retinoic acid modulates the radiosensitivity of head-and-neck squamous carcinoma cells grown in...

PII S0360-3016(02)02865-1

BIOLOGY CONTRIBUTION

RETINOIC ACID MODULATES THE RADIOSENSITIVITY OF HEAD-AND-NECK SQUAMOUS CARCINOMA CELLS GROWN IN COLLAGEN GEL

LORENZO ROSSI, PH.D.,*†AND RENZO CORVO, M.D.‡

*Laboratory of Comparative Oncology and ‡Division of Radiotherapy–Istituto Nazionale per la Ricerca sul Cancro; and †Department ofOncology, Genetics, and Biology—University of Genoa, Genoa, Italy

Purpose: Collagen gels are increasingly regarded as reliable scaffolds for studying cells in vitro, displaying thesame three-dimensional network of collagen fibers as encountered in vivo. As a contribution to therapeuticcontrol of head-and-neck cancer, we grew HSCO86 cells in collagen gel and assessed their behavior in thepresence of retinoic acid (RA) and radiation.Methods and Materials: The malignant epithelial cell line HSCO86 was isolated from a postirradiation humanoropharyngeal squamous carcinoma; it was EGFR-negative by immunocytochemical criteria. The cells wereembedded in hydrated collagen I at a density of 106 cells/mL, and on Days 8, 10, and 12 of culture, they weretreated with 10�5 M retinoic acid. Radiation was administered using two different schedules: simultaneously withRA in three daily doses totaling 10 Gy, or with a single dose of 8 Gy on Day 29 of culture, after the effects of RAhad taken place. Cell proliferation was evaluated by the MTT assay, whereas morphometric characteristics weredetected in the cultured gels directly or in the gels after they were fixed and stained with hematoxylin.Results: Contrary to growth in monolayer, where HSCO86 cells displayed a high proliferation rate, in collagengel only a tiny fraction of the cells, usually less than 0.02%, survived the environmental stress; these cellsspontaneously organized themselves into clonal multicellular spheroids growing up to 0.8 mm in diameter. Afterexposure to 10�5 M retinoic acid, cell proliferation first declined and then, about 15 days after treatment, itstarted to increase to a level far above that in the control group. This surge in proliferation was ascribed to theappearance of numerous fibroblast-like cells at the edge of the spheroids. These cells, called HSCO-F, were theresult of epithelial-to-mesenchymal conversion. When the gels were disaggregated by collagenase, and the cellswere seeded in monolayer, HSCO-F cells reversed their morphology into parental HSCO86 cells. Treatment ofcollagen gels with 10 Gy, fractionated in three daily doses, did not substantially affect the growth of HSCO86spheroids. However, when radiation was given simultaneously with RA, cell growth was significantly inhibited,both in terms of cell proliferation and size of spheroids (p < 0.0001 vs. untreated controls). This synergismapplied mainly to parental HSCO86 cells, because no significant damage was induced by radiation on theHSCO-F cells previously generated by treatment with RA.Conclusion: Differences in the radiosensitivity of HSCO86 and HSCO-F cells are surprising in view of theircommon origin; this suggests a scenario in which, to overcome a microenvironmental stress, head-and-neckcarcinoma cells can temporarily shift from an epithelial to a mesenchymal phenotype. In particular, morphologicand functional data suggested that HSCO-F cells were transformed into vascular endothelial cells whosecharacteristics included the following: (1) distinctive expression of Factor VIII and �1-integrin, not detected inparental HSCO86 cells; (2) active migration in the collagen network by extruded pseudopodia, frequentlyappearing as colonies of filamentous cells aligned along the radial axis of the spheroids; and (3) efficientcontraction of floating collagen gels. The implication of our study is that head-and-neck carcinomas may respondto RA treatment by selecting cell populations both resistant to radiation and capable of migrating inside theconnective tissue, mimicking the behavior of vascular capillaries. © 2002 Elsevier Science Inc.

