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[CANCER RESEARCH 38,3595-3598. 0008-5472/78/0038-OOOOS02.00 November 1978] In Vitro Analysis of the Response of Multicellular Tumor Spheroids Exposed to Chemotherapeutic Agents in Vitro or in V/Vo1 John M. Yuhas,2 Anne E. Tarleton, and James G. Harman Cancer Research and Treatment Mexico 87131. Center [J. M. Y., A. E. T., J. G. H.J and Department ABSTRACT Multicellular tumor spheroids (MTS) have been exposed to Chemotherapeutic agents in vitro (nitrogen mustard) or in vivo (cyclophosphamide) and analyzed in vitro in terms of altered growth patterns. Whether the MTS were ex posed in vitro or in vivo, the major effect of the drugs was to induce a dose-dependent lag period before the normal MTS growth rate resumed. Exposure of MTS in the perito neal cavity to i.v. injection cyclophosphamide results in patterns similar to the in vitro exposure system, except that a host anti-MTS reaction was detected. In combina tion, these two methods allow the study of the responses of these complex tumor forms to chemotherapy. INTRODUCTION One of the more perplexing problems in clinical cancer chemotherapy is the wide range of responses that a given drug or drug combination produces in apparently identical cases. Although both the patients and the tumors are indistinguishable in terms of standard criteria, one tumor will completely regress following treatment, whereas an other will progress not only after treatment but also during treatment. This individual variation in responsiveness can be attributed, at least in part, to variation in drug metabo lism (10), tumor-inhibiting (9) or tumor-promoting (5) fac tors, or the responsiveness of the tumor to the drug (4). Presently, it is difficult to evaluate the role of each of these (and other) factors, but the responsiveness of the tumorper se must play a major role. For evaluation of this role, however, it will be necessary to study the tumors without the confounding influences of the other factors, i.e., in vitro. For several years monolayer cultures have been used for this purpose, in spite of the fact that their only similarity to in vivo tumors is that the cell type is the same. Growing, 3-dimensional organized masses of tumor cells, which possess many of the characteristics of solid tumors in vivo, have been available, but they were grown under conditions (3, 7) that made Chemotherapeutic studies impractical, and very few tumors were thus adapted. More recently, we (11, 12) and others (6) have observed that MTS3 can be grown from a variety of solid tumors merely by placing the cells, in liquid medium, into plates that had been base coated with agar. These MTS lines show wide variations in growth rate 1 Research was supported by Contract NIH-N01-CB-74203 and Grant 1P30-CA-21074-01from the National Cancer Institute. 2 To whom requests for reprints should be addressed: at Cancer Research and Treatment Center, University of New Mexico, Albuquerque, N. M. 87131. 3 The abbreviations used are: MTS, multicellular tumor spheroids; EBME, Eagle's basal medium; HN2, nitrogen mustard; CYC, cyclophosphamide. Received April 24,1978; accepted July 28,1978. NOVEMBER of Radiology [J. M. Y.), University of New Mexico, Albuquerque, New and growth fraction (11), thereby offering a tool for the study of tumor-drug interactions. We report the develop ment of 2 assay methods whereby one can analyze the response of MTS in vitro following exposure to Chemother apeutic drugs in vivo or in vitro. MATERIALS AND METHODS Cells. Two cell lines were used in the present studies: a radiation-induced mammary carcinoma of the BALB/c mouse, MCa-11 (12); and the Line 1 lung carcinoma from the same mouse (12). The lines were maintained as monolayer cultures in EBME supplemented with 10% fetal calf serum,4 50 units penicillin per ml, 50 /*g streptomycin per ml (Grand Island Biological Co., Grand Island, N. Y.), and 10 /¿gsodium insulin per ml (Elanco Products Co., Indian apolis, Ind.). MTS. The methods for producing and studying the growth of MTS are the same as those described elsewhere (11, 12). Approximately 106 cells in 10 ml of EBME are plated in 100-mm