Cancer Research

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CANCER RESEARCH

VOLUME 18 SEPTEMBER 1958 NUMBER 8

Tissue Culture in Cancer Chemotherapy Screening*

ERICH HIRSCHBERG

(Institute of Cancer Research and Department of Biochemistry, Columbia University Collegeof Physicians and Surgeens, New York 8@,N.Y.)

The sponsorship of this Symposium represents on a secure basis and the availability of trans the first formal joint venture of the Tissue Culture plantable tumors facilitated the first attempts Association and the American Association for Can to develop systematic approaches to cancer chemo cer Research. As these two groups meet together therapy. today to appraise each other's problems and The year 1907 was an important one in both accomplishments, it is appropriate to recall that fields of investigation. Paul Ehrlich (@6) laid the tissue culture and experimental cancer research foundations of modern chemotherapy in his Har had their beginnings at about the same time and ben Lectures in London; Ross Harrison (40) made have developed over the years in close chronologi tissue culture a practical tool by explanting ner cal parallel and with increasing mutual interaction. vous tissues in a simple assembly and observing The history of both of these fields dates back their development in isolation. By 1910 and 1911, a little more than two generations, to the 1880's.' Alexis Carrel and Montrose T. Burrows (16, 17) During that decade, many attempts were made had started to maintain chick embryo fibroblasts to grow tissues or cells outside the body, as continuously in tissue culture and were cultivating illustrated by the work of Wilhelm Roux, who tumors in vitro; cancer chemotherapy also entered in 1885 achieved the isolation and maintenance a period of intense activity following the reports of the medullary plate of the chick embryo in of August von Wassermann and his associates warm saline solution (cf. 67). Four years later, (77, 78) on the inhibition of animal tumors by Arthur Hanau (39) carried out the first successful the intravenous or subcutaneous administration homologous transplantation of an animal tumor, of selenium and eosin. an achievement which signaled the start of modern This is not the occasion for a detailed account experimental oncology. By 1903, Carl Jensen (49) of the growth and development of the two fields was able to report that a mouse tumor could be during the period between the two World Wars. transplanted for nineteen successive generations; However, it is of interest to recall a few of the this important laboratory technic was thus put events of the past dozen years. In 1946, a notable Conference on Tissue Culture was convened in * Most of this material was presented at the 49th Annual Meeting of the American Association for Cancer Research in Hershey, Pa., which brought together a group Philadelphia, April 11, 1958, as part of a Symposium on â€œRe of active workers to evaluate the past, assess cent Contributions of Tissue Culture to Cancer Research.â€• future needs and objectives, and establish the The experimental work of the author and his associates men Tissue Culture Association. In 1953, Margaret tioned in this paper has been supported by U.S. Public Health Service Research Grants C-1894 and C-@33@and by the R. Murray and Gertrude Kopech (58) completed Charles Ulrick and Josephine Bay Gift for Research in Brain the monumental task of bringing together the Tumor Chemotherapy. tissue culture literature up to 1950 in a classified â€˜Auseful guide to the early history of tissue culture is found bibliography of some 15,000 references; a first in Parker (6@); the beginnings of experimental cancer research supplement covering the more recent literature have been fully detailed by Woglom (80), and the development is in preparation. In 1954, a conference on tissue of cancer chemotherapy has been traced by Dyer (@0). culture technics in pharmacology at the New Received for publication May 14, 1958. York Academy of Sciences (63) stimulated the 869 This on@