Collagen gel, Head-and-neck squamous cell carcinoma, Multicellular spheroids, Retinoic acid–induced radiationeffects, Epithelial-mesenchymal transition.

INTRODUCTION

Despite attempts at early diagnosis and treatment, head-and-neck squamous carcinomas continue to resist efforts to

defeat their malignant behavior. Surgery and radiotherapyare the current treatments for controlling local disease.Chemotherapy and/or radiosensitizer agents are commonlyadded to the radiotherapy regime in an attempt to increase

Reprint requests to: Lorenzo Rossi, Ph.D., Laboratory of Com-parative Oncology, Istituto Nazionale per la Ricerca sul Cancro,Largo Rosanna Benzi, 10-16132 Genoa, Italy. Tel: ��39 0105600200; Fax: ��39 010 5600208; E-mail: [email protected]

This work was supported in part by the Department of Oncology,Genetics, and Biology of the University of Genoa, Genoa, Italy.

Acknowledgments—We are grateful to Mrs. Anna Calabrese, Mrs.Giorgia Podesta, and Mr. Daniele Reverberi for technical assis-tance.

Received Nov 26, 2001, and in revised form Apr 1, 2002.Accepted for publication Apr 9, 2002.

Int. J. Radiation Oncology Biol. Phys., Vol. 53, No. 5, pp. 1319–1327, 2002Copyright © 2002 Elsevier Science Inc.Printed in the USA. All rights reserved

0360-3016/02/$–see front matter

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radiation efficacy and reduce systemic tumor dissemination(1). In general, however, the outcome is rather poor, and therisk of second primary tumors and mortality remains com-paratively high (2–4). A major problem with radiotherapy isthat a subset of malignant cells usually survives treatment,and after a lag period of 10 to 15 months, these cells start togrow again and spread, resulting in micrometastatic colo-nies (5, 6). One expedient, deemed to enhance therapeuticeffects and lower radiation doses, is modifying the behaviorof the malignant cells through induction of differentiation,with the assumption that fully differentiated carcinoma cellsare more sensitive to radiation effects than poorly differen-tiated cells are. This is largely conjectural, but the strategybased on biologic response modifiers to reverse carcinogen-esis is behind treatments associating the use of retinoidswith the chemoprevention of head-and-neck carcinomas(7–13).

Since the report by Bell et al. in 1979 (14), collagen gelshave found wide application in biology as a privileged invitro scaffold for growing a variety of cell types, essentiallyreproducing the three-dimensional (3D) environment foundin vivo. Perhaps the most remarkable property of this model,as compared to monolayer, is that collagen fibers, in their3D spatial arrangement, constrain cell behavior by limitingand directing cellular metabolic activities. Cell proliferationis usually impaired, and cells are forced to take a moreorganized morphology, frequently mimicking that of theparental tissue (15). Head-and-neck carcinomas are reportedto adapt well to growth in the collagen gel system, some-times preserving stromal and epithelial elements (16–18).

Studies using monolayer cultures have determined thatretinoic acid (RA), often in combination with interferon,sensitizes head-and-neck carcinoma cells to radiation treat-ment (19–21). To date, however, there are no reports deal-ing with this effect in collagen gel. Therefore, we assessedthe behavior of human oropharyngeal squamous carcinomacells in solidified collagen gels, alone and under treatmentwith RA and radiation. We confirmed not only RA’s role asa radiosensitizer, but also RA’s induction of a previouslyunnoticed phenotype, namely a reversible epithelial-to-mes-enchymal transition. Unlike the parental cells, these mesen-chymally transformed cells proved resistant to radiationexposure.

METHODS AND MATERIALS

Cell culturesThe human carcinoma cell line HSCO86, isolated from a

postirradiation squamous carcinoma of the oropharynx, re-tained epithelial morphology after more than 15 passages.Cells were maintained in RPMI-1640 (EuroClone) supple-mented with 10% fetal bovine serum (EuroClone). Themedium was enriched with 0.5% gentamycin and 2% L-glutamine (EuroClone), and cells were incubated at 37°C ina humidified incubator (5% CO2, 95% air). Confluent cul-tures were harvested with trypsin-EDTA (EuroClone) and

then counted and centrifuged before the cells were mixedwith collagen.

