Surface Morphology of Normal and Neoplastic Rat Cells Miles W. Cloyd, PhD, and Darell D. Bigner, MD, PhD
Nontumorigenic rat cells and their tumorigenic counterparts were studied with scanning electron microscopy under controlled conditions in vitro and with transmission electron microscopy after replantation in vivo to discern if external morphology reflected the cell's neoplastic state or the etiology of transformation. Interphase cells in six of seven nontumorigenic lines were flat and monolayered under confluent conditions and exhibited smooth, nonactive cell surfaces. A nontumorigenic cell line morphologically transformed with human adenovirus-2 consisted of spherical cells with blebbed surfaces. Cells from six tumorigenic lines transformed with avian sarcoma virus had highly active surfaces with many surface projections. Cells from two chemical carcinogen-transformed rat embryo lines were flat with no surface projections in subconfluent culture and rounded with only a few microvilli at high densities, but cells from a sarcoma chemically induced in an adult rat were villous. When villous cells were syngeneically replanted in vloo, they lost most microvilli. The external morphology of cells was influenced by a number of factors simultaneously, with no universal pattern associated with tumorigenic capacity or transforming agent. (Am J Pathol 88:29-52, 1977)
THE EXTERNAL SURFACES of cells appear to mediate the regulation of cellular growth and, perhaps, the immunologic consequences of neoplastic transformation.'4 Normally, cellular division is tightly controlled in the adult animal, and mitosis is only induced when needed (e.g., normal cellular replacement or healing of wounds). This control may be initiated at the cell surface from contacts with neighboring cells or surrounding environment, and it obviously requires intact cellular growth regulatory mechanisms. Surface function, in addition, probably determines cellular affinities by which normal cells can only have certain cells as neighbors. Neoplastic cells, in contrast, have lost this growth control and normal cellular affinities because of abnormalities in intemal mechanisms and resulting surface function. The architecture of the cell surface may reflect surface function or cellular behavior, and alterations of the plasmalemma may be the cause of the autonomous growth characteristics of neoplastic cells. If so, cell surFrom the Departments of Pathologp and Surgery, Duke University NMedical Center. Durham. North Carolina. Supported by Grants CA-i 1898 and CA-14651 from the National Cancer Institute; Dr. Bigner is the recipient of TIA Fellowship IFIINS11063 from the National Institute of Neurological Disease and Stroke and JFCF Aw-ard 2578 from the American Cancer Societh Accepted for publication M1arch 3, 1977. Address reprint requests to Dr. Darell D. Bigner. Department of Patholog-. Duke Unisersits \Nedical Center. Durham. NC 27710. 29
30
CLOYD AND BIGNER
American Journal of Pathology
face topography may be diagnostic of neoplastic transformation and/or its etiology. In order to investigate this hypothesis, the exterior morphology of normal cells and the cell surface alterations that occur upon neoplastic transformation by both viruses and chemical carcinogens were studied with fine structural techniques. Although a number of studies have attempted to characterize cell surface morphologic manifestations of neoplasia, the results have not been consistent. Early scanning electron microscopic (SEM) studies exploring surface morphology of normal and/or transformed cells in culture were compromised by poor preservation of cells.7-19 With improved methods for SEM, the surface features of cultured cells and tumor tissues have been reliably demonstrated,2027 but in most cases, rigid controls for SEM comparisons between normal and neoplastic cells in vitro or in vivo have not been provided. Accordingly, inherent in the experimental design of this study was the reduction of variables that might give unreliable conclusions. Only rat cells were used that were obtained from normal embryos and adult tissue of two inbred strains, from the same or similar cell lines transformed in vitro with tumor viruses or chemical carcinogens, and from induced subcutaneous tumors. Each line was well characterized as to its tumorigenicity in vivo; many were cloned; culture conditions were kept as nearly uniform as possible; and all cell lines were periodically checked for the presence of mycoplasma and were always found negative. Accurate preservation of cell surface detail was accomplished by fixation with isotonic buffered glutaraldehyde at 37 C, and critical point drying from liquid CO2. The architecture of transplanted neoplastic cell lines growing in vivo was also investigated in order to compare this morphology to that in vitro. At confluent culture conditions, distinct cellular shapes and surface morphologies were observed between nontumorigenic cells and their tumorigenic counterparts. However, there was no strict correlation of any morphologic pattern to the cell's tumorigenic capability or to the transforming agent. Materials and Methods Cells and Cell Culture Methods The cell lines used in this study are listed and described in Table 1. All are established lines derived from trypsinized embryos, kidneys, or subcutaneous tumors of inbred Fischer 344 (F-344) or BDIX rats. Many were cloned by plating very dilute cell suspensions in
wells of sterile Microtest II tissue culture plates (Falcon Plastics, Oxnard, Calif.) and selecting wells that contained single cells for further subculture. In vitro transformation of cells with avian sarcoma virus (ASV) was carried out by seeding 1 X 101 cells into 60-mm plastic petri dishes, incubating for 1 hour in medium containing 4 mg/ml polybrene, decanting polybrene medium, and adding 106 to 108 focus-forming units (FFU) of ASV.
