A CELL LINE FROM AN INDUCED CARCINOMA O F MOUSE RECTUM
L. M. FRANKS AND VALERIE J. HEMMINGS Department of Cellular Pathology, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, UK
PLATESXXI-XXIII
I N an accompanying paper (Franks et al., 1977) we have described the results obtained with cultures from normal mouse colon. In this paper we report on the culture of seven induced mouse tumours of large intestine and the establishment of a cell line from one of them. MATERIALS AND METHODS 1,2-dimethylhydrazine or methyl azoxymethanol acetate (MAM acetate) were used to induce tumours of colon and rectum in C57BL ICRF a' mice. Details are given by Defries (1976). The cell line described in this paper was established from a polypoid carcinoma of rectum from a 19-mth-old male mouse given four intraperitoneal injections of MAM acetate (each 0.2 mg in saline) at weekly intervals from 3 wk of age. Standard explant cultures (about 1 mm3) from six primary tumours and one transplanted rectal tumour were set up in plastic Petri dishes (Nunc (UK) Limited, Stafford, UK), in Waymouth's medium 752/1 with 15 per cent. calf serum, in 5 per cent C02 in air at 37°C. Medium was changed every 3 or 4 days using 10 per cent. calf serum. Selective trypsinisation and pipetting were used to remove " fibroblasts ". Details are given in the text. For electron microscopy, cells were fixed and processed as described previously (Franks et al., 1977). Cells are stored in liquid nitrogen in 10 per cent. dimethyl sulphoxide in culture medium.
RESULTS Tumour tissue was finely chopped with fine curved scissors and rinsed four times in culture medium containing 400 units/ml penicillin, 400 pg/ml streptomycin sulphate and 200 units/ml mycostatin (E. R. Squibb and Sons Ltd, Merseyside, UK) About 20 1-mm explants were plated into 50 mm Petri dishes in medium with antibiotics at one-quarter of the above concentrations. Cultures from four of the primary turnours were contaminated in the initial dishes and were discarded. Cultures from the remaining two tumours were maintained for five and nine transfer generations respectively. In these there was a progressive build up of a fungal infection and the cultures were eventually discarded. The cell line to be described (CMT93) was established from the fourth in-vivo transplant generation of a rectal carcinoma. Ten 50 mm Petri dishes were each innoculated with about 20 explants. Medium was changed Received 10 Feb. 1977; accepted 13 June 1977. J. PATH.-VOL.
124 (1978)
35
36
L. M. FRANKS AND VALERIE J. HEMMINGS
after 3 days. By 6 days clearly recognisable epithelial and mesenchymal cells had grown out from the explants. On days 7, 13, 20 and 32 the dishes were trypsinised and mesenchymal cells pipetted free, leaving predominantly epithelial cells attached to the substrate. Mesenchymal cell lines were established from the cells which were removed. On day 32 after initial trypsinisation to remove mesenchymal cells the epithelial cells from one dish were removed by a second trypsinisation and transferred to two Petri dishes. These cells gave rise to mixed epithelial and mesenchymal cell culture. After a further 24 days mesenchyme was removed by trypsinisation and the epithelium removed in the same way as before and transferred. The second transfer also produced a mixed culture, which was treated in the same way after 21 days. The cells in the third and subsequent passages were epithelial in morphology (figs. 1 and 2) and mesenchymal cells could not be recognised. At this stage the growth rate increased and the cells reached confluence after a 1 to 2 split in about 7 days. Since the cells grow in coherent clumps it is difficult to prepare single-cell suspensions for accurate cell counting, although the clumps can be removed for transfer by trypsinisation. Electron microscopy showed that the cells were closely packed and varied in shape, size and electron density (figs. 3. and 4). Many had no specific