Collagen gel preparationThe stock solution was prepared by dissolving 100 mg

acid-soluble Type I collagen from calf skin (ICN Biomedi-cals) in 40 mL sterile 0.1% acetic acid in distilled water.The final ready-to-use collagen solution was obtained from1 volume of a 2:1 mixture of 10� DMEM and 0.34 MNaOH mixed with 4 volumes of the stock solution. Thissolution, the complete collagen mixture serving as the three-dimensional substrate for cell growth, will not gel if kept inice. To study the effects of hyaluronic acid (HA) (Sigma),we prepared a stock solution of the polymer at a concen-tration of 5 mg/mL in phosphate-buffered saline (PBS), and0.2 mL of this solution was added to 0.8 mL collagen. Afterharvest, HSCO86 cells were resuspended in culture me-dium, counted, and adjusted to the chosen experimental cellnumber. The pellets were resuspended at 4°C in 1 mL of thecomplete collagen solution. A volume of 50 �L of this cellsuspension was dropped into each well of a six-well plate(Corning) and allowed to solidify in the incubator. After 1 h,4 mL of medium was added per well. To obtain floatinggels, anchored gels were suspended after 24 h with the aidof a sterile cell scraper. Media were changed three times aweek, and cultures were maintained for periods up to 2months.

Treatment with retinoic acidThe stock solution was prepared by dissolving All-Trans

Retinoic Acid (Sigma) in dimethylsulfoxide (DMSO) at aconcentration of 10�2 M. All the manipulations and treat-ments with this compound were done in the dark. After apreliminary study showing that, in terms of cell prolifera-tion, RA was mildly effective even at the high concentrationof 10�5 M, we selected this concentration to maximize theradiosensitivity effect. As a general strategy, and to ensurereproducibility of results, the gels were treated after theappearance of the first spheroids, which usually occurred onDays 5–7 of culture. Treatment with RA was repeated threetimes, on Days 8, 10, and 12 of culture.

IrradiationCell-containing gels were irradiated with 6-MV photons

using a linear accelerator (Varian, Palo Alto, CA). Irradia-tion was delivered with the gantry set at 180° and a dosedmax using a source-to-axis distance of 100 cm. Irradiationoccurred at a dose rate of 2.5 Gy/min in a flask placed on topof 1.5 cm of Plexiglas. This arrangement was used toprevent underdosing of cells as a result of the physical“buildup dose” effect. The gels were kept at room temper-ature for less than 5 min during the irradiation period. Therewere two treatment groups. In the first group, radiation wasgiven contemporaneously with RA, in that a total of 10 Gywas fractionated in three consecutive daily doses of 3.33 Gy onDays 9, 10, and 11 of culture. In the second group, the gels,previously treated with retinoic acid as reported above, were

1320 I. J. Radiation Oncology ● Biology ● Physics Volume 53, Number 5, 2002

irradiated with a single dose of 8 Gy on Day 29 of culture. Thegels assigned to control groups were exposed to the samemanipulations, except that they were not irradiated.

MTT assayTo measure cell proliferation in collagen gels, we used

the colorimetric method based on the 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay(Sigma). Briefly, 1 mg MTT (200 �L of a stock solution inPBS) was added to each tube containing a single gel in 1 mLserum-free medium, and the tubes were incubated at 37°Cfor 4 h. The medium was then removed by centrifugation,and 1 mL of collagenase (200 U/mL in serum-free M199)(Sigma) was added to each tube to allow digestion for 1 h at37°C. The collagen gel was then dissociated by vortexing.After being rinsed with PBS, the digested samples werecentrifuged at 5140 rpm for 15 min. The supernatants werediscarded, the pellets were dissolved in DMSO, and thesolutions were read in a Beckman DU-70 spectrophotome-ter at 570 nm. A standard curve was prepared using a knownconcentration of cells before each experiment. The produc-tion of formazan crystals, and therefore the intensity ofcolor after their dissolution, is proportional to the number ofviable cells.

