Comparative Chemotherapy Studies on Primary Short-Term Cultures of Human Normal, Benign, and Malignant Tumor Tissues a Five-Year Study*

Home , Chlorambucil

JEWEL PLUMMER COBB,~ DOROTHY G. WALKER, and JANE C. WRIGHT

(Department of Surgery, New York University, Medical School, the Fourth Surgical Division N.Y.U. of Bellevue Hospital, and the University Hospital, New York, N.Y.)

SUMMARY The cytological alterations in primary short-term tissue cultures of 196 malignant neoplasms, eight benign neoplasms, and fourteen normal tissues of human origin following a 96-hour exposure to several chemotherapeutic agents individually have been described and evaluated. Each replicate culture from a biopsy specimen responded in vitro as an individual entity. Cell populations were either sensitive or resistant. The test agents listed in order of their decreasing cytotoxic capacities in vitro were thio- TEPA, actinomycin D, chlorambucil, methotrexate, and phenylalanine mustard. Certain trends in cell responses were apparent and included: (a) the sensitivity of lymphosarcomas, Hodgkin's lymph nodes, and lymphomas of undetermined type to chlorambucil; (b) the sensitivity of lymphomas of undetermined type to thioTEPA; (c) the sensitivity of fibrosarcomas to actinomycin D, chlorambucil, and thioTEPA; (d) the sensitivity of certain carcinomas to methotrexate and phenylalanine mustard; (e) the resistance of lymphosarcomas to methotrexate and phenylalanine mustard; (f) the resistance of breast carcinomas to chlorambucil; and (g) the resistance of all melanomas to phenylalanine mustard. An exaggerated in vitro response to agents, observed for many malignant and benign neoplasms, was never observed in normal tissues. There was no relation between tissue culture response to drug and (a) growth rate in vitro prior to treatment, (b) primary or metastatic lesion, or (c) prior in vivo therapy. The potential role of chemotherapy studies on short-term primary human neoplastic tissue cultures was discussed.

This report deals with the tissue culture phase primary, short-term tissue cultures of biopsies of a long-range clinical and laboratory study of the freshly excised from the patient. effects of chemotherapeutic agents against cancer. The application of human cell cultures for eval- Data have been compiled for a 5-year period, on uation of the cytotoxic activity of new carcino- the direct action of actinomycin D, methotrexate static agents has been reported by a few investiga- (amethopterin), chlorambucil (CB 1548), thio- tors. A review of this subject has been presented TEPA, and phenylalanine mustard (CB 80~L5) on by Hirschberg (10). The effects of several antitumor agents on a group of primary and first- * This investigation was supported, in part, by research grants from the National Cancer Institute (C-~780, CY-s transfer human neoplastic tissue cultures were re- of the National Institutes of Health, and in part by the Damon ported by Antikajian et al. (1), Cobb (3), and Cobb Runyon Memorial Fund for Cancer Research, Inc. and Walker (4). Eagle and Foley (7, 8) reported Presented in part at the 51st Annual Meeting of the Ameri- the cytotoxicity of over 180 selected compounds can Associationfor Cancer Research, Chicago, Illinois, April 8, on a number of established human normal and 1960. neoplastic cell lines. McAllister et al. (1~) and t Present address: Sarah Iawren(e College, :PIcI::ville, Blakemore et al. (~) determined the cytotoxicity of N.Y. several antitumor agents on human HeLa, J-111, Received for publication July 1, 1960. and human kidney cell lines using an in vitro cyto- 583

