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[CANCER RESEARCH 40, 3665-3668. 0008-54 727 80 /0040-OOOOS02.00 October 1980] Limited Penetration of Methotrexate into Human Osteosarcoma Spheroids as a Proposed Model for Solid Tumor Resistance to Adjuvant Chemotherapy1 Gary W. West, Ralph Weichselbaum, and John B. Little Harvard School of Public Health, and Joint Center lor Radiation Therapy. Department ol Radiation Therapy. Harvard Medical School. Boston. Massachusetts 02115 ABSTRACT It is generally accepted that oxygen has a limited ability to diffuse into solid tumor masses. However, the question of the ability of chemotherapy agents to penetrate solid tumor masses has not been evaluated. This clearly would have an impact on the ability of chemotherapy to control microscopic disease during the "avascular" phase of growth. An attempt was made to evaluate the ability of methotrexate to penetrate solid tumor masses when grown in three dimensions (spheroids). Since methotrexate is used in the clinical management of human osteosarcoma, we chose this drug-tumor combination for our studies. This was done by growing human osteosarcoma cells into spheroids and exposing spheroids of various sizes to tritiated methotrexate. Audioradiographs were obtained from sections through the center of spheroids of various sizes. Our findings suggest that methotrexate has a limited ability to penetrate into avascular tumor masses when grown in three dimensions. This is most evident when the tumor masses are approximately 250 /im and larger in diameter. In addition, we compared the degree of penetration of methotrexate to the growth fraction of the tumor, as measured by tritiated thymidine, and found that the growth fraction was much greater than the fraction of cells reached by methotrexate. We conclude that the limited ability of methotrexate to penetrate solid tumor masses offers an alternative explanation for the limited effec tiveness of methotrexate when used as an adjuvant for osteo sarcoma. We question whether the established biochemical mechanisms for methotrexate resistance are comprehensive explanations for its limited clinical effectiveness. INTRODUCTION The goal of cancer chemotherapy is to eradicate clinically evident or microscopic métastases.The use of cytotoxic drugs to treat microscopic métastases after complete surgical re moval of the primary tumor is referred to as "adjuvant chemo therapy." MTX is used in combination with other drugs as an adjuvant to surgery in patients with osteosarcoma and appears to increase survival (3, 8). Nevertheless, 50% of patients with osteosarcoma die from widespread metastatic disease despite adjuvant treatment (19). The limited effectiveness of chemo therapy is frequently attributed to the known ability of tumor cells to develop resistance to a particular drug (11). To date, 3 mechanisms for tumor resistance to MTX' have been identi' This work was supported in part by Grant CA-2184802 from the National Cancer Institute. 1 The abbreviations used are: MTX. methotrexate; DHFR, dehydrofolate reductase; MTS. multicellular tumor spheroid(s): dThd, deoxythymidine; EBSS. Earle's balanced salt solution. Received October 1, 1979; accepted July 14. 1980. OCTOBER fied: (a) alterations in the carrier-mediated active transport of MTX across cellular membranes occur (1 7); (b) alterations in the structure of the target enzyme of MTX, DHFR occur, such that the high affinity for MTX is lost (6, 7); and, more recently, (c) selective gene amplification develops which results in an increase in DHFR production. Despite their value for providing biochemical bases for MTX resistance, it is important to recognize that these mechanisms were discovered by growing cells under conditions which per mit uniform exposure of cells to increasing concentrations of drug for prolonged periods of time. However, this is not the case for the clinical administration of MTX for adjuvant treat ment in which high doses of MTX are given for relatively short periods of time. Although in vitro or in vivo conditions of drug exposure can be manipulated to select or produce resistant cells, the number of resistant cells obtained are relatively rare compared to wild-type