Histology and immunohistochemistrySample gels were taken at random from the several

experimental groups and, after being fixed in formalin, wereinserted into an agarose support to keep track of their tinysize. Afterward, we processed them by following standardprocedures and staining them with hematoxylin and eosin.Selected histologic sections, as well as cell cultures usingLaboratory Tek 2-chamber slides (Nalge Nunc) fixed with3.5% paraformaldehyde in PBS, were immunostained usingthe avidin-biotin complex amplification system and anti-mouse and anti-rabbit secondary antibodies (Dako). Thefollowing primary antibodies were used: monoclonal anti-vimentin, anti–smooth muscle actin, and polyclonal anti–Factor VIII (Dako); monoclonal anti–pan-cytokeratins andpolyclonal anti–pan-catherin and anti-laminin (Sigma);monoclonal anti-EGFR, polyclonal anti–FGF-2, and anti-VEGF (Santa Cruz); and monoclonal anti–�1-integrin(Chemicon International).

Morphometric parameters and statistical analysisSpheroids were counted under an inverted microscope

and measured using the IMAGE analysis software, set tocalculate the projected areas in �2. A Prism 3 version servedas the basis for the statistical evaluation of results. Valuesare given as means � SEM. There were three to four gelsper group, each gel was cultured in a separate well, and allexperiments were repeated at least twice.

RESULTS

The behavior of HSCO86 cells in collagen gel differedstrikingly from behavior in monolayer. In monolayer, the

cells displayed a typical polarized, cuboidal morphologyand usually reached confluence after 3 to 4 days of culture(Fig. 1A). In collagen gel, they became rounded whileremaining quiescent, even at the relatively high density of2 � 106 cells/mL. Then, after a lag period of 4 to 5 days, afew of them, no more than 0.02%, started to proliferate,originating solid clonal spheroids up to 0.8 mm in diameter(Fig. 1B). Fully grown spheroids were made up of a centralnecrotic area and an external sheet, several cells thick,tightly arranged in palisades (Fig. 1C). In the presence ofHA, cell proliferation, as well as the number of spheroids,increased greatly compared to controls grown in collagenalone (p � 0.0001), although cell size and morphology didnot change (Table 1).

Effects of retinoic acidIn repeated experiments, it was found that the maximum

number of HSCO86 cells retrieved from the several RA-treated groups was comparable, around 2.5 � 106 cells/mL,regardless of the addition of HA to the collagen (Fig. 2).The effect of RA on the kinetics of the proliferation ofHSCO86 cells was biphasic (Fig. 3). Soon after exposure toRA (See “Methods and Materials”), cell proliferation wasinhibited, and on Day 18 of culture, the surviving fractionwas approximately 50% below that of the control group.However, after the cells were in culture 25 days, this effectstarted to reverse itself, and a significant increase in cellproliferation was achieved by Day 36 of culture (p � 0.0001vs. untreated control) (Fig. 3). This unexpected improve-ment in cell survival was the result of the appearance offibroblast-like cells on the edge of the spheroids, starting5–7 days after treatment with RA (Figs. 1D and 1E). Ap-parently, these cells, named HSCO-F, had undergone epi-thelial-to-mesenchymal transition. Their transitional naturewas confirmed when they were disaggregated from thecollagen gels by collagenase and cultured in monolayer.Here, after two to three passages, virtually all cells revertedinto the typical epithelial HSCO86 morphology (notshown). The few remaining HSCO-F cells had large, firmlyattached cytoplasms and did not proliferate anymore. Itshould be stressed, however, that in collagen gel, suchreversion did not occur, at least not during an observationperiod of up to 50 days. Instead, HSCO-F cells continued toproliferate, becoming filamentous and lining up radially tothe spheroids (Fig. 1F).