toxic metabolic inhibition (CMI) test. Toplin (16) coverslips. Several coverslips constituting four or employed a cytological cytotoxic rating system more explants were exposed to a specific drug and with treated HeLa or other human cell suspension constituted, therefore, one drug series. The rate 2 cultures for screening new agents. Schrek (15) and extent ~ of migration were recorded. Migration utilized a slide-chamber method for screening designates emigration of cells beyond the edge of chemotherapeutic agents against blood and lymph the explant independent of mitotic activity. node cells of leukemic patients and against normal Approximately 35 per cent of the specimens pre- cells as controls. McDonald and Cole (13) reported pared for culture were excluded from the study on the cultivation and testing of eighteen primary owing to either growth failure or absence of identi- cultures of human cancer tissue with several chem- fiable tumor cells. Lymphoma cell types were iden- otherapeutic agents in order to evaluate them for tified by the presence of a large number of imma- individual patient therapy. ture lymphocytic forms. The cellular components of Hodgkin's disease tissues were identified accord- MATERIALS AND METHODS ing to the morphological criteria described by Rei- Human neoplastic tissue was excised, or with- man et al. (14). Melanoma cells were identified by drawn (effusions), from patients with disseminated criteria described by Hu (11) and Cobb and or advanced neoplastic disease. Both primary and Walker (5). Carcinoma cells were identified in metastatic lesions from various anatomical sites vitro by the following criteria: (a) round vesicular were included. The diagnoses of all specimens were nucleus, (b) prominent large nucleoli, (c) often confirmed by microscopic examination. Thirty- polygonal to round shape, (d) marked basophilie two per cent of these specimens were obtained cytoplasm, and (e) variation in nuclear and also from patients who had had chemotherapy within total cell diameter within groups of cells. Sarcoma months prior to biopsy. Benign tumors were ob- cells were identified with some difficulty, but cri- tained at surgery for diagnostic and curative pur- teria used included: (a) rounder nucleus as com- poses. Normal tissues were obtained at surgery pared with fibroblasts, (b) bipolar to pear-shape in performed for (a) herniorraphy, (b) varicose-vein a plasma clot, (c) marked fibrinolysis, and (d) ligation and stripping, (c) excision of questionable swirling growth pattern. Cells with stromal ele- enlarged lymph node, or (d) removal of adjacent ments only were not included for study. The data "normal" tissue (as observed by histologic cri- reported represent studies of drug effects on teria) from patients with a diagnosed malignancy. healthy neoplastic cell types. The tissue described as type (d) may represent an The drug concentrations were as follows: aetino- altered metabolic type, but appeared normal by myein D, 1 X 10-4 ~g/ml; methotrexate, 0.1 mM; morphologic criteria. chlorambucil (CB 1348), 0.5 mM; thioTEPA, 0.1 Tissues were cut into 1- to ~-cu. mm. fragments mM; and phenylalanine mustard (CB 30~5), 0.1 and prepared in rooster plasma clots on coverslips mM. Experiments with 0.5 mM or 0.1 mM CB as multiple cultures. The coverslips were inserted 1348 and human tumor cells produced similar cy- in Porter flasks containing 1 ml. of nutrient me- totoxic responses. The doses employed were chosen dium and rotated in a roller drum at 1~ revolutions after establishment of the lowest concentration. per hour at 37 ~ C. Pleural or ascitic effusions were This caused a reduction in mitoses after r hours treated as cell suspensions on coverslips, as de- in the human carcinoma cell strain HeLa. Each scribed in an earlier report (4). The nutrient me- dose chosen represents an arbitrary concentration dium for the majority of the cultures was Eagle's known to damage several human neoplastic cell basal medium supplemented with ~0 per cent non- types. Replicate cultures were exposed in parallel dialyzed human serum plus one of several antibiot- series to one or several test agents individually, for ics. I Over a 5-year period a number of media were 96 hours. Each tissue type always had an equiva- investigated in an effort to improve growth. A de- lent control group. After 96 hours cultures were tailed list of these media will be presented elsewhere The migration rate is defined as the average distance cells (5). The medium for all cultures was renewed once emigrated from the explant and/or the average density of the or twice each week, depending upon the metabolism same cell population, per 7~-hour interval examinations. of the cultures. Only primary cultures (1-30 days 3 The extent of migration is defined as the approximate dis- in vitro) with identical tumor cell types, and a tance cells emigrated at 10-14 days and is a function of rate of minimum of 1,000 cells in the outgrowth, were migration. Linear measurements (taken from an average of used for control and drug-study series. Each speci- two diameters per explant) of the axes of outgrowth distances (in microns) were made at 100X magnification with an ocular men was cut into small fragments and cultured on micrometer. The cell population density was approximated by 1 Penicillin (100 units/ml) and streptomycin (~0 ~g/ml), or estimating the number of cells in two 150X fields of each out- neomycin (150 ~g/ml). growth, with the use of a Howard grid.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1961 American Association for Cancer Research. COBB et al.--Chemotherapy Studies on Human Tissue Cultures 585 fixed and stained with Jenner-Giemsa. The changes alteration of cytoplasmic processes resulting in a produced by drug treatment were evaluated by the spindle-shaped cell. The radiomimetic alkylating usual cytological criteria of cytotoxicity--i.e., agents, thioTEPA, chlorambucil, and phenyl- granulation, pyknosis, cytolysis, increased cellular alanine mustard produced damage cytologically debris, loss of staining capacity, nucleolar changes, similar to that observed after x-radiation, which mitotic reductions, and chromosomal aberrations. included giant cell formation, a decrease in mito- Cultures which exhibited damage were called sen- sis, and aberrant chromosomal structures (Figs. 8, sitive, whereas those with either equivocal or no 4). Detailed studies of these changes have been damage were called resistant. The extent of cyto- published (6). Damage to cells by all five agents toxicity was classified either as 1 + or 4 +. Over-all was cytologically nonspecific, in that all cell types cytolysis, also called an exaggerated response, was of a population were usually affected. called 4+, whereas mild changes were called 1 +. Summary of drug effects.--Two hundred and eighteen specimens from 188 patients (Table 1) RESULTS were grown in vitro and exposed to one or more Specific drug effects.--Specific cytotoxic changes agents for a total of 546 individual experiments. were observed with each drug type. The antibiotic The responses of all tissue types to the five test actinomycin D produced a decrease in mitosis and agents are summarized in Table ~. The average a reduction in rmcleolar size. Drug changes in a percentage of cells damaged (1+ or 4+ sensitiv- melanoma culture are seen in Figure ~. Control ity) was then used to rank the chemotherapeutic cells are seen in Figure 1. The antimetabolite, agents in decreasing order of effectiveness in vitro. methotrexate, produced a decrease in mitosis, and ThioTEPA produced cytotoxic changes in 71.6 per

TABLE 1 TYPES OF TISSUES GROWN AND TESTED in Vitro

No. No. ~0. No. Histological type Histological site specimens patients specimens patients

