Immunology
and Cell Biology ([991) 69. 2 0 5 - 2 1 3
A human colon cancer cell line established from collagen matrix cultures transplanted into nude mice M. V. AGREZ/ F. K. CHUA,* K. FAGAN,** J. W. HEATH* AND N. W. FERGUSON^ 'Stirgical Science and Anatomy. Faculty of Medicine. University of Newcastle and Departments of ''Anatomical Pathology and *'Cytogenetic.s. John Hunter Hcspital Newcastle, New South Wales. Australia (Submitted J May 1990. .Accepted for publication 6 June 1991.) Summary A new human colon cancer cell line (020588) has been derived by means of a combined in vitro matrix-f/i vivo xenograft technique. The tumour cell line is carcino-embryonic antigen positive, displays a marker chromosome and proliferates in chemically-defined scrum-free culture medium. The chemosensilivity pattern for the tumour cell line was similar to that observed for the parent tumour cells. The novel method used to establish this continuous human tumour cell line may have several advantages over standard techniques.
INTRODUCTION The identification of cellular and molecular mechanisms which regulate growth ofcolorec'al cancer in individual patients will be determined. at least in part, by the development of reliable and efficient means of establishing continuous human cell lines which display the same phenotypic characteristics as those of their parent tumours. Although the majority of primary colorectal cancer specimens are not adaptable to continuous in vitro passage using standard culture techniques, primary cultivation of tumour cells on collagen gels has been shown to facilitate the establishment of cell lines from less aggressive colonic neoplasms (1-4). An alternative approach is to develop cell lines from tumours which have been passaged as xenografts in immune-deficient mice and many researchers have found this model useful as a means to establish both colonic and other tumour cell lines in vitro (5-8). The present report describes a combined in vitro-in vivo technique in which tritlated thymidine incorporation by tumour cells was first determined in a collagen matrix culture system prior to transplantation of populated collagen gels into athymic nude mice. Subsequent conversion of the tumour xenograft to monolayer culture in vitro rapidly
yielded an adherent cell line which closely resembled the parent tumour and which proliferated in chemically-defined serum-free culture medium. MATERIALS AND M E T H O D S Tumour cells The tumour tissue was removed from the resected specimen of a poorly differentiated. Dukes' stage C colonic cancer and transported to the laboratory in standard tumour medium comprising Dulbecco's Modified Eagle's Medium (DMEM, Flow Laboratories, Sydney) supplemented with glutamine, insulin, hydrocortisone, transferrin, selenium, non-essential amino acids, 2-mercaptoethanol, antibiotics (penicillin, streptomycin, gentamycin and amphotericin P) and 10% fetal calf serum. The solid tumour specimen was minced with cross scalpels and enzymatically digested by incubating the minced tumour for 2 h at 37°C in standard tumour medium containing 0.3% collagenase (Boehringer-Mannheim, IN, USA). The tumour cells released into the medium were centrifuged and resuspended in standard tumour medium, filtered through a 42 \im pore diameter nylon mesh (A. R. Simpson Holdings, Vic. Australia)
Correspondence: Dr M. V. Agrez. Discipline of Surgical Science, Faculty of Medicine. David Maddison Building, Cnr King and Watt Streets, Newcastle, NSW 2300, Australia
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and cell viability estimated with 0.4% Trypan Blue solution. In vitro collagen matrix culture system Preparation of collagen matrices Native type 1 collagen was prepared by acetic acid extraction from rat tail tendons according to the method described by van Bockxmeer and Martin (9)., and protein concentration was estimated according to a modification ofthe Lowry method (Bio-RAD protein microassay (10). Bi-layer collagen gels were prepared in microtitre wells of 96-well microtitration plates (Linbro, Flow Laboratories. Sydney). Cell-free base layers consisted of equal volumes of collagen and 2.2 X concentrate standard culture medium (without fetal calf serum). The pH of the mixture was adjusted to 7.2-7.4 by addition of 1 mol/L sodium hydroxide and the collagen-media mix then supplemented with fetal calf serum. The final mixture containing collagen at a concentration of 1.2 mg/mL and 10% fetal calf serum was dispensed as 50 jiL aliquots into microtitre wells to form base layers, thereby preventing contact between cells and the plastic interface. The cell-containing upper collagen layer was prepared in the same way as the base layer. Tumour cells suspended in fetal calf serum were added to the collagen-media mix. Aliquots comprising 150 \iL of the cell-collagen-media mix were pipetted on top ofthe base layers and cultures incubated at 3T'C for 15 min to allow time for the collagen to gel before the addition of 100 nL of standard culture medium. Cell density and culture conditions
Tumour
cells were seeded into triplicate microtitre wells at a cell density in the range of 10-40X 10-^ viable cells per well. A further set of identical triplicate cultures was prepared at a tumour cell density of 40 X IO-* cells per gel for the purpose of xenograft passage. All gelled cultures within microtitre wells were then 'rimmed' at the plastic interface with a fine needle to facilitate later removal from the wells, and cultures were incubated at 37°C in 5% CO2 and 100% relative humidity for 48 h. With the exception of populated gels intended for xenograft passage, all tumour cells seeded in collagen were exposed to 2 jiCi of [^H]-thymldine (Amersham. Buckinghamshire, England) 24 h after initiation of cultures. Cells were harvested after a further 24 h by dissolution ofthe collagen matrix with 50 ^L of collagenase (Sigma, Cat. No. C5138: 15 mg/mL distilled water) before automated cell harvesting and measurement of radioactivity.