CELL SURFACE MORPHOLOGY
Vol. 88, No. 1 July 1977
31
Table 1-Cell Unes Studied
Cell line
Origin (rat, strain, tissue, cell line)
Treatment
Cloned
Infection in vitro with B-77-C ASV Infection in vitro with SR-D ASV Exposure in vitro to 3-methylcholanthrene
+
S-90 Cl3 S-278 C01
CDF F-44 embryo S-90 Cl,
S-06 Cl
S-90C01
H1240
MA F-344 embryo
H1241
MA F-44embryo
Exposureinvitroto7,12-
8617
MA F-344 embryo
Infection in vitro with human
S-204 Cl1
CDF F-344 sarcoma
S-205 S-202 Cl,
Kidneys of same rat as in S-204 CDF F-344 sarcoma
S-203 Cl, S-115 Cl,
Kidneys of same rat as in S-202
CDF F-344 sarcoma
S-325
CDF F-344 sarcoma
S-188 Cl, S-292 C14
BDIX kidneys S-188 Cl1
Infection in vitro with B-77-C
+
S-188 Cl1
S-280
BDIX embryo
ASV Infection in vitro with SR-D ASV -
+
S-295 Cl5
+ +
dimethylbenzanthracene advenovirus Type 2 Subcutaneous sarcoma induced in rat wihSR-D ASV Subcutaneous sarcoma induced in rat with SR-D ASV Scalp sarcoma induced in rat with B-77-C ASV Subcutaneous sarcoma induced in rat with 3-
+ + +
+
methyicholanthrene
+ -
The cells were then overlayed with soft agar, and foci that developed were selected for subculture and further cloning. F-344 rat embryo cell lines transformed in vitro with the chemical carcinogens 3-methylcholanthrene (MCA) and 7,12-dimethylbenzanthracene (DMBA) (lines H1240 and H1241, respectively) " and with human adenovirus type-2 (line 8617) were kindly provided by Dr. Paul J. Price, Microbiological Associates, Bethesda, Md. For morphologic study, cells were plated onto 1 sq cm sections of glass coverslips resting in the bottom of 35-mm plastic petri dishes and grown in 2 ml of 80% Richter's zinc option modification of Eagle's minimum essential medium (MEM) and 20% heat-inactivated fetal calf serum, supplemented with 10 mM Hepes buffer, 584 mg/liter glutamine, 50 jig/ml gentamycin, 100 units/ml penicillin G, and 0.5 mg/ml Amphotericin B. The adenovirus-transformed line, originally grown in Eagle's MEM without calcium in shaker culture,' was converted to monolayer culture and zinc-option medium. The cells were grown to both low and confluent densities, incubated from 48 to 120 hours at 37 C in a high-humidity 5% CO2 atmosphere, and then processed for SEM. All cells were checked repeatedly for mycoplasma contamination by culture and immunologic tests described by Barile.' Such testing involves incubation in mycoplasma agar of scraped cells and cell culture fluids from cultures grown longer than 3 days in antibiotic-free medium. Tum
Test Cells grown in large quantities were trypsinized, counted, centrifuged, and resuspended in phosphate-buffered saline at concentrations of 1 to 10 X 10 cells/0.1 ml and were
32
CLOYD AND BIGNER
American Journal of Pathology
injected subcutaneously over the back of syngeneic newborn rats or in the leg of adults. The time required for tumors to develop or the lack of tumor development were recorded, and only instances of progressive tumor growth were considered as positive (Tables 2 and
3). Scanning and Transmission Electron Microscopy
For both scanning (SEM) and transmission electron microscopy (TEM), cultured cells were rinsed with Puck's saline A and fixed in the plate for 30 minutes at 37 C with 2.5% glutaraldehyde buffered with 0.045 N sodium cacodylate (pH 7.3) and containing 0.05% CaC12 (390 mOsmoles). Following a rinse in 0.2 N cacodylate buffer, the cells were postfixed in 0.2 N cacodylate-buffered 1% Os04 for 15 minutes. For SEM the cells on coverslips were dehydrated through a graded series of ethanol or acetone, and critical point dried in liquid CO2, using a BOMAR SPC-900/EX critical point drying apparatus. A thin coating (100 X) of gold-palladium was evaporated onto the cells using a Film-Vac, Inc., Minicoater. For TEM the fixed cells were scraped from the coverslips with rubber policemen, transferred to glass centrifuge tubes, dehydrated through a graded series of ethanol, embedded in Epon 812, and routinely processed for TEM. Tissues from tumors induced subcutaneously with various cell lines were removed from rats perfused intravascularly with a cacodylate-buffered paraformaldehyde-glutaraldehyde fixative 31 as described previously,32 and were processed for TEM. The coverslips were examined with a Cwikscan/100 field emission electron microscope (Coates and Welter Instrument Corp.) and a Cambridge Stereoscan S4 scanning electron microscope, both operated at 15 kV, and photographs were taken with Kodak 4127 Commercial film. Observations and micrographs of sectioned material were made with a Hitachi HU-IIA TEM operated at 75 kV.
