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

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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] The in Vitro Interaction of Intraepithelial Neoplasia, Normal Epithelium, and Fibroblasts from the Adult Human Uterine Cervix1 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 37°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 AUGUST 1966 1641 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1966 American Association for Cancer Research. George D. Wübanks and Ralph M. Richart 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 37°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
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