In vivo culture
Under light ether anaesthesia a small subcutaneous pocket, sufficiently large enough to hold a 200 nL gel obtained from one microtitre well, was created in the flanks of athymic randombred Swiss nude mice (Animal Resource Centre, Perth, WA, Australia). After 48 h of in vitro growth, collagen gels were retrieved from microtitre wells by piercing with a hne gauge needle and one gel was transferred into each subcutaneous pocket with re-approximation of the skin using metal staples. Tumour xenografts were harvested 12 weeks later, mechanically disaggregated as described and the released cells and cell aggregates suspended in standard culture medium in 25 cm tissue culture flasks (Coming, New York, NY, USA). Growth characteristics ofthe cell line After the first subculture, standard culture medium was replaced with simplified culture medium comprising DMEM supplemented with glutamine, antibiotics (penicillin and streptomycin) and 10% fetal calf serum. The population doubling time ofthe cefl line established from the tumour xenograft was determined in the log phase of growth after seeding 2 X 10^ viable cells in simplified culture medium into replicate 25 cm^ tissue culture flasks. Cultures were fed every 3 days and cell counts were performed every 3 days for 3 weeks. In order to determine colony forming efficiency ofthe tumour cells within collagen gels, 40-80 X 10-^ cells were seeded into I mL collagen gels in 35 mm diameter culture dishes (Linbro). Replicate gels were prepared at each cell density tested and were overlain with 2 mL of simplified culture medium. Tumour cell eolonies >60 pm in diameter were enumerated after 3 weeks in culture using a Leitz inverted microscope fitted with a graticule in the eyepiece. The proliferative capacity of the tumour cell line was also assessed in collagen gel cultures prepared within microtitre wells as described for the primary tumour cell suspension. Uptake of [^H]-thymidine by cells cultured in collagen, and as an adherent monolayer on plastic, was compared in the absence and presence of fetal calf serum. In experiments performed under serumfree conditions, the cell line growing as an adherent monolayer in flask cultures was first harvested by exposure to 0.05% trypsin/0.02% ethylenediamine tetracetic acid (EDTA, Flow Laboratories) and washed once in simplified
USE OF COLLAGEN GELS TO ESTABLISH A TUMOUR CELL LINE
culture medium. The cell preparation was then washed three times in serum-free medium (standard tumour medium without fetal calf serum) before resuspension in serum-free medium and estimation of cell viability. Cells were seeded in collagen at a cell density of 2 X 10^ viable cells per well and pulsed for 24 h with l|iCi of [^H]thymidine before cell harvesting. Tumour cells intended for monolayer culture were seeded at the same cell density in 300 ^lL of culture medium per well apd were detached at the termination of experiments using 0.05% trypsin/ 0.02% EDTA solution. Monolayer flask cultures ofthe cell line were also maintained under serum-free conditions for periods of up to I year. This was achieved by means of repeated subculture at 3-4 week intervals with a 1:4 split at the time of each subculture. Subculture was performed by harvesting the cells with 0.05% trypsin/0.2% BDTA and incubating the tumour eell pellet at 37°C for 30 min in 0.1% soybean trypsin inhibitor (Sigma Chemical Company, St Louis. MO, USA) in serum-free medium. The tumour cells were then washed three times in serum-free medium before reseeding into 75 cm^ tissue culture flasks (Flow Laboratories). In vitro chemosensitivity testing Triplicate gels containing the primary tumour cells were prepared as described at a cell density of 30 X 10^ cells per wefl for testing against 5fluorouracil (final concentration in the well in the range of O.I-IO ^lg/mL) and mitomycin C (final concentration 0.4 |ig/mL). At the initiation of cultures. 25 ^L of standard culture medium containing the drug was added to each well so that exposure of cells to drugs was continuous for the duration ofthe experiment. In vitro chemosensitivity testing ofthe tumour cell line embedded in collagen matrices was performed in an identical manner to that described for the original tumour cell suspension, except for the cell density which was 2 X 10^ viable cells per gel. The cells were harvested after 48-72 h and drug effects, if any. were expressed as percentage inhibition of [^H]-thymidine uptake relative to control cultures. Tumorigenicity and immunostaining Tumorigenicity of the cell line was tested by inoculating 10^ cells subcutaneously into one flank of each of three athymic nude mice. Tumour xenografts were harvested 8 weeks later
207
and formalin-fixed paraffin-embedded sections were stained with haematoxylin and eosin. alcian blue, mucicarmine, and PAS-Diastase. Fixed tissue preparations of tumour xenografts derived from the cell line were exposed to antibodies against cytokeratins (39-59 kD, Beeton Dickinson, CA, USA and 48-61 kD Dako Corp.. CA, USA), vimentin (Dako), epithelial menibrane antigen (Biogenics, CA, USA) and carcino-embryonic antigen (Dako), and stained with peroxidase-conjugated secondary antibody according to standard techniques. Immunohistochemical staining of the primary tumour specimen and the established tumour cell line was carried out in the same manner.
Electron microscopy Tumour cells were fixed in situ in the culture dish in 5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer for 1 h. washed in buffer and post-fixed in 1% osmium tetroxide for I h. They were dehydrated in graded alcohols, infiltrated into 2-hydroxypropyl methacrylate. and embedded in Epon 812. Thin sections were cut with Diatome diamond knives, double-stained with uranyl acetate and lead citrate, and examined in a JEOL 100 CX transmission electron microscope.