features but in some areas acini were formed (fig. 4). The cells forming these acini and the cells at the free margins of the clumps still retained some of the differentiated characters of rectal epithelium. Junctional complexes were present, including true desmosomes (fig. 5) with a central electron-dense lamella and associated 10 nm filaments. The lumenal microvilli had a central core of fibrils and glycoprotein strands attached to the external lamella of the plasma membrane (figs. 6 and 7). Virus-like particles (C-type) were present in some of the cells. The overall pattern was similar in ultrastructure to that of the tumour from which the line was derived. The cells have been maintained for 16 transfer generations and are stored in liquid nitrogen. DISCUSSION In-vitro systems have not been used to any great extent in work on the aetiology or development of cancers of the large intestine, mainly because of the technical problems involved (see Franks, 1976, for review). Although shortterm cultures can be established from many tumours, including gut tumours (Ghose et al., 1957; Baldwin et al., 1973) these are almost invariably mixed cultures and most cannot be maintained indefinitely. However, six human cell lines (Kasten et al., 1965; Egan and Todd, 1972; Goldenberg et al., 1972; Tompkins et al., 1974; Fogh and Trempe, 1975; Tom et al., 1976) have been reported, although one (Kasten et al., 1965) is now thought to be contaminated with HeLa cells (Nelson-Rees and Flandermeyer, 1976). Martin et al. (1975) have established five cell lines from induced transplantable rat tumours (three colon and two duodenum) and Tan et al. (1976) have described two lines derived from dimethyl hydrazine-induced colon carcinomas in mice. Since the establishment of cell lines from tumours seems to be a rare event it is important
PLATE XXI
FRANKS AND HEMMINGS OF MOUSE RECTUM CARCINOMA
FIG. 1.
FIG.2.
Fro. 1.-Living FIG. 2.-A
culture (fourth transfer) CMT93 cells showing " epithelial " morphology. Phase contrast. x 84. higher power of the cells at the edge of a clump showing columnar shape and rather indefinite free margin. Phase contrast. x 280.
FIG.3.-Sheet of cells (fourth transfer) showing closely packed cells and microvilli along free margin. Junctional complexes can just be made out as darker lines between cells near the margin. x 2000.
PLATE XXII
FRANKS A N D HEMMINGS
CARCINOMA OF MOUSE RECTUM
FIG.4.-Part
of a cell sheet showing acinus formation with lumenal microvilli and dark junctional complexes near the lumen. The cells vary in electron density. x 10,OOO.
FRANKS AND HEMMINGS
PLATE XXIlI
CARCINOMA OF MOUSE RECTUM
FIG.5.
FIG.6.
FIG.5.-Part of a junctional complex from cells in fig. 3 showing two desmosomes with central electron-dense lamellae. x 144,000. FIG.6.-The free margin of cells in fig. 3 showing microvilli and attached glycoprotein strands x 30,000. FIG.7.-Lumenal margin of cells from an “acinus ” showing microvilli with central filaments and attached glycoprotein strands. x 118,OOO.
FIG.7.
CARCINOMA OF MOUSE RECTUM
37
that several different lines should be available for study since the established lines are derived from such a small number of tumours and may not be representative of the whole tumour group. The element of selection is emphasised by the fact that the one cell line which we have established was derived from only one out of ten Petri dishes originally set up, each containing at least 20 explants. The reasons for the failure of most normal and tumour cells to become established is not known (see Franks, 1976, for review) but the recent reports of Owens and collaborators (Owens, 1974; Owens et al., 1974; Owens et al., 1976) suggest that the proportion of tumours and tissues from which lines can be established may sometimes be increased by the use of special selection techniques (but see Franks et al., 1977). These workers and Tom et al. (1976) stress the importance of accurate characterisation of the cell lines, particularly since many cells tend to adopt a common morphological and biochemical pattern as a consequence of adaptation to in-vitro conditions (Wilson, 1973; Franks and Wilson, 1976). If cells retain specific morphological markers, as in the cell line we describe, identification is easy but in many cases evidence for identification seems to have been restricted to the label on the bottle. SUMMARY
