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In vitro studies of human tumor and embryonic tissue
In vitro studies of human tumor and embryonic tissue ARTHUR
ALLEN,
TELESFORO ALLAN
RAMON, JACOBSON,
MACLYN Los Angeles,
Tumor
M.D.
E.
WADE,
B.A. M.D. M.D.,
F.A.C.O.G.
California
tissue
obtained from 79 patient as biopsy or surgical specimens has been Control studies were performed to identify the original tumor during several generations of passage and to assess viability and continued virulence during replication. The patient’s tumor tissue cultures were categorized as to origin, cell type, and degree of differentiation. Chemotherapeutic agents in different concentrations and combinations were tested for eficacy on the various categories of tumors. Three salient findings were noted: (1) the almost consistent ability to culture human tumor tissue in the laboratory with reasonable assurance of maintaining its original characteristics as they were in vivo, (2) the variability of each patient’s tumor in its response to chemotherapeutic agents (i.e., one patient’s ovarian carcinoma culture will show greater susceptibility to the same drug than another patient’s culture of identical cell type and grade of ovarian carcinoma), (3) the availability of a technique for taking individual patients’ tumor specimens at operatitle procedures and usirzg the laboratory for culture and sensitivity testing to anticancer drugs.
cultured in vitro.
0 N E o F T H E problems in studying cancer and the susceptibility of tumors to chemotherapeutic agents has been the inadequate availability of laboratory facilities to test an individual patient’s tumor in vitro against various drugs, Th e chief obstacle is the difficulty in growing many types of tumors From the Department of Obstetrics Gynecology, Cedars-Sinai Medical Center, University of California. Los Angeles.
in the usual tissue culture fashion with any degree of certainty of replicating the patient’s own tumor.l In our laboratory we have been able to obviate this problem which has permitted further investigations of the tumors. Growing tumor cells of human tissue in vitro is not new.l, However, elaborate testing and replication studies have been designed to establish adequate control and thus obtain more reliable results. Once reproducible results were obtained consistently, testing with antitumor drugs was possible. Avenues for further investigation were also opened.
and
Supported by Hea!th, Education, and Welfare Grant RR05468, The Olincy Foundation, and The Western Cancer Institute. The Frank Lynch Memorial Essay, presented at the Thirty-ninth Annual Meeting of the Pacific Coast Obstetrical and Qvnecological Society, Harrison Hot Springs, British Columbia. Canada, October 3-7, 1972. Reprint requests: Arthur Allen, M.D., Cedars-Sinai Medical Center, Cedars Lebanon Hospital Division, 4833 Fountain Ave., Los Angeles. California
Methods
Tumor tissue from various types of carcinomas and sarcomas was obtained as sur,gical specimens and cultured in appropriate tissue culture media, either by trypsinization or by the Maitland procedure.* (A small piece
of
90029.
“Procedure
463
develop.-d
in our
laboratory.
464
Allen et al.
I 0
I 20 PERCENTAGE
Am.
I 40
I 60
I 80
GROWTH
I 100 RATE
Fig. 1. Plating efficiency of tumor cells. Twentyfour-hour-old cell cultures containing different numbers of cells from the same tumor and passage were trypsinized, plated, and incubated for 28 days at 37” C. in carbon dioxide. They were fed twice a week with Eagle’s minimum essential medium, Earle’s base media, and 10 per cent FBS.
of tissue is washed several times in Hanks balanced salt solution which contains antibiotics. The tissue is then very finely minced, suspended in the desired type of culture medium, and dispersed in tissue culture flasks that contain approximately 6 ml. of media. This is then incubated at 37’ C. in carbon dioxide.) Generations from P-O to P-10 were passed in our laboratory. Various tumor cells from the same generation were trypsinized and plated in 2 different sets. One set contained lo5 cells per plate, and the second set contained lo6 cells per plate. Proliferation of cells for both sets was done in medium L-15 and 10 per cent fetal bovine serum (FBS) and incubated at 4O C. They were fed every other day. Daily observations were kept. Microscopic comparison with phase contrast and classical light microscopy of living tissue cultures, as well as Giemsa- and eosinstained aliquots, yielded information as to the characteristic morphology’ of the tumor cells. Plating efficiency, a technique long used by tissue culture investigators,4 is shown in Fig. 1. Various tumor cells from the same generation were trypsinized and plated in 4
June 15, 1973 J. Obstet. Gynecol.
