent monolayers; however no mature intercellular contacts such as desmosomes could be found. Desmosomelike structures (DLS) were rare and small. Multicellular tumor spheroids of A431 cells (0.2 to 0.6 mm) showed the morphology of an undifferentiated carcinoma with polymorphous cells and a high mitotic rate throughout the period of growth (Figure 2a). Ultrastructurally tightly packed cells in the center of young MCTSs showed many membrane folds. Desmosomelike structures and some attenuated desmosomes were found (Figure 2b). During the period of MCTS growth, central necrosis developed, and more DLSs were found in the surrounding viable rim of cells. No mature desmosomes could be detected. Intermediate filaments were more frequent than in monolayer cells. Cellular debris was found in intercellular spaces, and cells increasingly showed membrane-bound vesicles of various sizes: autophagic vacuoles and microvesicular bodies. Although such cells

were frequent in old MCTSs (more than 30 days), a number of cells rich in polyribosomes, but having only a few other organelles, could still be seen. These cells, as well

as mitotic cells, were distributed through the total thickness of the viable rim. The size of xenograft tumors was 0.9 to 1.2 cm after 2 weeks of growth. Tumors grown for 4 weeks to diameters up to 1.6 cm contained large amounts of necrosis. The pattern in light microscopy was still that of an undifferentiated carcinoma (Figure 2c): nests of tumor cells were intercepted by small strands of stromal fibroblasts and capillaries. Xenograft tumors older than 2 weeks contained significant amounts of necrosis, more intercellular junctions, DLSs, attenuated desmosomes, and many mature desmosomes between tumor cells (Figure 2d). To summarize ultrastructural findings of the cell line A431 in different states of growth, we diagnosed a poorly differentiated squamous cell carcinoma. Signs indicating squamous differentiation in monolayers were hard to find and were confined to intermediate filaments. In MCTSs, attenuated desmosomes provided a further marker of differentiation. The diagnosis of squamous carcinoma was certain in xenograft tumors, in which true desmosomes and thick bundles of intermediate filaments were numerous.

Morphology of CaSki Cells Monolayer cells grown exponentially and as confluent plateau-phase cells showed a cuboidal epithelial pattern. The ultrastructure of the cells showed many polyribosomes and thin bundles of intermediate filaments in the cytoplasm. No epithelial type intercellular junctions could be identified. Multicellular tumor spheroids of CaSki cells ranged from 0.2 mm (4 days) to 0.8 mm (about 25 days) in average diameter. Young MCTS showed tightly packed cells that were characterized by abundant polyribosomes and few organelles at the ultrastructural level. Central necrosis developed after 10 days and the viable rim of MCTS showed an increasing epithelial pattern with prickle cells and flattened surface cells (Figure 3a). Electron microscopy revealed many intercellular contacts, DLSs, as well as true desmosomes with tonofilaments radiating into the dense membranous plaques (Figures 3b and c). Intracellular keratin or keratin pearls could not be seen in MCTSs grown up to 30 days. A marked difference was found in CaSki xenografts. Only 1 of 30 tumor cell injections (axilla, 2 X 107/0.1 ml) resulted in tumor growth 5 months after injection. This tumor grew to a diameter of 1.0 cm after 3 weeks, when it was excised. Light microscopy showed a carcinoma with single-cell keratinization and keratin pearls (Figure 3d). Intermediate filaments were significantly more frequent than in the in vitro cultures, and true desmosomes connected the tumor cells, result-

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AlP September 1990, Vol. 13 7, No. 3

Figure 2. Signs ofsquamous differentiation in MCTS and xenograft tumors of A431 cells. a: MCTS grown in EMfor 30 days, diameter approximately 0.8 mm, showing an undiferentiated epithelial tumor, with central necrosis on the bottom of the picture and many mitoses in the viable rim (LM, MBA, original magnification X 175. b: MCTS grown in EMfor 30 days, showing an undifferetntiated epithelial tumor with intense intercellular membranefolditng (upper half of picture) and rare attenuated desmosomes (close to bottom of picture) (TEM, original magnification X 11, 050). c: A431 xenograft tumor at 4 weeks, with epithelial tumor nests and sparse intratumor stromal cords (LM, MBA, original magniicationi X 175). d: A431 xenograft tumors at 4 weeks, showing large mature desmosomes and increased and thick intermediate filamenzt bundles (TEM, original mag-