A tissue culture cell line has been established from one out of seven induced carcinomas of mouse large intestine. The cells have an epithelial morphology and form tumour acini in vitro. Electron microscopy shows that the cells have retained some differentiated characters, including junctional complexes and microvilli with attached glycoprotein strands both in the acini and at the free margins of the cell sheets. Our thanks are due to Mr G. Leach and the Photographic Department of the Imperial Cancer Research Fund for the photographic prints. REFERENCES M. M., JONES,J. S . P., AND LANGMAN, M. J. S. 1973. CellBALDWIN,R. W., EMBLETON, mediated and humoral immune reactions to human tumours. Znt. J. Cancer, 12, 73. DEFRIES, E. A. 1976. The effects of age and carcinogen treatment on adult mouse colon epithelium. Ph.D. thesis, University of London. EGAN,M. L., AND TODD,C. W. 1972. Carcinoembryonicantigen: Synthesis by a continuous line of adenocarcinoma cells. J. natl. Cancer Znst., 49, 887. FOGH, J., AND TREMPE, G. 1975. New human tumor cells lines. In Human tumor cells in vitro, edited by J. Fogh, Plenum Press, New York, pp. 115-160. FRANKS, L. M. 1976. Cell and organ culture techniques applied to the study of carcinoma of colon and rectum. Path. Europ., 11, 167. FRANKS, L. M., HAMILTON, E., AND HEMMINGS, V. J. 1977. " Epithelial " foci of presumptive neural origin in cultures of normal mouse colon. J. Path., 124, 19-22. FRANKS, L. M., AND WILSON,P. D. 1977. Origin and ultrastructure of cells in vitro. Int. Rev. Cytol., 48, 55. GHOSE, T., NAIRN,R. C., AND CERINI,M. 1968. Persistence of gastrointestinal-specific antigens in primary culture of human colon epithelium. Exptl. Cell Res., 49, 513. GOLDENBERG, D. M., PAVIA,R. A., BANSEN, H. J., AND VENDEVOORDE, J. P. 1972. Synthesis of carcinoembryonic antigen in vitro. Nature (New Biol.), 239, 189.
38
L. M. FRANKS AND VALERIE J. HEMMZNGS
KASTEN, F. H., STRASSER, F. F., AND TURNER, M. 1965. Nucleolar and cytoplasmic ribonucleic acid inhibition by excess thymidine. Nature, 207, 161. MARTIN,F., KNOBEL, S., MARTIN, M., AND BORDES, M. 1975. A carcinofetal antigen located on the membrane of cells from rat intestinal carcinoma in culture. Cancer Res., 35, 333-337. NELSON-REES, W. A., AND FLANDERMEYER, R. R. 1976. HeLa cultures defined. Science, 191, 96. OWENS,R. B. 1974. Glandular epithelial cells from mice: A method for selective cultivation. J. natl. Cancer Znst., 52, 1375. OWENS,R. B., SMITH,H. S., AND HACKEIT,A. J. 1974. Epithelial cell cultures from normal glandular tissue of mice. J. natl. Cancer Znst., 53, 261. OWENS, R. H., SMITH,H. S., NELSON-REES, W. A., AND SPRINGER, E.L. 1976. Epithelial cell cultures from normal and cancerous human tissues. J . natl. Cancer Znst., 56, 843. TAN,M. H., HOYOKE, E. D., AND GOLDROSEN, M. H. 1976. Murine colon adenocarcinomas: Methods for selective culture in vitro. J. natl. Cancer Znst., 56, 871. TOM,B. H., RUTZKY,L. P., JAKSTYS, M. M., OYASU,R.,KAYE,C. I., AND KAHAN, B. D. 1976. Human colonic adenocarcinoma cells. 1. Establishment and description of a new line. In Vitro, 12, 180. TOMPKINS, W. A. F., WATRACH, A. M., SCHMALE, J. D., SCHULTZ, R.M., AND HARRIS,J. A. 1974. Cultural and antigenic properties of newly established cell strains derived from adenocarcinomas of the human colon and rectum. J. natl. Cancer Inst., 52, 1101. WILSON, P. D. 1973. Reversible and irreversible effects of tissue culture on enzyme patterns of spontaneous mouse tumours and mouse and human embryo tissues. Cancer Res., 33, 375.