different sets as follows: 100, 500, 1,000, and 5,000 cells per plate in Eagle’s minimal essential medium, Earle’s base, and 10 per cent FBS, incubated at 370’ C. in carbon dioxide, and fed twice each week. Readings and observations were done daily and kept until they reached confluency. Tritiated uridine labeling’. j Leas performed on 30 different tumor cell lines in various generations. Once the tumor cells reached 100 per cent growth (conffuency) in a 75 cm.” tissue culture flask,* they were exposed for 48 hours at 37’ C. in a maintenance medium containing 20 PC per milliliter of Hs-uridine. The supernatant fluid was then centrifuged? for 10 minutes at 10,000 r.p.m. at 4’ C. to remove all floating cells or cellular debris. The remaining supernatant was then centrifuged for 2 hours at 30,000 r.p.m. at 4’ C. The resulting pellet was resuspended in l/40 volume of O.OlM TRISS at pH 7.4. The concentrated material was layered on top of a preformed 15 to 60 per cent linear sucrose gradient in TNE buffer (O.OlM TRIS, O.lM NaCl, and O.OOlM EDTA,§ pH 7.4) and centrifuged at 40,000 r.p.m. for 3 hours at 4’ C. with the SW 50.1 rotor. A gradient range maker of a known specific gravity was incorporated and 30 different fractions were collected, containing 7 drops to a fraction, from the bottom of the tube. These were precipitated with cold 5 per cent trichloracetic arid (TCA! and then placed onto Whatman GF/A glass fiber papers. These were washed in 5 per cent TCA, drid out, then assayed for radioactivity by liquid scintillation counting (Beckman model LS-350t). Sucrose density measurements were determined with a Zeiss refractometer.!/ Chromosome counts and karyotypes to further confirm replication of the tumor cells are being performed, but the results are not available at the time of this prepublication. *Falcon
Plastics,
tBeckman Instruments,
Div.
ultracrntrifuge Inc.,
Fullerton,
of
Bioquest. L2-65B,
Oxnard, rotor
California.
$2-Amino-2-(hydro~ymethyl)-l.3-propanediol. KEthylrnedinitrilo-tetra-acrfic (ICarl
Zriw.
Inc..
Nrw
acid. York.
Nm
York.
type
California. 30, Beckman
Volume Number
116 4
After several passages we could assure ourselves that we were dealing with tumor cells that demonstrated characteristics1 similar to those of the original surgical specimens. We then undertook further studies. A number of chemotherapeutic agents were employed to determine the possible SUSceptibility and specific concentrations of an agent thought to be most suitable for an individual patient’s tumor. Manners of testing response to chemotherapeutic agents were essentially similar to what we have previously described. The drugs used were the following: Ara-C (cystosine arabinoside, The Upjohn Co., Kalamazoo, Michigan), Alkeran (melphalan, Burroughs Wellcome & Co. [U. S. A.] Inc., Research Triangle Park, North Carolina) , Cortone (cortisone acetate, Merck Sharp & Dohme, Div. Merck & Co., Inc., West Point, Pennsylvania) , Cytoxan (cyclophosphamide, Mead Johnson Laboratories, Div. of Mean Johnson & Co., Evansville, Indiana), 5-fluorouracil (cyclophosphamide, Roche Labs., Div. Hoffmann-La Roche Inc., Nutley, New Jersey), Premarin (conjugated estrogens, Ayerst Labc., Div. Amer. Home Products Corp., New York, New York), Luteroid and testosterone (progesterone, Invenex Pharmaceuticals, San Francisco, California) . Not all of these drugs were used on all tumors that were tested; however, controls were employed with the use of: (1) the same tumor of the same generation with media and no drug, and (2) embryonic tissue cultures of the same generation with drug of the same concentration as used in the tumor studies. The end point was considered to be the percentage of cells destroyed after a given period of time. The same parameters were used for the controls. In all cases, 24-hour-old cultures, 3 x 10” cells per plate, were treated with the appropriate drugs in the following concentrations+‘? : 0.5 mg. per milliliter (10-l) , 0.05 mg. per milliliter (lo+), 0.005 mg. per milliliter ( 10m3), 0.0005 mg. per milliliter (10-l)) and 0.00005 mg. per milliliter ( 10-5) with
Tumor
0
2
4
and
6 NUMBER
6
embryonic
IO OF DAYS
12
tissue
14
465
16
Fig. 2. Growth characteristic of tumor cells. Cells were established by the Maitland procedure in Eagle’s minimum essential medium and 10 per cent FBS and incubated at 37” C. in a gas mixture of 5 per cent carbon dioxide and 95 per cent oxygen. 4 plates per dilution in maintenance media (Eagle’s minimum essential medium and Earle’s base) and 5 per cent FBS* in an environment of 95 per cent oxygen and 5 per cent carbon dioxide at 37’ C. The control set differed only in that it received no drug. In Part 2, tumor tissue cultures grown and treated exactly as above were exposed to 20 PC of H”-uridine for 24 hours. The supernatant was collected in the same manner as previously described for H3-uridine labeling. Results
These studies demonstrate the ability to grow tumor cells in culture and reproduce the same tumor cells after several generations. Fig. 2 demonstrates that individual tumors have particular growth characteristics.“-F Human embryonic tissue was cultured to compare with cultures of human tumors. The growth characteristics can also be shown when compared with their multiplication role as noted in Fig. 3. Population density was compared in different generations. Fig. 4 demonstrates that the population density is very characteristic, illustrating a number of tumors in the P-2, P-3, and P-4 generations. The standard deviation of these generations is only 1.2 days. It should be noted from Figs. 1 and 2 that the tumor cells differ little in morphology from the parent generation after several passages. Figs. *Flow
Labm atorics,
Inglewood.
