The in Vitro Interaction of Intraepithelial Neoplasia, Normal Epithelium, and Fibroblasts from the Adult Human Uterine Cervix1

(CANCER RESEARCH 26 Part 1, 1641-1647, August 1966]

GEORGE D. WILBANKS- AND RALPH M. RICHART

Departments of Pathology and Obstetrics and Gynecology, College of Physicians and Surgeom, Columbia University, and the Obstetrical and Gynecological Service (The Sloane Hospital) of the Presbyterian Hospital, New York, New York

Summary in vitro phase-contrast time-lapse cinematographic observations of the interaction of the earliest morphologically recognizable The interaction of tissue-cultured cells derived from normal forms of neoplasia in the human uterine cervix (dysplasia and squamous epithelium, normal connective tissue, and intra- carcinoma-in-situ) with the normal tissues that usually surround epithelial neoplasia of the adult human uterine cervix was studied them, and the interaction between normal cervical epithelial and using phase-contrast time-lapse cinematography. The fibroblasts connective tissue cells. exhibited typical, passive, contact inhibition to other fibroblasts but not to the normal epithelial cells or cells from intraepithelial neoplasia. The normal epithelial cells grew as an adherent sheet Materials and Methods and exhibited an active type of contact inhibition with other nor All cells used in this study were derived from expiants of adult mal cells but no contact inhibition in association with fibroblasts human cervix. The full details of the technic used have been or neoplastic cells. The cells derived from intraepithelial neoplasia previously reported (14, 15). The essentials of the technic for the behaved in a manner intermediate between those derived from growth of squamous epithelium consist of delineating the areas normal epithelium and those derived from invasive carcinoma. In of normal epithelium, dysplasia, or carcinoma-in-situ in vivo these cells there was a striking lack of adhesiveness and an using a colpomicroscope (16) and the Toluidine blue staining absence of contact inhibition, but no actual invasion of normal reaction (13). A biopsy site is chosen to lie totally within the area epithelium or fibroblasts was observed, and the cells remained of neoplasia or normal epithelium, a punch biopsy is performed, essentially as a monolayer. It was suggested that the term "contact inhibition" not be used alone in describing these and Yi of the biopsy is fixed for histologie examination. The epithelim of the remaining half is separated from the stroma phenomena but be modified according to whether it is of the under a dissecting microscope, finely minced, and placed in fibroblastic or passive type or the active, epithelial type. culture under perforated cellophane. The fibroblast cultures are prepared by cutting away the epithelium and immediately Introduction subjacent stroma under a dissecting microscope and mincing The extensive literature on the surface properties and inter only the remaining connective tissue. All cells were grown on glass at 37Â°Cusing Eagle's minimal actions of normal and malignant cells was recently reviewed by Abercrombie and Ambrose (1). Previous studies of cell-cell essential media with 15% fetal calf serum as the fluid phase and interrelationships have examined the interaction of similar cells, 5% CO2 and air as the gas phase. The cells from the cervical cells from different species, cells from different stages of develop intraepithelial neoplasia (dysplasia and carcinoma-in-situ) and ment, or those not normally found in apposition. Studies of the the fibroblasts were transferred periodically using trypsin- in vitro interaction between 2 types of adult human tissues Versene and a rubber policeman. The cinematographic observa normally in apposition are meager. Cells from various human tions were made following 2 or more successive transfers. The and animal invasive cancers, both spontaneous and induced, original expiant cultures of normal epithelium were used for have been studied alone and in combination with other tissues cinematography because the success of subculturing these cells in vitro (I, 12, 17,18, 21), but no studies involving cells from pre- was unpredictable. invasive neoplasms have been published. This paper reports For time-lapse cinematography, the concentration of the cell suspensions obtained at transfer was adjusted to approximately 25,000 cells/ml. The suspensions of the 2 cell types to be studied 1This investigation was supported by USPHS Research Grant No. CA-07060-02,USPHS Fellowship No. l-F3-CA-23,432-01, and were placed as large drops in separately marked areas of a special Career Award No. 7-K3-CA 13,975-03from the National Cancer culture chamber (14) and coalescence prevented until cell Institute. attachment had occurred. When the cells had attached to the 2USPHS Special Fellow. Present address: Department of glass, additional medium was added and changed 3 times per Obstetrics and Gynecology, Duke University Medical Center, week. In the studies involving normal epithelium the fibroblasts Durham, N. C. and neoplastic cells were plated onto the plate containing the Received for publication December 30, 1965. original monolayer, after removal of the cellophane. Tris-buffered