L. M. FRANKS AND VALERIE J. HEMMINGS Department of Cellular Pathology, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, UK
PLATESXXI-XXIII
I N an accompanying paper (Franks et al., 1977) we have described the results obtained with cultures from normal mouse colon. In this paper we report on the culture of seven induced mouse tumours of large intestine and the establishment of a cell line from one of them. MATERIALS AND METHODS 1,2-dimethylhydrazine or methyl azoxymethanol acetate (MAM acetate) were used to induce tumours of colon and rectum in C57BL ICRF a' mice. Details are given by Defries (1976). The cell line described in this paper was established from a polypoid carcinoma of rectum from a 19-mth-old male mouse given four intraperitoneal injections of MAM acetate (each 0.2 mg in saline) at weekly intervals from 3 wk of age. Standard explant cultures (about 1 mm3) from six primary tumours and one transplanted rectal tumour were set up in plastic Petri dishes (Nunc (UK) Limited, Stafford, UK), in Waymouth's medium 752/1 with 15 per cent. calf serum, in 5 per cent C02 in air at 37°C. Medium was changed every 3 or 4 days using 10 per cent. calf serum. Selective trypsinisation and pipetting were used to remove " fibroblasts ". Details are given in the text. For electron microscopy, cells were fixed and processed as described previously (Franks et al., 1977). Cells are stored in liquid nitrogen in 10 per cent. dimethyl sulphoxide in culture medium.
RESULTS Tumour tissue was finely chopped with fine curved scissors and rinsed four times in culture medium containing 400 units/ml penicillin, 400 pg/ml streptomycin sulphate and 200 units/ml mycostatin (E. R. Squibb and Sons Ltd, Merseyside, UK) About 20 1-mm explants were plated into 50 mm Petri dishes in medium with antibiotics at one-quarter of the above concentrations. Cultures from four of the primary turnours were contaminated in the initial dishes and were discarded. Cultures from the remaining two tumours were maintained for five and nine transfer generations respectively. In these there was a progressive build up of a fungal infection and the cultures were eventually discarded. The cell line to be described (CMT93) was established from the fourth in-vivo transplant generation of a rectal carcinoma. Ten 50 mm Petri dishes were each innoculated with about 20 explants. Medium was changed Received 10 Feb. 1977; accepted 13 June 1977. J. PATH.-VOL.
124 (1978)
35
36
L. M. FRANKS AND VALERIE J. HEMMINGS
after 3 days. By 6 days clearly recognisable epithelial and mesenchymal cells had grown out from the explants. On days 7, 13, 20 and 32 the dishes were trypsinised and mesenchymal cells pipetted free, leaving predominantly epithelial cells attached to the substrate. Mesenchymal cell lines were established from the cells which were removed. On day 32 after initial trypsinisation to remove mesenchymal cells the epithelial cells from one dish were removed by a second trypsinisation and transferred to two Petri dishes. These cells gave rise to mixed epithelial and mesenchymal cell culture. After a further 24 days mesenchyme was removed by trypsinisation and the epithelium removed in the same way as before and transferred. The second transfer also produced a mixed culture, which was treated in the same way after 21 days. The cells in the third and subsequent passages were epithelial in morphology (figs. 1 and 2) and mesenchymal cells could not be recognised. At this stage the growth rate increased and the cells reached confluence after a 1 to 2 split in about 7 days. Since the cells grow in coherent clumps it is difficult to prepare single-cell suspensions for accurate cell counting, although the clumps can be removed for transfer by trypsinisation. Electron microscopy showed that the cells were closely packed and varied in shape, size and electron density (figs. 3. and 4). Many had no specific features but in some areas acini were