cells (12). Therefore, if these transport and enzymic mechanisms were the sole determinants of MTX resistance, one might predict adjuvant MTX therapy to prove more successful. Moreover, many other clinical studies on various cytotoxic agents for a variety of solid tumor cell types have been less effective than preliminary in vitro studies pre dicted (22). Therefore, other mechanisms of drug resistance probably exist. It is known that solid tumors require adequate diffusion of nutrients for tumor growth (2, 18), but the diffusion (whether passive or facilitated) of cytotoxic agents into solid tumor masses has not been investigated. Certainly, for solid tumors, adequate drug diffusion to all cells is fundamental for cytotoxicity. The recent development of simple techniques for the growth of MTS in vitro has permitted us to investigate drug diffusion as a possible limitation to chemotherapy (5, 21 ). Since MTX is used extensively in the treatment of human osteosar coma, we chose this drug-tumor combination for our investi gations. We report here experimental evidence to support the hypothesis that diffusion gradients to cytotoxic agents develop relatively early during the growth of solid tumors in vitro (i.e., with a diameter of approximately 250 firn), and we propose that either passive or facilitated diffusion is an important variable in determining the effectiveness of cytotoxic chemotherapy. MATERIALS AND METHODS Differences of MTX Diffusion in Human Osteosarcoma Cells Grown in 2 Versus 3 Dimensions. Extracellular MTX enters cells by actively traversing the cell membrane in asso ciation with a carrier (facilitated diffusion) (4, 20). Because of its high affinity for DHFR, all intracellular MTX is bound to DHFR until its binding sites are saturated. Initially, the drug moves only intracellularly (influx), and its rate of entry is de- 1980 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. 3665 G. W. West et al. pendent on both extracellular drug concentration and mem brane permeability. However, once all DHFR binding sites are saturated, exchangeable or (nonbound) intracellular MTX ac cumulates, and an equilibrium is established. Since DHFR is concentrated in the cytoplasm, little or no MTX enters the nucleus (16). This characteristic of MTX allowed us to identify the diffusion patterns of MTX and to distinguish its diffusion from that of thymidine. In all experiments, a standard exposure concentration of 0.33 x 10~6 M high-specific activity (30 Ci/ mmol) [3H]MTX at 10 ¿iCi/mlwas used. The exposure concen tration of [3H]dThd (49.5 Ci/mmol) at 10 /iCi/ml was 0.2 x 10~6 M. We chose this concentration and specific activity to simulate therapeutic concentrations and to insure adequate autoradiography. Human osteogenic sarcoma tumor cells were obtained from Dr. E. Lloyd of the Argonne National Laboratory with permission from Dr. Jörgen Fogh of the Sloan-Kettering Memorial Cancer Center (13). Cells were grown in Eagle's minimal essential medium supplemented with 10% fetal calf serum (Microbiolog ical Associates, Bethesda, Md.), 9 mg-D-glucose per liter, 0.66 mg sodium pyruvate per liter and 50 fig gentamicin per ml (Schering Corp., Kenilworth, N. J.) at 37°in an atmosphere of 95% air:5% CO2. For the [3H]MTX uptake in monolayer exper iments, exponentially growing stock cultures were treated with 0.25% trypsin in calcium- and magnesium-free EBSS for 3 min. Two-hundred and fifty dissociated cells were counted in a hemocytometer, plated in 35-mm Falcon tissue dishes, allowed to grow to colonies of various sizes, and then exposed to 0.33 x 10~6 M [3H]MTX (30 Ci/mmol; Moravek Biochemicals, City of Industry, Calif.) at 10 ftCi/ml for 48 hr. Radioactive medium was safely discarded, and colonies were washed 5 times with EBSS at 4°. This procedure should remove all extracellular MTX but should have no effect on intracellular (enzyme-bound) MTX (9). The effectiveness of the wash procedure was deter mined by measuring the amount of radioactivity in a sample of the final wash. No radioactivity (above background levels) was detected. Colonies were fixed for 10 min with methanoliacetic acid (3:1), dried for 4 hr, and then dipped into NTB-2 (Kodak) nuclear track