interest of experimental chemotherapists in this inhibition, the comments of several other reviewers useful investigative tool. Two years ago, the De (1, 15, 18, 50, 53, 57, 61, 71, 7@) have been of cennial Review Conference on Tissue Culture at great assistance. Woodstock, Vt. (79), bore witness to the vastly increased scope and versatility of these technics SYSTEMATIC APPLICATION OF TISSUE in many diverse areas of investigation. CULTURE TO SCREENING In experimental cancer chemotherapy, there During the last decade, much information has has been a similar period of intense and ever been gathered on the effects of a variety of chemi expanding research effort and periodic critical cal compounds on tumor cells in tissue culture, assessment. In the summers of 1945 and 1946, with or without normal cell controls. Many of conferences dealing with the current status of the these experiments, however, have been concerned field were held at Gibson Island; much of this with one or two agents at a time; only a handful material was published in 1947 (56). Helen Dyer's of studies have attempted a systematic explora very useful â€œIndex of Tumor Chemotherapyâ€• tion of larger series, thus permitting a more direct (@0) appeared in 1949. A concerted national pro evaluation of in vitro technics as screening tools. gram in cancer chemotherapy was initiated by A brief outline of the various systems which have the U.S. Public Health Service in 1953, and the been employed in this way will provide an indica Cancer Chemotherapy National Service Center tion of the diversity of approaches while furnishing @ began operations years later (@8). A number some background information for the general corn of conferences at the New York Academy of rnents which will be made later. Sciences and elsewhere afforded an evaluation Investigatioiu of Biesele and associales.â€”An im of particular types of chemotherapeutic agents, pressive series of publications (@â€”14,47, 48, 74) e.g., folic acid antagonists and other antimetabo has provided data on the in vitro effects of about lites (65, 81), 6-mercaptopurine (64), and alkylat 300 compounds, particularly purines, purine nu ing agents (51), and of various tools and technics cleosides, pyrirnidines, benzirnidazoles, antifolics, used for the selection of potential agents of chemo and amino acid antagonists. In most of these therapeutic interest (41, 73). studies, short-term primary explants of a trans ofcourse,asbothoftheseinvestigatiyeareas, plantable mouse tumor, principally mouse Sar tisssue culture and cancer chemotherapy, have coma 180, T@41, or Ma887, were placed into roller expanded and flourished, more and more points tubes; in addition to several tumor fragments, of contact between them have become apparent. each tube also contained explants of mouse em The availability of compounds with a documented bryonic skin as the normal control. The medium inhibitory effect on tumor growth has been of consisted of balanced salt solution, chick embryo great value in studies of the growth and behavior extract, human placental serum, and horse serum. of cells in vitro; problems dealing with the chemis The agent to be tested was incorporated into try of mitosis and the mechanism of action of the medium at several concentrations, ranging mitotic poisons have concerned investigators in generally from 0.1 to 4.0 millimoles/mi, and al both fields; inter-comparisons of the direct in lowed to act on the tumor and embryonic cells vitro effects and the often indirect in vivo action for 24 hours. Morphological evidence of damage of chemical agents on cells have proved a fruitful was then sought, and counts of mitotic and pyknot area of research; the applicability of tissue culture ic nuclei were often carried out to obtain a more technics to the search for carcinost.atic and car quantitative result. cinolytic agents has become an important question The main objective of these experiments has in cancer chemotherapy screening methodology. been to find compounds with selective or differ Each of these topics merits review and thorough ential toxicity for tumor cells. Several agents, discussion, but only the last will be considered notably 2,6-diaminopurine, 6-mercaptopurine, and in detail in this survey. No systematic attempt other 2- and 6- substituted purines, caused signif has been made to deal with the multitudinous icant damage to tumor cells at concentrations investigations in which nontumor cells or tissues which were entirely nontoxic to the embryonic (rat fibrocytes, chick and mouse embryonic tissues, controls, but most of them exhibited no such organ cultures, normal cell lines, and so on) were selectivity (cf. 3). A second objective has been exposed to various chemical agents. The pages to study the mechanism of action of these com to follow will deal primarily with those recent pounds by appropriate reversal experiments with studies in which tissue cultures of human and presumed normal metabolites. Experiments with animal tumors have been used; in considering the purine antagonists, in particular, have contributed relevance of these in vitro results to in vivo tumor to our understanding of nucleic acid synthesis and