plastic Petri dishes that have previously been base coated with 0.75% Noble agar (Difco Laborato ries, Inc., Detroit, Mich.) in complete EBME. Within 9 and 6 days, respectively, MCa-11 and line 1 MTS measuring 250 to 350 /j.m develop and are readily isolated for study. Following treatment the MTS are returned to individual agar-based 16-mm wells along with 1 ml of complete EBME. The MTS are sized at least 3 times weekly on a dissecting microscope, and the medium is changed twice weekly. The MTS "cure" experiments were performed by placing MTS in standard 16-mm wells after treatment. At 2-day intervals after treatment, the wells, each containing 1 MTS, were examined with both dissecting and phase-contrast microscopes. At each inspection each well was scored as positive or negative for detectable outgrowth. The lack of detectable outgrowth is equated with cure in this system. Drugs. Two drugs were purchased commercially and used in the present studies: HN2 (Mustargen; Merck Sharp and Dohme, West Point, Pa.); and CYC (Cytoxan; Mead Johnson & Co., Evansville, Ind.). Immediately before use the drugs were diluted with complete EBME for the in vitro exposures or with 0.9% NaCI solution for in vivo injections. Two-month-old female BALB/c mice served as the hosts for the in vivo assays. For the in vitro exposures, 12 to 18 MTS were placed in 60-mm agar-based dishes along with 5 ml of complete medium for a 24-hr pretreatment. This pretreatment was used to avoid the variable and unknown effects of crowding ' Fetal calf serum was dialyzed and absorbed on dextran-coated charcoal (1) to reduce endogenous insulin and steroid hormones to as low a level as possible. 1978 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1978 American Association for Cancer Research. 3595 J. M. Yuhas et al. these data is to calculate delay in time to reach a given size versus drug concentration. Over the range of 400 to 700 ¿¿m, the shape and slope of the curve were independent of the final size chosen for analysis and were linear, with a slope of 14.5 ±1.36 (S.E.) days delayed/pig/ml. This inde pendence of size chosen for analysis is a reflection of the fact that the drug has induced a dose-dependent lag period but has not altered the growth rate once growth is resumed (Chart 1). Similar studies involving a variety of drugs, MTS, and exposure schedules have been performed in our laboratory and will be reported elsewhere. In our experience the shape of the dose-response curve and its slope are a complex product of the interaction among the drug, MTS, and exposure pattern especially for more complex drugs, which operate at a specific phase of the cell cycle. The range of the sensitivity estimates (slopes) has not exceeded 10% in RESULTS our experience thus far, for a given drug/MTS combination. Chart 1 is a plot of the growth of 300-^m MCa-11 MTS as The responses of MTS to chemotherapeutic agents can a function of time after a 1-hr exposure to graded doses of also be quantitated in terms of cure. Cure in the present HN2. In actual practice the MTS ranged from 250 to 325 context is defined as the inability to show growth, in standard tissue culture plates, by some presettime. Chart 2 /urn. Since the diameter increase per day is a linear function (12), we normalized all groups to an initial size of 300 ¿¿m.is a plot of the percentage of 300-/¿mMCa-11 MTS, which The primary effect of exposure to HN2 is to produce a dose- showed viable cell outgrowth in standard tissue culture dependent increase in the "lag" period before the normal dishes at varying periods after treatment as a function of growth rate resumes (Chart 1). As had been used in the past the concentration of HN2, which they were exposed to for 1 hr. As time after exposure increases, the HN2 concentration for in vivo experiments (8), a convenient way to analyze required for 50% cure increases to a maximum of 1.28 /u,g/ ml at Day 17 (Chart 2) and afterward (data not shown). This is a reflection of the fact that the time required to produce a detectable outgrowth is inversely related to the number of 700 viable cells in the transferred MTS, which is in turn inversely related