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MEM3 was utilized during the time-lapse cinematographic of fibroblasts derived from the connective tissue beneath areas of observations. The films were made at magnifications of 100- intraepithelial neoplasia and those beneath normal epithelium. 250 X and frame intervals of 20-60 sec using a Zeiss Plankton The cells derived from cervical intraepithelial neoplasia had a microscope with phase-contrast optics. The cultures were main distinctive morphologic appearance which varied from patient to tained at 37Â°Cduring photography. patient (15). These morphologic differences were due primarily to variations in cellular shape and in the degree of cell cohesion. The Results cells from dysplasia and carcinoma-in-situ were similar and had an epithelioid appearance. In contrast to the closely adherent The normal epithelial cells grew as a confluent sheet, each cell pattern of the sheets of normal epithelium, however, each cell border being intimately juxtaposed to the neighboring cells and functioned as an individual unit, and cytoplasmic membrane firmly adherent to them. There was little apparent independent activity was prominent, involving a large portion of the cellular movement of these cells, but the entire monolayer appeared to border. In contrast to movement in the fibroblast cultures, move as a unit. There was no visible cytoplasmic membrane cellular movement in CIN cultures was more random and was activity at the margins of the cells that lay within the confluent seldom in any single direction for a sustained period. Although sheet, but there was abundant, undulating, cytoplasmic activity cytoplasmic activity decreased when cell contacts were made and in the cells that formed the periphery of the sheet. In several cell-cell associations were formed, these associations were always instances a small number of cells became separated from the sparse and were easily separated when the cells moved away from primary sheet and these cells, which had not demonstrated one another. Permanent cell contacts were seldom made even in peripheral cytoplasmic activity while within the monolayer, crowded cultures, yet there was more cytoplasmic overlapping became as active as those at the margin of the monolayer. The than in the cultures of normal epithelial cells or fibroblasts cells in these separated clumps remained closely adherent, (Fig. 2). however, and in each instance, contact was reestablished with the In 3 separate experiments, the junction of a monolayer of major cell sheet, whereupon peripheral cytoplasmic activity normal epithelium with fibroblasts was studied. In each experi ceased, intimate cell-cell contacts were reformed and the prodigal ment the culture of normal epithelium was derived from a clump rejoined the main epithelial sheet (Fig. 6). different individual. One patient's fibroblasts were plated onto 2 In the normal cell cultures areas developed in which the nuclei cultures of normal epithelium and those from another individual became smaller, motion of the cytoplasmic organelles ceased, and onto the 3rd. mitosis no longer occurred. These cells bore a resemblance to the In each of these junction experiments, the cytoplasmic activity superficial cells of the normal cervical epithelium, and were interpreted as having "differentiated" in vitro. Such "differ at the edge of the epithelial sheet appeared unaltered as the 2 cell entiated" cells remained within the monolayer in the areas of types approached each other. Similarly, the behavior of the fibroblasts also appeared unaltered until actual contact with the actively dividing cells, but no longer influenced the cytoplasmic epithelium occurred. At the time of contact the fibroblasts activity of neighboring cells. The active cells adjacent to those adhered momentarily to the undulating edge of the epithelial which had differentiated behaved like those at the periphery of sheet and, although some appeared to withdraw following the cell sheet and their cytoplasmic undulations overlapped the "differentiated" cells freely. contact, this was not a consistent response and the majority were pushed aside or detached from the glass by the edge of the The fibroblasts, in contrast to the normal squamous epithelium, advancing sheet. When the epithelial sheet made contact with a did not form a completely adherent cellular sheet nor were they crowded group of fibroblasts, the edge advanced at a slower rate observed to participate in mass movements as did the epithelial than when contact with individual fibroblasts was made, but the cells. They were larger, more elongated and angular than the same withdrawal and/or detachment of the fibroblasts occurred. squamous cells, and tended to move in 1 direction, preceded by There was no apparent change in the peripheral cytoplasmic an undulating membrane localized at 1 pole of the cell. While in activity of the epithelium. Gradually, as the epithelial sheet motion, portions of the cytoplasm of the fibroblasts often re advanced through the fibroblast cultures, rounded cells and mained adherent to the glass and stretched as a long projection debris accumulated at the advancing edge and a slight indenta at the tail of the cell. When the attachment to the glass was released, this projection abruptly "snapped" toward the leading tion developed in the epithelial sheet. In this area the epithelial monolayer appeared to be compressed (Fig. 5). portion of the cell. The junction of a monolayer derived from normal epithelium When 1 fibroblast made contact with the trailing portion of and CIN from the same patient were studied in 2 experiments, another, the 1st cell continued in the same direction after the and from different patients in 3 experiments. In each of these other fibroblast had moved on, frequently crossing over the studies, no changes in the activity of the undulating edge of the trailing cytoplasmic strands of the 2nd cell. Upon contact with epithelial sheet occurred as it approached the neoplastic cells nor the leading portion of another fibroblast, however, a mobile cell upon contact with the neoplastic cells. Similarly, there was no stopped momentarily and then moved in another direction. alteration in the random movements of the neoplastic cells as the Locomotion was strikingly decreased as the culture became normal sheet approached and the neoplastic cells made no with crowded, and in such cultures cellular overlapping was generally drawal response or change in movement patterns upon contact not observed (Fig. 1). No differences were noted between cultures with the normal monolayer. Although momentary contacts between the 2 cell types occurred, stable attachments were not 3The abbreviations used are: MEM, minimal essential media; noted and the neoplastic cells in both sparse and dense cultures CIN, cervical intraepithelial neoplasia. were pushed aside or detached by the advancing epithelial edge.