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1958 American Association for Cancer Research. HIRSCHBERGâ€”Ti&@Ue Culture in Chemotherapy Screening 871 its blockade by compounds like 6-mercaptopurine Re8ulis obtained by Wright, Cobb,andassociates.â€” or 2,6-diaininopurine. An attempt to employ tissue culture in a more Some of the more recent studies (7â€”9)have direct approach to the problems of clinical chemo been extended to human cell strains of cancer therapy, by-passing animal tumors entirely, has and normal tissue origin grown in the semisyn been made (19, 82, 83) by using a biopsy sample thetic medium of Eagle (cf. below). In general, of a patient's tumor for the in vitro tests and corn the results with these materials were similar to paring the response of these primary explants those reported earlier with mouse tumors on a with the clinical response of the same patient nonsynthetic medium, but the human cell lines to the same drug. Patients with a wide variety often appeared less sensitive. of neoplastic diseases were used. Standard tissue As regards the comparison of these in vitro culture technics were employed, the agent being results with the anticancer effects of these corn added to the nonsynthetic medium at the smallest pounds in vivo, it was pointed out repeatedly concentration producing inhibition of outgrowth (e.g., 1, 4) that the correlation has not been in chick or mouse heart fibroblasts, which served particularly impressive. Selective toxicity to tumor as the normal controls. Morphological evidence cells in tissue culture, it appears, does not neces of cytotoxicity was used as the criterion for in sarily or predominantly go along with a satis vitro effects. In more than half of the experiments, factory therapeutic index in the whole animal. a good correlation of tissue culture results with Experiment, of EiChOT-TLandcollaborators.â€”This clinical response was found; fifteen of nineteen group of investigators (27) used short-term pri instances of clinical improvement were accom mary explants of mouse mammary carcinoma 72J panied by pronounced inhibition of the tumor and glioma 8110 in roller tubes, with mouse em in vitro, and, in nine of sixteen cases which failed bryonic skin explants in separate control tubes. to improve, the tissue culture results were also The cells were fed a medium consisting of horse negative. Triethylene melamine and triethylene serum and chick embryo extract for 24â€”48hours; thiophosphoramide were used most frequently; this was then replaced by a solution of 0.004 preliminary results with several other agents of per cent serum albumin containing the agent clinical interest were included. to be tested at various concentrations. After 48 Investigation8 ofEagle and Foley.â€”In the studies hours' exposure, cytological evidence of damage which have been outlined and in most other tissue was noted. The tissue culture response to eight culture experiments related to cancer chemother compounds including triethylenethiophosphor apy, tissue explants were grown in a plasma clot amide and 6-mercaptopurine was compared with in media composed of various natural materials, the effect of the same compounds on the same particularly plasma and tissue extracts. The recent two transplantable tumors in the mouse. work of Eagle and Foley (22â€”24)represents a In this short series of compounds, the cor radical departure from this procedure. On the relation of in vitro and in vivo inhibition of the basis of extensive investigations (cf. 21, 2,5, other same tumor was very close. In tissue culture, references in 23), it was found that a variety the glioma responded to five of the eight agents, of long established cell lines of normal or neoplastic the mammary tumor only to three. There was origin could be grown as a monocellular layer no differential effect on tumors in comparison directly on a glass surface in a relatively defined to the normal tissue control. or semisynthetic medium. The latter contained Studies of Fjelde et alâ€”Several recent reports thirteen essential amino acids, seven vitamins, (29â€”32, 70) have outlined experiments carried salts, glucose, and 10 per cent human serum or out with long established strains of human tumor 5â€”10per cent horse serum for human and mouse cell lines H.S. #1, H.Ep.#1, or H.Ep. #2 in Porter cell lines, respectively. An inoculum of about flasks on a nonsynthetic medium consisting of 200,000 cells was used, and growth was allowed balanced salt solution, chick embryo extract, hu to proceed in culture flasks for 5â€”8days in the man serum, human placental serum, and horse presence and absence of various levels of the serum. No normal cell controls were employed. compound to be tested. Chemical determination The effect of seven compounds on the growth of the protein content of flasks at the beginning of these cells was assessed by measuring the total and end of the experiment provided a quantitative tissue culture area at the beginning of the experi measure of cell multiplication and, consequently, ment and 7 days later by an area! growth pro of the inhibitory effect of the agent. This was jection technic. The minimum inhibitory concen expressed in terms of an â€œID50,â€•theconcentration trations ranged from 108 mg/m! for 2-azaadenine at which 50 per cent inhibition of growth was to 10@ mg/mi for 6-mercaptopurine. achieved.