I ing in a prickle cell layer (Figure 3f). The finding of keratin pearls in light microscopy is supported by the ultrastructure of keratinized cells showing keratohyalin and partly pyknotic nuclei (Figure 3e). In summary, the CaSki cell line represents a poorly differentiated carcinoma in vitro. Signs indicating epithelial differentiation were more pronounced than in the different experimental growth states of A431 cells. The tumor, grown in vivo in nude mice, was diagnosed as a moderately differentiated keratinizing carcinoma.

Immunohistochemistry The reaction of A431 cells with different antibody clones against CKs is shown in Table 2. A431 cells showed a negative staining pattern with the antibody against CK 19

nification X23,400).

well as with the antibody against CKs 10 and 11 in monolayers, MCTS, and xenografts in nude mice. The polykeratin antibody KL1 stained all A431 cells, showing an increase in intensity from exponential to plateau monolayers (Figure 4a). A uniformly positive staining pattern of all cells could be seen in monolayers, MCTS, and xenografts with the antibody against CK 18. Finally, the antibody against CKs 13 and 16 did stain some of the A431 cells in monolayers (Figure 4b); however all cells in MCTS and xenograft tumors, as judged from a minimum of three sections per sample, did stain positively (Figures 4c and d). The reaction of CaSki cells with different antibody clones against CK is shown in Table 3. CaSki monolayers showed a positive staining pattern with the antibodies against CKs 18 and 19 as well as with the clone KL1 against a broad spectrum of CKs. The latter showed a as

Differentiation Patterns in 2D and 3D Culture Systems

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A/P September 1990, Vol. 13 7, No. .3

-~~~~~~~zf'

*

I*

..

I

.W

4dial-,...

idL

*L

9: A.,. ':

Au..

Figure 3. Signs ofsquamous di#ferentiation in MCTS and xenograft tumors of CaSki cells, a: MCTS grown for 21 das in EM, diameter approximately 0.8 mm, shouwinig epithelial pattern uith prickle cells (LM, MBA, X360). b, c: MCTS grown for 21 days in EM, showing large mnature desmosomes with typical tonofilaments radiating into dense membrane plaques (inset c) (TEM, b: X 17,800; c: X 55, 100). d: Xenograft tumor at 4 weeks with epithelial pattern, invasive growth (nerve and muscle on the left side of the picture), development of single-cell keratinization, and keratin pearls. (LM, MBA, X 175). e, f: Xenograft tumor at 4 weeks, showing true keratiniization with mature desmosomes (insetf) causing a prickle cell appearance (TEM, e: X3, 000;f X22,050).

732 Knuechel et al A/P Septemiber 1990, Vol. 137, No..3

Table 3. Distribution ojAntibodiesAgainst CGjtokeratins in CaSki Cells*

Table 2. Distribuitioi ofAnitibodies Againist Cj'tokeratins in A431 Cells.*

Clone

KL1 8.60 8.12 CK5 4.62

Against

Against cytokeratin

Monolayer

MCTSt

Xenograft

poly

++

++

++ ++ ++

10,11 13,16 18 19

++ ++

+ ++

-

cytokeratin

Monolayer

MCTSt

Xenograft

KL1

poly

++

++

8.60 8.12 CK5 4.62

10,11 13,16 18 19

+ +

+ +

+++ ++ + + +

Clone

*Semiquantitative evaluation of immunofluorescence staining. -, negative; +, weakly positive; ++, positive. t Multicellular tumor spheroid.