Approximately 70% of the spheroids displayed this phe-notype. The immunohistochemical profiles of HSCO86 andHSCO-F cells are summarized in Table 2. Both cells werenegative to vimentin, FGF-2, and EGFR and positive tolaminin and VEGF. Differences were seen with respect tostaining with other antibodies: HSCO86 cells stained posi-tive with pan-cytokeratin and pan-cadherin antibodies andnegative with Factor VIII and �1-integrin antibodies,whereas the reverse was true for HSCO-F cells (Table 2).Despite a certain ambiguity in the interpretation of thesefindings, two other functional assays were strongly sugges-tive of the identification of HSCO-F cells with mesenchy-

1321Radiosensitivity of oropharyngeal squamous carcinoma cells in collagen gel ● L. ROSSI AND R. CORVO

mal cells. The first is that they migrated actively on theplastic surface surrounding anchored collagen gels. Forexample, when HSCO-F–containing gels were disaggre-gated, and the cells were embedded in fresh collagen gels,migration increased with time, and on Day 8 of culture,

there was an average of 45 HSCO-F cells/gel scattered onthe plastic surface surrounding the edge of the gels, com-pared to none in the gels containing untreated parentalHSCO86 cells (Fig. 4A). The second assay concerns thewell-known capability of fibroblasts in contracting floating

Fig. 1. Effects of culture conditions on the behavior of HSCO86 cells. (A) Morphologic organization in monolayer: Thepolarized epithelial architecture of the cells is apparent; (B) Sharp, multicellular spheroids derived from the spontaneousorganization of the cells in collagen gel; (C) Histologically, the spheroids were made up of an extended central necroticarea surrounded by a multistratified sheet of epithelial cells; (D) General view of a spheroid after it was exposed to 10�5

M retinoic acid. Densely packed filamentous buds are distributed all around the spheroid’s surface; (E) A highermagnification of the filamentous cells; (F) After being stained with hematoxylin, filaments showed up as bipolar cellsaligned next to each other and radial to the spheroid. Photographs A, B, D, and E were taken directly in vivo, and C andF were taken after staining with H&H and hematoxylin, respectively. Actual magnifications: B and D, 30�; A and E,150�; C, 190�; and F, 290�.


collagen gels. Indeed, in this model system, HSCO-F cellscontracted the gels much faster than the HSCO86 cells did.This is shown in Fig. 4B, where it is indicated that by Day6 in culture, the average diameter of the HSCO-F–contain-ing gels was down 50% compared to the initial diameter,whereas the diameters of the HSCO86-containing gels weredown only 10%.

Response to ionizing radiationThe effects of radiation on HSCO86 cell proliferation and

radiation’s possible synergism with retinoic acid wereprobed with the MTT assay performed on Day 24 of culture;the results are summarized in Fig. 5. In control groups, theaverage cellular density was 1.3 � 106 cells/mL in collagenalone and 3.1 � 106 cells/mL in collagen with HA. Irradi-ation with 10 Gy fractionated in three daily doses did notaffect the growth of HSCO86 cells, which remained at thesame level as that found in controls, irrespective of stromalcomposition. On the other hand, in response to treatmentwith RA, the proliferation of HSCO86 cells stabilized at a

density of roughly 2.5 � 106 cells/mL, no matter what thecomposition of the stroma and, therefore, the rate of cellproliferation in control groups (Fig. 5). In any case, theconcomitant exposure to radiation and RA caused a syner-gistic inhibition of HSCO86 cell proliferation, resulting in acell density averaging 0.8 � 106 cells/mL and 1.3 � 106

cells/mL in the groups without and with HA, respectively(in both cases p � 0.0001 vs. controls) (Fig. 5). The en-hanced radiosensitivity induced by RA on HSCO86 cellswas further demonstrated by the reduction in the projectedarea of HSCO86 spheroids. As shown in Fig. 6, spheroids inthe control group continued to enlarge during the observa-tion period of 21 days in culture. At this time, their averagearea was 2 � 105 �2, compared to 1.2 � 105, 1.9 � 105, and0.5 � 105 �2 in RA-, RAD-, and RA�RAD–treated groups,respectively. Therefore, the combined action of RA andradiation most effectively inhibited the growth of HSCOspheroids, even when compared with the group with RAalone (p � 0.0001 vs. controls). In contrast, the number ofspheroids somewhat declined with time compared to thecontrol, although the trend was not significantly different inthe several treatment groups (data not shown).