Results Tumorigenic Capacity of Cultured Rat Cell Lines
The property of tumorigenicity was determined for each cell line by its capacity to induce progressively growing tumors when injected into syngeneic rats. Table 2 lists seven cell lines that did not induce tumors when syngeneically injected and the conditions of testing used. Five of the lines originated from normal rat tissue (embryos or adult kidneys) (cell lines S90 Cl3, S-280, S-203 Cli, S-205, and S-188 Cli); one was derived from a tumor (line S-202 Cl7); and another was an adenovirus-transformed embryo line (8617). For a cell line to be considered nontumorigenic, there had to be no tumors resulting from syngeneic injection of at least 1 X 106 cells during a 4- to 11-month observation period. The nine rat cell lines that were tumorigenic are listed in Table 3. All were either cell lines transformed in vitro with ASV or chemical carcinogens or lines derived from ASV or chemical carcinogen-induced tumors. Cell lines S-278 Cl2 and S-306 Cl1 were derived from nontumorigenic F344 embryo line (S-90 C13) by in vitro infection with- Bratislava 77, Subgroup C (B-77-C), and Schmidt-Ruppin, Subgroup D (SR-D), strains of ASV, respectively, and lines S-292 C14 and S-293 C15 were similarly
CELL SURFACE MORPHOLOGY
Vol. 88, No. 1 July 1977
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34
CLOYD AND BIGNER
American Journal of Pathology
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Nontumorigenic rat cells and their tumorigenic counterparts were studied with scanning electron microscopy under controlled conditions in vitro and with transmission electron microscopy after replantation in vivo to discern if external morphology reflected the cell's neoplastic state or the etiology of transformation. Interphase cells in six of seven nontumorigenic lines were flat and monolayered under confluent conditions and exhibited smooth, nonactive cell surfaces. A nontumorigenic cell line morphologically transformed with human adenovirus-2 consisted of spherical cells with blebbed surfaces. Cells from six tumorigenic lines transformed with avian sarcoma virus had highly active surfaces with many surface projections. Cells from two chemical carcinogen-transformed rat embryo lines were flat with no surface projections in subconfluent culture and rounded with only a few microvilli at high densities, but cells from a sarcoma chemically induced in an adult rat were villous. When villous cells were syngeneically replanted in vloo, they lost most microvilli. The external morphology of cells was influenced by a number of factors simultaneously, with no universal pattern associated with tumorigenic capacity or transforming agent. (Am J Pathol 88:29-52, 1977)
THE EXTERNAL SURFACES of cells appear to mediate the regulation of cellular growth and, perhaps, the immunologic consequences of neoplastic transformation.'4 Normally, cellular division is tightly controlled in the adult animal, and mitosis is only induced when needed (e.g., normal cellular replacement or healing of wounds). This control may be initiated at the cell surface from contacts with neighboring cells or surrounding environment, and it obviously requires intact cellular growth regulatory mechanisms. Surface function, in addition, probably determines cellular affinities by which normal cells can only have certain cells as neighbors. Neoplastic cells, in contrast, have lost this growth control and normal cellular affinities because of abnormalities in intemal mechanisms and resulting surface function. The architecture of the cell surface may reflect surface function or cellular behavior, and alterations of the plasmalemma may be the cause of the autonomous growth characteristics of neoplastic cells. If so, cell surFrom the Departments of Pathologp and Surgery, Duke University NMedical Center. Durham. North Carolina. Supported by Grants CA-i 1898 and CA-14651 from the National Cancer Institute; Dr. Bigner is the recipient of TIA Fellowship IFIINS11063 from the National Institute of Neurological Disease and Stroke and JFCF Aw-ard 2578 from the American Cancer Societh Accepted for publication M1arch 3, 1977. Address reprint requests to Dr. Darell D. Bigner. Department of Patholog-. Duke Unisersits \Nedical Center. Durham. NC 27710. 29
30
CLOYD AND BIGNER
American Journal of Pathology