I. Malignant lymphomas: V. Malignant neoplasms of nervous Lymphosarcoma 14 10 system: Hodgkin's disease 17 12 Neuroblastoma 1 1 Chronic lymphatic leukemia 1 1 Glioblastoma Mycosis fungoides 8 2 Ependymoma 1 1 Giant follicular lymphoblastoma 1 1 Malignant lymphoma, undeter- Total: 4 4 mined type 9 8 Reticulum-cell sarcoma 8 2 VI. Miscellaneous malignant neoplasms: Multiple myeloma 1 1 Total: 48 86 Wilm's tumor 1 1 Mesothelioma 1 1 II. Sarcomas: Undiagnosed pelvic tumor 1 1 Fibrosarcoma 9 9 Osteogenic sarcoma 2 2 Total: 4 4 Kaposi's sarcoma 8 2 Rhabdomyosarcoma 2 2 VII. Benign tumors: Liposarcoma 1 1 Craniopharyngioma 1 1 Synovial sarcoma 2 1 Neurofibroma Leiomyosarcoma 1 1 Acoustic neuroma 1 1 Sclerosing hemangioma 1 1 Total: 20 18 Benign giant-cell tumor 1 1 Angiolipoma 1 1 III. Melanomas: 24 21 Pigmented nevus 1 1 IV. Carcinomas: Total: 8 8 Skin 4 4 Respiratory system 11 9 VIII. "Normal" Alimentary system and associ- Lymph node 10 10 ated structures 29 27 Spleen 1 1 Male and female reproductive, Granulation tissue urinary system 15 18 Bile duct 1 1 Breast 25 19 Metastases, undetermined his- Total: 14 14 togenesis 7 7 Miscellaneous types 5 4

Total: 96 83

cent of all cultures tested with that agent, while trexate. The latter culture types were also sensitive chlorambucil damaged only 45.8 per cent of all to actinomycin D and thioTEPA. Breast carcino- cultures tested. Actinomycin D produced damage mas were resistant to chlorambucil. The number of in 70 per cent or more cultures of sarcomas, malig- the remaining specific tissue types was too small nant neoplasms of the nervous system, and normal for evaluation. tissues. Damage occurred with chlorambucil in There was a specific and individual response to many cultures of lymphomas and melanomas, and each test agent for each replicate culture from one in all cultures of normal tissues. Damage by thio- biopsy specimen. Hence, the relation between the TEPA was observed in a significant number of sensitivity or resistance of an individual culture to sarcomas, malignant neoplasms of the nervous one test agent and the response of sister cultures to system, and benign neoplasms. Striking instances a second and third test agent was investigated of cellular resistance were noted in sarcoma and (Table 4). Sixty-five per cent of specimens treated melanoma cultures exposed to methotrexate, and in vitro with thioTEPA and also with phenylalanine in melanomas exposed to phenylalanine mustard. mustard (as parallel tissue cultures) displayed

TABLE 2 RRSTONSE OF TISSUE CL%TLrRE CELLS TO TEST AGENTS* (PER CENT CELLS DAMAGED)

PIt ENyI.,a Ln~'INE CHLORA3&BU CIL TOTAl, Ac~o~rrc'~ D METHO~ TE MUSTARD (CB 1848) THIoTEPA No. No. (CB 8025) TISSUE TYPE SPECI- EXPERI- MENS MENT8 No. Per cent No. Per cent No, Per cent No. Per cent No. Per cent tested positive tested positive tested positive tested positive tested positive .,, , I. Lymphomas 48 147 35 54.3 39 37.9 36 66.6 33 63.6 24 35.7 II. Sarcomas 20 44 14 71.4 6 16.6 50.0 13 69.2 3 66.6 ItI. Melanomas 24 66 16 62.5 11 27.2 81. 22 59.1 6 0 IV. Carcinomas 96 213 56 58.9 34 52.9 41 46.3 68 52.9 14 64.28 V. Malignant neoplasms of nervous system 4 18 75.0 4 75.0 4 25.0 4 100.0 2 50.0 VI. Misc. malignant neoplasms 4 10 3 66.6 3 33.3 3 100.0 1 100.0' VII. Benign neoplasms 8 15 4 50.0 1 100.0" 3 66.6 6 66.6 1 0* VIII. "Normal" tissues 14 33 10 70.0 4 50.0 6 100.0 8 61.5 6 50.0

Average per cent cells damaged 63.8 51.4 58.7 71.6 45.8

* One specimen tested with a specific drug.

These totals were then analyzed by individual similar responses. These responses included both tissue types (Table 3). Although the individual sensitivity and resistance. groups were small, nevertheless certain combina- Eighty per cent of specimens severely damaged tions were suggestive of a differential sensitivity or (exaggerated response, 4 -4- S) by one of the agents resistance in some series. Lymphosarcomas, Hodg- were also mildly damaged (1 + S) by specific sec- kin's disease, and lymphomas of undetermined ond agents as cited in the bottom section of Table type were most sensitive to chlorambucil. Lym- 4. phomas, type undetermined by pathological cri- The relations between an exaggerated response teria, were also sensitive to thioTEPA. Lympho- and type of tissue cultured, and test agent, are sarcomas were resistant to methotrexate and summarized in Table 5. An exaggerated response is phenylalanine mustard. Fibrosarcomas were more defined as total cytolysis with loss of cellular in- sensitive to direct drug action than other sarcoma tegrity after a 96-hour treatment with a test agent. types. They were often damaged in vitro by actino- An example of this response is seen in Figure 6. An mycin D, chlorambucil, and thioTEPA. A signifi- exaggerated response was observed in 11.~ per cent cant number of melanomas were resistant to up to 26.6 per cent of culture of lymphomas, sar- methotrexate and phenylalanine mustard. Car- comas, melanomas, carcinomas, miscellaneous ma- cinomas of the gastrointestinal tract, reproductive lignant neoplasms (Figs. 5, 6), and benign neo- and urinary systems were most sensitive to metho- plasms. This type response was never observed in