Karyotype determination Chromosomes were produced according to the method described by Gibas (11). In brief, tumour cells growing as an adherent monolayer were re-fed with fresh growth medium 24 h prior to the addition of Colcemid (0.5 ng/niL: Grand Island Biological Co., NY, USA). One to four hours later they were harvested by subjecting the cells to hypotonic treatment directly in the culture flasks, i.e. the medium was discarded and the hypotonic solution (pH 7.4) containing KCI (3 g/L), EDTA (0.2 g/L) and HEPES (4.8 g/L) was added for 30 min at 37°C. This suspension was transferred to a test tube, centrifuged at 1000 rev/min for 5 min and resuspended in fixative (3 methanol : 1 glacial acetic acid). After three further changes of fixative, slides were prepared by dropping the cell suspension onto cold, wet slides. Trypsin-Giemsa banding was performed and the cell line was categorized by the karyotypic pattern present in the majority of cells.
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RESULTS In vitro-in vivo culture system The ability ofthe primary tumour cells to incorporate [3H]-thymidine when cultured within 3dimensional collagen gels for 48 h is shown in Fig. I. Linearity was observed between increasing cell number seeded and DNA synthesis as estimated by [^HJ-thymidine uptake. Transplantation ofthe collagen gels into the flanks of athymic mice once active DNA synthesis in vitro had been established resulted in the development of subcutaneous tumours > 1 cm in diameter after 3 months. Tumour cells obtained by mechanical disaggregation ofthe tumour xenograft adapted rapidly to growth in vitro as an adherent monolayer of tightly packed polygonal cells (Fig. 2) and the morphology ofthe cells remained unchanged during repeated subculture over a 12 month period. The tumour cefl line, designated 020588, was repeatedly tested at 3 monthly intervals for the presence of myco-
20
30
40
Cell numbnr x
Fig. I. Uptake of [^HJ-thymidine by primary tumour cells seeded at increasing cell density within collagen gels and cultured for 48 h.
i
J
100 jum
Fig. 2. Phase contrast photomicrograph of 020588 cells showing a colony of small pleomorphic cells growing as an adherent monolayer on plastic (left). Photomicrograph of 020588 cells after 21 days in culture showing a tightly packed colony derived from a single cell suspension seeded within a collagen gel (right). Magnification identical to the colonies shown growing as an adherent monolayer.
USE OF COLLAGEN GELS TO ESTABLISH A TUMOUR CELL LINE
209
plastic was 48 h. In three-dimensional collagen matrices the tumour cells formed tightly packed colonies, as shown in Fig. 2. with a plating efficiency of 2%. Thymidine incorporation by the cell line during 10 days in culture is shown in Fig. 3. In the presence of fetal calf serum [-''H]thymidine uptake was similar for cells cultured either as an adherent monolayer on plastic or within a collagen matrix. However, in chemically-defined serum-free medium, thymidine uptake by cells cultured in collagen ceased after 4 days as shown in Fig. 3. In contrast, DNA synthesis by the tumour cells, growing as an adherent monolayer in the absence of fetal calf serum, approximated that observed in the presence of serum factors after 10 days in culture. This was consistent with our observation that the cell line growing in serum-free medium could be subcultured indefinitely (1 year) by means of 1:4 split from flask cultures at regular intervals. Fig. 3. Uptake of [^Hl-thyraidine by 020588 cells seeded at a cell density of 2 X 10-'' cells within collagen gets and on plastic, and cultured for 10 days. [^H]thymidine uptake: monolayer growth on plastic in the presence of fetal calf serum ( ——) monolayer growth on plastic in chemically-defined serum-free medium (—A—); growth in collagen matrices in the presenceof fetal calf serum { o ); and growth in collagen matrices in chemically-defined, serum-free
Exposure of the cell line to 5-fluorouraciI (10 M-g/mL) and mitomycin C (0.4 |jg/mL) resulted in >80% inhibition of [^HJ-thymidine uptake for both agents and reflected the chemosensitivity patterns observed for the original primary tumour cell suspension as shown in Table 1.
medium
Histochemistry and morphology
(••-••).
plasma according to the method of Chen and was free of contamination (12). Growth characteristics of the cell line
The cell doubling time of 020588 cells during the log phase of growth as an adherent monolayer on
The parent tumour stained positively for mucin and heterogeneous staining was observed for epithelial membrane antigen, cytokeratin and carcino-embryonic antigen. These heterogeneous staining patterns were maintained in thetumour xenograftand cell line derived therefrom as shown for carcino-embryonic antigen (Fig. 4). Vimentin was not identified in any of
Table I Chemosensitivity patterns for the primary tumour cell suspension and the established eell line. Primary tumour cell suspension (3X 10^ cells/gel) Control 5-Fluorouracil 0.1 j^g/mL 1 |ig/mL Mitomycin C 0.4 fig/mL
Established cell line (2X103 cells/gel)
5436 ±227
10984 ± 480
7582 ± 286(0)%* 1555±9(71%) 1020± 177(81%)
16259 ±894(0%) 4120 ±234 (62%) 1545 ±215 (86%)
1403± 148 (74%)
1521 ± 2 3 (86%)
Results expressed as mean value for [^H]-thymidine uptake in triplicate gels± s.e.m. •Percentage inhibition = (control count-drug count) control count
210
M.V. AGRE2£7/(L.