emulsion diluted 1:1 in complete darkness. Emul sion-covered plates were dried overnight, exposed at 4°in a desiccator light-sealed box for 21 days, developed with Kodak D19-B (5 min), and then fixed, and photomicrographs were taken. For spheroid formation, approximately 105 cells were har vested from monolayer stock cultures and seeded with 15 ml of complete medium (i.e.. Eagle's minimum essential medium supplement as in the monolayer experiments) into 100-mm Falcon dishes that had been previously base coated (2 to 3 mm) with 0.75% Noble agar(Difco Laboratories, Detroit, Mich.) in complete medium to prevent cell attachment (21). These dishes were then incubated (95% air:5% CO2 at 37°) for approximately 2 weeks for spheroids 50 /im in diameter and approximately 5 weeks for speroids 250 /im in diameter. Five ml of medium were exchanged twice per week for feeding. After appropriate incubation times, all spheroids were approx imately 50 or 250 jum in diameter. Approximately 15 to 20 of these spheroids were transferred to 35-mm Falcon dishes base coated with Noble agar and complete medium as described above for exposure to 0.33 x 10~6 M [3H]MTX for 48 hr. Similarly, approximately 15 to 20 of the 250-fim spheroids were exposed to 0.2 x 10~6 M [3H]dThd for 48 hr. After these 3666 exposures, the respective radioactive media were safely dis carded, and the spheroids were washed 9 to 10 times with EBSS at 4°to ensure removal of all extracellular [3H]MTX or [3H]dThd. The effectiveness of the wash procedure was deter mined in a manner similar to that for the monolayer [3H]MTX uptake experiments. The spheroids were then fixed in 2.5% gluteraldehyde for 30 min, washed again 5 to 6 times with EBSS at 4°to prevent negative chemography during autora diography, dehydrated in 70, 80, 90, and 100% ethanol for 15 min in each dilution, and embedded in a Beem capsule, in accordance with the instruction for the Polysciences JB-4 embedding kit. A Sorvall JB-4 glass microtome was used to cut 2-/im sections from the center of the spheroids. These sections were mounted on glass slides and processed for autoradiography, as in the monolayer experiments. The autoradiographs were then stained with hemotoxylin and eosin, and photomicrographs were taken. RESULTS Fig. '\A is an autoradiograph of a small colony of human osteosarcoma cells grown in monolayer after exposure to 0.33 x 10~6 M [3H]MTX for 48 hr. As expected for cells grown in monolayer, [3H]MTX appears to be distributed uniformly in the cytoplasm of all cells. Since the cells were washed thoroughly after exposure to [3H]MTX, all extracellular [3H]MTX should be absent; therefore, we interpret this autoradiograph to be an accurate representation of intracellular MTX bound to DHFR. Fig. 1/\ indicates that cells exposed to this concentration of [3H]MTX maintain their ability to transport and take up MTX, which is an important requirement for drug diffusion when tumor cells are grown in 3 dimensions. If the cells are allowed to grow to larger colony sizes (data not shown), there is more crowding of cells but a similar distribution of [3H]MTX. Thus, in 2-dimensional growth, there are no limitations to drug diffusion since the surface of each cell is always exposed to equal concentrations of MTX. Under these conditions, MTX would be expected to have a marked cytotoxic effect. Fig. 1S is an autoradiograph of a 2-p.m section taken from the center of an MTS of human osteosarcoma cells grown to approximately 50 ¿imin diameter and exposed, as in Fig. 14, to 0.33 x 10~6 M [3H]MTX for 48 hr. Although [3H]MTX is not homogeneously distributed throughout the MTS, evidence of uptake can be seen both peripherally and centrally within the spheroidal mass. Thus, MTX reaches all cells comprising tumor spheroids of this size, and all cells within the mass are vulner able to its cytotoxic effect. Although cytoplasmic uptake is not as distinct as in Fig. 1/\, [3H]MTX appears to surround the nuclei of the cells comprising the tumor mass, and no evidence of [3H]MTX within the nuclei can be seen. If the MTS are allowed to grow to approximately 100 /im and then exposed to [3H]MTX, the drug tends to concentrate toward the periphery of the tumor mass, but