* Semiquantitative evaluation of immunofluorescence staining; negative; +, weakly positive; ++, positive; +++, strongly positive. t Multicellular tumor spheroid.

focally more intensive staining pattern in the xenograft tumor, which could not be seen in MCTSs or monolayers. Antibodies against CKs 10 and 1 1 as well as CKs 13 and 16 were negative in monolayers and MCTSs in CaSki cells (Figures 5a and b); however both stained positively in the xenograft tumor (Figures 5c and d). Both antibodies showed strong focal staining next to weakly positive cells and negatively stained cells. These strongly positive areas were found in proximity to stromal elements when staining was carried out with the antibody against CKs 13 and 16, and more in the center of tumor nodules when antibodies against CKs 10 and 1 1 were applied. Staining with the antibody against CKs 10 and 11 resulted in a more intense staining pattern than with the antibody against CKs 13 and 16.

(Table 4). As shown on sections, only some of the A431 cells were positive in monolayers stained with the antibody 8.12. A significant increase was seen from exponential monolayers (approximately 67%) to plateau monolayers (79%) to MCTSs (approximately 97%) (P < 0.01). Figure 6 demonstrates this increase in positivity (Figures 6b to d) and the reappearance of negative cells after replating of MCTS cells as monolayers (Figure 6e). It also shows that antigen expression was unrelated to the cellcycle state, which was confirmed by calculating the mean fluorescence within windows for separate cell-cycle phases (data not shown). In xenograft tumors in nude mice, tumor cells could be analyzed separately from host cells by using bitmaps for the different cell populations. Therefore the level of positivity of tumor cells could be defined and compared with that of the in vitro cultures (Table 4). Xenograft tumor cells of the A431 cell line did not show significant changes in CK pattern after 2 and 4 weeks of growth. The percentage of positive CaSki cells stained with anti-CK antibodies was also quantified by flow cytometry (Table 4). When antibodies CK5, 4.62, and KL1 were used, levels of positivity in monolayer and MCTSs were

Flow Cytometry Evaluation of indirect immunofluorescence staining by flow cytometry supported the data obtained by immunohistochemistry of sections. In addition this method provides a precise quantitation of positive and negative cells

Figure 4. Indirect immunolluorescence staining of cytokeratins in differenit experimenital states ofA431 cells (exposure tinie and printing conditions idenitical for all photographs). a: Confluent monolayer stainied uith clote KL1 showing weakly positive cells (upper left quadrant of picture) niext to stronglj positive cells (remainder of picture) (original magnificaX50). b: Similar confluent tioni stained uwith clotne 8.12 showinig negative cells (upper third ofpicture) and positii'e cells (remainder of picture) (original

monolayVer

I

magnfication X50). c: MCTSgrown in EM for 14 days, diameter approximately 0.5 mm, stained uwith clone 8.12 showing a ho:-

~

mogeneously, positive staininlg patterrn (originial magnificatiotn X50). d: Xenioinnssude mice stained with graft tumor clonie 8.12 showing a homogeneously positiie staining of tu mor cells as well as positive epidermal staining of n ude motuse epidermal cells (original magnification X20).

Differentiation Patterns in 2D and 3D Culture Systems 733 A/P September 1990, Vol 137, No. 3

Figure 5. Incdirect immunofluorescence staininlg of cytokeratins in different experimental states of CaSki cells (exposure time and printinlg coniditions idenitical in all photographs except a, in which exposure time was douibled to demonstrate conitours of negative cells. a: Confluent monolayer stainied with clonie 8.60 showinig a negative pattern (originial magnzifcation X-50). b: MCTS grown ]br 14 days in EM, diameter

approximately 0.6 mm, stainied with clonte 8. 12 shou'ing a negative staining pattern (original magniftcationi X50). c: Xenograft

g, 1-

~F

5.

i

tuimor stained with clone 8. 12 shouwing intenise focal staining close to stromal cells (originial magnificationt X20). d: Xenograft tuimor staitned with clone 8.60 shouwinlg intense staining in areas ofkeratinizationi (origitnal magnification X20).

constantly high. Antibodies 8.12 and 8.60 were negative in all in vitro cultures of CaSki cells.