Fig. 4. Immunostaining for carcino-embryonie antigen expressed by 020588 cells cultured as an adherent monolayer on plastic.
the preparations. At the ultrastructural level, the cell line resembled typical epithelial cells with abundant microvilli projecting from the cell surface (Fig. 5). Karyotype
Fifty well-spread metaphases were counted and two karyotype patterns defined, one near diploid(--80%)andonene3rtetraploid(~20%of cells), with 82-88 chromosomes present. The modal number was 44 chromosomes with the following numerical changes as shown in Fig. 6: additional chromosomes 5, 20 and a marker, monosomy for chromosomes 10, 12, 14. 15, 22 and loss of the Y-chromosome. In addition, consistent structural abnormalities included: del Ip36.l,del3p25,del4q31.8p+, 17+, I8q-and Xq"*. The tetraploid cells showed the above abnormalities in the same proportions.
DISCUSSION The technique of using immune-deficient mice as a means of establishing human tumour cell lines in tissue culture is well recognized and it is thought that adaptation to growth in the nude mouse contributes in some way to the ability of tumour cells to be converted to in vitro culture (8). The histochemical and ultrastructural features of the cell line designated 020588 were consistent with its origin from a colonic carcinoma. Moreover, the chemosensitivity pattern for the tumour cell line reflected that observed for the cells obtained from the parent tumour and cytogenetic analysis showed deletion of the long arm (q) of chromosome 18 which is a consistent Ending in sporadic colorectal cancer (13). There were insufficient cells obtained from the initial tumour specimen to attempt simul-
USE OF COLLAGEN GELS TO ESTABLISH A TUMOUR CELL LINE
•^>ii"
211
"''1^
Fig. 5. Transmission electron microscopy of 020588 culli grown on a plastic substrate. Microvilli (mv) project from the epithelial cell surface into the extracellular environment (E). The plasma membranes of adjacent cells interdigitate closely (arrows), and are joined by desmosomes (arrowheads). Cytoplasmic filaments (f) are abundant. Ribosomes and rough endoplasmic reticulum (R); nucleus (N); vacuole (V); bar= 1 ^m. The microvilli contain fine filaments. Arrow indicates glycocalyx, bar = 0.5 ^m (inset).
taneous establishment of the cell line using standard techniques. Common problems encountered in establishing cell lines using standard culture methods include fibroblast overgrowth, microbial contamination and a requirement for large numbers of viable tumour cells (3,5.6,14). Possible advantages of the combined in vitro-in
vivo method reported in the present study include: the inability of colon fibroblasts to proliferate within three-dimensional collagen gels (15); the identification of microbial contamination within populated collagen gels prior to xenograft passage; and a requirement for cell number approximating one-hundredth of that
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Fig. 6. Karyotype: modal number 44; additional chromosomes 5. 20 and a marker; monosomy lor chromosomes 10, 12. 14, 15. 22 and loss of the Y-chromosome; del Ip36.!,del 3p25, del 4q3I, 8p + . I7p + , I8q^ and Xq + .
normally required to establish either human colorectal cancer xenografts or in vitro cell lines (6,7). One other attempt to generate a tumour xenograft from populated collagen gels based on uptake of tritiated thymidine by the primary tumour cells has also been successful (to be reported). It has to be recognized, however, that tumour cells have a lag phase when first established in culture and lack of significant thymidine incorporation during the first 48 h may not necessarily militate against the successful establishment ofa collagen gel xenograft. It remains to be established whether the routine use of a short-term collagen matrix assay could optimize selection of tumour specimens intended for xenograft passage. The new colon cancer cell line reported herein is able to proliferate indefinitely in the absence of serum and thus may offer a useful tool with which to study autocrine growth-regulating mechanisms in bowel cancer.
ACKNOWLEDGEMENTS This study was jomtly supported by a Royal Australasian College of Surgeons Foundation Research Grant, the New South Wales State Cancer Council and the Jane Durbin Trust Fund, Royal Newcastle Hospital, Australia.
REFERENCES 1. McBain. J. A.. Weese, J. L., Meisner, L. F., Wolberg W. H. and Willson, J. K. V. 1984. Establishment and eharactcrisation of human coloreetal eancer cell lines. Cancer Res. 44: 5813-5821. 2. Willson, J, K. V.. Bittner. G. N.. Oberley, T. D. and Weese, J. L. 1985. Primary and continuous eullure of human colonic adenomas, carcinomas and metastases. Amer. Assoc. Cancer Res. 26: 24.
USE OF COLLAGEN GELS TO ESTABLISH A TUMOUR CELL LINE 3. Leibovitz. A., Stinson. J. C . McCombs, W. B.. HI, McCoy, C. E., Mazur. K. C. and Mabry, N. D. 1976. Classification of human colcrectal adenocarcinoma cell lines. Cancer Res. 36: 45624569. 4. Willson, J. K. V., Bittner, G. N., Oberley. T. D.. Meisner, L. F. and Weese. J. L. 1987. Ceil culture of human colon adenomas and carcinomas. Cancer Re.s. 47: 2704-2713. 5. van der Bosch. J., Masui, H. and Sato. G. 1981. Growth characteristics of primary tissue cultures from heterotransplanted human colorectal carcinomas in serum-free medium. Cancer Res. 41: 611-681. 6. Park, J-G., Oie, H. K., Sugarbaker, P. H. ei al. 1987. Characteristics of cell lines established from human colorccta! carcinoma. Cancer Res. 47: 6710-6718. 7. Walton, J.. Winterbourne, D., Fiennes. A.. Harris. P.. Hermon-Taylor, J. and Grant. A. 1985. Human tumour cell lines established in vitro from tumours after long-term passage as nude mouse xenografts. Comparative fingerprinting of their Concanavalin-A acceptor glycoproteins Br. J. Cancer 51: 675-680. 8. Rae-Venter, B. and Reid, L. M. 1980. Growth of human breast carcinomas in nude mice and
9.
