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(CANCER RESEARCH 37. 3228-3237, September 1977] Light-Microscopic Morphology of Cell Types Cultured during Preneoplasia from Foreign Body-reactive Tissues and Films1 Kenneth H. Johnson, Lance C. Buoen, Inge Brand, and K. Gerhard Brand Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Minnesota, St. Paul 55108 ¡K.H. J.¡,and Department Medical School, University of Minnesota, Minneapolis, Minnesota 55455 [L. C. B., I. B., K. G. B.¡ SUMMARY Cells isolated in vitro from preneoplastic foreign body (FB)-reactive capsule tissue or surfaces of FB segments from mice were studied and found to conform to one of four cell-type categories on the basis of light-microscopic mor phology, pattern of in vitro appearance, in vitro topographi cal relationships, and certain karyotype similarities. Euploid type I (macrophage-like) and II (fibroblast-like) cells pre dominated in primary cultures and early passages (pas sages 1 and 2) of cells derived from FB-reactive capsule tissue. The observation of small numbers of type III cells (unidentified cell type with unknown karyotype characteris tics) in passages 1 and 2 of cells from FB-reactive capsule origin coincided with the deterioration of euploid type II cell populations and preceded the observation of type IV (endothelial-like) cells. Type IV cells had a pronounced growth advantage over cell types I, II, and III, resulting in cultures composed only of type IV cells after three passages. Cul tures derived from cells attached to the surfaces of FB segments also conformed to the criteria established for type IV cells. Of the four cell types identified in this study, type IV cells were determined to have special importance regarding the nature of the progenitor cell in FB tumorigenesis, in that they were aneuploid and eventually produced homologous sarcomas when injected as a suspension into compatible hybrid recipient mice. These findings are consistent with our earlier reported hypothesis implicating certain cells of the microvasculature as the likely progenitor cells from which FB sarcomas are derived. INTRODUCTION of Microbiology, and electron microscopy have elucidated morphological features which suggest that the progenitor cell is a multipotential mesenchymal cell type derived from the local micro vasculature (14). More recent investigations in our laboratory have concen trated on the in vitro isolation and study of FB-induced capsule- and/or film-attached cells from film-capsule com plexes removed at various stages postimplantation (8). The potential usefulness of these in vitro techniques for obtain ing further information regarding the nature of the progeni tor cell and various aspects of neoplastic transformation appears promising because of the preliminary findings that: (a) initial cultures consisting predominantly of euploid fibroblast- and macrophage-like cells were often gradually outgrown by morphologically distinct cells with specific aneuploid karyotypes which were identical with, or closely related to, tumors derived from corresponding portions of Tilm-capsule complex left in mice; (b) the cultured aneu ploid cells frequently gave rise to homologous sarcomas when implanted in hybrid recipient mice; and (c) the neo plastic cell determinants were stable in vitro as they were in vivo, but preneoplastic cell maturation was arrested during culture, regardless of the number of passages in vitro. The latter studies have thus demonstrated that preneoplastic cells from which sarcomas arise many months later can be isolated and expended in vitro. This provides the opportu nity to obtain preneoplastic cell preparations from FB-reac tive tissue and films for analytical studies at defined stages of preneoplastic maturation. The primary objective of this investigation was to charac terize and identify morphologically the various cell types isolated in vitro from preneoplastic FB-reactive tissues and films. The induction of sarcomas in mice by s.c. implantation of various FB2 materials has been used in our laboratory as a model to elucidate preneoplastic events concerning, espe cially, the origin and identification of the progenitor cells and the nature of neoplastic transformation. These studies have provided information regarding the appearance time and location of preneoplastic clones of cells in FB-reactive tissue and on implant surfaces (6, 7). Studies of FB-induced sarcomas utilizing special histological staining techniques 1 Supported by USPHS Grant CA 10712 from the National Cancer Institute. 