Cells of MCTSs that

tion

studies'14

cells with

Discussion

were

dissociated still showed

un-

differentiated viable tumor cells in both cell lines. Prolifera-

a

confirmed that these

similar

were

cell-cycle distribution

to

mainly cycling

MCTS in earlier om MCTS ne-

stages of growth. Freyer, using extractdst crosis on monolayers, did not see a significant shift in cell-

Epidermal Growth Factor Effects Rounding of cells as seen in surface cells of MCTSs has been described previously for monolayer A431 cells and was interpreted as a consequence of tyrosine phosphorylation in certain cytoskeletal proteins.13 Morphologically, rounding was associated with formation of many microvilli and a decrease of intercellular contacts, resulting in broader intercellular spaces. Mitosis could be seen frequently in the center of MCTSs grown in EM, which is in accordance with our previous finding of high proliferative fractions in those MCTS as shown by application of proliferation-associated antigens.14 Necrosis in MCTS from both cell lines grown in EM occurred later than in MCTSs grown in regular medium; MCTSs grown in EM also reached a larger average diameter, a finding that is even more pronounced in A431 MCTSs when serum concentration is raised to 20%, as shown in the study of Ng et al.'5 The observation that necrosis is significant for MCTS growth limitation correlates with results of Freyer,'6 who demonstrated the direct growth-inhibiting effect of necrosis on MCTS diameter. Fifteen different normal and cancer cell lines were tested in that study, and MCTS volume at saturation varied by a factor of 67 under identical growth conditions. This variation in size at saturation of growth showed no correlation with the monolayer doubling time, clonogenic efficiency, or cell density per unit volume of MCTS for the different cell lines. Persistence of the viability and intactness of MCTSs grown in EM contrasted with the disaggregation of MCTSs grown in RM (A431 MCTS after 2 weeks, CaSki-MCTS after 3 weeks).

N 0 MROFP-

z

2 LU

~c

I-i2 n

LUz C.) 0 -i LL

e

FLUORESCENCE INTENSITY-DNA Figure 6. Three-dimensionial plots (X, DNA-fluorescence, lin-

ear scale; Y, FITC-Jluorescenice, logarithmic scale) of diferent in vitro culturestates ofA43I cell stained uith the anticytokeratiml clonie 8. 12 for cytokeratins 13 and 16. The height of the peak inidicates the number of cells. a: Monolayer (2 days); conItrol, anticytokeratin clonie 4.62. b: Monolayer (2 days); stained uwith anticytokeratin clone 8.12. c: Monolayer (10 days), stainced u'ith 8.12. d: MCTS (14 days), diameter approximatelj 0. 5 mm, stained uith 8.12. e: Monolayer (6 das) from replated suspensions ofjMCTS (14 dajs), stainied u'ith 8. 12.

734 Knuechel et al All' Septemnber 199(, Vol 137, No. 3

Table4. Percenztage of Positility in A431 and CaSki CellsforDiferenitAniticytokeratin Anitibodies, Ilcluding Stanidard Error of the Meani Antibody clone Kll 8.12 8.60 CK5 4.62 Sample 67.2 ± 1.9 95.9+ 2.2 94.1 + 1.9 A431 EML 92.0 + 8.7 79.0 ± 4.2 93.5 ± 0.7 A431 PML 96.6± 2.1 88.4 ± 1.7 97.6± 1.6 A431 MCTS 93.5+ 2.1 92.3i 1.8 95.7 ± 1.9 A431 Xeno 97.3 + 0.4 96.7 + 2.5 95.5 + 3.5 CaSki EML 97.3 ± 1.4 97.5 ± 0.7 96.5 ± 2.1 CaSki PML 93.5 + 1.9 93.0 + 2.8 96.6 ± 0.4 CaSki MCTS ND ND ND ND ND CaSki xeno EML, exponential monolayer; PML, plateau monolayer; xeno, xenograft tumor; ND, not done;-, negative.

cycle distribution pattern in cells, which showed overall growth inhibition due to the necrotic material. Finally, after long periods of growth in EM, increased signs of epithelial differentiation could be seen in MCTS: specifically, mature desmosomes and thicker intermediate filament bundles. Our hypothesis is that cell-cell interaction in the three-dimensional structure leads to reduced proliferation and enhanced production of extracellular matrix components and formation of desmosomes. Parallels are seen in the study of Rieber et al,17 showing increased amounts of desmosomes and dense intercellular contacts with the addition of EGF in A431 cells grown in collagen gels.