10.
11. 12.
13. 14. 15.
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subsequent establishment in tissue culture. Cancer Res. 40: 95-100. van Bockxmeer, F. M. and Martin, C. E. 1982. Measurement of cell proliferation and cell mediated contraction in three-dimensional hydrated collagen matrices. / Tiss. Cull. Meth. 7: 163-7. Lowry, O. H., Rosebrough, N. J,, Farr, A. L. and Randall, R. J. 1951. Protein measurement with the Folin Phenol Reagent. / Biol. Chem. 193: 265-75. Gibas, L. M., Gibas, Z. and Sandberg, A. A. 1984. Technical aspects of cytogenetic analysis of human solid tumours. Karyograin 10: 25-27. Chen, T. R. 1975. Microscopic demonstration of mycoplasma contamination in cell cultures and cell culture media. Tissue Cull. Assoc. Manual I: 229-232. Kern, S. E.. Fearon, E. R., Tersmette, K. W. F. el al. 1989. Allelic loss in colorectal carcinoma. JA.M.-\ 261: 3099-3103. Franks, L. M. 1976. Cell and organ culture techniques applied to the study of carcinoma of colon and rectum. Palhol. Eur. 11: 167-177. Agrez, M. V. 1989. Human colon cancer and fibroblast cell lines cultured in and on collagen gels. Ausl. NZJ. Surg. 59: 415-420.
and Cell Biology ([991) 69. 2 0 5 - 2 1 3
A human colon cancer cell line established from collagen matrix cultures transplanted into nude mice M. V. AGREZ/ F. K. CHUA,* K. FAGAN,** J. W. HEATH* AND N. W. FERGUSON^ 'Stirgical Science and Anatomy. Faculty of Medicine. University of Newcastle and Departments of ''Anatomical Pathology and *'Cytogenetic.s. John Hunter Hcspital Newcastle, New South Wales. Australia (Submitted J May 1990. .Accepted for publication 6 June 1991.) Summary A new human colon cancer cell line (020588) has been derived by means of a combined in vitro matrix-f/i vivo xenograft technique. The tumour cell line is carcino-embryonic antigen positive, displays a marker chromosome and proliferates in chemically-defined scrum-free culture medium. The chemosensilivity pattern for the tumour cell line was similar to that observed for the parent tumour cells. The novel method used to establish this continuous human tumour cell line may have several advantages over standard techniques.
INTRODUCTION The identification of cellular and molecular mechanisms which regulate growth ofcolorec'al cancer in individual patients will be determined. at least in part, by the development of reliable and efficient means of establishing continuous human cell lines which display the same phenotypic characteristics as those of their parent tumours. Although the majority of primary colorectal cancer specimens are not adaptable to continuous in vitro passage using standard culture techniques, primary cultivation of tumour cells on collagen gels has been shown to facilitate the establishment of cell lines from less aggressive colonic neoplasms (1-4). An alternative approach is to develop cell lines from tumours which have been passaged as xenografts in immune-deficient mice and many researchers have found this model useful as a means to establish both colonic and other tumour cell lines in vitro (5-8). The present report describes a combined in vitro-in vivo technique in which tritlated thymidine incorporation by tumour cells was first determined in a collagen matrix culture system prior to transplantation of populated collagen gels into athymic nude mice. Subsequent conversion of the tumour xenograft to monolayer culture in vitro rapidly
yielded an adherent cell line which closely resembled the parent tumour and which proliferated in chemically-defined serum-free culture medium. MATERIALS AND M E T H O D S Tumour cells The tumour tissue was removed from the resected specimen of a poorly differentiated. Dukes' stage C colonic cancer and transported to the laboratory in standard tumour medium comprising Dulbecco's Modified Eagle's Medium (DMEM, Flow Laboratories, Sydney) supplemented with glutamine, insulin, hydrocortisone, transferrin, selenium, non-essential amino acids, 2-mercaptoethanol, antibiotics (penicillin, streptomycin, gentamycin and amphotericin P) and 10% fetal calf serum. The solid tumour specimen was minced with cross scalpels and enzymatically digested by incubating the minced tumour for 2 h at 37°C in standard tumour medium containing 0.3% collagenase (Boehringer-Mannheim, IN, USA). The tumour cells released into the medium were centrifuged and resuspended in standard tumour medium, filtered through a 42 \im pore diameter nylon mesh (A. R. Simpson Holdings, Vic. Australia)
Correspondence: Dr M. V. Agrez. Discipline of Surgical Science, Faculty of Medicine. David Maddison Building, Cnr King and Watt Streets, Newcastle, NSW 2300, Australia
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and cell viability estimated with 0.4% Trypan Blue solution. In vitro collagen matrix culture system Preparation of collagen matrices Native type 1 collagen was prepared by acetic acid extraction from rat tail tendons according to the method described by van Bockxmeer and Martin (9)., and protein concentration was estimated according to a modification ofthe Lowry method (Bio-RAD protein microassay (10). Bi-layer collagen gels were prepared in microtitre wells of 96-well microtitration plates (Linbro, Flow Laboratories. Sydney). Cell-free base layers consisted of equal volumes of collagen and 2.2 X concentrate standard culture medium (without fetal calf serum). The pH of the mixture was adjusted to 7.2-7.4 by addition of 1 mol/L sodium hydroxide and the collagen-media mix then supplemented with fetal calf serum. The final mixture containing collagen at a concentration of 1.2 mg/mL and 10% fetal calf serum was dispensed as 50 jiL aliquots into microtitre wells to form base layers, thereby preventing contact between cells and the plastic interface. The cell-containing upper collagen layer was prepared in the same way as the base layer. Tumour cells suspended in fetal calf serum were added to the collagen-media mix. Aliquots comprising 150 \iL of the cell-collagen-media mix were pipetted on top ofthe base layers and cultures incubated at 3T'C for 15 min to allow time for the collagen to gel before the addition of 100 nL of standard culture medium. Cell density and culture conditions
Tumour