2 The abbreviations used are: FB, foreign body; MGG. May-GrunwaldGiemsa. Received January 3. 1977; accepted June 13, 1977. 3228 MATERIALS AND METHODS Primary cultures or various passages of 5 different cell cultures isolated in vitro from preneoplastic capsules or implant surfaces were utilized for this light-microscopic study (Table 1). Five mice (CBA/J, CBA/H, and C57BL6/J x C57BL6/J-bgJbg' F, hybrid) were implanted s.c. in the left flank area with either single hydrophilic, 15- x 22-mm Millipore filters (0.025-/nm pore size), manufactured by Millipore Filter Corp., Bedford, Mass., or single 15- x 22- x 0.2-mm unplasticized vinyl chloride-vinyl acetate copolymer films (Busse, Great Neck, N. Y.). The basic procedures used for CANCER RESEARCH VOL. 37 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research. Cell Types Cultured in Preneoplastic FB Tumorigenesis Table 1 Procedural details and derivation of cell cultures used for in vitro light-microscopic studies Original donor mouse data Implant data Cell culture data Culture vivo identifica source of tionACulturepno.PrimaryPassage assage cellsCapsuleCapsule implantation time code (mos.)555 no.90229022 (mm)15 xO.215 x22 B 1 Passage 2 Passage 3 Passage 4 Passage 5In x22 Capsule VCVA 15 x 22 15 x22 Capsule VCVA 15 x 22 Capsule VCVA 15 x 22 CapsuleMaterialVCVA"VCVA VCVASize Passage 2 Film CDEFPrimaryPassage VCVA xO.2 xO.2 xO.2 x 0.2 xO.2Total 15 x 22 x 0.2 5 5 5Mouse strain(C57BL6/J x C57BL6/Jbgjbgj) normal F, hybrid Normal F, hybrid Normal F, hybrid 9022 M Normal F, hybrid 9022 M Normal F, hybrid 9022 M 9022SexMM MMouseNormal F, hybrid 9022 M (C57BL6/J x C57BL6/Jbg-'bg-') normal F, hybrid 2215 x 2Passage x2215 2Passage x2215 BA/ H x 22 x 0.21.51210e9097910090946129FFFMCBA/JCBA/JCBA/JC 6CapsuleCapsuleCapsuleFilmMF"MFMFVCVA15 " CVA, unplasticized vinyl chloride-vinyl acetate copolymer films. b MF, Millipore filter (0.025-/im pore size). r Film/capsule complex transferred in situ after 3 months implantation in Mouse 6129 to a CBA/H-T6 Mouse (6521); after 4 months in Mouse 6521, film and attached cells were transferred to a (C57BL10/ScSn x CBA/H-T6) F, hybrid mouse (6941) for an additional 3 months. Cultured cells (cell line F) were of original donor mouse genotype (6129). FB implantation are the same as those detailed in our earlier publications (5, 6). Preneoplastic FB segments or FB-reactive capsule tissue for culture were excised at 1, 1.5, 2, 5, and 10 months after implantation. Details of procedures used for surgical exci sion of FB segments or FB-reactive tissue for culture or transfer during preneoplasia are the same as those previ ously reported (5, 6). For cell culture (8), preneoplastic, FB-reactive capsule tissue was minced, treated for 30 min with 0.3% collagenase at 37°on a magnetic stirrer, and washed with balanced salt solution. These cells were seeded into 35-mm plastic cul ture dishes (with an 11- x 22-mm glass coverslip on the floor of each dish) containing McCoy's 5A modified medium with antibiotics and 20% newborn calf serum. Cell-laden film implants were placed directly into 35-mm culture dishes containing the culture medium. The cultures were passed routinely (after trypsinization) as soon as monolayers reached confluence (i.e., after approximately 4 to 6 days for capsule-derived primary cultures and 3 to 5 weeks for filmattached primary cultures). Each culture was expanded into 2 culture dishes. Glass coverslips (with attached cells) on the floors of the culture dishes were removed from the cultures for morphological and karyological studies when the cultures had reached monolayer confluence. The coverslip from 1 of the paired culture dishes was scored and divided so that one-third was placed in methanol fixative for subsequent staining with MGG for light microscopy, and two-thirds were placed in 3% buffered glutaraldehyde for electron microscopy. Cells attached to the coverslip in the 2nd culture dish were uti lized for karyological studies (5). SEPTEMBER 1977 RESULTS Cell Culture A (primary culture and passages 1 to 5), derived from preneoplastic capsule tissue enveloping a plastic film implanted for 5 months, was used as the proto type for identification and morphological characterization of FB-reactive cell types and cell populations present in vitro at various stages of culture (Table 1). Primary cultures or selected passages of 5 other cell cultures (Cultures B to F), derived either from FB-reactive capsule tissue or filmattached cells, were studied for comparison and confirma tion of observations made with Cell Culture A. Four morphologically distinguishable categories of cell types, arbitrarily identified by Roman numerals I to IV, were isolated in vitro from preneoplastic FB-reactive tissues or FB segments (Table 2). All cells identified and placed in a particular category had a number of common morphologi cal features which were demonstrably different from other categories of cells (Table 2). Morphology, Time of in Vitro Appearance, in Vitro Topo graphical Relationships, and Karyotype Characteristics of Cell Types Isolated from FB-reactive Tissues or Films Cell Type I: Macrophage-like Cells Morphology. Type I cells (Figs. 1 and 2) were predomi nantly round to spindle or fusiform in shape. Stellate (tripo lar) shapes were less commonly observed. The terminal extremities of relatively elongated bipolar type I cells tended to fan out, forming a fishtail appearance. The cell margins at these fanned-out extremities were indistinct. Type I cells had either 1 or 2 nuclei surrounded by a 3229 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research. K. H. Johnson et al. Table 2 Morphology, in vitro topographical relationships, culture from time FB-induced of in vitro preneoplastic appearance,capsules karyotype, andand filmstumorigenicity of cell types isolated in Time of in vitro appearance of cell population Morphological characteristics FB-revitro topo FB-immic mar graphical rela active cap ginsDistinctCytoplasmic modificationsMany tionshipsAttachedtissuePR KaryotypeNot plant surface sule (3-8)" basoto to observed philic cytospindleCytoplascally lo film or sur P-1 (0-3) plasmic cated ; face of type P-2 (0-3)From dense hetgranulesNucleiAsymmetri II cellsFrom erochromatinIn Cell Type1Cell shapeRound Irregularly stellate to fusiform Indistinct feathered edges None Smooth contour;. pink matrix; 1-8 chromacenters Overlapped cells and crisscross growth pattern III Elongated fusiform; few stellate Distinct Cytoplasmic blebs on surface of occasional cells Symmetrically lo cated; margination of heterochromatin IV Polygonal to stellate Relatively distinct Many cytoplasmic blebs on cells at in termediate stages of passage Many nuclear blebs; nu merous chromacenters; vi olet-purple matrix " Numbers in parentheses, percentage; PR, primary; PR (92-97) P-1 (95) P-2 (87-98) genicityNT» EuploidTumori Not observed Euploid NT Located on P-1 (2) film surface P-2 (2-10) close to type II cells or on sur face of Type II cells; formed syncytiallike ar range ments Not observed Not yet established NT Formed pave- P-3 (100) ment-like P-4 (100) P-5 (100) monolayer with little or no cell overlap P-2 (100) P-6 (100) Aneuploid P, passage. 6 NT, In vivo tumorigenicity not tested. c Cells injected s.c. as a suspension into compatible hybrid recipient mice. smooth, contoured nuclear membrane. Nuclei were charac teristically at least slightly eccentrically placed within the cells, even in round cell shapes. The nuclei were character ized by the presence of uniformly dense heterochromatin that made it impossible to delineate the presence of nu cleoli. The cytoplasm of type I cells was uniformly and relatively intensely basophilic. Numerous basophilic Cytoplasmic granules (Fig. 2) and occasional vacuoles were characteris tically present, especially concentrated in juxtanuclear areas. Time of in Vitro Appearance. Type I cells were observed as 1 of the 2 cell types predominating in confluent monolayers of primary cultures of preneoplastic FB-reactive capsule tissue (Cultures A and C) that had been removed after 1.5 (Culture C) and 5 (Culture A) months of implantation. Type I cells represented 8% (Culture A) and 3% (Culture C) of the cells in these primary cultures. This cell type represented 3% of the cells from passage 1 of Culture A but was not observed in confluent monolayers of any of the subsequent 3230 passages studied from Culture A (passages 2 to 5) and was not observed in a confluent monolayer of passage 2 of Culture D. Three % of the cells from the confluent monolayer stage of passage 2 of Culture E were identified as type I cells. Type I cells were not observed in confluent monolayers of passage 2 of Culture B or passage 6 of Culture F, both derived from film-attached cells. [Primary cultures of filmattached cells were not studied in these experiments, but we have demonstrated the presence of macrophage-type cells on implant surfaces in many previous experiments (7, 13).] In Vitro Topographical Relationships. Type I cells were either attached directly to the coverslip surface or were located on the surface of thin cytoplasmic extensions of type II cells also present in primary cultures and early pas sages. Direct cell-to-cell contact between type I cells was rarely seen, even when they were present in colonies or foci. Chromosomes. 