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As in the junction with fibroblasts, rounded cells and debris tion that differed from that seen in the fibroblast cultures. The collected at the edge of the epithelial sheet. In no instance was epithelial cells upon contact actively established intimate, lasting invasion of the epithelial sheet by the neoplastic cells observed. cell-to-cell adhesions accompanied by cessation of cell membrane No differences were noted in the behavior of the junction of cells activity, and no attempt at withdrawal after initial contact from the same individual or from different individuals (Fig. 4). occurred, nor did directional changes in movement occur. These The junction of neoplastic cells and fibroblasts from the same 2 types of cellular response to contact are strikingly different patient were studied in 2 experiments and from different patients qualitatively, and, in our opinion, both should not be termed in 5 experiments. No alterations in the activity and behavior of contact inhibition without further qualification. We propose the cells in approaching cultures were noted. The neoplastic cells instead that 2 types of contact inhibition be distinguishedâ€”the did not withdraw upon contact with the fibroblasts but continued fibroblastic (passive) type and the epithelial (active) type. to exhibit randomly oriented movement and the fibroblasts did This distinction will emphasize the strikingly different responses not form cellular associations or alter their pattern of motion of these 2 types of tissue to contact. upon contact with the neoplastic cells. Upon contact with a In addition to interacting with each other by alterations in neoplastic cell which interrupted the path of motion of a fibro- mobility and cytoplasmic membrane activity, cells interact with blast, the fibroblast detoured around it and continued moving their substrate by forming contacts of variatile strengths. Coman as before; in no instance did a fibroblast completely overlap a (6) and Berwick and Coman (4) measured the adhesion between neoplastic cell. Moving cells at the peripheral junction of the neo cells and between cells and their substrate by determining the plastic cell and fibroblast cultures intermingled indiscriminately, force required to separate such adhesions and found a difference and the motion of both cell types gradually decreased as the between the 2. The tendency of the cell to remain attached to the cultures became crowded. In certain areas the neoplastic cells were substrate they termed "stickiness," and they differentiated this completely surrounded by fibroblasts, but no cellular adhesions property from the "adhesiveness" of intercellular contacts. between the 2 cell types occurred even under crowded conditions. These authors found that normal cells were extremely "adherent" Occasionally, a neoplastic cell was observed covering a portion of but were not very "sticky," whereas malignant cells were very the cytoplasm of a fibroblast but no actual invasion of a fibroblast "sticky" but not very "adherent." Such alterations appear to culture by the CIN cells was observed. The behavior of the cells occur at a relatively early stage in neoplasia as suggested by the from the same patient and from different patients appeared to be decrease in adhesiveness noted within 1 hr after the exposure of identical (Fig. 3). the epidermis to carcinogen (5) and by the lack of contact inhibition early in certain viral infections in vitro (17, 19), al Discussion though the observation that the epithelium surrounding an experimentally-produced wound exhibits a decreased adhesive The interaction between cells of similar and dissimilar origin ness for several mm around the wound (11) suggests that this has been discussed in detail in recent reviews (1, 7, 9, 20, 21). change may occur in normal cells under certain circumstances. Cell movement in mammalian cells is accompanied by undulating In our studies the normal adult squamous epithelium was contacts between the cell membrane and the substrate surface (8). strikingly adhesive in vitro and exhibited contact inhibition of the These undulations involve the entire cell but appear to be epithelial type. Small groups of epithelial cells, when separated "controlled" by the leading undulating membrane of a single from the main cell sheet, readily reestablished intimate contact cell (1) or the leading undulating edge of a sheet of cells (1, 11). with the main masts of cells. The portions of the monolayer In tissue cultures of embryonic chick heart fibroblasts (2) and in which had "differentiated," and in which virtually all morpho urodele wound healing (11) it has been noted that when the logically evident cellular activity ceased, were fully overlapped undulating membrane of a motile fibroblast or epithelial sheet by the undulating cytoplasmic membrane of adjacent cells. meets another similar cell or sheet that the motion of the cells Although the fibroblasts were not adhesive, they exhibited in that direction of travel ceases, and, in the case of the fibro definite inhibition of the passive type. The cells from CIN were blasts, the direction of motion is altered and the cell moves much less adhesive than those derived from normal epithelium or off in another direction. When the cell becomes surrounded by connective tissue, and there appeared to be no contact inhibition other cells, active motion ceases. This phenomenon has been between individual neoplastic cells. termed "contact inhibition" (1) and appears to depend on direct Although the normal cells consistently exhibited contact contact and not to be mediated by a diffusible substance (2). inhibition to others of their type whether derived from the same Contact inhibition was originally defined following observa patient or from different patients, there was a total lack of re tions on embryonic chick heart fibroblasts (2) and later applied to sponse between fibroblasts and epithelium, whether derived from epithelial cells from in vivo wound healing studies (3, 11). It is the same or different patients or from normal or neoplastic apparent that the phenomenon of contact inhibition operates in epithelium. This lack of response might be anticipated because of adult human cervical fibroblasts in a manner similar to that the qualitative differences in contact inhibition in the fibroblastic observed in embryonic cells. In our studies normal adult cells and epithelial cells. It would be of interest to know if epithelial exhibited typical inhibition with temporary cessation of move cells from other organs react in a fashion similar to those of the ment of individual fibroblasts on contact, followed by a re- uterine cervix and whether epithelial cells of different origins will establishment of movement in another direction. Only when there respond to each other by the active type of contact inhibition. was no free area in which to move did the fibroblasts become In contrast to the normal fibroblasts and epithelial cells, the inhibited to further motion by contact. The normal epithelial cells derived from intraepithelial neoplasia did not exhibit cells in our system, however, exhibited a type of contact inhibi contact inhibition of any type. In this respect they resemble