The response of this test system was assessed or long established cell lines derived from them. first (28) by exposing each of seven cell lines Studies with human and mouse brain tumors to fifteen compounds, including thirteen of known at Columbia University.â€”For the last half-dozen in vivo carcinostatic activity (four antifolics, two years, the author has participated in a collabora dihydrotriazines, azaserine, stylomycin, 6-mercap tive program2 (36, 37, 42, 43, 45, 46, 59) of topurine, actinomycin D, triethylene melamine, experimental cancer chemotherapy encompassing triethylenethiophosphoramide, and Daraprim), tissue culture studies, the assessment of carcino and two folic acid analogs of known in vivo in static activity of a variety of compounds in a effectiveness. Four of the cell lines were of neo spectrum of transplantable mouse tumors, at plastic origin (human HeLa, KB, J, and mouse tempts to determine tissue distribution and mech 5-180), and three were derived originally from anism of action of some of these agents, and normal human tissues. Several interesting findings clinical evaluation where indicated. The animal emerged from these comparisons. All seven cell tumor spectrum has included primarily the brain lines exhibited essentially the same response to tumor Glioma 26, mammary adenocarcinomas 755 any one of the fifteen compounds. The thirteen and E 0771, Sarcoma 180, the Ehrlich ascites carcinostatic compounds all were highly cytotoxic carcinoma, and the ascitic form of leukemia L1210. in vitro, i.e., their ID50 was low; the two inactive Compounds have been tested routinely at the maxi compounds also were devoid of activity in tissue mum tolerated dose and evaluated statistically culture. The cytotoxicity of the active compounds on the basis of the difference in tumor weights varied over a 2000-fold range, with aminopterin, of treated and control groups of ten to twenty actinomycin D, and A-methopterin at the upper animals each. In general, it has been assumed, and triethylenethiophosphoramide and Daraprim as a working hypothesis, that compounds with at the lower end. reproducible activity against a variety of trans More recently, this comparison has been ex plantable tumors are of greater potential clinical tended to 180 compounds of known activity or interest than those which cause significant inhibi inactivity in vivo and of widely differing chemical tion of only a single tumor in this or similar constitution. One normal and one neoplastic cell spectra of experimental neoplasms. line were employed ; there was no significant dif The in vitro experiments have utilized primary ference in susceptibility to inhibition between short-term explants of human glioblastoma multi them. In general, the active tumor inhibitors forme and of the mouse brain tumor. Normal as a group were significantly more cytotoxic than controls are generally omitted. The explants are the compounds without carcinostatic activity in carried in roller tubes for about 10 days until animal tumor systems, but about one in five uniform growth is obtained, then transferred to of the active agents failed to meet the criteria Maximow slides, where they are maintained for for a positive in vitro effect (inhibition at an 7â€”10days in a plasma clot by the double cover ID50 of 10@ gm/mi or less), and a similar propor slip lying-drop method. The feeding fluid consists tion of the inactive compounds exhibited activity of human placental serum, chick embryo extract, in the tissue culture test. ox serum ultrafiltrate, and balanced salt solution. The pronounced similarity of response of normal A solution or fine suspension of the compound and neoplastic cell lines in this system has been to be tested is prepared to contain 10 times the one of the most intriguing features of these results, desired concentration, and a tenfold dilution is particularly if Biesele's success in demonstrating then made with feeding fluid. The medium is differential toxicity of a number of carcinostatic renewed twice during the experiment. The cultures purines to short-term tumor explants in a different are examined daily for morphological evidence culture system is recalled and if it is kept in of damage and fixed and stained at the end mind that positive carcinostatic activity in vivo of the test. The damage rating involves four presupposes a difference in response between the â€˜This long-range program has been conducted by Dr. tumor and the normal host tissues. One of the Alfred Gellhorn of the Department of Medicine, Dr. Margaret possible explanations revolves around the idea R. Murray of the Department of Anatomy, and Drs. J. Law that these long established lines of cells of liver rence Pool and Joseph Ransohoff of the Department of Neuro logical Surgery. The able assistance, at various times, of Mrs. or conjunctiva or intestinal epithelium can no Edith Peterson, Mrs. Ruth F. Eising, and Miss Annelies Herr longer behave as normal cells. Quite recently, mann in the tissue culture studies, Mrs. Martha Osnos in however, Foley and Eagle (34) demonstrated that tissue distribution and biochemical experiments, and Miss normal human and animal cells in first in vitro Alice Kells, Miss Gloria Semente, and Mrs. Shirley Brindle in the animal tumor tests is gratefully acknowledged. The author passage gave the same response to nineteen agents is particularly indebted to Drs. Gellhorn and Murray for many of varied effectiveness as did recently isolated helpful discussions.