formed (fig. 4). The cells forming these acini and the cells at the free margins of the clumps still retained some of the differentiated characters of rectal epithelium. Junctional complexes were present, including true desmosomes (fig. 5) with a central electron-dense lamella and associated 10 nm filaments. The lumenal microvilli had a central core of fibrils and glycoprotein strands attached to the external lamella of the plasma membrane (figs. 6 and 7). Virus-like particles (C-type) were present in some of the cells. The overall pattern was similar in ultrastructure to that of the tumour from which the line was derived. The cells have been maintained for 16 transfer generations and are stored in liquid nitrogen. DISCUSSION In-vitro systems have not been used to any great extent in work on the aetiology or development of cancers of the large intestine, mainly because of the technical problems involved (see Franks, 1976, for review). Although shortterm cultures can be established from many tumours, including gut tumours (Ghose et al., 1957; Baldwin et al., 1973) these are almost invariably mixed cultures and most cannot be maintained indefinitely. However, six human cell lines (Kasten et al., 1965; Egan and Todd, 1972; Goldenberg et al., 1972; Tompkins et al., 1974; Fogh and Trempe, 1975; Tom et al., 1976) have been reported, although one (Kasten et al., 1965) is now thought to be contaminated with HeLa cells (Nelson-Rees and Flandermeyer, 1976). Martin et al. (1975) have established five cell lines from induced transplantable rat tumours (three colon and two duodenum) and Tan et al. (1976) have described two lines derived from dimethyl hydrazine-induced colon carcinomas in mice. Since the establishment of cell lines from tumours seems to be a rare event it is important
PLATE XXI
FRANKS AND HEMMINGS OF MOUSE RECTUM CARCINOMA
FIG. 1.
FIG.2.
Fro. 1.-Living FIG. 2.-A
culture (fourth transfer) CMT93 cells showing " epithelial " morphology. Phase contrast. x 84. higher power of the cells at the edge of a clump showing columnar shape and rather indefinite free margin. Phase contrast. x 280.
FIG.3.-Sheet of cells (fourth transfer) showing closely packed cells and microvilli along free margin. Junctional complexes can just be made out as darker lines between cells near the margin. x 2000.
PLATE XXII
FRANKS A N D HEMMINGS
CARCINOMA OF MOUSE RECTUM
FIG.4.-Part
of a cell sheet showing acinus formation with lumenal microvilli and dark junctional complexes near the lumen. The cells vary in electron density. x 10,OOO.
FRANKS AND HEMMINGS
PLATE XXIlI
CARCINOMA OF MOUSE RECTUM
FIG.5.
FIG.6.
FIG.5.-Part of a junctional complex from cells in fig. 3 showing two desmosomes with central electron-dense lamellae. x 144,000. FIG.6.-The free margin of cells in fig. 3 showing microvilli and attached glycoprotein strands x 30,000. FIG.7.-Lumenal margin of cells from an “acinus ” showing microvilli with central filaments and attached glycoprotein strands. x 118,OOO.
FIG.7.
CARCINOMA OF MOUSE RECTUM
37
that several different lines should be available for study since the established lines are derived from such a small number of tumours and may not be representative of the whole tumour group. The element of selection is emphasised by the fact that the one cell line which we have established was derived from only one out of ten Petri dishes originally set up, each containing at least 20 explants. The reasons for the failure of most normal and tumour cells to become established is not known (see Franks, 1976, for review) but the recent reports of Owens and collaborators (Owens, 1974; Owens et al., 1974; Owens et al., 1976) suggest that the proportion of tumours and tissues from which lines can be established may sometimes be increased by the use of special selection techniques (but see Franks et al., 1977). These workers and Tom et al. (1976) stress the importance of accurate characterisation of the cell lines, particularly since many cells tend to adopt a common morphological and biochemical pattern as a consequence of adaptation to in-vitro conditions (Wilson, 1973; Franks and Wilson, 1976). If cells retain specific morphological markers, as in the cell line we describe, identification is easy but in many cases evidence for identification seems to have been restricted to the label on the bottle. SUMMARY
