Cancer Chemother Pharmacol (1990) 26:198- 204
ancer hemotherapyand harmacology © Springer-Verlag 1990
Slow penetration of anthracyclines into spheroids and tumors: a therapeutic advantage? Ralph E. Durand Medical Biophysics Unit, B. C. Cancer Research Centre, 601 West 10th Avenue,Vancouver,BC V5Z 1L3, Canada Received 23 October 1989/Accepted 18 January 1990
Summary. Despite clear evidence that the effective penetration of the anthracycline antibiotics into experimental tumors or multicell spheroids is poor, these drugs exhibit clinical activity against a variety of solid tumors. In an attempt to understand this apparent contradiction, we used the Chinese hamster V79 spheroid system and flow cytometry techniques for intra-spheroid pharmacological studies of doxorubicin and daunomycin. Our results indicate that the slow delivery of the anthracyclines to the inner cells of spheroids is due to the rapid binding of the drug by ceils in the outer layers. After exposure, the anthracyclines are retained much more effectively when ceils remain in intact spheroids than when the spheroids have been dispersed, resulting in considerably more cytotoxicity in situ. This result indicates a need for considerable caution in attempting to predict the anti-tumor efficacy of drugs by using either conventional cell-culture systems, spheroids that have been disaggregated immediately post-exposure, or excision assays of tumors from experimental animals. Furthermore, our results suggest the need for a critical evaluation of the significance of the multidrug resistance (MDR) phenotype for cells surrounded by other drug-containing cells as opposed to single ceils in drug-free culture medium.
Introduction The anthracycline antibiotics were first developed as antineoplastic agents in the 1960s and are now widely used due to their activity against a variety of solid and hematological malignancies [ 16]. The continued use of the best-known derivative doxorubicin (Adriamycin) may be somewhat surprising, in view of its rather severe acute (alopecia, vomiting, myelosuppression) and late (cardiac) toxic effects. These, however, can be minimized with appropriate Offprqnt requests to: R. E. Durand
dose scheduling. Perhaps even more surprising is laboratory evidence suggesting that the anthracyclines should be of limited potential: tumor cells efficiently develop resistance to this family of drugs [1, 3, 14, 16, 22]; furthermore, the anthracyclines are one of the few classes of antineoplastic agents that penetrate very poorly into solid tissue masses ([7, 10, 12, 13, 15-21, 30; reviewed in [17]). Clearly, these inconsistencies between clinical and laboratory observations suggest that our understanding of the anti-tumor activity of the anthracyclines is incomplete. We [7, 8], like other investigators [13, 17-21, 24-26, 30-32], have often touted the value of spheroids as a good model for evaluating drug penetration, based in part on results obtained using doxorubicin. Admittedly, it has often been hard to rationalize the poor delivery of this drug with its readily demonstrated clinical efficacy; at least in our own case, we have generally adopted the fairly trivial explanation that clinical response is based not on a single treatment but, rather, on repeated drug exposures over the course of time. Our recent interest in multifraction treatments of the spheroid system with common chemotherapeutic agents (including the anthracyclines) led to some rather unexpected observations that stimulated the present studies. Spheroid regrowth and repopulation generally proceeds quite rapidly following non-ablative drug exposures. However, with the anthracyclines we have found that the net number of surviving cells often decreases during the intervaI between the termination of one treatment and the initiation of the next (manuscript in preparation). That observation may not be overly surprising; doxorubicin intercalates into cellular DNA and remains for long periods of time (a phenomenon observed and described by radiotherapists as a "recall" reaction; see [2, 4, 5]). If a drug is not rapidly cleared from the cell, a potential for continuing toxicity obviously remains [35]. The question addressed in the current report was whether the post-exposure environment of the cell could influence the retention of anthracyclines and their associated cytotoxicity (essentially, an evaluation of post-exposure pharmacology at the cellular level). Clearly, one
199
might expect quite a different rate of elimination of the drug if the cell is surrounded by other drug-containing cells, as opposed to the more typical in vitro case in which an isolated cell sits in a sea of culture medium that provides a high intracellular-to-extracellular drug concentration gradient. An obvious extension of the significance of these results concerns the role of the multidrug resistance (MDR) phenomenon (activated drug efflux) in cases in which the immediate extracellular environment is another tumor cell rather than culture medium.
Materials and methods Cell line and culture techniques. Chinese hamster V79-171b lung cells were used exclusively in these studies. The cells were routinely maintained as monolayers on plastic petri dishes with biweekly subcultivation in Eagle's minimal essential medium supplemented with 10% fetal calf serum. To initiate spheroid growth, asynchronous cells were removed from the plastic growth dishes by trypsinization. Spinner culture flasks (250 ml) were then inoculated with 2 x 106 cells in 150 ml medium plus 5% serum and were stirred at 180 rpm; the gas phase comprised 5% CO2 in air. During the growth of the spheroids, the medium was first changed on day 3 and then daily thereafter (spent medium was completely removed and replaced with fresh medium by sedimentation of the spheroids to the bottom of the flask, followed by removal of the medium by aspiration). Spheroids were used after 7 - 1 2 days of growth, which produced spheroids with a diameter of 400-800 ~m.