When the gels were treated with RA as usual, and were

Table 1. Growth characteristics of HSCO86 cells in collagen gel with and without the addition of hyaluronic acid*

Stromal supportMTT (number of cells/mL

� 106)Size of the spheroids(area in �2 � 104)

Number ofspheroids/gel

Collagen 4.35 � 0.14† 27.57 � 3.81 5.00 � 0.71Collagen � hyaluronic acid 7.31 � 0.29‡ 21.07 � 1.46§ 43.0 � 2.92‡

* The density of the collagen was 2.5 mg/mL. Hyaluronic acid was mixed with collagen at the concentration of 1 mg/mL. Cells wereseeded at the density of 1 � 106 cells/mL. The evaluation was made on Day 12 of culture.

† Values are given as means � SD of 4 to 6 gels per group.‡ p � 0.0001 compared to control cells in collagen alone.§ Not significant.

Fig. 2. Influence of HA on the growth of HSCO86 cells in collagengel. HA was mixed with the collagen at a concentration of 1mg/mL, and cells were embedded in this mixture at a density of106 cells/mL. On Days 8, 10, and 12 of culture, the gels weretreated with 10�5 M retinoic acid. Cell survival was evaluated onDay 25 of culture by the MTT assay. Values are means � SEM(bars) of triplicate incubations.

Fig. 3. Kinetics of proliferation of untreated and RA-treatedHSCO86 cells embedded in anchored collagen gels at a density of106 cells/mL (MTT assay). RA was administered at a concentra-tion of 10�5 M on Days 8, 10, and 12 of culture. Values aremeans � SEM (bars) of triplicate incubations.


then irradiated with 8 Gy on Day 29 of culture, a time whenHSCO-F cells were already numerous, the outcomechanged radically in that the combined action of the twoagents did not affect cell proliferation. The results are re-ported in Fig. 7, which shows cell proliferation as evaluatedon Day 36 of culture by the MTT assay. There was anaverage of 9 � 106 cells/mL in the untreated control and12 � 106 cells/mL and 7 � 106 cells/mL in the RA- andradiation-treated groups, respectively. By comparison, therestill were 8 � 106 cells/mL in the group receiving thecombined treatments, demonstrating that, in this instance,the two agents had no synergistic effects. Morphologicobservations confirmed that irradiation damaged mostly theparental HSCO86 cells, whereas HSCO-F cells were stillalive and growing at the time of MTT assay (not shown).

DISCUSSION

In this study it was shown that, unlike growth in mono-layer, where the cells displayed high vitality and continuousproliferation, HSCO86 cells in collagen gels rarely outlivedforced growth; even at a density as high as 2 � 106 cells/mL, well over 99% of them died out soon after seeding, andsurvival of the remaining few was bound to their capabilityto multiply and spontaneously organize into multicellularspheroids. By unanimous consensus, it was decided thatmulticellular spheroids are the structures in vitro mostclosely resembling the multicellular spatial organization invivo (22). Indeed, as demonstrated by the clonal reorgani-zation attained in our approach, growth in collagen gel

seems to offer novel opportunities to characterize head-and-neck carcinomas, here exemplified by HSCO86 cells, apostirradiation-derived oropharyngeal squamous carcinomacell line. At the same time, the clonal organization high-lights a common property of the collagen gel system,namely, its capability of constraining cell proliferation andmorphology, mimicking the homeostatic events occurringin vivo, where physiologic conditions ensure the regulatedbehavior of cells, specified by organ- and tissue-dependentfactors.