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PUB(v ajnjino jo g aßsssed'-ß-a)anssij aA|;oBaj -gd Luojj paAjJsp ssjnuno jo saßessedpaouBAps u| suso •s.ot'L JOs.o/ agi Al 3dA; q;|M pajeposse AIUOLULUOO aj3M 'sjsqujnu aujos u; sjaqujnu aujosoLUOJijo peu. AIUOIUWOO ISOLUsnao asau.1 -oujojqo pio|dej;ai U.JJMpajEjoossE '¡apnu aßjeiAJSA'lus •p|0|dnauB A|qB|JBAU|aja/w snao AI adAj. -saujosouiojiio -jBddB saujijaLuos os|e BJBMSUJJDJ pajBapnujq jnq 'snapnu •(OL pue IBAOoj punoj 'aßjB|'a|ßu|SE psg A||Bnsn SUBOAl adAi 6 'sß|j)dB|j9AOnao jo dn Bu^d jo aouapma ou jo amii MUM •S||30|| aouanijuoo o\ MajßA||BO!isuapBJBU.osnao AI sdAi 'snao u 3dA; OJ JSEJ1.UOOU| SUjßjBLU||30 p3U|J3p-||3/V\ 3JOLU pBq A||BO|} •/e )3 uosuyop - Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research. Cell Types Cultured in Preneoplastic FB Tumorigenesis a less proliferative but functionally active stage. If type III and/or IV cells were present in these early stages of growth in vitro, they were obscured by the initial rapid proliferation of fibroblast-like type II cells. Aneuploid type IV cells were demonstrable when the growth of type II cells subsided. Passage at this point resulted in the appearance of signifi cant numbers of type IV cells approximately midway through the passage (e.g., at 5 to 6 days of passage 3 of Culture A) and a pure population of aneuploid type IV cells at the stage of monolayer confluency (e.g., at 11 days of passage 3 of Culture A). The possible relationship of type III cells to aneuploid type IV cells is not clearly understood on the basis of these lightmicroscopic studies alone. The observation of type III cells in culture coincided with the deterioration of euploid fibro blast-like populations (cell type II) and the 1st appearance of type IV cells. The disappearance of type III cells coincided with the progressive and rapid expansion of aneuploid type IV cells. Insufficient numbers of type III cells in mitosis precluded any conclusions regarding the karyotype charac teristics of the cell type. On the basis of the observations of this study, it is possi ble to propose at least 3 hypotheses that could account for the changing predominance of cell types: (a) small numbers of preneoplastic type IV cells were present in primary cul tures but were obscured during this period by the rapid proliferation of fibroblast-like cells (i.e., type II cells); (b) type III cells, present in small numbers in early cultures, represent the progenitor cells from which type IV aneuploid cells develop under selective and preferential conditions provided in the in vitro environment; and (c) type IV cells, although recognized in the study to be distinct from type II cells on the basis of light-microscopic morphology and growth characteristics, represent in vitro aneuploid variants of the earlier-appearing, euploid, fibroblast-like cells (type II cells). Further information concerning the possible relation ships and distinctions between cell types II, III, and IV is provided by our electron-microscopic studies of these cell types (to be reported in a subsequent paper). As was discussed earlier, all cells in the 2 cultures derived from cells attached to the surfaces of FB implants (i.e., passage 2 of Culture B and passage 6 of Culture F) con formed to the criteria established for type IV cells. These observations possibly indicate that the surface of FB im plants in these cases provided the same preferential stimu lus for development and growth of type IV cells before culture (i.e., in vivo) as the cell cultures provided for these cells in vitro. Consequently, type IV cells already present on the FB surface at the time of excision apparently had an early growth advantage in vitro (e.g., passage 2 of Culture B) over other cell types also possibly present on the FB segments. In that the nature and origin of cell types cannot be