cells were seeded into triplicate microtitre wells at a cell density in the range of 10-40X 10-^ viable cells per well. A further set of identical triplicate cultures was prepared at a tumour cell density of 40 X IO-* cells per gel for the purpose of xenograft passage. All gelled cultures within microtitre wells were then 'rimmed' at the plastic interface with a fine needle to facilitate later removal from the wells, and cultures were incubated at 37°C in 5% CO2 and 100% relative humidity for 48 h. With the exception of populated gels intended for xenograft passage, all tumour cells seeded in collagen were exposed to 2 jiCi of [^H]-thymldine (Amersham. Buckinghamshire, England) 24 h after initiation of cultures. Cells were harvested after a further 24 h by dissolution ofthe collagen matrix with 50 ^L of collagenase (Sigma, Cat. No. C5138: 15 mg/mL distilled water) before automated cell harvesting and measurement of radioactivity.
In vivo culture
Under light ether anaesthesia a small subcutaneous pocket, sufficiently large enough to hold a 200 nL gel obtained from one microtitre well, was created in the flanks of athymic randombred Swiss nude mice (Animal Resource Centre, Perth, WA, Australia). After 48 h of in vitro growth, collagen gels were retrieved from microtitre wells by piercing with a hne gauge needle and one gel was transferred into each subcutaneous pocket with re-approximation of the skin using metal staples. Tumour xenografts were harvested 12 weeks later, mechanically disaggregated as described and the released cells and cell aggregates suspended in standard culture medium in 25 cm tissue culture flasks (Coming, New York, NY, USA). Growth characteristics ofthe cell line After the first subculture, standard culture medium was replaced with simplified culture medium comprising DMEM supplemented with glutamine, antibiotics (penicillin and streptomycin) and 10% fetal calf serum. The population doubling time ofthe cefl line established from the tumour xenograft was determined in the log phase of growth after seeding 2 X 10^ viable cells in simplified culture medium into replicate 25 cm^ tissue culture flasks. Cultures were fed every 3 days and cell counts were performed every 3 days for 3 weeks. In order to determine colony forming efficiency ofthe tumour cells within collagen gels, 40-80 X 10-^ cells were seeded into I mL collagen gels in 35 mm diameter culture dishes (Linbro). Replicate gels were prepared at each cell density tested and were overlain with 2 mL of simplified culture medium. Tumour cell eolonies >60 pm in diameter were enumerated after 3 weeks in culture using a Leitz inverted microscope fitted with a graticule in the eyepiece. The proliferative capacity of the tumour cell line was also assessed in collagen gel cultures prepared within microtitre wells as described for the primary tumour cell suspension. Uptake of [^H]-thymidine by cells cultured in collagen, and as an adherent monolayer on plastic, was compared in the absence and presence of fetal calf serum. In experiments performed under serumfree conditions, the cell line growing as an adherent monolayer in flask cultures was first harvested by exposure to 0.05% trypsin/0.02% ethylenediamine tetracetic acid (EDTA, Flow Laboratories) and washed once in simplified
USE OF COLLAGEN GELS TO ESTABLISH A TUMOUR CELL LINE
culture medium. The cell preparation was then washed three times in serum-free medium (standard tumour medium without fetal calf serum) before resuspension in serum-free medium and estimation of cell viability. Cells were seeded in collagen at a cell density of 2 X 10^ viable cells per well and pulsed for 24 h with l|iCi of [^H]thymidine before cell harvesting. Tumour cells intended for monolayer culture were seeded at the same cell density in 300 ^lL of culture medium per well apd were detached at the termination of experiments using 0.05% trypsin/ 0.02% EDTA solution. Monolayer flask cultures ofthe cell line were also maintained under serum-free conditions for periods of up to I year. This was achieved by means of repeated subculture at 3-4 week intervals with a 1:4 split at the time of each subculture. Subculture was performed by harvesting the cells with 0.05% trypsin/0.2% BDTA and incubating the tumour eell pellet at 37°C for 30 min in 0.1% soybean trypsin inhibitor (Sigma Chemical Company, St Louis. MO, USA) in serum-free medium. The tumour cells were then washed three times in serum-free medium before reseeding into 75 cm^ tissue culture flasks (Flow Laboratories). In vitro chemosensitivity testing Triplicate gels containing the primary tumour cells were prepared as described at a cell density of 30 X 10^ cells per wefl for testing against 5fluorouracil (final concentration in the well in the range of O.I-IO ^lg/mL) and mitomycin C (final concentration 0.4 |ig/mL). At the initiation of cultures. 25 ^L of standard culture medium containing the drug was added to each well so that exposure of cells to drugs was continuous for the duration ofthe experiment. In vitro chemosensitivity testing ofthe tumour cell line embedded in collagen matrices was performed in an identical manner to that described for the original tumour cell suspension, except for the cell density which was 2 X 10^ viable cells per gel. The cells were harvested after 48-72 h and drug effects, if any. were expressed as percentage inhibition of [^H]-thymidine uptake relative to control cultures. Tumorigenicity and immunostaining Tumorigenicity of the cell line was tested by inoculating 10^ cells subcutaneously into one flank of each of three athymic nude mice. Tumour xenografts were harvested 8 weeks later
207
and formalin-fixed paraffin-embedded sections were stained with haematoxylin and eosin. alcian blue, mucicarmine, and PAS-Diastase. Fixed tissue preparations of tumour xenografts derived from the cell line were exposed to antibodies against cytokeratins (39-59 kD, Beeton Dickinson, CA, USA and 48-61 kD Dako Corp.. CA, USA), vimentin (Dako), epithelial menibrane antigen (Biogenics, CA, USA) and carcino-embryonic antigen (Dako), and stained with peroxidase-conjugated secondary antibody according to standard techniques. Immunohistochemical staining of the primary tumour specimen and the established tumour cell line was carried out in the same manner.