main criteriaâ€”inhibition of migration, inhibi sixteen compounds which had the highest activity tion of mitosis, nuclear changes, and cytoplasmic in vitro. It is clear that under the particular changes (cf. 36, 59). The minimum inhibitory conditions chosen for these comparative studies concentration is obtained by determining the effect @ TABLE of various levels; for routine comparisons, lack of cytotoxicity at 1 X 103M is considered a nega In Vitro ACTIVITY OFCOMPOUNDS tive result, even though it is recognized that, WITH GREATEST in Vise with certain compounds, activity at higher con AcTIVITY centrations may still be of physiological signif Alkylating agents: HN@ ++ icance. Myleran 0* About 150 compounds have been examined Nonane 0 Benzimidazole mustard 0 in brain tumor tissue culture by this procedure. Fluorobenzyl mustard ++ Table 1 summarizes the results obtained with Purines and pyrimidines: 6.Mercaptopurmne 0 8.Azaguanine 0 TABLE 1 Thioguanine 0 SUMMARY OF CoMPARATIVE in Vitro AND in Vise TESTS 5-Fluorouracil 0 Thiaxanthenone Active in piv@@ Miracil D + Againat Benzimidazole atNo.10'u10'u@819419181819171@1@1@1010%1Active invitro >1 Tu 5,6-Dichioro. + Type of compound I Tumor mor Miscellaneous: Alkylating agents 16 5 N-Methylformamide 0 Quinolines 4 0 N.Methylacetamide 0 Thiaxanthenones 6 1 Urethan ++ Acridines 8 0 Hydrocortisone 0 @ Benzimidazoles 1 Ethionine 0 Purines and pyrim. Coichicine ++++ idines 9 4 4 1,5-Diaminobiuret 0 18 9 3 8 7 Miscellaneous * 0 = inactive at 10'at. Total: 115 79 45 43 18 TABLE S 115 compounds which at this writing have also been studied in some detail in the mouse tumor In Vivo ACTIVITY OFCOMPOUNDSWITH spectrum in vivo. Since one of the features of GREATEST in Vitro ACTIVITY this program has been a continuing interest in Compound Tumora inhibited (mm. inbibitory concn.) In Vivo structure-activity relationships, most of these com About @X10Â¼: pounds fall into one of a few chemical classes, Acridine A Ehrlich with nitrogen- and sulfur-containing heterocyclic B Ehrlich C structures predominating. The miscellaneous com D pounds include many which were studied inten E Ehrlich F sively in a recent investigation of diverse systems G* for cancer chemotherapy screening (35). QuinolineA 755 Most of the quinolines, thiaxanthenones, and About 4X1O'u: acridines exhibited pronounced in vitro cytotoxici Quinoline B* 755 C ty, but only one compound, the thiaxanthenone D Miracil D, has shown significant carcinostatic E activity against more than one of these experimen N Mustard A 755 tal neoplasms in vivo (44). Conversely, the over-all Thiaxanthenone A B L1@10 tissue culture response to alkylating agents, ben C zimidazoles, purines, pyrimidines, and the group * These two compounds, quinacrine and of miscellaneous agents was much less striking, camoquin, have also been evaluated in a small although these groups included a considerable series of patients with glioblastoma multiforme and found to be essentiallyinactive(to be number of compounds with clear-cut activity in published). the mouse and some with clinical usefulness as well. Table 2 details the in vitro activity of the there has been little correlation between the ability eighteen compounds which affected more than of an agent to damage brain tumor cells in tissue one of the experimental tumors in vivo to a signif culture and its ability to provide a useful thera icant extent. Table 3 provides the complementary peutic index when tested against a variety of data on the in vivo results obtained with the transplantable neoplasms growing in the mouse.