evidence of central [3H]MTX can still be seen (data not shown). However, if the MTS are allowed to continue to grow in size before exposure to [3H]MTX, the difference between central and peripheral distribution of the drug increases until almost all the drug is concentrated to the outermost peripheral cell layers, as dem onstrated in Fig. 1C. Fig. 1C is an autoradiograph of a 2-p.m section taken from the center of an MTS allowed to grow to approximately 250 CANCER RESEARCH VOL. 40 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. Limited Diffusion and Drug Resistance jum in diameter before exposure to [3H]MTX for 48 hr. In this size range of MTS, drug diffusion is essentially limited to the outer 2 to 3 cellular layers of the spheroidal mass. In fact, on higher magnification (data not shown), we can detect no evi dence of [3H]MTX above background levels in the center of the spheroid. Also, as in Fig. 15, the drug appears to surround the nuclei of the peripheral cells, which we interpret to be cytoplasmic concentration. Thus, in spheroids of this size, the peripheral cells are most vulnerable to the cytotoxic effects of MTX, while the central cells, which comprise the largest part of the spheroid mass, have either a limited vulnerability to the drug or may even be completely protected from the cytotoxic agent. Since it can be argued that the central cells of MTS in the 250-jum range are not viable, the fact that MTX does not diffuse to reach these cells has little relevance to its ability to eradicate métastasesof this size. To answer this question, we exposed human osteosarcoma MTS in this size range to 0.2 x 10~6 M [3H]dThd (10 /xCi/ml) for 48 hr to ensure continuous labeling over approximately 2 doublings. Fig. 1D is an autoradiograph of a 2-fim section taken through the center of a human osteosarcoma MTS grown to approxi mately 280 /im in diameter and then exposed to [3H]dThd. When this autoradiograph is compared to that in Fig. 1C, it can be seen that both the inner and outer cells of the MTS are capable of DNA synthesis, as indicated by the marked nuclear uptake of [3H]dThd throughout the MTS. Thus, the inner cells not reached by MTX because of its limited diffusion (either passive or facilitated) are viable. Stated differently, MTX does not reach all the cells that make up the growth fraction of the tumor mass. DISCUSSION chemotherapy should be very effective and perhaps curative. However, once tumor cell populations develop 3-dimensional growth configurations (linear growth phase), MTX is no longer uniformly distributed to all cells within the tumor mass (Fig. 1B). The cytotoxic effect of MTX would be expected to become increasingly ineffective with increasing tumor size until a diffu sion gradient develops (Fig. 1C) during the late "linear" or "dormant" phase of tumor growth, at which time only the most peripheral outer layer of cells effects of MTX. Since Fig. 1D are also viable, MTX would not tumors in this phase of growth. ized and "breaks-out" of the is vulnerable to the cytotoxic indicates that the central cells be expected to be curative for If the tumor becomes vascular dormant phase, growth again increases rapidly, and the effectiveness of cytotoxic agents may then be limited by the magnitude of the number of tumor cells. However, in this circumstance, the disease usually be comes clinically evident and patients are no longer considered candidates for standard "adjuvant chemotherapy" regimens. The maximum size of tumors during the "avascular" period of tumor growth is well below the level that is detectable clinically by available technology. Nevertheless, tumors of this size have heterogeneous growth characteristics and develop heteroge neous microenvironments which not only regulate their growth but also affect the ability of cytotoxic agents to diffuse uniformly to all viable cells. Our experiments indicate that MTX diffusion (either passive or facilitated) into solid tumors of micrometastatic size is dependent on the relative size of the tumor mass. Although the transport and enzyme mechanisms already iden tified for MTX resistance are important biological findings with profound clinical implications, the passive or facilitated