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cells of invasive cancer (1). In contrast to invasive cancer (1, 10, 9. Eagle, H. Metabolic Controls in Cultured Mammalian Cells. 12, 22), however, the cells of CIN did not overlap each other or Science, 148; 42-51, 1965. the normal cells nor did they invade the normal epithelial sheets 10. Gey, G. O. Some Aspects of the Constitution and Behavior of or dense fibroblast cultures. In these respects they resemble Normal and Malignant Cells Maintained in Continuous Cul ture. In: Harvey Lectures, Ser. 50, 1954-55, pp. 154-229. New normal epithelium. Taken as a whole, these observations suggest that surface changes in the cells of CIN occupy an intermediate York: Academic Press, Inc., 1956. 11. Lash, J. W. Studies on Wound Closure in Urodele. Exptl. position between those of normal and invasive cancer cells. It is Zool., 128: 13-28, 1955. possible that the additional alterations in surface properties that 12. Leighton, J., Kalla, R. L., Klein, L, and Belkin, M. Patho- characterize invasive cancer are acquired at the time of the genesis of Tumor Invasion. I. Interaction between Normal transition from the intraepithelial to the invasive stage and that Tissues and Transformed Cells in Tissue Culture. Cancer the acquisition of these characteristics is an essential alteration Res., 19: 23-27, 1959. for invasion to occur. 13. Richart, R. M. A Clinical Staining Test for the in vivo De lineation of Dysplasia and Carcinoma-in-Situ. Am. J. Obstet. Acknowledgments Gynec., 86: 703-12, 1963. 14. â€” â€”. A Method for the Growth of "Pure" Cervical Epi The authors wish to acknowledge the excellent technical as thelium in Vitro. Ibid., 88: 710-14, 1964. sistance of Mr. G. A. Cottral and Miss Gerda Netta. 15. â€” â€”. The Growth Characteristics in Vitro of Normal Epi thelium, Dysplasia, and Carcinoma-in-Situ of the Uterine References Cervix. Cancer Res., 24: 662-69, 1964. 1. Ahercrombie, M., and Ambrose, E. J. The Surface Properties 16. . Colpomicroscopic Studies of Cervical Intra-epithelial of Cancer Cells. A Review. Cancer Res., 22: 525-48,1962. Neoplasia. Cancer, 19: 395-405, 1966. 2. Abercrombie, M., and Heaysman, J. E. M. Observations of 17. Sachs, L., and Medina, D. In Vitro Transformation of Normal the Social Behavior of Cells in Tissue Culture. II. Mono- Cells by Polyoma Virus. Nature, 189: 457-58. 1961. layering of Fibroblasts. Exptl. Cell Res., 6: 293-306, 1954. 18. Santesson, L. Characteristics of Epithelial Mouse Tumor 3. Arey, L. B. Wound Healing. Physiol. Rev., 16: 327-406, 1936. Cells in Vitro and Tumor Structures in Vivo. A Comparative 4. Berwick, L., and Coman, D. R. Some Chemical Factors in Study. Acta Path, et Microbiol. Scand., 24 (Suppl.): 1-237, Cellular Adhesions and Stickiness. Cancer Res., 82: 982-86, 1935. 1962. 19. Temin, H. M., and Rubin, H. Characteristics of an Assay for 5. Coman, D. R. Reduction in Cellular Adhesiveness upon Con Rous Sarcoma Virus and Rons Sarcoma in Tissue Culture. tact with a Carcinogen. Ibid., 20; 1202-04, 1960. Virology, 6: 669-88, 1958. 6. â€”.Adhesiveness and Stickiness: Two Independent Prop 20. Weiss, L. Cell Movement and Cell Surfaces. A Working Hy erties of the Cell Surface. Ibid., 21: 1436-38, 1961. pothesis. J. Theoret. Biol., 2: 236-50, 1962. 7. Curtis, A. S. G. Cell Contact and Adhesions. Biol. Rev., 37: 21. Weiss, P. Guiding Principles in Cell Locomotion and Cell Ag 82-129, 1962. gregation. Exptl. Cell Res., 8 (Suppl.): 260-81, 1961. 8. â€” â€”. The Mechanism of Adhesions of Cells to Glass. A 22. Wolff, E., and Schneider, N. La Culture d'un Sarcome de Study by Interference Reflection Microscopy. J. Cell Biol., Souris sur des Organes de Poulet explantes in Vitro. Arch. 20: 199-215, 1964. Anat. Microscop. Morphol. Exptl., 46: 173-97, 1957.