A detailed analysis of some of these findings RELEVANCE OF TISSUE has been or will be made elsewhere, but it is CULTURE RESULTS pertinent to mention here that, even within a particular chemical group, this correlation was A casual observer may ask in all fairness why often not impressive; Miracil D, for instance, comparative studies like the one just outlined the most active tumor-inhibitor among the thia have been continued for so protracted a period xanthenones studied to date, was only one tenth of time in the face of the patent failure of the as active in tissue culture as at least twelve of tissue culture data to mirror in vivo activity. its congeners. Another example is given in Table Another, less interested in assessing past per 4, which indicates that in a small series of closely formance than in charting future courses of action, related benzimidazole derivatives the brain tumor properly may inquire whether any extensive fur cytotoxicity in vitro correlates well with relative ther tissue culture observations should be made antiviral activity and to some extent with relative in the current quest for compounds of clinical toxicity to mice, as determined by the maximum usefulness and, if so, which particular in vitro tolerated dose in a 5-day chronic toxicity test, technic should now be selected. The answer to but that none of these data bear any demonstrable these questions hinges on a consideration of some

TABLE 4

COMPARATIVE RESULTS IN A SERIES OF BENZIMIDAZOLE DERIVATIVES Rel. antiviral Rel. tumor Rel. mouae Tumorl inhibited activity@ cytotoxicityt toxicity@ in vivo Benzimidazole1115-Chlorobenzimidazole5455,6.Dichlorobenzimidazole1455,6-Dichlorobenzimidazole E0771@

755,Ehrlich ribofuranosidell9@@O4,5,6-Trichlorobenzimidazole # ribofurano.sidefi7605010

* Data of Tamm (76). t Data obtained with human and mousebrain tumors in vitro;for details, cf. text. @ Based on the reciprocal of the maximum tolerated dose in mice. Â§Dataof Sugiura (75). # Data reported by Kissman etat. (5@). 1We are indebted to Dr. K. Folkers of Merck and Co. for these compounds.