face topography may be diagnostic of neoplastic transformation and/or its etiology. In order to investigate this hypothesis, the exterior morphology of normal cells and the cell surface alterations that occur upon neoplastic transformation by both viruses and chemical carcinogens were studied with fine structural techniques. Although a number of studies have attempted to characterize cell surface morphologic manifestations of neoplasia, the results have not been consistent. Early scanning electron microscopic (SEM) studies exploring surface morphology of normal and/or transformed cells in culture were compromised by poor preservation of cells.7-19 With improved methods for SEM, the surface features of cultured cells and tumor tissues have been reliably demonstrated,2027 but in most cases, rigid controls for SEM comparisons between normal and neoplastic cells in vitro or in vivo have not been provided. Accordingly, inherent in the experimental design of this study was the reduction of variables that might give unreliable conclusions. Only rat cells were used that were obtained from normal embryos and adult tissue of two inbred strains, from the same or similar cell lines transformed in vitro with tumor viruses or chemical carcinogens, and from induced subcutaneous tumors. Each line was well characterized as to its tumorigenicity in vivo; many were cloned; culture conditions were kept as nearly uniform as possible; and all cell lines were periodically checked for the presence of mycoplasma and were always found negative. Accurate preservation of cell surface detail was accomplished by fixation with isotonic buffered glutaraldehyde at 37 C, and critical point drying from liquid CO2. The architecture of transplanted neoplastic cell lines growing in vivo was also investigated in order to compare this morphology to that in vitro. At confluent culture conditions, distinct cellular shapes and surface morphologies were observed between nontumorigenic cells and their tumorigenic counterparts. However, there was no strict correlation of any morphologic pattern to the cell's tumorigenic capability or to the transforming agent. Materials and Methods Cells and Cell Culture Methods The cell lines used in this study are listed and described in Table 1. All are established lines derived from trypsinized embryos, kidneys, or subcutaneous tumors of inbred Fischer 344 (F-344) or BDIX rats. Many were cloned by plating very dilute cell suspensions in
wells of sterile Microtest II tissue culture plates (Falcon Plastics, Oxnard, Calif.) and selecting wells that contained single cells for further subculture. In vitro transformation of cells with avian sarcoma virus (ASV) was carried out by seeding 1 X 101 cells into 60-mm plastic petri dishes, incubating for 1 hour in medium containing 4 mg/ml polybrene, decanting polybrene medium, and adding 106 to 108 focus-forming units (FFU) of ASV.
CELL SURFACE MORPHOLOGY
Vol. 88, No. 1 July 1977
31
Table 1-Cell Unes Studied
Cell line
Origin (rat, strain, tissue, cell line)
Treatment
Cloned
Infection in vitro with B-77-C ASV Infection in vitro with SR-D ASV Exposure in vitro to 3-methylcholanthrene
+
S-90 Cl3 S-278 C01
CDF F-44 embryo S-90 Cl,
S-06 Cl
S-90C01
H1240
MA F-344 embryo
H1241
MA F-44embryo
Exposureinvitroto7,12-
8617
MA F-344 embryo
Infection in vitro with human
S-204 Cl1
CDF F-344 sarcoma
S-205 S-202 Cl,
Kidneys of same rat as in S-204 CDF F-344 sarcoma
S-203 Cl, S-115 Cl,
Kidneys of same rat as in S-202
CDF F-344 sarcoma
S-325
CDF F-344 sarcoma
S-188 Cl, S-292 C14
BDIX kidneys S-188 Cl1
Infection in vitro with B-77-C
+
S-188 Cl1
S-280
BDIX embryo
ASV Infection in vitro with SR-D ASV -
+
S-295 Cl5
+ +
dimethylbenzanthracene advenovirus Type 2 Subcutaneous sarcoma induced in rat wihSR-D ASV Subcutaneous sarcoma induced in rat with SR-D ASV Scalp sarcoma induced in rat with B-77-C ASV Subcutaneous sarcoma induced in rat with 3-
+ + +
+
methyicholanthrene
+ -