to the HN2 concentration. The plateauing of the HN2 concentration required to inhibit outgrowth by Day 17 and afterward suggests that all cells that are capable of outgrowth do so by this time. The time at which cure rates plateau depends on the MTS line studied, but it appears 600 independent of the drug under consideration. As will be discussed in a subsequent report, however, cures are difficult to achieve with cycle-specific and phase-specific drugs unless the exposure time is prolonged far beyond 1 hr. in the plates used to mass produce the MTS. The groups of MTS were then transferred to similar plates containing the appropriate concentrations of the respective drugs and returned to the incubator for 1 hr. Following treatment the MTS were removed from the drugs, washed twice with complete medium, and placed individually in 16-mm agarbased wells along with 1 ml of medium. For in vivo exposure of MTS, approximately 30 MTS were injected i.p. in 0.5 ml of EBME. Fifteen to 30 min later, the drugs were injected i.V.; 4 or 24 hrs later, the mice were killed and the MTS were harvested from the peritoneal cavity by means of a sterile Pasteur pipet. Fifty % of the MTS can usually be retrieved. Following washing as de scribed previously, the MTS were transferred to 16-mm agar-based wells with 1 ml of EBME. 500 100 400 300 025 5 10 15 20 DAYS POST-TREATMENT 25 30 Chart 1. Growth of MCa-11 MTS as a function of time after exposure in vitro to graded concentrations of HN2 (N, 12/group); •¿ 0 ^g/ml; O, 0.25 fig/ml; x , 0.5 /¿g/ml;A, 1.0 /¿g/ml. 3596 050 075 1OO 125 ISO 175 200 Chart 2. Percentage of SOO-^m MCa-11 MTS cured by a 1-hr in vitro exposure to graded concentrations of HN2 (H/V,). Cure is defined as the lack of detectable viable cell outgrowth when the MTS are placed in standard tissue culture wells. Inspection for outgrowth was made at 2-day intervals, starting on Day 1 after treatment, through Day 21. After Day 17 the data remained constant. •¿, assayed on Day 9; O, assayed on Day 13; and x, assayed on Day 17. CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1978 American Association for Cancer Research. Chemotherapy Similar studies (data not shown) in which MCa-11 MTS were exposed to HN2 and placed in agar-based wells yielded a similar estimate of the concentration of HN2 (1-hr exposure) required for a 50% cure; i.e., all MTS exposed to I /¿g/ml regrew within 30 days, whereas none of the MTS exposed to 2 /¿g/mldid so. The strict in vitro system described previously (in vitro exposure and in vitro analysis), although providing a high level of control and precision, cannot simulate the rise, plateau, and fall of drug concentrations in vivo without requiring esoteric apparatus or a knowledge of the propor tionality constants relating the effects of exposure to a constant concentration versus exposure to a rising and falling one (2). A second problem of the strict in vitro system is that it requires the use of additional biological supple ments (e.g., liver microsomes) to allow the study of drugs such as CYC, which require metabolic activation (2). Lastly, the in vitro system is not a perfect duplication of in vivo environments, which includes such factors as hormones, ¡mmunologically reactive effectors, etc. Although it is our hope to eventually simulate the in vivo conditions in vitro, it will first be necessary to develop an understanding of the in vivo patterns so that they can be duplicated in vitro. Toward this end we have been studying the in vitro growth of MTS, which were exposed to chemotherapeutic agents in vivo. In brief, MCa-11 MTS are injected into the peritoneal cavity of syngeneic BALB/c mice, and 15 to 30 min later, the drug is injected i.v. Four or 24 hr later, the mice are killed and the MTS are harvested from the peritonal cavity, placed in agar-based wells along with 1 ml of medium, and followed as described previously. In the absence of drug injection, MCa-11 MTS, which had been placed in the peritoneal cavity of syngeneic mice, showed a lag period in growth of 3 to 7 days, after which the normal growth rate resumed (Chart 3a). With another syngeneic tumor, Line 1 (Chart 3b), no