California
466
Allen et al. Am.
Junr J. Obstet.
15, 1973 (:ynecol.
6X10’
0
24
48
96
NUMBER
120
144
168
i
OF HOURS
Fig. 3. Multiplication role of tumor cells. All cells were plated from the same passage number or generation P-l in Eagle’s minimum essential medium and 5 per cent FBS incubated at 37” C. in carbon dioxide. Every 24 hours, the cells were counted until they reached confluency.
Fig. hour-old
0
20
40
60
PERCENTAGE POPULATION
80
100
OF DEATH
Fig. 4. Population density of tumor cells. Proliferation of cells was done in media L-15 and 10 per cent FBS. The cells were incubated at 4” C. and fed twice a week. After 4 days, they reach confluency. They start to degenerate thereafter at the rate of 15 to 25 per cent each day.
5 and 6 compare embryonic tissue culture to an ovarian carcinoma after several passages. The embryonic tissue cells attain a more spindlelike appearance when contrasted with those of the tumor cells. Fig. 2 demonstrates that a small number of cells from Generations P-2 and P-4 had similar plating efficiencies at various ages. There is a small standard deviation of 2 days. At any point in any generation from P-O through P-9, the tumor& 13-18 were tested with appropriate chemotherapeutic agents. One ovarian tumor of particular interest was studied up to the ninth generation of passage with the fol-
5.
Ovarian culture
carcinoma P-2. Twenty-fourin maintenance medium.
lowing drugs : conjugated estrogens plus melphalan, progesterone, conjugated estrogens, cyclophosphamide, and melphalan. The tritiated uridine uptake, as well as visual estimate for confinning destruction of tumor cells. is remarkably consistent for each of the generations. However, the specie? activity of a drug or combination of drugs’“, *’ showed a large variation. In this particular series, melphalan was found to be the most effective single agent regardless of the concentration of drugs. After fractionation and counting in the scintillation counter, it was noted that the tumor cells of this ovarian tumor produced a concentrate with a buoyant density of approximately 1.16 Gm. per cubic centimeter. This specific activity was very characteristic of certain of the tumors tested. This degree of H3-uridine uptake can be equated with an active virulent ribonucleic acid (RNA) virus.” The embryonic control cells showed no uptake at this density. The specific activity of H3-uridine uptake was determined for many tumors, as well as
Tumor
Fig. 6. Human embryo P-2. Twenty-four-hour-old
human embryo cultures in different generations. We found that, in certain tumors in generations P-O through P-9, the rate of H”-uridine uptake up to P-9 was consistently the same in each generation. Individual patients’ tumors were tested through several generations by passage against different concentrations of chemotherapeutic agents. It was noted that more information could be obtained regarding the specificity of action against an individual patient’s tumor than against a group of similar tumors. In addition, it was also learned that the specificity of action against tumors within a single class is not necessarily the same for each lesion despite their similar origin and morphology. The time period for destruction of cells varied with both the type of cells being tested and the concentration of drugs.” However, it was notable that with human embryo tissue cultures there was no destruction of cells after 8 days, whereas with tumor cells, as shown in Fig. 7, there was frequently up to 100 per cent destruction in 5 days. Usually, the higher the concentration of the anticancer drug, the more rapid leas the cell destruction. However, this was not universally true, suggesting further that certain drugs--independent of their concentra-
and
embryonic
culture in maintenance
tissue
467
medium.