determined with absolute certainty by their light-micro scopic morphology in culture, the 4 cell types observed in this in vitro study were arbitrarily identified by Roman nu merals to avoid confusion that would result from designa tion of specific names without further ultrastructural and histochemical conformation. However, on the basis of lightmicroscopic studies of MGG-stained preparations, certain SEPTEMBER 1977 morphological and growth characteristics of the cell types observed in this study could be correlated with in vitro features reported for several specific cell types. The size (15- to 50-jiim diameter), shape (round to spin dle), and presence of numerous cytoplasmic granules and/or vacuoles characteristic of type I cells are consistent with functionally active macrophages. Type II cells, which tended to grow in overlapping and crisscross arrays, were observed as large (up to 400 /¿m long) stellate to fusiform cells with indistinct feathered edges, numerous fibrillar cytoplasmic components, and closely associated extracellular deposits of collagen-like material. These morphological and growth characteristics are generally consistent with those described for fibroblasts or fibroblast-like cells in vitro. These characteristics of type II cells also conform in general to those described for smooth muscle cells in vitro, but type II cells did not form distinct bands of parallel cells which are characteristic of smooth muscle cells (10). The presence of collagen forma tion is not adequate evidence to distinguish between fibro blasts and smooth muscle cells in vitro because smooth muscle cells (like fibroblasts) have been reported to secrete collagen (15, 17). It is thus apparent that an accurate mor phological distinction between fibroblasts and smooth muscle cells in vitro must be made with the electron micro scope (17). Type III cells characteristically were very slender (approxi mately 10 /xm in diameter), bipolar cells up to 200 ¡¿m long that often formed linear cords of overlapping cells. These cells had a single symmetrically located nucleus with heav ily marginated heterochromatin. Stellate type III cells, with 3 or 4 finely tapered cytoplasmic processes that provided points of cell-to-cell contact, were also apparent. Attempts to specifically identify type III cells based on these lightmicroscopic studies alone are admittedly speculative, but these cells do appear to have some features in common with certain cells isolated in vitro from retinal capillaries. Retinal capillaries present in expiants of retina cultured in vitro produce cord-like outgrowths of cells (1) that some what resemble the overlapping cell networks observed in type III cells in culture. These cells, derived from retinal capillaries and identified as primitive vascular mesenchymal cells, were demonstrated to have the capacity to differen tiate into endothelial cells and intramural pericytes (1). Type IV cells were polygonal to stellate and, in contrast to type II cells, had relatively short cytoplasmic processes, more clearly defined cell margins, and a more homogenous cytoplasm with less fibrillar banding. Type IV cells were also generally more uniform in size and shape as compared to type II cells and often contained cytoplasmic vacuoles pre sumed to be lipid. The growth characteristics of type IV cells were significantly different from type II cells in that the former cells grew to confluence, forming a monolayer with little or no evidence of piling up or cell overlap. These morphological features and the tendency to form monolayers of uniformly spaced cells in pavement-like or mosaic patterns are consistent with the characteristics reported for in vitro cultured endothelial cells (2, 11, 12, 18, 19). Cytoplasmic blebs were commonly observed with type IV cells in intermediate stages of passage (passages 3 and 4) of 3233 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research. K. H. Johnson et al. Culture A and only occasionally with type III cells. Cytoplasmic blebs were not present in later passages of Culture A, nor were they present in cultures of type IV cells obtained from film-attached cells (passage 2 of Culture B and pas sage 6 of Culture F). In that type IV cells were demonstrated in this and earlier studies to be aneuploid cells capable of producing tumors when injected into compatible recipient mice, it is possible that the cytoplasmic blebs associated with these cells