Electron microscopy Tumour cells were fixed in situ in the culture dish in 5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer for 1 h. washed in buffer and post-fixed in 1% osmium tetroxide for I h. They were dehydrated in graded alcohols, infiltrated into 2-hydroxypropyl methacrylate. and embedded in Epon 812. Thin sections were cut with Diatome diamond knives, double-stained with uranyl acetate and lead citrate, and examined in a JEOL 100 CX transmission electron microscope.
Karyotype determination Chromosomes were produced according to the method described by Gibas (11). In brief, tumour cells growing as an adherent monolayer were re-fed with fresh growth medium 24 h prior to the addition of Colcemid (0.5 ng/niL: Grand Island Biological Co., NY, USA). One to four hours later they were harvested by subjecting the cells to hypotonic treatment directly in the culture flasks, i.e. the medium was discarded and the hypotonic solution (pH 7.4) containing KCI (3 g/L), EDTA (0.2 g/L) and HEPES (4.8 g/L) was added for 30 min at 37°C. This suspension was transferred to a test tube, centrifuged at 1000 rev/min for 5 min and resuspended in fixative (3 methanol : 1 glacial acetic acid). After three further changes of fixative, slides were prepared by dropping the cell suspension onto cold, wet slides. Trypsin-Giemsa banding was performed and the cell line was categorized by the karyotypic pattern present in the majority of cells.
M.V. AGREZ ETAL.
208
RESULTS In vitro-in vivo culture system The ability ofthe primary tumour cells to incorporate [3H]-thymidine when cultured within 3dimensional collagen gels for 48 h is shown in Fig. I. Linearity was observed between increasing cell number seeded and DNA synthesis as estimated by [^HJ-thymidine uptake. Transplantation ofthe collagen gels into the flanks of athymic mice once active DNA synthesis in vitro had been established resulted in the development of subcutaneous tumours > 1 cm in diameter after 3 months. Tumour cells obtained by mechanical disaggregation ofthe tumour xenograft adapted rapidly to growth in vitro as an adherent monolayer of tightly packed polygonal cells (Fig. 2) and the morphology ofthe cells remained unchanged during repeated subculture over a 12 month period. The tumour cefl line, designated 020588, was repeatedly tested at 3 monthly intervals for the presence of myco-
20
30
40
Cell numbnr x
Fig. I. Uptake of [^HJ-thymidine by primary tumour cells seeded at increasing cell density within collagen gels and cultured for 48 h.
i
J
100 jum
Fig. 2. Phase contrast photomicrograph of 020588 cells showing a colony of small pleomorphic cells growing as an adherent monolayer on plastic (left). Photomicrograph of 020588 cells after 21 days in culture showing a tightly packed colony derived from a single cell suspension seeded within a collagen gel (right). Magnification identical to the colonies shown growing as an adherent monolayer.
USE OF COLLAGEN GELS TO ESTABLISH A TUMOUR CELL LINE
209
plastic was 48 h. In three-dimensional collagen matrices the tumour cells formed tightly packed colonies, as shown in Fig. 2. with a plating efficiency of 2%. Thymidine incorporation by the cell line during 10 days in culture is shown in Fig. 3. In the presence of fetal calf serum [-''H]thymidine uptake was similar for cells cultured either as an adherent monolayer on plastic or within a collagen matrix. However, in chemically-defined serum-free medium, thymidine uptake by cells cultured in collagen ceased after 4 days as shown in Fig. 3. In contrast, DNA synthesis by the tumour cells, growing as an adherent monolayer in the absence of fetal calf serum, approximated that observed in the presence of serum factors after 10 days in culture. This was consistent with our observation that the cell line growing in serum-free medium could be subcultured indefinitely (1 year) by means of 1:4 split from flask cultures at regular intervals. Fig. 3. Uptake of [^Hl-thyraidine by 020588 cells seeded at a cell density of 2 X 10-'' cells within collagen gets and on plastic, and cultured for 10 days. [^H]thymidine uptake: monolayer growth on plastic in the presence of fetal calf serum ( ——) monolayer growth on plastic in chemically-defined serum-free medium (—A—); growth in collagen matrices in the presenceof fetal calf serum { o ); and growth in collagen matrices in chemically-defined, serum-free
Exposure of the cell line to 5-fluorouraciI (10 M-g/mL) and mitomycin C (0.4 |jg/mL) resulted in >80% inhibition of [^HJ-thymidine uptake for both agents and reflected the chemosensitivity patterns observed for the original primary tumour cell suspension as shown in Table 1.