Drugs and treatment. Doxorubicin and daunomycin were purchased from Sigma Chemical Co. (St. Louis, Mo.) and stock solutions were prepared using sterile water. The desired drug concentration was achieved by dilution into the normal growth medium of the spheroids. The clinical formulation of etoposide (Bristol Laboratories of Canada, Toronto, Ontario) was diluted directly into spheroid growth medium as required. Drug exposures lasted for either 1 or 2 h; for "delayed assay" experiments, the drug-containing medium was removed at the end of the exposure period by sedimentation of the spheroids followed by removal of the medium by aspiration, two washes, and the addition of fresh growth medium. Additional washes were done hourly for at least the first 6 h post-exposure.
Cell-sorting procedures. Our cell-sorting techniques are based on the general principles of slow diffusion and the rapid, essentially irreversible binding of Hoechst 33342 as it penetrates into the spheroid mass. The external cells of the spheroids fluoresce very brightly, whereas little stain reaches (and is bound by) the innermost cells (staining at the outside of the spheroid is >100-fold that obtained deep in its interior; e.g. [7]). When the stained spheroid is reduced to a monodispersed single-cell suspension, a fluorescence-activated cell sorter can be used asceptically to recover individual cells containing predetermined levels of the Hoechst stain (therefore, cells that were at any desired depth within the spheroids at the time of staining). The sorted cells are then placed in normal culture dishes for determination of their viability by the conventional clonogenicity assay [8-10]. Our cell sorter is a dual laser Becton Dickinson FACS 440 (Becton Dickinson Immunochemistry Systems, Mountainview, Calif.). Hoechst 33342 (Sigma) was used at 2/.tM by direct addition of the dye to the drag-containing flask for the final 20 rain of drug exposure. At the end of drug exposure, excess drug and stain were removed by aspiration after the spheroids had sedimented to the bottom of a collection tube. After three washes, the spheroids were reduced to a single-cell suspension using 0.25% trypsin at 37"C for 10-12 min with continuous agitation.
The stained, disaggregated single-cell suspension was then resuspended in a mixture of growth medium and trysin ( >1 3: 1, v/v) and maintained at 4"C to inhibit stain and drug efflux during the sorting procedure (typically
ancer hemotherapyand harmacology © Springer-Verlag 1990
Slow penetration of anthracyclines into spheroids and tumors: a therapeutic advantage? Ralph E. Durand Medical Biophysics Unit, B. C. Cancer Research Centre, 601 West 10th Avenue,Vancouver,BC V5Z 1L3, Canada Received 23 October 1989/Accepted 18 January 1990
Summary. Despite clear evidence that the effective penetration of the anthracycline antibiotics into experimental tumors or multicell spheroids is poor, these drugs exhibit clinical activity against a variety of solid tumors. In an attempt to understand this apparent contradiction, we used the Chinese hamster V79 spheroid system and flow cytometry techniques for intra-spheroid pharmacological studies of doxorubicin and daunomycin. Our results indicate that the slow delivery of the anthracyclines to the inner cells of spheroids is due to the rapid binding of the drug by ceils in the outer layers. After exposure, the anthracyclines are retained much more effectively when ceils remain in intact spheroids than when the spheroids have been dispersed, resulting in considerably more cytotoxicity in situ. This result indicates a need for considerable caution in attempting to predict the anti-tumor efficacy of drugs by using either conventional cell-culture systems, spheroids that have been disaggregated immediately post-exposure, or excision assays of tumors from experimental animals. Furthermore, our results suggest the need for a critical evaluation of the significance of the multidrug resistance (MDR) phenotype for cells surrounded by other drug-containing cells as opposed to single ceils in drug-free culture medium.