FIG. 1. Cervical fibroblast culture. Note 2 fibroblasts in center of photograph which are in contact end-to-side. Cytoplasmic activity at area of contact has ceased in Fig. lo, direction of motion of approaching cell is reversed and in succeeding frames this cell withdraws in direction opposite of its approach. Twenty-five min between photographs. (Phase-contrast. Enlarged from 16-mm movie film. Orig inal magnification, X 200.) FIG. 2. Culture of cervical intraepithelial neoplasia. Note relatively limited movement and relative lack of cell contacts. Note also that although cell contacts are made, they are not stable and are disrupted as cells move away. Fifty min between photomicrographs. (Phase-contrast. Enlarged from 16-mm movie film. Original magnification, X 200.) FIG. 3. Mixed culture of cervical fibroblasts and cervical intraepithelial cells abutting against it. Seventy-five min between photo micrographs. (Phase-contrast. Enlarged from 16-mm movie film. Original magnification, X 200.) FIG. 4. Culture of normal cervical epithelium in lower left corner of culture plate onto which cells of cervical epithelial neoplasia have been plated. Note lack of contact inhibition between these cell types. Whereas normal epithelial cells are in intimate contact, intraepithelial cells are not and their contact as in lower right corner of Fig. 4a does not affect cytoplasmic membrane activity of either. Note also that many of the separated intraepithelial cells are dislodged from glass by advancing confluent normal monolayer and be come compressed or piled up at its edge. Fifty min between frames. (Phase-contrast. Enlarged from 16-mm movie film. Original mag nification, X 200.) FIG. 5. Mixed culture of normal cervical epithelium and cervical fibrohlasts. Note absence of contact inhibition between these cell types. Particularly evident in areas of contact between normal cells on left and fibroblasts on right at middle edge of normal monolayer in Fig. 2a, 6. Note gradual compression and undermining of fibroblasts by normal sheet as monolayer advances across field. Twen ty-five min between photomicrographs. (Phase-contrast. Enlarged from Ki-mm movie film. Original magnification, X 200.) FIG. 6. Culture of normal cervical epithelium. A number of cells have separated from monolayer, a highly unusual occurrence. In lower center, 1 single cell touches normal clump, forms intimate contacts, and becomes incorporated into clump. Entire clump and most other cells later became incorporated into parent monolayer. Twenty-five min between frames. (Phase-contrast. Enlarged from 16-mm movie film. Original magnification, X 200.)

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1966 American Association for Cancer Research. The in Vitro Interaction of Intraepithelial Neoplasia, Normal Epithelium, and Fibroblasts from the Adult Human Uterine Cervix

George D. Wilbanks and Ralph M. Richart

Cancer Res 1966;26:1641-1647.

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