relationship to the carcinostatic activity of these general aspects of cancer chemotherapy screening, compounds in these particular transplantable tu which may be discussed briefly at this point. mors. Objectives of screening.â€”It is instructive as well The inability of quinacrine to affect tumor as diverting to consult Webster's unabridged dic growth significantly in vivo despite its pronounced tionary (60) for a serviceable definition of a cytotoxicity in vitro has been subjected to further â€œscreen.â€•Thisis a word of many meanings, with analysis with the aid of a sensitive assay method seventeen different entries relevant to subjects for this representative of the acridine series. Com from archeology and cricket to motion pictures parative determinations in brain tumor cell ex and physics. It goes without saying that the plants after exposure to the minimum inhibitory proper meaning for our uses is embodied best concentration and in tumors collected from mice by the second definition (â€œadevice used to sepa or patients after courses of quinacrine administra rate coarser from finer partsâ€•) and that the first tion with various dosage regimens demonstrated and principal meaning of the word (â€œthatwhich that the lowest drug levels which are effective shelters or conceals from viewâ€•) is to be avoided in tissue culture cannot be attained routinely with care. in vivo without prohibitive toxicity to the host A cancer chemotherapy screen, then, should (45). Information gained by this type of experi permit the sifting of large numbers of compounds, ment not only helps to explain discrepancies be with the retention of those which will exert a tween the results of different screening technics significant beneficial effect on cancer in man. It but often aids also in the design or selection of is axiomatic that in theory the cancer patient chemotherapeutic agents which may have a more would provide the most suitable screen, but for suitable distribution pattern and consequently a many valid reasons this test system cannot be better therapeutic index in vivo. employed extensively in practice and a secondary

screen must be selected. In trying to evaluate cancer and the advancement of cancer chemo these ancillary approaches, it cannot be em therapy. phasized too often that at present the correlation Intercompari.8ons ofin vitro reault@.â€”Thesurvey of the response of any laboratory screen with that of tissue culture screening studies which has been of the disease in man is most tenuous. The over presented here has shown that, up to the present, whelming majority of the many thousands of only a few different systems have been exposed compounds examined by one or more experimental to more than a handful of chemicals. Some of screening technics have never been tested in the the test system@shave been designed to seek corn clinic ; where results in human cancer are available, pounds with selective toxicity to tumor cells; it is apparent that the response to treatment others have been tailored to establish the minimum differs with the type of neoplasm, the state of cytotoxic concentration of various agents under the disease, the status of the patient, and numer specified culture conditions. Even with the sparse ous other factors. It is, of course, true that most comparative information now available, discrep of the chemotherapeutic agents now available ancies among various in vitro results are beginning for the clinical treatment of cancer were found to appear. These differences may be a function first in one or another of the experimental screens, of the nature of the culture medium ; for ex and this fact is a strong and telling argument for ample, compounds such as 6-mercaptopurine, 8- the continuation of screening in the laboratory. azaguanine, thioguanine, and 1,9-dimethanesul Not infrequently, however, one may be tempted fonyloxynonane were quite active inhibitors of to claim a special preference for a particular long established cell lines on a semisynthetic me screening technic because it gives a positive re dium (22) but failed to affect primary explants sponse to many agents of clinical usefulness; this of brain tumor cells on a nonsynthetic medium temptation must be resisted, since the few corn at concentrations of 10@i or lower (Table 2); pounds which significantly affect neoplastic growth 5-fluorouracil, another agent without effect on in man have so wide a spectrum of biological the brain tumor cultures, was recently shown activity that almost every screening system can to be a powerful inhibitor of H.Ep. #1 cells in make this claim with equal justification. Eagle's medium (66) ; A-methopterin inhibited the Intercomparison of in vivo results.â€”By far the growth of Sarcoma 180 in Eagle's basal medium, most common screening systems are the trans but this inhibition could be prevented by the plantable rodent tumors whose successful estab addition of hypoxanthine + thymidine + glycine lishment by Hanau and Jensen was mentioned even if the folic acid antagonist was added at at the beginning of this paper. A wide variety concentrations 10,000 times that ordinarily re of experimental tumors have been employed over quired for complete inhibition (38). The nature the years, and, while many compounds have been of the tumor may also play a role, as shown tested only against a single tumor, many inter by the significantly greater effectiveness of 8- comparisons among different tumors have also azaguanine against Brown-Pearce tumor cells than been carried out. These studies have shown that against glioblastoma cells in the same tissue cul there are extensive differences in the response ture medium (38). As more tests are done and of different tumors to the same compound (cf. other tissue culture systems are added (e.g., 54, 85, 72, and many others). No single transplantable 68), additional discrepancies will undoubtedly neoplasm has emerged which has an unusual ca emerge. pacity to predict the clinical usefulness of chemo Comparison of in vitro and in vivo results.â€” therapeutic agents, and it is the current policy Since well under 1000 of the more than 25,000 of major screening operations including those compounds tested in vivo have been examined guided by the Cancer Chemotherapy National in vitro at this writing, there is as yet no real Service Center to test compounds against several basis for comparison. However, if a tentative transplantable tumors side by side, without letting conclusion is to be drawn, the correlation of the the results in one tumor determine the extension response of tumors in tissue culture and in the of the tests to the others. animal has, in general, not been impressive. This Numerous other in vivo systems have become has been illustrated adequately in the survey of available or are in the process of development the brain tumor studies; but other instances may (cf. 73). The examination of their response to also be cited, e.g., the failure of the test system many of the compounds which have already been of Eagle and Foley (22) to respond to hydrocorti tested in the more conventional systems cannot sone, Myleran, or N-methylformamide; the early but add significantly to our understanding of data of McConnell at al. (55) demonstrating that