a
CONTROL
0
2
4
INCUBATION IN
6 PERIOD
DAYS
Fig. 7. Cervical carcinoma versus Am-C. Twentyfour-hour-old cell culture, 3 x 105 cells per plate, was treated with Ara-C as shown under DATA and incubated at 37” C. Carbon dioxide in Eagle’s minimum essential medium and 5 per cent FBS controls indicate no cell destruction. tions-arc not effective in destruction of tumor that is actively reproducing. There were trvo principal methods used to assess the action of the anticancer drugs. One was to estimate and count visually the number of living cells. Figs. 8 and 9 illustrate the results attained with the use of tritiated uridine as previously described to provide us with information as to the remaining virulence” of tumor cultures to which drugs or chemotherapeutic agents have been added and those used as controls, as well as hurnan embryo cultures (Fig. 7). In the embryo
468
Allen et al.
June 15, 1973 Am. J. Obstet. Gynecol.
Fig. 8. Ovarian carcinoma P-6. No drugs. Used as a control in maintenance medium. Fig. 9. Ovarian carcinoma P-6 treated with 0.5 mg. per milliliter of melphalan after 3 days in maintenance medium.
cultures, incorporation of H3-uridine and chemotherapeutic drugs together, for a 24 hour incubation period, failed to demonstrate any uptake suggestive of viral particles. It could be that the chemotherapeutic agents may suppres? the viral particle RNA H3uridine incorporation in tumor cultures. Comment The growth of human tumor cells in tissue culture of various types, including carcinoma of the cervix, breast, endometrium, ovary, lung, and sarcoma, were studied. It has been found by controlled experiments that these tumor cells can be passed from generation to generation, up to 10 generations in some tumors, without loss of cell characteristics or apparent virulence. The data involving the susceptibility of tumor tissue cells to destruction by chemotherapeutic agents offer a new approach to dealing with the problem of treatment of tumors for which chemotherapy appears to be all that remains available in the thera-
peutic armamentarium at our disposal, However, a rational approach to the use of chemotherapy on an individual basis can be attained in a relatively short period of time, perhaps as little as 3 weeks, and appropriate chemotherapy can be instituted. This would certainly be of value to clinicians who are repeatedly faced with the problem of the patient whose responsiveness to various anticancer drugs can only be determined by their use subsequently over varying periods of time. We must emphasize, of course, in this study that we are dealing with in vitro cells even though one of the qualifications that we strove for was to continue a tumor cell line as it was obtained from the patient. We do know that modification in vivo of tumor tissue does occur and that any sample of a tumor tissue taken at operation is not necessarily representative of all, or even a majority, of the tumor cells that this patient has at the time the surgical specimen is obtained or subsequently. We appreciate that there are other means and methods which
Volume Number
116 4
can be employed to ensure even more stringent controls. However, we offer these techniques as a start in the direction of a valuable experimental approach to solving a difficult clinical problem which has now come to the point where patients at our hospital are treated with chemotherapeutic agents based upon tumor tissue culture susceptibility to these drugs. When results are obtained after studying the patients and their re-
Tumor
and
embryonic
tissue
469
sponses, it will certainly be of great interest to note the value of this work. Of course, other phases of testing are underway. Animal inoculation studies with tumor concentrates are currently in the experimental stage. Ultimately, the techniques employed and the reliable reproducibility of results must help advance our goals toward appropriate treatment of an individual’s malignancy.
REFERENCES
1. 2.
Clarkson, B.: Cancer Res. 27: 2483, 1967. Foley, G. E., Lazarus, H., Ferber, G., Uzman, B. G., and Adams, R. A.: Studies of Human Leukemia Cells in Vitro, Baltimore, 1968, The Williams & Wilkins Company, pp. 6597. Puck, T. T., Marcus, P. E., and Cieciora, S. J.: J. Exp. Med. 103: 273, 1956. Somers, K., and Kit, S.: Virology 46: 774, 1971. Todaro, G., Zeve, V., and Aaronson, S.:
12.
Nature 226: 1047, 1972.
17.
Skipper, H. E., Schabel, F. M., Jr., and Wilcox, W. S.: Cancer Chemother. Rep. 51: 125, 1967. 7. DeWys, W. D.: Cancer Res. 32: 369, 1972. 8. Bruce, W. R., Meeker, B. E., and Valeriote, F. A.: J. Natl. Cancer Inst. 37: 233, 1966. 9. DeWys, W. D., Humphreys, S. R., and Goldin, A.: Cancer Chemother. Rep. 52: 229, 1968. 10. Straus, M. J., and Goldin, A.: Cancer Chemother. Rep. 56: 25, 1972. 11. Borsin, J,, and Whitmore, G. F.: Cancer Res. 29: 737. 1969.