cultures of malignant neoplasms of the nervous cessive biopsies were alike in pathological type al- system and normal tissues. The highest percentage though excised from different sites. In most in- of severely damaged cultures was observed follow- stances a comparison of responses was possible ing exposure to thioTEPA. The lowest percentage only between two successive biopsies. Three pa- was observed in cultures exposed to methotrexate. tients (with lymphosarcoma, melanoma, and The constancy of a sensitive or resistant re- breast carcinoma) had three or more successive sponse to a test agent by successive biopsies from biopsies. The type of response was then recorded the same patient was investigated (Table 6). Suc- for all the biopsies of the patient and counted as

TABLE 3 RESPONSE OF NORMAL, BENIGN, AND MALIGNANT CELLS*

Tissue types Actinomycin D Methotrexate Chlorambucil ThioTEPA Phenylalanine mustard I. Lymphomas: Lymphosarcoma 5/11 3/10 9/12 5/9 2/5 Hodgkin's disease 7/13 3111 8113 6111 1/5 Chronic lymphatic leukemia 1/1 0/0 1/i 0/1 o/o Mycosis fungoides 1/1 1/1 o/1 1/1 o/o Giant follicular lymphosarcoma o/o 0/0 olo 1/1 o/o Reticulum-cell sarcoma 2/2 2/2 i/2 2/2 112 Malignant lymphoma, undetermined type 317 215 5/7 618 112 II. Sarcomas: Fibrosarcoma 4/5 112 4/~ 5/7 ~/~ Osteogenic sarcoma o/o o/1 1/2 0/o Kaposi's sarcoma 1/2 o/2 0/2 2/3 o/o Rhabdomyosarcoma 2/2 o/o o/o o/o 01o Liposarcoma 1/1 o/o o/o o/o o/o Synovial sarcoma 1/1 o/2 o/1 1/1 o/1 Leiomyosarcoma 0/1 o/o o/o o/o o/o III. Melanomas 10/16 3/11 9/11 1S/22 o16 IV. Carcinomas: Skin s14 1/3 SlS 214 ~/~ Respiratory 3/5 s16 318 1tl Alimentary system 11121 5/5 711s 9118 2/3 Reproductive and urinary system 5/7 417 911o 1/~ Breast 5/14 219 812o 3/5 Undetermined histogenesis I/3 o/1 0/3 3/5 o/o Miscellaneous types (thyroid and malignant ca. of parotid) s/4 011 o/o 2Is 0/1 V. Nervous system: Neuroblastoma 1/1 1/1 1/1 111 0/1 Glioblastoma 2/2 2/2 0/2 2/2 0/0 Ependymoma 0/1 o/1 0/1 1/1 1/1 VI. Miscellaneous malignant neoplasms', Mesothelioma oll o/o 1/1 1/1 o/o Wilm's tumor 1/1 o/o 0/1 o/o o/o Undiagnosed tumor of pelvis 1/1 o/o 0/1 1/1 1/1 Multiple myeloma o/o o/o 0/0 1/1 o/o VII. Benign neoplasms: Neurofibroma 1/2 o/o 1/1 1/2 o/o Craniopharyngioma 0/0 o/o 1/1 1/1 o/o Acoustic neuroma 0/1 1/1 0/1 1/1 o/1 Angioplioma 1/1 o/o 0/0 1/1 o/o Pigmented nevus 0/0 o/o 0/0 o/1 o/o VIII. "Normal" tissues: Normal lymph node s/6 212 2/2 3/6 1/3 Spleen 1/1 oto 1/1 o/o o/ Granulation tissue 2/2 o/~ 2/2 2/2 1/~ Bile duct 1/1 o/o 1/1 o/o 1/1

* The response in each case is given as the total no. positive/total no. experiments.

TABLE 4 one comparison. As indicated in Table 6, 65 per RELATION OF LIKE VERSUS UNLIKE RESPONSES cent of carcinoma multiple biopsy specimens, each OF CULTURES TO A SECOND TEST AGENT* tested only once with each drug, showed a constant response; the other groups showed incon-