medium
Histochemistry and morphology
(••-••).
plasma according to the method of Chen and was free of contamination (12). Growth characteristics of the cell line
The cell doubling time of 020588 cells during the log phase of growth as an adherent monolayer on
The parent tumour stained positively for mucin and heterogeneous staining was observed for epithelial membrane antigen, cytokeratin and carcino-embryonic antigen. These heterogeneous staining patterns were maintained in thetumour xenograftand cell line derived therefrom as shown for carcino-embryonic antigen (Fig. 4). Vimentin was not identified in any of
Table I Chemosensitivity patterns for the primary tumour cell suspension and the established eell line. Primary tumour cell suspension (3X 10^ cells/gel) Control 5-Fluorouracil 0.1 j^g/mL 1 |ig/mL Mitomycin C 0.4 fig/mL
Established cell line (2X103 cells/gel)
5436 ±227
10984 ± 480
7582 ± 286(0)%* 1555±9(71%) 1020± 177(81%)
16259 ±894(0%) 4120 ±234 (62%) 1545 ±215 (86%)
1403± 148 (74%)
1521 ± 2 3 (86%)
Results expressed as mean value for [^H]-thymidine uptake in triplicate gels± s.e.m. •Percentage inhibition = (control count-drug count) control count
210
M.V. AGRE2£7/(L.
Fig. 4. Immunostaining for carcino-embryonie antigen expressed by 020588 cells cultured as an adherent monolayer on plastic.
the preparations. At the ultrastructural level, the cell line resembled typical epithelial cells with abundant microvilli projecting from the cell surface (Fig. 5). Karyotype
Fifty well-spread metaphases were counted and two karyotype patterns defined, one near diploid(--80%)andonene3rtetraploid(~20%of cells), with 82-88 chromosomes present. The modal number was 44 chromosomes with the following numerical changes as shown in Fig. 6: additional chromosomes 5, 20 and a marker, monosomy for chromosomes 10, 12, 14. 15, 22 and loss of the Y-chromosome. In addition, consistent structural abnormalities included: del Ip36.l,del3p25,del4q31.8p+, 17+, I8q-and Xq"*. The tetraploid cells showed the above abnormalities in the same proportions.
DISCUSSION The technique of using immune-deficient mice as a means of establishing human tumour cell lines in tissue culture is well recognized and it is thought that adaptation to growth in the nude mouse contributes in some way to the ability of tumour cells to be converted to in vitro culture (8). The histochemical and ultrastructural features of the cell line designated 020588 were consistent with its origin from a colonic carcinoma. Moreover, the chemosensitivity pattern for the tumour cell line reflected that observed for the cells obtained from the parent tumour and cytogenetic analysis showed deletion of the long arm (q) of chromosome 18 which is a consistent Ending in sporadic colorectal cancer (13). There were insufficient cells obtained from the initial tumour specimen to attempt simul-
USE OF COLLAGEN GELS TO ESTABLISH A TUMOUR CELL LINE
•^>ii"
211
"''1^
Fig. 5. Transmission electron microscopy of 020588 culli grown on a plastic substrate. Microvilli (mv) project from the epithelial cell surface into the extracellular environment (E). The plasma membranes of adjacent cells interdigitate closely (arrows), and are joined by desmosomes (arrowheads). Cytoplasmic filaments (f) are abundant. Ribosomes and rough endoplasmic reticulum (R); nucleus (N); vacuole (V); bar= 1 ^m. The microvilli contain fine filaments. Arrow indicates glycocalyx, bar = 0.5 ^m (inset).
taneous establishment of the cell line using standard techniques. Common problems encountered in establishing cell lines using standard culture methods include fibroblast overgrowth, microbial contamination and a requirement for large numbers of viable tumour cells (3,5.6,14). Possible advantages of the combined in vitro-in
vivo method reported in the present study include: the inability of colon fibroblasts to proliferate within three-dimensional collagen gels (15); the identification of microbial contamination within populated collagen gels prior to xenograft passage; and a requirement for cell number approximating one-hundredth of that
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M. V. AGREZ ET AL.
Fig. 6. Karyotype: modal number 44; additional chromosomes 5. 20 and a marker; monosomy lor chromosomes 10, 12. 14, 15. 22 and loss of the Y-chromosome; del Ip36.!,del 3p25, del 4q3I, 8p + . I7p + , I8q^ and Xq + .
normally required to establish either human colorectal cancer xenografts or in vitro cell lines (6,7). One other attempt to generate a tumour xenograft from populated collagen gels based on uptake of tritiated thymidine by the primary tumour cells has also been successful (to be reported). It has to be recognized, however, that tumour cells have a lag phase when first established in culture and lack of significant thymidine incorporation during the first 48 h may not necessarily militate against the successful establishment ofa collagen gel xenograft. It remains to be established whether the routine use of a short-term collagen matrix assay could optimize selection of tumour specimens intended for xenograft passage. The new colon cancer cell line reported herein is able to proliferate indefinitely in the absence of serum and thus may offer a useful tool with which to study autocrine growth-regulating mechanisms in bowel cancer.
ACKNOWLEDGEMENTS This study was jomtly supported by a Royal Australasian College of Surgeons Foundation Research Grant, the New South Wales State Cancer Council and the Jane Durbin Trust Fund, Royal Newcastle Hospital, Australia.
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