13. 14.
15. 16.
18.
19.
20. 21.
Salick, H., Flax, H., and Hobbs, J. D.: Lancet 161: 1198, 1972. Wade, M. E., and Williams, H. M.: Conn. Med. 31: 426, 1967. Dexter, D. L., Wolberg, W. H., Ansfield, F. J., Helson, L., and Heidelberger, C.: Cancer Res. 32: 247, 1972. Heidelberger, C.: Progr. Nucleic Acid Res. Mol. Biol. 4: 1, 1965. Skipper, H. E., Thomson, J. R., and Bell, M.: Cancer Res. 14: 1964. 1954. Ansfield, F. J., and’ Ramirez, G.: Cancer Chemother. Rep. 55: 205, 1971. Laster, W. R., Jr., Mayo, J. G.: SimpsonHarrem, L., Griswald, D. P., Jr:, Lloyd, H. H., Schubel, F. M., Jr., and Skrpper, H. E.: Cancer Chemother. Rep. 53: 169, 1969. Pearson, J. W., Pearson, W. R., Gibson, W. T., Chermann, J. C., and Chirigos, M. A.: Cancer Res. 32: 904, 1972. Kinne, D. W., and Humphrey, E. W.: Cancer Chemother. Rep. 56: 53, 1972. Schwartz, D. B., Zbar, B., Gibson, W. T., and Chirigos, M. A.: Int. J. Cancer 8: 320, 1971.
ALLEN,
TELESFORO ALLAN
RAMON, JACOBSON,
MACLYN Los Angeles,
Tumor
M.D.
E.
WADE,
B.A. M.D. M.D.,
F.A.C.O.G.
California
tissue
obtained from 79 patient as biopsy or surgical specimens has been Control studies were performed to identify the original tumor during several generations of passage and to assess viability and continued virulence during replication. The patient’s tumor tissue cultures were categorized as to origin, cell type, and degree of differentiation. Chemotherapeutic agents in different concentrations and combinations were tested for eficacy on the various categories of tumors. Three salient findings were noted: (1) the almost consistent ability to culture human tumor tissue in the laboratory with reasonable assurance of maintaining its original characteristics as they were in vivo, (2) the variability of each patient’s tumor in its response to chemotherapeutic agents (i.e., one patient’s ovarian carcinoma culture will show greater susceptibility to the same drug than another patient’s culture of identical cell type and grade of ovarian carcinoma), (3) the availability of a technique for taking individual patients’ tumor specimens at operatitle procedures and usirzg the laboratory for culture and sensitivity testing to anticancer drugs.
cultured in vitro.
0 N E o F T H E problems in studying cancer and the susceptibility of tumors to chemotherapeutic agents has been the inadequate availability of laboratory facilities to test an individual patient’s tumor in vitro against various drugs, Th e chief obstacle is the difficulty in growing many types of tumors From the Department of Obstetrics Gynecology, Cedars-Sinai Medical Center, University of California. Los Angeles.
in the usual tissue culture fashion with any degree of certainty of replicating the patient’s own tumor.l In our laboratory we have been able to obviate this problem which has permitted further investigations of the tumors. Growing tumor cells of human tissue in vitro is not new.l, However, elaborate testing and replication studies have been designed to establish adequate control and thus obtain more reliable results. Once reproducible results were obtained consistently, testing with antitumor drugs was possible. Avenues for further investigation were also opened.
and
Supported by Hea!th, Education, and Welfare Grant RR05468, The Olincy Foundation, and The Western Cancer Institute. The Frank Lynch Memorial Essay, presented at the Thirty-ninth Annual Meeting of the Pacific Coast Obstetrical and Qvnecological Society, Harrison Hot Springs, British Columbia. Canada, October 3-7, 1972. Reprint requests: Arthur Allen, M.D., Cedars-Sinai Medical Center, Cedars Lebanon Hospital Division, 4833 Fountain Ave., Los Angeles. California
Methods
Tumor tissue from various types of carcinomas and sarcomas was obtained as sur,gical specimens and cultured in appropriate tissue culture media, either by trypsinization or by the Maitland procedure.* (A small piece
of
90029.
“Procedure
463
develop.-d
in our
laboratory.
464
Allen et al.
I 0
I 20 PERCENTAGE
Am.