TOTAL stant responses to the same test agent. It was of AGENT NO. LIKE USLmE interest, however, that three biopsies from a lym- DRUG RE- RE" phosarcoma patient, removed at different time pe- EXPERI- SPONSE SPONSE First Second MENTS riods, showed constant responses to three of four agents tested. Constant responses were then re- Actinomycin D Methotrexate ---7-~--- 41 31 Actinomycin D Chlorambucil 91 47 44 lated to the test agents. Methotrexate produced Actinomycin D ThioTEPA 104 58 46 the same response (including both sensitivity and Actinomycin D Phenylalanine 37 17 20 mustard resistance) in all multiple biopsy types tested. It Methotrexate Chlorambucil 69 29 40 Methotrexate ThioTEPA 77 42 35 TABLE 5 Methotrexate Phenylalanine 36 14 22 mustard EXAGGERATED RESPONSE RELATED TO PATHOLOGICAL Chlorambucil ThioTEPA 82 39 43 TISSUE TYPE AND CHEMOTHERA- Chlorambucil Phenylalanine 35 14 mustard PEUTIC AGENTS ThioTEPA Phenylalanine 49 32 17 mustard No. ex- Per cent Total no. aggerated tests exaggerated * Data cite comparisons between replicate cultures each response response treated with only one test agent. Four of four specimens which exhibited marked cytotoxic Pathological tissue type: changes (4+S) after treatment with methotrexate were slightly Lymphomas 23 147 15.6 damaged (1+S) by chlorambucil. Sarcomas 5 44 11.3 Three of three specimens which exhibited marked cytotoxic Melanomas 9 66 13.6 changes (4+S) after treatment with methotrexate were slightly Carcinomas 24 213 11.2 damaged (1+S) by actinomycin D. Malignant neoplasms of Fourteen of eighteen specimens which exhibited marked nervous system 18 cytotoxic changes (4+S) after treatment with thioTEPA were Miscellaneous malig- slightly damaged (1+S) by actinomycin D. nant neoplasms 9 22.2 Seven of eight specimens which exhibited marked cytotoxie Benign neoplasms 15 26.6 changes (4+S) after treatment with thioTEPA were slightly "Normal" tissues 33 0 damaged (1+S) by phenylalanine mustard. Eight of nine specimens which exhibited marked cytotoxie ThioTEPA 30 160 18.7 changes (4+S) after treatment with chlorambucil were slightly Chlorambucil 15 109 13.8 damaged (1 +S) by actinomycin D. Actinomycin D 13 141 9.2 Three of three specimens which exhibited marked cytotoxie Phenylalanine mustard 4 48 8.3 changes (4+S) after treatment with phenylalanine mustard Methotrexate 5 91 5.4 were slightly damaged (1 +S) by thioTEPA.

TABLE 6 RESPONSE OF MULTIPLE BIOPSIES*

Correlation of No. No. No. com- No, like responses Tissue type patients specimens parisons constant between any two specimens

Relation by pathological type: Lymphomas 15 20 10 m Sarcomas 4 2 2 + Melanomas 6 4 2 Carcinomas 14 17 11 + Trem

Relation by test agent: Actinomycin D 9 20 9 4 Methotrexate 9 20 9 9 + Chlorambucil 10 22 10 4 ThioTEPA 13 29 13 7 Phenylalanine mustard 2 4 2 41

* Time interval between excisions of biopsies ranged from 3 weeks to 28 months.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1961 American Association for Cancer Research. COBB et al.--Chemotherapy Studies on Human Tissue Cultures 589 was impossible to evaluate specific in vivo therapy, trexate, caused pyknosis to form narrow, elongated given before or between biopsies, in relation to cells. All three alkylating agents, thioTEPA, constant responses owing to the small sampling of chlorambucil, and phenylalanine mustard, pro- patients with identical drug therapy. duced changes that were qualitatively similar and A comparison was made between rate and ex- resembled changes seen after x-radiation--namely, tent of migration and growth in vitro prior to aberrant chromosome structures and giant cells. treatment and subsequent response for all normal, The compounds in decreasing order of effective- malignant, and benign neoplastic tissues tested. ness were: thioTEPA, actinomycin D, chlorambu- There was no detectable correlation between the cil, methotrexate, and phenylalanine mustard. prior growth rate in vitro and response to any test Whereas tumor types in general responded indi- agent other than that expected due to chance. All vidually to the test agents, certain trends were solid tissues were handled and cultured in an iden- apparent: tical manner, regardless of the tissue type. There- a) the sensitivity of lymphosarcomas, Hodg- fore, differences in cytotoxic responses as described kin's, and lymphomas of undetermined type to di- for individual specimens apparently were not re- rect exposure to chlorambucil; flections of either differences in growth rates of b) the sensitivity of lymphomas to thioTEPA; cultures prior to drug addition or methods of cul- c) the sensitivity of fibrosarcomas to direct ex- ture. posure to actinomycin D, chlorambucil, and thio- To investigate the relation between the response TEPA; of primary versus metastatic tissues, the responses d) the sensitivity of certain carcinoma types to of 30 culture series of primary melanoma and car- methotrexate, actinomycin D, and thioTEPA; cinoma lesions were compared with the responses e) the resistance of lymphosarcomas to metho- of 30 randomly selected culture series of metastatic trexate and phenylalanine mustard; lesions of the same type. This group did not in- f) the resistance of breast carcinomas to chlor- clude any primary and metastatic tumors from the ambucil; and same patient. There was no significant difference g) the resistance of all melanomas tested to in sensitivity or resistance to any agent based on phenylalanine mustard. lesion type in the group evaluated. Normal treated tissue cultures never displayed The relation of prior in vivo therapy to cellular the severe cytotoxic changes that were observed in response in vitro was investigated. Prior treatment some cultures of tumor tissues exposed to identical included administration of carcinostatic agents drug concentrations. These data may be useful in within ~ months prior to biopsy and/or radiation clinical procedures involving the use of agents ad- therapy within 7 months prior to biopsy. The re- ministered at the tissue site as, for example, in sponses of 154 specimens excised from patients perfusions. with prior in vivo therapy were compared with the Studies based on a large group of malignant neo- responses of 311 specimens from patients with no plasms indicated that there was no relation be- prior therapy. All specimens were excised from tween migration and growth in tissue culture and patients treated with one or several of the drugs cell response to drug. This conclusion was further tested in vitro in this study. An analysis of the supported by the consistent responses to drugs of experimental data failed to reveal over-all sensi- three successive biopsies from a patient with lym- tivity or resistance as a result of prior therapy. phosarcoma, despite their different rates of growth in vitro. There was no relation between tissue cul- DISCUSSION ture response based on primary or metastatic le- The five agents under investigation produced sion type. A more definitive analysis awaits a cellular changes in a wide variety of human benign study of drug effects based on the in vitro responses and malignant tumors and normal tissues in vitro. of primary and metastatic tissues from the same They responded, in the main, as individual tissue patient. specimens. Cells were either sensitive or resistant Of interest was the absence of a relation between to each agent individually, except in a few cases prior in vivo therapy and drug response in vitro. which responded in a similar manner to thioTEPA Apparently, tissues which survive therapy and and phenylalanine mustard. All agents tested pro- grow in vitro are able to respond like untreated tis- duced direct objective cytological changes. All five sues. The effect of prior therapy seems to be more agents caused mitotic inhibition and cytolysis. closely related to success or failure of growth in The antibiotic, actinomycin D, produced nu- vitro, according to the observations of Cobb and cleolar reduction. The antimetabolite, metho- Walker (5) on a series of melanomas.