diffu sion of MTX into solid tumors of micrometastatic size may be of equal clinical importance or at least offers another explana tion for the variation of the efficacy of MTX adjuvant chemo therapy in patients with primary osteosarcoma. We propose that micrometastases can be thought of as the prevascular or avascular period of tumor growth, first described ACKNOWLEDGMENTS by Folkman (1 ). This period of early tumor growth is divided into 3 phases: (a) a brief phase of exponential growth (0 to 150 The authors wish to thank John Nove and Anne Schmidt for their expert /im in diameter) during which all the cells have equal access to technical assistance. Dr. Hatsumi Nagasawa provided superb technical direction as well as stimulating discussion. nutrients; (6) a second phase of linear growth (150 to 500 /¿m in diameter) beginning with the appearance of central necrosis, probably due to a limitation of oxygen diffusion; and (c) a REFERENCES "dormant phase" which begins when growth plateaus (1 to 2 mm in diameter) and beyond which a tumor cannot grow unless it becomes vascularized. Without vascularity, tumor growth in all of these phases is "self-regulated" by the centrifugal diffu sion (passive or facilitated) of catabolites (growth inhibitors) and the centripetal diffusion (passive or facilitated) of nutrients (2). This description of early tumor growth can be used as a model for micrometastases in which the regulation of cell kill is greatly determined by how well cytotoxic agents can diffuse (passive or facilitated) centripetally to the center of the tumor mass, just as growth is greatly determined by the passive or facilitated diffusion of nutrients. Our observations of MTX diffusion are significant in that they may represent drug tumor interactions during the different phases of early or "prevascular" metastatic tumor growth in vivo. For example, cells grown in monolayer (Fig. 1/\) may be analogous to the very earliest phase (exponential growth) of métastases during which MTX is uniformly distributed to all cells and is highly cytotoxic. 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The histological structure of some human lung cancers and the possible implications for radiotherapy. Br. J. Cancer. 9. 539-549, 1955. 19. Uribe-Botero, G., Russell, W. O.. Sutow, W. W.. and Martin. R. G. Primary osteosarcoma of bone: a clinicopathologic investigation of 243 cases, with necropsy studies in 54 (Abstract). J. Am. Med. Assoc., 238. 1576, 1977. 20. Wilbrandt, W., and Rosenberg, T. The concept of carrier transport and its corollaries in pharmacology. Pharmacol. Rev.. 13. 109-183, 1961. 21. Yuhas. J. M., Li. A. P.. Martinex, A. O., and Ladman, A. J. A simplified method for production and growth of multicellular tumor spheroids. Cancer Res., 37. 3639-3643, 1977. 22. Zubrod, C. G. Selective toxicity of anticancer drugs: Presidential Address. Cancer Res., 38: 4377-4384, 1978. v*. *** ¿r• / T *s "• -' - -v • . •*' •< > * »*•*> , *• 200JJ Fig. 1. A, autoradiograph of a small colony of human osteogenic tumor cells grown in monolayer and then exposed to 0.33 x 10~6 M [3H]MTX at 10/iCi/ml for 48 hr. 8 and C. autoradiographs of 2-jim sections taken from the center of human osteosarcoma tumor cells grown into MTS with diameters of approximately 50 and 250 (im. respectively, and then exposed to 0.33 x 10~6 M [3H]MTX at 10 /iCi/ml for 48 hr. D, autoradiograph of a 2-fim section taken from the center of a human osteogenic sarcoma MTS grown to a diameter of approximately 280 ¡anand then exposed to 0.2 x 10"6 M [3H]dThd at 10 /iCi/ml for 48 hr. Except for exposure to [3H)dThd, these spheroids were grown and processed identically to those in ßand C. 3668 CANCER RESEARCH VOL. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1980 American Association for Cancer Research. 40 Limited Penetration of Methotrexate into Human Osteosarcoma Spheroids as a Proposed Model for Solid Tumor Resistance to Adjuvant Chemotherapy Gary W. West, Ralph Weichselbaum and John B. Little Cancer Res 1980;40:3665-3668. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/40/10/3665 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 18, 2017. © 1980 American Association for Cancer Research.