An interesting finding of our study was the loss of epi-thelial morphology of the HSCO86 cells lining the bordersurface of the spheroids treated with RA, and their breakinginto spindle-shaped and fibroblast-like cells. The emergenceof these cells just at the interface with the collagen fibers,out of otherwise regular, compacted spheroids, brings tomind the cellular process known as epithelial-mesenchymaltransition (EMT), by which is meant the reversible conver-sion of epithelial cells into mesenchyme-like cells (23, 24).This transitory phenotypic change has been recognized asan important feature of morphogenetic processes and tissueremodeling during embryogenesis and as an early effectorof metastasis, particularly prominent at the invasive front oftumors (25, 26). The behavior of the mesenchymal cellsderived in the present approach, called HSCO-F cells, re-sembled the behavior of dermal fibroblasts in their capabil-ity to migrate from the 3D environment of the collagensupport into the surrounding plastic surface (27). Moreover,when embedded within floating collagen gels, HSCO-Fcells contracted the collagen fibers, exactly as expected

Table 2. Immunohistochemical characterization of HSCO86 squamous carcinoma cells and their mesenchymally derived HSCO-F cells

Cells Laminin Vimentin Pan-cytokeratin Pan-cadherinFactorVIII EGFR VEGF

Integrin�1 FGF-2

Smooth muscleactin

HSCO � � � � � � � � � �HSCO-F � � � � � � � � � �

Fig. 4. Comparative behavior of HSCO86 (filled squares) and HSCO-F (empty squares) cells embedded in (A) anchoredand (B) floating collagen gels at a density of 106 cells/mL. (A) Rate of cell migration on the plastic surface, and (B) rateof contraction of the floating collagen gels. In each case, values represent means � SEM (bars) of quadruplicateincubations.


from normal fibroblasts (28). No such behavior was dis-played by the parental HSCO86 cells that, by any account,were functionally and morphologically epithelial cells.

The mechanisms underlying these effects of RA onHSCO86 cells were only minimally addressed in thepresent approach by immunocytochemical techniques.However, the factors determining the appearance of EMTcells have been examined in several models (29 –31).Perhaps the model best fitting our data is that stressingthe role of the cadherin family of proteins as determi-nants of EMT cell transition. Maintenance of epithelialmorphology seems to require especially the integrity ofadherens junctions, of which E- and N-cadherins are theprincipal components (32). Loss or inappropriate expres-sion of E- or N-cadherin may induce epithelial cells toacquire mesenchymal characteristics, and, on the oppo-site end, exogenous expression of E-cadherin in cells

with a mesenchymal appearance may cause them to un-dergo a morphologic transition into epithelia (33–36).RA has been found to interfere with the cadherin– cateninsignaling pathway, resulting in the disturbance and reor-ganization of the cytoskeletal proteins involved in theemergence of fibroblast-like phenotypes (37, 38). Thefinding that immunostaining with poly cadherin was pos-itive in HSCO86 cells and negative in HSCO-F cellssupports this mechanism.

Considering the peculiarity of the response of HSCO86cells to RA, we speculate that the emergence of HSCO-Fcells mimicked specialized mesenchymal cells, possiblyvascular endothelial cells, of the kind found in the granula-tion tissue during wound healing (39–40). Circumstantialevidences for this hypothesis, supported also by recent dataindicating that head-and-neck carcinoma cells express an-giogenic factors (41), are as follows: (1) HSCO-F cellslacked the intermediate filament vimentin and FGF-2, butexpressed the endothelial growth factor VEGF and distinc-tively expressed Factor VIII and particularly �1-integrin,confirming previous reports on the capability of RA toinduce this family of proteins in vascular cells (42); (2)HSCO-F cells dissociated from the spheroids and activelyinfiltrated the collagen. Morphologically, this process wascharacterized by extruded filopodia at the distal tip of thecytoplasm, and it was likely dependent on the expression of�1-integrin; (3) While infiltrating the collagen fibers,HSCO-F cells stretched and elongated radially to the sphe-roids, and were frequently aligned next to each other infilamentous elements; (4) Although definitively measurable,the performance of HSCO-F cells in terms of cell migrationand gel contraction was quantitatively weaker than expectedif compared with normal connective tissue fibroblasts, aneffect tentatively blamed on vimentin deficiency. This pos-sibility is supported by recent data showing that fibroblastsfrom mice deficient in vimentin were severely disabled in