Although a rationale exists for the in vitro test- 5. . Studies on Human Melanoma Cells in Tissue Cul- ing of promising chemotherapeutic agents of ture. I. Growth Characteristics and Cytology. Cancer Re- search, 20: 858-67, 1960. freshly excised human tumor tissue, the data re- 6. COBB,J. P.; WALXER, D. G.; and WRIGHT, J. C. Observa- ported here demonstrate some inconsistent re- tions on the Action of Triethylene Thiophosphoramide sponses to the same drug in repeat biopsies for four within Individual Cells. Acta Unio Internat. contra can- of the five agents tested. Factors which may influ- crum, 16:567-83, 1960. ence the response of repeat biopsies may involve 7. EAGLE, H., and FOLEY, J. E. Cytotoxic Action of Car- cinolytic Agents in Tissue Culture. Am. J. Med., 21: 739- variations in the physiology of the tumor in vivo at 49, 1956. the time of excision which cannot be evaluated. 8. --. Cytotoxicity in Human Cell Cultures as Primary The relation between in vitro and in vivo drug ef- Screen for Detection of Anti-Tumor Agents. Cancer Re- fects in animals and human tumors has been varied search, 18:1017-25, 1958. 9. GOI~MB, F. M.; WRIGHT, J. C.; COBB, J. P.-" GUMPORT, (14). However, some investigators have reported S. L.; POSTEL, A.; and SAFAm, D. The Chemotherapy of good correlations (9, 15, 17). Cautious clinical ex- Human Solid Tumors by Perfusion Technics. Proc. Am. trapolation from tissue culture data is in order Assoc. Cancer Research, 3: 92, 1960. pending additional culture-clinical correlation re- 10. I-ImscHBmtG, E. Tissue Culture in Cancer Chemotherapy sults. Such an extended study is now in progress. 4 Screening. Cancer Research, 18:869-78, 1958. 11. Hu, F. Cytological Studies of Human Figment Cells in Tissue Culture. In: Figment Cell Biology, pp. 147-58. New ACKNOWLEDGMENTS York: Academic Press, 1959. The authors are grateful to Miss Marcia Rowan and Mr. 12. McALLISTER,R.; GRV~mEa, P. W.; ComE~, L. L.; and Carlton H. Nadolney for their technical assistance. BmKEMORE, W. S. A Quantitative Technique for the Measurement of in Vitro Cytotoxieity of 5-Fluor-2-Deoxy- REFERENCES uridine (FUDR), Nitrogen Mustard (HN~), and Other 1. ANTIKAJIAN, G.; W~UOHT, L. T.; P~ER, J. I.; and Antitumor Agents. Cancer, 12:938-48, 1959. WEI~TP~UB, S. The Effect of Triethyleue Melamine, 13. McDoNAIZ~, G. 0., and COLE, W. H. The Use of Human Aureomycin, and Some 4-Amino Derivatives of FoIic Acid Tumors in Primary Culture for Testing Anticancer Com- on Tissues in Vitro. J. Nat. Cancer Inst., 12:269--74, 1951. pounds. Surgical Forum, 10: 67-71, 1959. 2. BLAKEMORE, W. S.; McKENNA, J. M.; MxCALLmTER, 14. R~rMAN, M. S.; STERN, I. R.; FORD, M. Z.; and HOSTER, H. A. Studies in Hodgkin's Syndrome. XI. The Influence R. M.; and CORmLL, L. L. The Cytotoxic Metabolic In- hibition Test for Screening Compounds for Antitumor Ac- of Normal Serum and Hodgkin's Serum on Cellular Growth tivity. Proc. Am. Assoc. Cancer Research, 3:96, 1960. and Morphology in Tissue Culture. Cancer Research, 10: 3. COBB, J. P. Tissue Culture Observations of the Effects of 467-78, 1950. Chemotherapeutic Agents on Human Tumors. Trans. N.Y. 15. SCHRm~, R. A Method for Screening Chemotherapeutic Acid. Sc., II, 17: 257-49, 1955. Agents against Normal and Leukemic Cells of Man. Proc. 4. COBB, J. P., and WAI,KER, D. G. Effect of Actinomycin D Am. Assoc. Cancer Research, 3:148, 1960. 16. ToPxzN, I. A Tissue Culture Cytotoxicity Test for Large- on Tissue Cultures of Normal and Neoplastic Cells. J. Nat. Cancer Inst., 21: 263--77, 1958. Scale Cancer Chemotherapy Screening. Cancer Research, 19: 959-65, 1959. 4 j. C. Wright, J. P. Cobb, S. L. Gumport, F. M. Golomb, 17. WPaOHT, J. C.; COBB, J. P.; GUMPORT, S. L.; GOLO~B, D. Safadi, D. G. Walker, and C. Nadolney. Further Investiga- F. M.; and SAFXDI, D. Investigation of the Relation be- tions of the Relation between Clinical and Tissue-Culture tween Clinical and Tissue-Culture Response to Chemo- Response to Chemotherapeutic Agents on Human Cancer (to therapeutic Agents on Human Cancer. New Eng. J. Med., be published). 257:1207-11, 1957.