I 40
I 60
I 80
GROWTH
I 100 RATE
Fig. 1. Plating efficiency of tumor cells. Twentyfour-hour-old cell cultures containing different numbers of cells from the same tumor and passage were trypsinized, plated, and incubated for 28 days at 37” C. in carbon dioxide. They were fed twice a week with Eagle’s minimum essential medium, Earle’s base media, and 10 per cent FBS.
of tissue is washed several times in Hanks balanced salt solution which contains antibiotics. The tissue is then very finely minced, suspended in the desired type of culture medium, and dispersed in tissue culture flasks that contain approximately 6 ml. of media. This is then incubated at 37’ C. in carbon dioxide.) Generations from P-O to P-10 were passed in our laboratory. Various tumor cells from the same generation were trypsinized and plated in 2 different sets. One set contained lo5 cells per plate, and the second set contained lo6 cells per plate. Proliferation of cells for both sets was done in medium L-15 and 10 per cent fetal bovine serum (FBS) and incubated at 4O C. They were fed every other day. Daily observations were kept. Microscopic comparison with phase contrast and classical light microscopy of living tissue cultures, as well as Giemsa- and eosinstained aliquots, yielded information as to the characteristic morphology’ of the tumor cells. Plating efficiency, a technique long used by tissue culture investigators,4 is shown in Fig. 1. Various tumor cells from the same generation were trypsinized and plated in 4
June 15, 1973 J. Obstet. Gynecol.
different sets as follows: 100, 500, 1,000, and 5,000 cells per plate in Eagle’s minimal essential medium, Earle’s base, and 10 per cent FBS, incubated at 370’ C. in carbon dioxide, and fed twice each week. Readings and observations were done daily and kept until they reached confluency. Tritiated uridine labeling’. j Leas performed on 30 different tumor cell lines in various generations. Once the tumor cells reached 100 per cent growth (conffuency) in a 75 cm.” tissue culture flask,* they were exposed for 48 hours at 37’ C. in a maintenance medium containing 20 PC per milliliter of Hs-uridine. The supernatant fluid was then centrifuged? for 10 minutes at 10,000 r.p.m. at 4’ C. to remove all floating cells or cellular debris. The remaining supernatant was then centrifuged for 2 hours at 30,000 r.p.m. at 4’ C. The resulting pellet was resuspended in l/40 volume of O.OlM TRISS at pH 7.4. The concentrated material was layered on top of a preformed 15 to 60 per cent linear sucrose gradient in TNE buffer (O.OlM TRIS, O.lM NaCl, and O.OOlM EDTA,§ pH 7.4) and centrifuged at 40,000 r.p.m. for 3 hours at 4’ C. with the SW 50.1 rotor. A gradient range maker of a known specific gravity was incorporated and 30 different fractions were collected, containing 7 drops to a fraction, from the bottom of the tube. These were precipitated with cold 5 per cent trichloracetic arid (TCA! and then placed onto Whatman GF/A glass fiber papers. These were washed in 5 per cent TCA, drid out, then assayed for radioactivity by liquid scintillation counting (Beckman model LS-350t). Sucrose density measurements were determined with a Zeiss refractometer.!/ Chromosome counts and karyotypes to further confirm replication of the tumor cells are being performed, but the results are not available at the time of this prepublication. *Falcon
Plastics,
tBeckman Instruments,
Div.
ultracrntrifuge Inc.,
Fullerton,
of
Bioquest. L2-65B,
Oxnard, rotor
California.
$2-Amino-2-(hydro~ymethyl)-l.3-propanediol. KEthylrnedinitrilo-tetra-acrfic (ICarl
Zriw.
Inc..
Nrw
acid. York.
Nm
York.