are linked to a phase of neoplastic transfor mation. Cytoplasmic blebs were not associated with the euploid cell types that predominated in the primary cultures and early passages of Culture A. The possibility that cyto plasmic blebbing is linked to the process of neoplastic transformation is further supported by scanning electronmicroscopic studies of in vitro transformed BALB/3T3 mouse embryo cells (16). In the latter studies, SV40, murine sarcoma virus, and spontaneous transformants of the BALB/3T3 cell line were shown to have numerous 1.0 to 5.0/nm cytoplasmic blebs not present in the parent cell line. Also in the context of the present study, BALB/3T3 cells, which were originally considered to be fibroblast cells, are now considered to have morphological and growth charac teristics that are more consistent with cells derived from small blood vessels (i.e., endothelial cells or pericytes) (9, 16). The morphological and growth characteristics reported for BALB/3T3 cells are in fact not unlike those of the type IV cells identified in the present study. Recent experiments have further demonstrated that BALB/3T3 cells, which cannot divide in vitro unless at tached to a solid substrate and are nontumorigenic when inoculated s.c. into syngeneic mice, produce tumors (ma lignant hemangioendotheliomas) when they are inoculated s.c. after being attached to glass beads (3) or plastic films (4). These findings, which demonstrate that BALB/3T3 cells are tumorigenic in animals when attached to a solid sub strate, provide an additional link of similarity between BALB/3T3 cells and our in vitro isolated type IV cells. He mangioendotheliomas or hemangiosarcomas are in fact va rieties of sarcomas induced by the experimental implanta tion of plastic or glass films (14). The findings of this in vitro study are considered to be consistent with our earlier reported hypothesis (14) impli cating cells of the local microvasculature as the likely pro genitor or parent cells from which FB sarcomas are derived. REFERENCES 1. Ashton. N. Oxygen and the Growth and Development of Retinal Vessels. in Vivo and in Vitro Studies. Am. J. Ophthalmol., 62: 412-435, 1966. 2. Blose. S. H., and Chacko, S. In Vitro Behavior of Guinea Pig Arterial and Venous Endothelial Cells. Develop.. Growth Differentiation. 17: 153-165. 1975. 3. Boone, C. W. Malignant Hemangioendotheliomas Produced by Subcuta neous Inoculation of BALB/3T3 Cells Attached to Glass Beads. Science, 188: 68-70. 1975. 4. Boone, C. W., Takeichi, N.. Parangpe, M., and Gilden. R. Vasoformitive Sarcomas Arising from BALB/3T3 Cells Attached to Solid Substrates. Cancer Res..36: 1626-1633. 1976. 5. Brand. K. G., Buoen, L. C., and Brand. I. Carcinogenesis from Polymer Implant: New Aspects from Chromosomal and Transplantation Studies during Premalignancy. J. Nati. Cancer Inst., 39. 663-679, 1967. 6. Brand, K. G.. Buoen, L. C., and Brand. I. Foreign Body Tumorigenesis: Timing and Location of Preneoplastic Events. J. Nati. Cancer Inst., 47: 829-836, 1971. 7. Brand. K. G., Buoen, L. C., Johnson, K. H., and Brand, I. Etiological Factors, Stages, and the Role of the Foreign Body in Foreign Body Tumorigenesis: A Review. Cancer Res.. 35: 279-286. 1975. 8. Buoen, L. C.. Brand, I., and Brand. K. G. Foreign-Body Tumorigenesis: In Vitro Isolation and Expansion of Preneoplastic Clonal Cell Popula tions. J. Nati. Cancer Inst.. 55: 721-723, 1975. 9. Franks, L. M.. and Cooper. T. W. The Origin of Human Embryo Lung Cells in Culture: A Comment on Cell Differentiation in Vitro Growth and Neoplasia. Intern. J. Cancer, 9: 19-29. 1972. 10. Gimbrone, M. A.. Jr., and Cotran, R. S. Human Vascular Smooth Muscle in Culture. Growth and Ultrastructure. Lab. Invest.,33: 16-27. 1975. 11. Gimbrone, M. A., Jr., Cotran, R. S., and Folkman, J. Human Vascular Endothelial Cells in Culture. Growth and DNA Synthesis. J. Cell Biol.,60: 673-684. 1974. 12. Jaffe, E. A., Nachman, R. L.. Becker. C. G., and Minick, C. R. Culture of Human Endothelial Cells Derived from Umbilical Veins. J. Clin. Invest.. 52: 2745-2756, 1973. 13. Johnson, K. H., Ghobrial. H. K. G., Buoen, L. C., Brand, I., and Brand. K. G. Foreign-Body Tumorigenesis in Mice: Ultrastructure of the Preneo plastic Reaction. J. Nati. Cancer Inst.,49: 1311-1319, 1972. 14. Johnson, K. H., Ghobrial, H. K., Buoen, L. C., Brand, K. G., and Brand, I. Nonfibroblastic Origin of Foreign Body Sarcomas Implicated by Histological and Electron Microscopic Studies. Cancer Res.. 33: 3139-3154, 1973. 