FIG. 1.--Melanoma cells in 13-day control culture. Note culture of an anaplastic skin carcinoma exposed for 96 hours to number and size of nucleoli. Jenner-Giemsa, X470. chlorambucil (classified as a 1+ response). Jenner-Giemsa, FIG. 2.--Melanoma cells in 13-day culture exposed for 96 X900. hours to aetinomyein D. Note decreased number and size of FIG. 4.--Scattered chromosomes in metaphase stage in 7- nucleoli, and contracted cytoplasmic processes (classified as a day culture of an anaplastie skin carcinoma exposed for 96 1+ response). Jenner-Giemsa, )<470. hours to chloramhucil (classified as a 1+ response). Jenner- FiG. 3.--Lagging chromosomes in anaphase stage in 7-day Giemsa, X900.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1961 American Association for Cancer Research. COBB et al.--Chemotherapy Studies on Human Tissue Cultures 589 was impossible to evaluate specific in vivo therapy, trexate, caused pyknosis to form narrow, elongated given before or between biopsies, in relation to cells. All three alkylating agents, thioTEPA, constant responses owing to the small sampling of chlorambucil, and phenylalanine mustard, pro- patients with identical drug therapy. duced changes that were qualitatively similar and A comparison was made between rate and ex- resembled changes seen after x-radiation--namely, tent of migration and growth in vitro prior to aberrant chromosome structures and giant cells. treatment and subsequent response for all normal, The compounds in decreasing order of effective- malignant, and benign neoplastic tissues tested. ness were: thioTEPA, actinomycin D, chlorambu- There was no detectable correlation between the cil, methotrexate, and phenylalanine mustard. prior growth rate in vitro and response to any test Whereas tumor types in general responded indi- agent other than that expected due to chance. All vidually to the test agents, certain trends were solid tissues were handled and cultured in an iden- apparent: tical manner, regardless of the tissue type. There- a) the sensitivity of lymphosarcomas, Hodg- fore, differences in cytotoxic responses as described kin's, and lymphomas of undetermined type to di- for individual specimens apparently were not re- rect exposure to chlorambucil; flections of either differences in growth rates of b) the sensitivity of lymphomas to thioTEPA; cultures prior to drug addition or methods of cul- c) the sensitivity of fibrosarcomas to direct ex- ture. posure to actinomycin D, chlorambucil, and thio- To investigate the relation between the response TEPA; of primary versus metastatic tissues, the responses d) the sensitivity of certain carcinoma types to of 30 culture series of primary melanoma and car- methotrexate, actinomycin D, and thioTEPA; cinoma lesions were compared with the responses e) the resistance of lymphosarcomas to metho- of 30 randomly selected culture series of metastatic trexate and phenylalanine mustard; lesions of the same type. This group did not in- f) the resistance of breast carcinomas to chlor- clude any primary and metastatic tumors from the ambucil; and same patient. There was no significant difference g) the resistance of all melanomas tested to in sensitivity or resistance to any agent based on phenylalanine mustard. lesion type in the group evaluated. Normal treated tissue cultures never displayed The relation of prior in vivo therapy to cellular the severe cytotoxic changes that were observed in response in vitro was investigated. Prior treatment some cultures of tumor tissues exposed to identical included administration of carcinostatic agents drug concentrations. These data may be useful in within ~ months prior to biopsy and/or radiation clinical procedures involving the use of agents ad- therapy within 7 months prior to biopsy. The re- ministered at the tissue site as, for example, in sponses of 154 specimens excised from patients perfusions. with prior in vivo therapy were compared with the Studies based on a large group of malignant neo- responses of 311 specimens from patients with no plasms indicated that there was no relation be- prior therapy. All specimens were excised from tween migration and growth in tissue culture and patients treated with one or several of the drugs cell response to drug. This conclusion was further tested in vitro in this study. An analysis of the supported by the consistent responses to drugs of experimental data failed to reveal over-all sensi- three successive biopsies from a patient with lym- tivity or resistance as a result of prior therapy. phosarcoma, despite their different rates of growth in vitro. There was no relation between tissue cul- DISCUSSION ture response based on primary or metastatic le- The five agents under investigation produced sion type. A more definitive analysis awaits a cellular changes in a wide variety of human benign study of drug effects based on the in vitro responses and malignant tumors and normal tissues in vitro. of primary and metastatic tissues from the same They responded, in the main, as individual tissue patient. specimens. Cells were either sensitive or resistant Of interest was the absence of a relation between to each agent individually, except in a few cases prior in vivo therapy and drug response in vitro. which responded in a similar manner to thioTEPA Apparently, tissues which survive therapy and and phenylalanine mustard. All agents tested pro- grow in vitro are able to respond like untreated tis- duced direct objective cytological changes. All five sues. The effect of prior therapy seems to be more agents caused mitotic inhibition and cytolysis. closely related to success or failure of growth in The antibiotic, actinomycin D, produced nu- vitro, according to the observations of Cobb and cleolar reduction. The antimetabolite, metho- Walker (5) on a series of melanomas.