such lag effect of a 24-hr peritoneal exposure was observed. This sensitivity of the MCa-11 MTS to peritoneal exposure is barely detecta ble, if the peritoneal exposure is limited to 4 hr (data not shown), and does not appear to be due to the trauma of the procedure. Chart 4 is a plot of the delay in growth for MCaII MTS due to a 24-hr peritoneal exposure in syngeneic BALB/c mice that had been given a 250 mg/kg injection of CYC between 1 and 15 days earlier. Injection of CYC 1 to 2 days before placing the MCa-11 MTS in the peritoneal cavity resulted in a reduction of this peritoneal exposure effect. Between 5 and 9 days after CYC, the peritoneal exposure effect was even more pronounced than it was in controls, but by Day 15 after CYC, it had returned to normal. Chart 5 is a plot of the growth (in agar-based wells) of MCa-11 MTS as a function of time after they have been exposed, in the peritoneal cavity of BALB/c, for 15 to 30 min prior to and for 4 hr after the i.v. injection of graded doses of CYC. Controls received no drug injection but were placed in the peritoneal cavity for 4 hr. A fraction of each of the groups treated with CYC failed to grow during the 30day observation period. This fraction increased directly with drug dose (Chart 5) and was not included in the regrowth calculations. As was the case for the strict in vitro system (Chart 1), increasing drug doses served to increase the lag period before the normal growth rate resumed. The plot of and MTS 400 350 -100 0 DAYS Chart 3. Growth of MCa-11 and line 1 MTS after a 24-hr exposure in the peritoneal cavity of syngeneic female BALB/c mice ( •¿ ) compared to the growth of similar MTS that remained in vitro (O). Data are plotted as growth since the sizes of the 2 lines differed at the time of analysis, a, MCa-11, 275 to 325 ¿im;b, line 1, 400 to 450 ^m. Size was not a factor in this comparison since subsequent studies showed a similar effect for MCa-11 MTS over the size range of 200 to 550 urn 8 12 510 6 8 DAYS POST Chart 4. Delay in growth of MCa-11 MTS, due to a 24-hr exposure in the peritoneal cavity of BALB/c mice, as a function of the time since the mice had received an i.v. injection of CYC (250 mg/kg). , delay observed in mice that had never received CYC. delay in time to reach sizes of 400 or 500 /¿mversus injected drug dose was linear, with a slope of 0.08 ±0.004 day/mg/ kg. DISCUSSION As a complex tissue composed of tumor cells in varying physiological states, MTS do not provide the precise esti mates of the kinetics of drug-induced cell killing that one NOVEMBER 1978 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1978 American Association for Cancer Research. 3597 J. M. Yuhas et al. 550- 4 6 8 10 12 14 POST-TREATMENT (DAYS) 16 Chart 5. In vitro growth of 300-jim MCa-11 MTS, which had been placed in the peritoneal cavity of BALB/c mice 15 to 20 min prior to and had been allowed to remain for 4 hr after i.v. injections of O(O), 50 (x), 100(«),or 200 (A) mgCYC per kg. obtains in monolayer experiments. In fact we have not identified which subpopulations of cells in the MTS are being injured or whether this injury is cell killing, mitotic delay, both, or neither. Admittedly, the drug responses of MTS must eventually be described in more precise terms, but their major advantage is their similarity to in vivo tumors (3, 7, 11,12). Whether this similarity will allow prediction of in vivo tumor responses remains to be determined. Each of the 2 methods for exposing MTS, described previ ously, possesses certain advantages and disadvantages. The strict in vitro system allows precise quantitation of drug dose, but in doing so it fails to parallel the oscillating drug concen trations that an in situ tumor would encounter in v/Vo.The in vivo exposure system produces the rising and falling drug kinetics, but the drug dose to the peritoneal contents may or may not reflect or be proportional to the drug dose to an in situ tumor. This is a particularly important consideration in any comparison involving one type of MTS and a series of drugs. The relationship between the amount of drug delivered to an in situ tumor and that delivered to the