Fig. 5. Synergistic effects of RA and radiation on the proliferationof HSCO86 cells. Cells were embedded in anchored collagen gels,with and without HA, at a density of 106 cells/mL. The gels weretreated with 10�5 M retinoic acid on Days 8, 10, and 12 of culture,and on Days 9, 10, and 11, they were irradiated with 3.33 Gy. MTTassay performed on Day 24 of culture. Values are means � SEM(bars) of triplicate incubations.

Fig. 6. Projected area in �2 of the same HSCO86 spheroids asdetected in the treatment groups in Fig. 5. Approximately 10spheroids/gel were measured at random using the NCI IMAGEprogram. Values are means � SEM (bars) of quadruplicate incu-bations.

Fig. 7. Effects of irradiation on HSCO86 cells previously exposedto RA. Cells were embedded in anchored collagen gels at a densityof 106 cells/mL. On Days 8, 10, and 12 of culture, they weretreated with 10�5 M retinoic acid, and on Day 29, they wereirradiated with a single dose of 8 Gy. MTT assay performed onDay 36 of culture. Values are means � SEM (bars) of triplicateincubations.


their capacity to migrate and to contract a 3D collagennetwork, and, as a result, these animals had impaired heal-ing (43). Consistent with reports stressing two of its appar-ently unrelated properties, namely antiangiogenic activityand the capability to accelerate wound healing (44), ourapproach indicates that RA may have played the role of awound-healing modulator, in that it induced HSCO86 cellsto turn into mesenchymal cells capable of mechanicallycontracting collagen fibers. Contrary to monolayer, whereHSCO-F cells fully reversed to parental cells, inside the 3Denvironment of the collagen gel, HSCO-F cells apparentlystabilized their phenotype, because no reversion to HSCO86cells was seen after up to 2 months in culture. By this strictcriterion, HSCO-F cells should be regarded as terminallydifferentiated cells. However, more investigation is neededto better define the parameters driving the transformation ofHSCO86 cells into HSCO-F cells, especially because theprocess was not accompanied by the expression of FGF-2,a marker of mesenchymal cells.

Retinoic acid combined with radiation treatments resultedin the synergistic inhibition of HSCO86 cells grown incollagen gel, measured in terms of cell survival and ofmorphometric parameters. The capability of RA to increasethe radiosensitivity of head-and-neck carcinoma cells hasbeen emphasized, and our results confirm and extend pub-lished reports (21). In addition, however, we dealt with the

new finding that RA promoted epithelial-to-mesenchymaltransformation, so that at any given time after RA treatment,the system was compounded by the simultaneous presenceof epithelial and mesenchymal cells in the same gel. Wasthere any cell-selective toxicity of radiation in this complexenvironment? According to our data, HSCO-F cells contin-ued to proliferate, even after exposure to radiation, at a timewhen large tears of necrosis infiltrated HSCO86 spheroids.This may indicate that HSCO86 cells, by converting to amesenchymal phenotype, escaped damage from radiationeffects, an inference consistent with reports that fibroblastsderived from squamous cell carcinomas are more radiore-sistant than the epithelial component (45). If transferred invivo, these results would suggest a biphasic sensitivity ofepithelial carcinoma cells to radiation. Inside the primarytumor, they would display high radiosensitivity. However,during metastatic migration to locoregional and distantsites, these same tumor cells, now turned into mesenchy-mal-like cells, would exert a strong resistance to radiationeffects, showing up undisturbed at their destination, wherethey would eventually resume epithelial morphology. Inaddition to the known effects of radiation on solid tumors,the observation that a head-and-neck malignant epithelialcell line can be transformed into radioresistant cells by RA,a powerful morphogen, offers new insight into ways tocontrol head-and-neck carcinomas.

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