Morphologic Criteria for Epithelial Differentiation A431 cells were originally derived from an epidermoid carcinoma of the vulva.7 In the original paper, morphologic correspondence between the original tumor and tumors grown from cells of the established cell line in NIH-Swiss mice was reported. A spinous cell-like growth is described by most authors.9 In our experiments, A431 cells showed a mixture of cuboidal and more spindle-type cells, along with transitions between those two cell types. This pattern has been found to be stable in our laboratory for several years, and no selection of one cell type, by, for example, constant exposure to EGF, could be observed (Dr. T. Kwok, oral personal communication, July 20, 1989). Ultrastructurally, in A431 monolayer cells, we found polyribosome-rich cells with only a few organelles, ie, rough endoplasmic reticulum, mitochondria, and small Golgi complexes. This structure represents undifferentiated, rapidly proliferating cells and is confirmed by other ultrastructural descriptions of A431 cells.18 19 However, Wynford-Thomas et al18 describe tight junctions and desmosomes in confluent monolayer cultures, whereas our investigations and those of Boonstra19 only show intense membrane folding and lack of mature intercellular contacts. Attenuated desmosomes in A431 -MCTS were an

additional hint of epithelial differentiation not found in monolayers. Even more pronounced signs of squamous differentiation were found in xenograft tumors, which showed quantitative changes in intermediate filaments and larger and more mature forms of desmosomes. A similar gradual increase of morphologic signs of squamous differentiation was found in CaSki cells from monolayers to MCTSs to xenograft tumors. CaSki cells were derived from bowel metastasis of an epidermoid carcinoma of the cervix.8 The original report does not mention elements of keratinization, as were found in the xenograft tumor in our study. Conclusions cannot be drawn with any certainty because only one tumor was found; however a recent extended study of growth of tumors in nude mice provides a possible explanation for our observation.20 In this paper, reduced clonogenicity and lack of proliferation were found when tumors, which had shown late onset in mice, were transferred back into tissue culture. Adaptation to the host had changed the phenotype of these tumor cells. This change did not seem to be simply related to the residual immunity in nude mice because tumors with the longest delay in onset showed comparatively low numbers of infiltrating host cells. The one CaSki xenograft tumor that grew within the time of our experiments had a growth delay of 5 months, in contrast to less than 2 weeks for A431 cells. Infiltration by host cells was similar in the CaSki and A431 tumors.

Cytokeratins The CK pattern of the female genital tract can be divided into two main groups. The upper genital tract (oviduct, endometrium, endocervix) and the mesothelium are characterized by CKs of simple epithelia, ie, 7, 8, 18, and 19, whereas CKs of nonkeratinizing stratified epithelium are found in the lower genital tract. Tumors of the female genital tract correspondingly have two major groups of CK patterns: the pattern of ovarian and endometrial carcinoma, which is identical to those of normal cells, and the