peritoneal contents may vary widely, among a series of drugs, thereby leading to a false estimate of which drug would be most effective for the tumor growing in situ if the MTS responses were compared in terms of injected dose. Determination of the relative amounts of drugs delivered to in situ tumors and peritoneal contents are presently in progress, and it should eventually prove possible to adjust the injected dose for each drug so that the kinetics of drug appearance and disappearance in the peritoneal cavity mimic the kinetics at the in situ tumor site. Although the advantages of using a common host are obvious, this/n vivo exposure approach cannot be used for tumors other than those of host origin unless it is known that the pharmacokinetics of the drug are similar in the host species and in the species of tumor origin. Likewise, it will be necessary to determine whether xenogenic tumors suffer 3598 the effects of cross-species immunological reactions, and if so, how these might be abrogated. We have, however, initiated pilot studies with human breast cancer MTS, such as those derived from the MDA-361 cell line (13), and have been able to develop drug dose-response curves similar to those presented previously, by using a 4-hr i.p. exposure in both mice and rats. In summary, the systems described previously provide a simple means of studying the response of very complex tumor cell populations to chemotherapeutic agents. Both in vitro and in vivo exposure methods can contribute to our understanding of these responses, with each providing a different aspect of the process. However, before it will be possible to compare the responses of MTS versus those of in situ tumors within a species, the pharmacokinetics of each drug must be understood in terms of tissue and site differences. Further, use of this system to study MTS from other than the host species will require a full knowledge of the species differences in pharmacokinetics, as well as any cross-species natural immunity. REFERENCES 1. Armelin, H. A., Wishikawa, K., and Sato, G. H. Control of Mammalian Cell Growth in Culture; The Action of Protein and Steroid Hormones as Effector Substances. In: B. Clarkson and R. Baserga (eds.), Control of Proliferation in Animal Cells, pp. 97-104. Cold Spring Harbor, N. Y.: Long Island Biological Association, 1974. 2. Bagley, C. M., Jr., Bostick, F. W., and DeVita, V. T. Clinical Pharmacol ogy of Cyclophosphamide. Cancer Res., 33: 226-233, 1973. 3. Folkman, J., Hachberg, M., and Knighton, D. Self Regulation of Growth in Three Dimensions. In: B. Clarkson and R. Baserga (eds.), Control of Proliferation in Animal Cells, pp. 833-842. Cold Spring Harbor, N. Y.: Long Island Biological Association, 1974. 4. Jackson, R. C., Hart, L. I., and Harrap, K. R. 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Rapid Arrest of DNA Synthesis by W,O2'-Dibutyryl Cyclic Adenosine 3',5'-Monophosphate in Cultured Hepatoma Cells. Cancer Res.,33: 1331-1338, 1973. 10. Wan, S. H., Haffman, D. H., Azarnoff, D. L., Stephens, R., and Hoogstraten, B. Effect of Route of Administration and Effusions on Methotrexate Pharmacokinetics. Cancer Res., 34: 3487-3491, 1974. 11. Yuhas, J. M., and Li, A. P. The Growth Fraction as the Major Determinant of Multicellular Tumor Spheroid Growth Rates. Cancer Res., 38: 15281532, 1978. 12. Yuhas, J. M., Li, A. P., Martinez, A. O., and Ladman, A. J. A Simplified Method for the Production and Growth of Multicellular Tumor Sphe roids. Cancer Res., 37: 3639-3643, 1977. 13. Yuhas, J. M., Tarleton, A. E., and Molzen, K. B. Multicellular Tumor Spheroid Formation by Breast Cancer Cells Isolated from Different Sites. Cancer Res.. 38. 2486-2491, 1978. CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1978 American Association for Cancer Research. In Vitro Analysis of the Response of Multicellular Tumor Spheroids Exposed to Chemotherapeutic Agents in Vitro or in Vivo John M. Yuhas, Anne E. Tarleton and James G. Harman Cancer Res 1978;38:3595-3598. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/38/11_Part_1/3595 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1978 American Association for Cancer Research.