type
California. 30, Beckman
Volume Number
116 4
After several passages we could assure ourselves that we were dealing with tumor cells that demonstrated characteristics1 similar to those of the original surgical specimens. We then undertook further studies. A number of chemotherapeutic agents were employed to determine the possible SUSceptibility and specific concentrations of an agent thought to be most suitable for an individual patient’s tumor. Manners of testing response to chemotherapeutic agents were essentially similar to what we have previously described. The drugs used were the following: Ara-C (cystosine arabinoside, The Upjohn Co., Kalamazoo, Michigan), Alkeran (melphalan, Burroughs Wellcome & Co. [U. S. A.] Inc., Research Triangle Park, North Carolina) , Cortone (cortisone acetate, Merck Sharp & Dohme, Div. Merck & Co., Inc., West Point, Pennsylvania) , Cytoxan (cyclophosphamide, Mead Johnson Laboratories, Div. of Mean Johnson & Co., Evansville, Indiana), 5-fluorouracil (cyclophosphamide, Roche Labs., Div. Hoffmann-La Roche Inc., Nutley, New Jersey), Premarin (conjugated estrogens, Ayerst Labc., Div. Amer. Home Products Corp., New York, New York), Luteroid and testosterone (progesterone, Invenex Pharmaceuticals, San Francisco, California) . Not all of these drugs were used on all tumors that were tested; however, controls were employed with the use of: (1) the same tumor of the same generation with media and no drug, and (2) embryonic tissue cultures of the same generation with drug of the same concentration as used in the tumor studies. The end point was considered to be the percentage of cells destroyed after a given period of time. The same parameters were used for the controls. In all cases, 24-hour-old cultures, 3 x 10” cells per plate, were treated with the appropriate drugs in the following concentrations+‘? : 0.5 mg. per milliliter (10-l) , 0.05 mg. per milliliter (lo+), 0.005 mg. per milliliter ( 10m3), 0.0005 mg. per milliliter (10-l)) and 0.00005 mg. per milliliter ( 10-5) with
Tumor
0
2
4
and
6 NUMBER
6
embryonic
IO OF DAYS
12
tissue
14
465
16
Fig. 2. Growth characteristic of tumor cells. Cells were established by the Maitland procedure in Eagle’s minimum essential medium and 10 per cent FBS and incubated at 37” C. in a gas mixture of 5 per cent carbon dioxide and 95 per cent oxygen. 4 plates per dilution in maintenance media (Eagle’s minimum essential medium and Earle’s base) and 5 per cent FBS* in an environment of 95 per cent oxygen and 5 per cent carbon dioxide at 37’ C. The control set differed only in that it received no drug. In Part 2, tumor tissue cultures grown and treated exactly as above were exposed to 20 PC of H”-uridine for 24 hours. The supernatant was collected in the same manner as previously described for H3-uridine labeling. Results
These studies demonstrate the ability to grow tumor cells in culture and reproduce the same tumor cells after several generations. Fig. 2 demonstrates that individual tumors have particular growth characteristics.“-F Human embryonic tissue was cultured to compare with cultures of human tumors. The growth characteristics can also be shown when compared with their multiplication role as noted in Fig. 3. Population density was compared in different generations. Fig. 4 demonstrates that the population density is very characteristic, illustrating a number of tumors in the P-2, P-3, and P-4 generations. The standard deviation of these generations is only 1.2 days. It should be noted from Figs. 1 and 2 that the tumor cells differ little in morphology from the parent generation after several passages. Figs. *Flow
Labm atorics,
Inglewood.
California
466
Allen et al. Am.
Junr J. Obstet.
15, 1973 (:ynecol.
6X10’
0
24
48
96
NUMBER
120
144
168
i
OF HOURS
Fig. 3. Multiplication role of tumor cells. All cells were plated from the same passage number or generation P-l in Eagle’s minimum essential medium and 5 per cent FBS incubated at 37” C. in carbon dioxide. Every 24 hours, the cells were counted until they reached confluency.
Fig. hour-old
0
20
40
60
PERCENTAGE POPULATION
80
100
OF DEATH
Fig. 4. Population density of tumor cells. Proliferation of cells was done in media L-15 and 10 per cent FBS. The cells were incubated at 4” C. and fed twice a week. After 4 days, they reach confluency. They start to degenerate thereafter at the rate of 15 to 25 per cent each day.
5 and 6 compare embryonic tissue culture to an ovarian carcinoma after several passages. The embryonic tissue cells attain a more spindlelike appearance when contrasted with those of the tumor cells. Fig. 2 demonstrates that a small number of cells from Generations P-2 and P-4 had similar plating efficiencies at various ages. There is a small standard deviation of 2 days. At any point in any generation from P-O through P-9, the tumor& 13-18 were tested with appropriate chemotherapeutic agents. One ovarian tumor of particular interest was studied up to the ninth generation of passage with the fol-
5.
Ovarian culture
carcinoma P-2. Twenty-fourin maintenance medium.