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1958 American Association for Cancer Research. 876 Cancer Research Vol. 18, September, 1958 emetine and S. marcescens polysaccharide both age caused by a chemical to tumor cells in different damaged Sarcoma 37 in vivo but only the former types of tissue culture. Differences abound in affected the same tumor in vitro; or the recent the intercomparisons of experimental with human report of Schindler and Welch (69) of the failure neoplasms and of in vitro data with both. The rule of Sarcoma 180 cells in tissue culture to respond of this microcosm, as of any other, seems to to 6-azauracil, a potent inhibitor of this tumor be diversity and variety. in vivo, because of the inability of the cultured It may be submitted that too much effort cells to carry out the required conversion of 6- has gone into looking for similarities of response azauracil to 6-azauridine. of different ancillary screening methods, in the There are, of course, many reasons for discrep virtual absence of a primary yardstick by which ancies between in vitro and in vivo results. For they might be judged. What is needed is the one thing, the toxicity of a compound to tumor simultaneous examination of large series of com cells, the primary criterion of activity in tissue pounds of widely varying chemical structure and culture, is only one of the several component biological activity in different in vitro systems, parts of the therapeutic index which is the basis in ascites cells, in solid transplantable neoplasms, of the demonstration of carcinostatic activity in in spontaneous tumors, in heterologous human the animal. Moreover, an agent after administra transplants, and in every other system which tion to the tumor-bearing host may be transformed can contribute meaningful information on their rapidly into a derivative with greater or lesser carcinostatic or carcinolytic activity under every effectiveness against tumor cells or with higher reasonable set of conditions. At the present im or lower toxicity to normal cells; its distribution perfect state of our knowledge, the most fruitful and localization in tissues and body fluids and approach to the complexities of cancer chemother its rate of excretion are crucial factors in an apy may well be the imaginative exploitation in vivo test but are of little significance in tissue of the discrepancies which are bound to emerge culture; problems of cumulative toxicity and the from these comparisons. If cancer chemotherapy development of resistance plague the chemother screening is designed with these objectives in apist working with animal tumors or cancer pa mind, it is clear that tissue culture will continue tients but are of small import in the in vitro to be an integral part of the investigative effort studies; tumor-host interactions and indirect, en in this challenging field. docrine-mediated effects may play a vital role in the response of the neoplasm to chemotherapy but cannot, of course, be reproduced in tissue REFERENCES culture; cells may be endowed with quite different 1. BnmEi@s,J. J. Tissue Culture Screening of Agents in Ex permeability to drugs when bathed by body fluids perimental Cancer Chemotherapy. In: M. B. VisscHmi or by tissue culture feeding fluids; the chemical (ed.), Methods in Medical Research, 4:@7@â€”80.Chicago: Year Book Publishers, Inc., 1951. compound may be more stable in one of these Q . 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Erich Hirschberg

Cancer Res 1958;18:869-878.

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