15. Layman, D. L., and Titus, J. L. Synthesis of Type I Collagen by Human Smooth Muscle Cells in Vitro. Lab. Invest., 33: 103-107, 1975. 16. Porter. K. R.. Todaro. G. T.. and Fönte,V. Scanning Electron Micro scope Study of Surface Features of Viral and Spontaneous Transformants of Mouse BALB/3T3 Cells. J. Cell Biol.. 59: 633-642, 1973. 17. Rossi, G. L.. Alroy, J., and Rothenmund. S. Morphological Studies of Cultured Swine Aorta Media Explants. Virchows Arch. Abt. BZellpathol., 72: 133-144, 1973. 18. Slater, D. N., and Sloan, J. M. The Porcine Endothelial Cell in Tissue Culture. Atherosclerosis. 21: 259-272, 1975. 19. Wechezak, A. R., and Mansfield. P. B. Isolation and Growth Characteris tics of Cell Lines from Bovine Venous Endothelium. In Vitro. 9: 39-45, 1973. Fig. 1. Primary culture of FB-reactive tissue origin. The monolayer is composed predominantly of type II cells and a few type I cells (arrows). MGG, x 256. Fig. 2. Focus of type I cells (round, spindle, and stellate forms) from a primary culture of FB-reactive tissue. The eccentrically placed nuclei contain uniformly dense heterochromatin. Numerous basophilic cytoplasmic granules are evident. MGG, x 1024. Fig. 3. Large stellate type II cell from a primary culture of FB-reactive tissue. The smooth-contoured nucleus contains 2 nucleoli and multiple, smaller chromacenters. Faintly visible fibrillar components (arrows) are evident in the less basophilic cytoplasmic matrix. MGG, x 1024. Fig. 4. Dense culture (passage 1 of FB-reactive tissue origin) composed predominantly of overlapping and unidirectional type II cells with early appearance of a few typical fusiform type III cells (arrows). MGG. x 256. Fig. 5. High-magnification photomicrograph of 2 fusiform type III cells present in Fig. 4. These cells have hyperchromatic nuclei that are symmetrically located with the cell cytoplasm. The cytoplasm of type III cells is more intensely basophilic than the cytoplasm of the underlying type II cells. MGG, x 640. Fig. 6. Two closely associated type III cells (passage 1 of FB-reactive tissue origin) located on the surface of thin cytoplasmic extensions of large underlying type II cells. A few clear vacuoles (arrows) are evident in the cytoplasm of the type III cells. MGG, x 1024. Fig. 7. Nonconfluent monolayer of polygonal type IV cells (passage 3 of FB-reactive tissue origin). These cells have more well-defined cell margins and more homogeneously basophilic cytoplasm than type II cells observed in earlier passages. A large number of intensely basophilic blebs (arrow) are evident on the cytoplasmic membrane of 1 type IV cell. MGG. x 384. Fig. 8. High-magnification photomicrograph of 2 type IV cells (passage 4 of FB-reactive tissue origin). Irregularities in nuclear membrane contour are manifested by areas of slight indentation or evagination to form nuclear blebs. The cytoplasm is characterized by numerous clear vacuoles, distinct margins, and basophilic blebs (arrows) concentrated on the extremities of pseudopodia. MGG. x 640. Fig. 9. Confluent monolayer composed of uniformly spaced type IV cells (passage 5 of FB-reactive tissue origin). There is little or no evidence of poling up or cell overlap. A significant degree of irregularity in nuclear membrane contour is apparent, and cytoplasmic lipid vacuoles are apparent in some type IV cells. MGG, x 205. Fig. 10. High-magnification photomicrograph of uniformly spaced type IV cells (passage 5 of FB-reactive tissue origin) without directional orientation. MGG, x 512. 3234 CANCER RESEARCH VOL. 37 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research. Cell Types Cultured in Preneoplastic FB Jumorigenesis • * I? ^ ,v \ i * If '• *'è ß S» M-J •* \ SEPTEMBER 1977 3235 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research. K H. Johnson 3236 et al. CANCER RESEARCH Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research. VOL. 37 Cell Types Cultured in Preneoplastic FB Tumorigenesis r SEPTEMBER 1977 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research. 3237 Light-Microscopic Morphology of Cell Types Cultured during Preneoplasia from Foreign Body-reactive Tissues and Films Kenneth H. Johnson, Lance C. Buoen, Inge Brand, et al. Cancer Res 1977;37:3228-3237. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/37/9/3228 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1977 American Association for Cancer Research.