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pattern of squamous carcinoma cells, which is highly complex. The nonkeratinizing squamous carcinoma of the cervix has been characterized as showing a complex pattern of CKs,6 including CKs 5, 6, 7, 8, 13, 14, 15, 17, 18, and 19. According to information in the literature, CaSki cells represent a nonkeratinizing squamous carcinoma, but no reports are available on the CK pattern of this cell line. Our results show positive staining for CKs 18 and 19, which are CKs of simple epithelial type.5 Cytokeratin 13 was negative in vitro (Figure 5b), although after a long growth delay period in nude mice, the CaSki xenograft tumor stained positively with antibodies against CKs 13, 10, and 1 1. This indicated mature keratinization and confirmed the ultrastructural findings. Cytokeratin 13 is a marker for stratified epithelium,5 also indicating higher differentiation. Because CKs are known to be a stable feature of a tissue type, this change is interpreted as a major difference in the phenotype of the tumor, as discussed above. The cell line A431 has a complex CK pattern that has been shown to be stable in tissue culture but that lacks correlation with identified patterns of specific normal cell types.9 A431 cells show CKs 5, 7, 8, 13, 15, 17, and 18, as well as trace amounts of CKs 6, 14, and 16, but lack CK 19. This pattern was shown by two-dimensional gelelectrophoresis techniques and is interpreted as indicating rapid proliferation as well as the presence of tissue type markers, which are maintained in culture.9 Immunohistochemical staining of A431 cells was consistent with electrophoretic results, showing positive staining for CKs 13, 16, and 18, and with the polykeratin-antibody KL1, and a negative reaction for CK 19. The distribution of the antibodies was identical in vitro and in vivo. However the level of positive staining with the antibody 8.12 varied in a reproducible way. A negative and a positive population of cells in monolayers was paralleled by a weakly positive and strongly positive staining using the antibody KL1 (Figures 4a and b). We interpreted this effect as dependent on the degree of cell-cell interaction because we saw an increase in positive cells from exponential monolayers to confluent monolayers to MCTSs. Also the effect was reversible: we observed recurrence of negative cells after replating (Figure 6). Various observations on rapid changes in the detection of CKs in a cell population can be found in the literature21-24 and can be explained by epitope masking. Masking often is related to different functional states of the cell, such as the cell-cycle state.21 Franke et al21 describe masking of the antigen to CK antibody 8.13 in interphase cells of the kangaroo rat cell line PtK2, in contrast to positive staining in other cell-cycle states. Comparing Figures 6b and c, a cell-cycle relation of the antibody masking is likely in A431 cells. While the exponential monolayer (Figure 6b) shows similar amounts of cells in various cell-cycle phases in positive and nega-

tive cells, the number of negative cells in plateau monolayers (Figure 6c) indicates relatively fewer cells in the Sand G2/M-phase of the cell cycle in the negative cell population than in the positive cells. Other functional relations observed in connection with masking are cell metabolism22 or cell differentiation.22'24 The CK-distribution patterns of neither cell line were altered by addition of EGF to the medium. Short-term studies on the influence of EGF on cytoskeletal structures showed no change in microtubules and intermediate filaments, but changes in the actin-associated microfilament system have been reported.25 Comparisons of the differentiation states of tissue cultures indicated differences due to growth in three-dimensional structure and further changes when the same cells were grown in nude mice. Epidermal growth factor affected in vitro three-dimensional tumor spheroids differently than monolayers. Initial reduction of intercellular contact caused by EGF coincided with a higher viability and larger average diameters of the spheroids, findings that may be more relevant to the in vivo situation. Changes in EGF-receptor phosphorylation and production of extracellular matrix components are under further investigation to understand the differences observed. Immunohistochemistry and flow cytometry provided information at the cell level to characterize differentiation states. Immunohistochemistry with a panel of anti-CK antibodies helped to relate the differentiative state of cells to features such as cell shape, cell density, or cell-cell contact, for instance, with stromal cells. The advantage of flow cytometry is the rapid quantification of a marker in many cells, allowing easy monitoring of cell populations. Flow cytometric measurements will find increasing application in tumor diagnosis because multiparameter analysis enables us to relate different markers to each other or to the DNA distribution of the cell population. Although tissue culture conditions were standardized, deviations and similarities were found when comparing our data with the literature. Results obtained under standardized conditions in our laboratory were constant and reproducible. The variation observed in the different experimental tissue culture states of cloned cell lines indicates that we are working with functionally different experimental tumors. Our results emphasize the need to define tissue differentiation to be able to evaluate influences on tumors of growth-inhibiting or growth-promoting agents, and to relate findings to in vivo cultures.

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Acknowledgments The authors thank Gertrude Nielsen, Kim Wiltse, Kathy Maltby, and Anne Paxhia for technical assistance. The electron microscopy was performed at the Ultrastructure Facility of Experimental Pathology, and the flow cytometric analysis was performed at the cell-sorting facility of the University of Rochester Cancer Center, supported by grant CA 11198.