A tissue culture cell line has been established from one out of seven induced carcinomas of mouse large intestine. The cells have an epithelial morphology and form tumour acini in vitro. Electron microscopy shows that the cells have retained some differentiated characters, including junctional complexes and microvilli with attached glycoprotein strands both in the acini and at the free margins of the cell sheets. Our thanks are due to Mr G. Leach and the Photographic Department of the Imperial Cancer Research Fund for the photographic prints. REFERENCES M. M., JONES,J. S . P., AND LANGMAN, M. J. S. 1973. CellBALDWIN,R. W., EMBLETON, mediated and humoral immune reactions to human tumours. Znt. J. Cancer, 12, 73. DEFRIES, E. A. 1976. The effects of age and carcinogen treatment on adult mouse colon epithelium. Ph.D. thesis, University of London. EGAN,M. L., AND TODD,C. W. 1972. Carcinoembryonicantigen: Synthesis by a continuous line of adenocarcinoma cells. J. natl. Cancer Znst., 49, 887. FOGH, J., AND TREMPE, G. 1975. New human tumor cells lines. In Human tumor cells in vitro, edited by J. Fogh, Plenum Press, New York, pp. 115-160. FRANKS, L. M. 1976. Cell and organ culture techniques applied to the study of carcinoma of colon and rectum. Path. Europ., 11, 167. FRANKS, L. M., HAMILTON, E., AND HEMMINGS, V. J. 1977. " Epithelial " foci of presumptive neural origin in cultures of normal mouse colon. J. Path., 124, 19-22. FRANKS, L. M., AND WILSON,P. D. 1977. Origin and ultrastructure of cells in vitro. Int. Rev. Cytol., 48, 55. GHOSE, T., NAIRN,R. C., AND CERINI,M. 1968. Persistence of gastrointestinal-specific antigens in primary culture of human colon epithelium. Exptl. Cell Res., 49, 513. GOLDENBERG, D. M., PAVIA,R. A., BANSEN, H. J., AND VENDEVOORDE, J. P. 1972. Synthesis of carcinoembryonic antigen in vitro. Nature (New Biol.), 239, 189.
38
L. M. FRANKS AND VALERIE J. HEMMZNGS
KASTEN, F. H., STRASSER, F. F., AND TURNER, M. 1965. Nucleolar and cytoplasmic ribonucleic acid inhibition by excess thymidine. Nature, 207, 161. MARTIN,F., KNOBEL, S., MARTIN, M., AND BORDES, M. 1975. A carcinofetal antigen located on the membrane of cells from rat intestinal carcinoma in culture. Cancer Res., 35, 333-337. NELSON-REES, W. A., AND FLANDERMEYER, R. R. 1976. HeLa cultures defined. Science, 191, 96. OWENS,R. B. 1974. Glandular epithelial cells from mice: A method for selective cultivation. J. natl. Cancer Znst., 52, 1375. OWENS,R. B., SMITH,H. S., AND HACKEIT,A. J. 1974. Epithelial cell cultures from normal glandular tissue of mice. J. natl. Cancer Znst., 53, 261. OWENS, R. H., SMITH,H. S., NELSON-REES, W. A., AND SPRINGER, E.L. 1976. Epithelial cell cultures from normal and cancerous human tissues. J . natl. Cancer Znst., 56, 843. TAN,M. H., HOYOKE, E. D., AND GOLDROSEN, M. H. 1976. Murine colon adenocarcinomas: Methods for selective culture in vitro. J. natl. Cancer Znst., 56, 871. TOM,B. H., RUTZKY,L. P., JAKSTYS, M. M., OYASU,R.,KAYE,C. I., AND KAHAN, B. D. 1976. Human colonic adenocarcinoma cells. 1. Establishment and description of a new line. In Vitro, 12, 180. TOMPKINS, W. A. F., WATRACH, A. M., SCHMALE, J. D., SCHULTZ, R.M., AND HARRIS,J. A. 1974. Cultural and antigenic properties of newly established cell strains derived from adenocarcinomas of the human colon and rectum. J. natl. Cancer Inst., 52, 1101. WILSON, P. D. 1973. Reversible and irreversible effects of tissue culture on enzyme patterns of spontaneous mouse tumours and mouse and human embryo tissues. Cancer Res., 33, 375.