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1961 American Association for Cancer Research. FIG. 5.--Wihn's tumor cells in l~-day control culture. Jenner-Giemsa, )<470. FiG. 6.--Wilm's tumor cells in 1"2-day culture exposed for 96 hours to actinomycin D. Note marked cell damage classified as a 4+ response. Jenner-Giemsa, )<470.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1961 American Association for Cancer Research. COBB et al.--Chemotherapy Studies on Human Tissue Cultures 589 was impossible to evaluate specific in vivo therapy, trexate, caused pyknosis to form narrow, elongated given before or between biopsies, in relation to cells. All three alkylating agents, thioTEPA, constant responses owing to the small sampling of chlorambucil, and phenylalanine mustard, pro- patients with identical drug therapy. duced changes that were qualitatively similar and A comparison was made between rate and ex- resembled changes seen after x-radiation--namely, tent of migration and growth in vitro prior to aberrant chromosome structures and giant cells. treatment and subsequent response for all normal, The compounds in decreasing order of effective- malignant, and benign neoplastic tissues tested. ness were: thioTEPA, actinomycin D, chlorambu- There was no detectable correlation between the cil, methotrexate, and phenylalanine mustard. prior growth rate in vitro and response to any test Whereas tumor types in general responded indi- agent other than that expected due to chance. All vidually to the test agents, certain trends were solid tissues were handled and cultured in an iden- apparent: tical manner, regardless of the tissue type. There- a) the sensitivity of lymphosarcomas, Hodg- fore, differences in cytotoxic responses as described kin's, and lymphomas of undetermined type to di- for individual specimens apparently were not re- rect exposure to chlorambucil; flections of either differences in growth rates of b) the sensitivity of lymphomas to thioTEPA; cultures prior to drug addition or methods of cul- c) the sensitivity of fibrosarcomas to direct ex- ture. posure to actinomycin D, chlorambucil, and thio- To investigate the relation between the response TEPA; of primary versus metastatic tissues, the responses d) the sensitivity of certain carcinoma types to of 30 culture series of primary melanoma and car- methotrexate, actinomycin D, and thioTEPA; cinoma lesions were compared with the responses e) the resistance of lymphosarcomas to metho- of 30 randomly selected culture series of metastatic trexate and phenylalanine mustard; lesions of the same type. This group did not in- f) the resistance of breast carcinomas to chlor- clude any primary and metastatic tumors from the ambucil; and same patient. There was no significant difference g) the resistance of all melanomas tested to in sensitivity or resistance to any agent based on phenylalanine mustard. lesion type in the group evaluated. Normal treated tissue cultures never displayed The relation of prior in vivo therapy to cellular the severe cytotoxic changes that were observed in response in vitro was investigated. Prior treatment some cultures of tumor tissues exposed to identical included administration of carcinostatic agents drug concentrations. These data may be useful in within ~ months prior to biopsy and/or radiation clinical procedures involving the use of agents ad- therapy within 7 months prior to biopsy. The re- ministered at the tissue site as, for example, in sponses of 154 specimens excised from patients perfusions. with prior in vivo therapy were compared with the Studies based on a large group of malignant neo- responses of 311 specimens from patients with no plasms indicated that there was no relation be- prior therapy. All specimens were excised from tween migration and growth in tissue culture and patients treated with one or several of the drugs cell response to drug. This conclusion was further tested in vitro in this study. An analysis of the supported by the consistent responses to drugs of experimental data failed to reveal over-all sensi- three successive biopsies from a patient with lym- tivity or resistance as a result of prior therapy. phosarcoma, despite their different rates of growth in vitro. There was no relation between tissue cul- DISCUSSION ture response based on primary or metastatic le- The five agents under investigation produced sion type. A more definitive analysis awaits a cellular changes in a wide variety of human benign study of drug effects based on the in vitro responses and malignant tumors and normal tissues in vitro. of primary and metastatic tissues from the same They responded, in the main, as individual tissue patient. specimens. Cells were either sensitive or resistant Of interest was the absence of a relation between to each agent individually, except in a few cases prior in vivo therapy and drug response in vitro. which responded in a similar manner to thioTEPA Apparently, tissues which survive therapy and and phenylalanine mustard. All agents tested pro- grow in vitro are able to respond like untreated tis- duced direct objective cytological changes. All five sues. The effect of prior therapy seems to be more agents caused mitotic inhibition and cytolysis. closely related to success or failure of growth in The antibiotic, actinomycin D, produced nu- vitro, according to the observations of Cobb and cleolar reduction. The antimetabolite, metho- Walker (5) on a series of melanomas.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1961 American Association for Cancer Research. Comparative Chemotherapy Studies on Primary Short-Term Cultures of Human Normal, Benign, and Malignant Tumor Tissues−−a Five-Year Study

Jewel Plummer Cobb, Dorothy G. Walker and Jane C. Wright

Cancer Res 1961;21:583.

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