lowing drugs : conjugated estrogens plus melphalan, progesterone, conjugated estrogens, cyclophosphamide, and melphalan. The tritiated uridine uptake, as well as visual estimate for confinning destruction of tumor cells. is remarkably consistent for each of the generations. However, the specie? activity of a drug or combination of drugs’“, *’ showed a large variation. In this particular series, melphalan was found to be the most effective single agent regardless of the concentration of drugs. After fractionation and counting in the scintillation counter, it was noted that the tumor cells of this ovarian tumor produced a concentrate with a buoyant density of approximately 1.16 Gm. per cubic centimeter. This specific activity was very characteristic of certain of the tumors tested. This degree of H3-uridine uptake can be equated with an active virulent ribonucleic acid (RNA) virus.” The embryonic control cells showed no uptake at this density. The specific activity of H3-uridine uptake was determined for many tumors, as well as
Tumor
Fig. 6. Human embryo P-2. Twenty-four-hour-old
human embryo cultures in different generations. We found that, in certain tumors in generations P-O through P-9, the rate of H”-uridine uptake up to P-9 was consistently the same in each generation. Individual patients’ tumors were tested through several generations by passage against different concentrations of chemotherapeutic agents. It was noted that more information could be obtained regarding the specificity of action against an individual patient’s tumor than against a group of similar tumors. In addition, it was also learned that the specificity of action against tumors within a single class is not necessarily the same for each lesion despite their similar origin and morphology. The time period for destruction of cells varied with both the type of cells being tested and the concentration of drugs.” However, it was notable that with human embryo tissue cultures there was no destruction of cells after 8 days, whereas with tumor cells, as shown in Fig. 7, there was frequently up to 100 per cent destruction in 5 days. Usually, the higher the concentration of the anticancer drug, the more rapid leas the cell destruction. However, this was not universally true, suggesting further that certain drugs--independent of their concentra-
and
embryonic
culture in maintenance
tissue
467
medium.
a
CONTROL
0
2
4
INCUBATION IN
6 PERIOD
DAYS
Fig. 7. Cervical carcinoma versus Am-C. Twentyfour-hour-old cell culture, 3 x 105 cells per plate, was treated with Ara-C as shown under DATA and incubated at 37” C. Carbon dioxide in Eagle’s minimum essential medium and 5 per cent FBS controls indicate no cell destruction. tions-arc not effective in destruction of tumor that is actively reproducing. There were trvo principal methods used to assess the action of the anticancer drugs. One was to estimate and count visually the number of living cells. Figs. 8 and 9 illustrate the results attained with the use of tritiated uridine as previously described to provide us with information as to the remaining virulence” of tumor cultures to which drugs or chemotherapeutic agents have been added and those used as controls, as well as hurnan embryo cultures (Fig. 7). In the embryo
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June 15, 1973 Am. J. Obstet. Gynecol.
Fig. 8. Ovarian carcinoma P-6. No drugs. Used as a control in maintenance medium. Fig. 9. Ovarian carcinoma P-6 treated with 0.5 mg. per milliliter of melphalan after 3 days in maintenance medium.
cultures, incorporation of H3-uridine and chemotherapeutic drugs together, for a 24 hour incubation period, failed to demonstrate any uptake suggestive of viral particles. It could be that the chemotherapeutic agents may suppres? the viral particle RNA H3uridine incorporation in tumor cultures. Comment The growth of human tumor cells in tissue culture of various types, including carcinoma of the cervix, breast, endometrium, ovary, lung, and sarcoma, were studied. It has been found by controlled experiments that these tumor cells can be passed from generation to generation, up to 10 generations in some tumors, without loss of cell characteristics or apparent virulence. The data involving the susceptibility of tumor tissue cells to destruction by chemotherapeutic agents offer a new approach to dealing with the problem of treatment of tumors for which chemotherapy appears to be all that remains available in the thera-
peutic armamentarium at our disposal, However, a rational approach to the use of chemotherapy on an individual basis can be attained in a relatively short period of time, perhaps as little as 3 weeks, and appropriate chemotherapy can be instituted. This would certainly be of value to clinicians who are repeatedly faced with the problem of the patient whose responsiveness to various anticancer drugs can only be determined by their use subsequently over varying periods of time. We must emphasize, of course, in this study that we are dealing with in vitro cells even though one of the qualifications that we strove for was to continue a tumor cell line as it was obtained from the patient. We do know that modification in vivo of tumor tissue does occur and that any sample of a tumor tissue taken at operation is not necessarily representative of all, or even a majority, of the tumor cells that this patient has at the time the surgical specimen is obtained or subsequently. We appreciate that there are other means and methods which
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can be employed to ensure even more stringent controls. However, we offer these techniques as a start in the direction of a valuable experimental approach to solving a difficult clinical problem which has now come to the point where patients at our hospital are treated with chemotherapeutic agents based upon tumor tissue culture susceptibility to these drugs. When results are obtained after studying the patients and their re-
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sponses, it will certainly be of great interest to note the value of this work. Of course, other phases of testing are underway. Animal inoculation studies with tumor concentrates are currently in the experimental stage. Ultimately, the techniques employed and the reliable reproducibility of results must help advance our goals toward appropriate treatment of an individual’s malignancy.
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