The cells were then overlayed with soft agar, and foci that developed were selected for subculture and further cloning. F-344 rat embryo cell lines transformed in vitro with the chemical carcinogens 3-methylcholanthrene (MCA) and 7,12-dimethylbenzanthracene (DMBA) (lines H1240 and H1241, respectively) " and with human adenovirus type-2 (line 8617) were kindly provided by Dr. Paul J. Price, Microbiological Associates, Bethesda, Md. For morphologic study, cells were plated onto 1 sq cm sections of glass coverslips resting in the bottom of 35-mm plastic petri dishes and grown in 2 ml of 80% Richter's zinc option modification of Eagle's minimum essential medium (MEM) and 20% heat-inactivated fetal calf serum, supplemented with 10 mM Hepes buffer, 584 mg/liter glutamine, 50 jig/ml gentamycin, 100 units/ml penicillin G, and 0.5 mg/ml Amphotericin B. The adenovirus-transformed line, originally grown in Eagle's MEM without calcium in shaker culture,' was converted to monolayer culture and zinc-option medium. The cells were grown to both low and confluent densities, incubated from 48 to 120 hours at 37 C in a high-humidity 5% CO2 atmosphere, and then processed for SEM. All cells were checked repeatedly for mycoplasma contamination by culture and immunologic tests described by Barile.' Such testing involves incubation in mycoplasma agar of scraped cells and cell culture fluids from cultures grown longer than 3 days in antibiotic-free medium. Tum
Test Cells grown in large quantities were trypsinized, counted, centrifuged, and resuspended in phosphate-buffered saline at concentrations of 1 to 10 X 10 cells/0.1 ml and were
32
CLOYD AND BIGNER
American Journal of Pathology
injected subcutaneously over the back of syngeneic newborn rats or in the leg of adults. The time required for tumors to develop or the lack of tumor development were recorded, and only instances of progressive tumor growth were considered as positive (Tables 2 and
3). Scanning and Transmission Electron Microscopy
For both scanning (SEM) and transmission electron microscopy (TEM), cultured cells were rinsed with Puck's saline A and fixed in the plate for 30 minutes at 37 C with 2.5% glutaraldehyde buffered with 0.045 N sodium cacodylate (pH 7.3) and containing 0.05% CaC12 (390 mOsmoles). Following a rinse in 0.2 N cacodylate buffer, the cells were postfixed in 0.2 N cacodylate-buffered 1% Os04 for 15 minutes. For SEM the cells on coverslips were dehydrated through a graded series of ethanol or acetone, and critical point dried in liquid CO2, using a BOMAR SPC-900/EX critical point drying apparatus. A thin coating (100 X) of gold-palladium was evaporated onto the cells using a Film-Vac, Inc., Minicoater. For TEM the fixed cells were scraped from the coverslips with rubber policemen, transferred to glass centrifuge tubes, dehydrated through a graded series of ethanol, embedded in Epon 812, and routinely processed for TEM. Tissues from tumors induced subcutaneously with various cell lines were removed from rats perfused intravascularly with a cacodylate-buffered paraformaldehyde-glutaraldehyde fixative 31 as described previously,32 and were processed for TEM. The coverslips were examined with a Cwikscan/100 field emission electron microscope (Coates and Welter Instrument Corp.) and a Cambridge Stereoscan S4 scanning electron microscope, both operated at 15 kV, and photographs were taken with Kodak 4127 Commercial film. Observations and micrographs of sectioned material were made with a Hitachi HU-IIA TEM operated at 75 kV.
Results Tumorigenic Capacity of Cultured Rat Cell Lines
The property of tumorigenicity was determined for each cell line by its capacity to induce progressively growing tumors when injected into syngeneic rats. Table 2 lists seven cell lines that did not induce tumors when syngeneically injected and the conditions of testing used. Five of the lines originated from normal rat tissue (embryos or adult kidneys) (cell lines S90 Cl3, S-280, S-203 Cli, S-205, and S-188 Cli); one was derived from a tumor (line S-202 Cl7); and another was an adenovirus-transformed embryo line (8617). For a cell line to be considered nontumorigenic, there had to be no tumors resulting from syngeneic injection of at least 1 X 106 cells during a 4- to 11-month observation period. The nine rat cell lines that were tumorigenic are listed in Table 3. All were either cell lines transformed in vitro with ASV or chemical carcinogens or lines derived from ASV or chemical carcinogen-induced tumors. Cell lines S-278 Cl2 and S-306 Cl1 were derived from nontumorigenic F344 embryo line (S-90 C13) by in vitro infection with- Bratislava 77, Subgroup C (B-77-C), and Schmidt-Ruppin, Subgroup D (SR-D), strains of ASV, respectively, and lines S-292 C14 and S-293 C15 were similarly
CELL SURFACE MORPHOLOGY
Vol. 88, No. 1 July 1977
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34
CLOYD AND BIGNER
American Journal of Pathology
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