Introduction The anthracycline antibiotics were first developed as antineoplastic agents in the 1960s and are now widely used due to their activity against a variety of solid and hematological malignancies [ 16]. The continued use of the best-known derivative doxorubicin (Adriamycin) may be somewhat surprising, in view of its rather severe acute (alopecia, vomiting, myelosuppression) and late (cardiac) toxic effects. These, however, can be minimized with appropriate Offprqnt requests to: R. E. Durand
dose scheduling. Perhaps even more surprising is laboratory evidence suggesting that the anthracyclines should be of limited potential: tumor cells efficiently develop resistance to this family of drugs [1, 3, 14, 16, 22]; furthermore, the anthracyclines are one of the few classes of antineoplastic agents that penetrate very poorly into solid tissue masses ([7, 10, 12, 13, 15-21, 30; reviewed in [17]). Clearly, these inconsistencies between clinical and laboratory observations suggest that our understanding of the anti-tumor activity of the anthracyclines is incomplete. We [7, 8], like other investigators [13, 17-21, 24-26, 30-32], have often touted the value of spheroids as a good model for evaluating drug penetration, based in part on results obtained using doxorubicin. Admittedly, it has often been hard to rationalize the poor delivery of this drug with its readily demonstrated clinical efficacy; at least in our own case, we have generally adopted the fairly trivial explanation that clinical response is based not on a single treatment but, rather, on repeated drug exposures over the course of time. Our recent interest in multifraction treatments of the spheroid system with common chemotherapeutic agents (including the anthracyclines) led to some rather unexpected observations that stimulated the present studies. Spheroid regrowth and repopulation generally proceeds quite rapidly following non-ablative drug exposures. However, with the anthracyclines we have found that the net number of surviving cells often decreases during the intervaI between the termination of one treatment and the initiation of the next (manuscript in preparation). That observation may not be overly surprising; doxorubicin intercalates into cellular DNA and remains for long periods of time (a phenomenon observed and described by radiotherapists as a "recall" reaction; see [2, 4, 5]). If a drug is not rapidly cleared from the cell, a potential for continuing toxicity obviously remains [35]. The question addressed in the current report was whether the post-exposure environment of the cell could influence the retention of anthracyclines and their associated cytotoxicity (essentially, an evaluation of post-exposure pharmacology at the cellular level). Clearly, one
199
might expect quite a different rate of elimination of the drug if the cell is surrounded by other drug-containing cells, as opposed to the more typical in vitro case in which an isolated cell sits in a sea of culture medium that provides a high intracellular-to-extracellular drug concentration gradient. An obvious extension of the significance of these results concerns the role of the multidrug resistance (MDR) phenomenon (activated drug efflux) in cases in which the immediate extracellular environment is another tumor cell rather than culture medium.
Materials and methods Cell line and culture techniques. Chinese hamster V79-171b lung cells were used exclusively in these studies. The cells were routinely maintained as monolayers on plastic petri dishes with biweekly subcultivation in Eagle's minimal essential medium supplemented with 10% fetal calf serum. To initiate spheroid growth, asynchronous cells were removed from the plastic growth dishes by trypsinization. Spinner culture flasks (250 ml) were then inoculated with 2 x 106 cells in 150 ml medium plus 5% serum and were stirred at 180 rpm; the gas phase comprised 5% CO2 in air. During the growth of the spheroids, the medium was first changed on day 3 and then daily thereafter (spent medium was completely removed and replaced with fresh medium by sedimentation of the spheroids to the bottom of the flask, followed by removal of the medium by aspiration). Spheroids were used after 7 - 1 2 days of growth, which produced spheroids with a diameter of 400-800 ~m.
Drugs and treatment. Doxorubicin and daunomycin were purchased from Sigma Chemical Co. (St. Louis, Mo.) and stock solutions were prepared using sterile water. The desired drug concentration was achieved by dilution into the normal growth medium of the spheroids. The clinical formulation of etoposide (Bristol Laboratories of Canada, Toronto, Ontario) was diluted directly into spheroid growth medium as required. Drug exposures lasted for either 1 or 2 h; for "delayed assay" experiments, the drug-containing medium was removed at the end of the exposure period by sedimentation of the spheroids followed by removal of the medium by aspiration, two washes, and the addition of fresh growth medium. Additional washes were done hourly for at least the first 6 h post-exposure.
Cell-sorting procedures. Our cell-sorting techniques are based on the general principles of slow diffusion and the rapid, essentially irreversible binding of Hoechst 33342 as it penetrates into the spheroid mass. The external cells of the spheroids fluoresce very brightly, whereas little stain reaches (and is bound by) the innermost cells (staining at the outside of the spheroid is >100-fold that obtained deep in its interior; e.g. [7]). When the stained spheroid is reduced to a monodispersed single-cell suspension, a fluorescence-activated cell sorter can be used asceptically to recover individual cells containing predetermined levels of the Hoechst stain (therefore, cells that were at any desired depth within the spheroids at the time of staining). The sorted cells are then placed in normal culture dishes for determination of their viability by the conventional clonogenicity assay [8-10]. Our cell sorter is a dual laser Becton Dickinson FACS 440 (Becton Dickinson Immunochemistry Systems, Mountainview, Calif.). Hoechst 33342 (Sigma) was used at 2/.tM by direct addition of the dye to the drag-containing flask for the final 20 rain of drug exposure. At the end of drug exposure, excess drug and stain were removed by aspiration after the spheroids had sedimented to the bottom of a collection tube. After three washes, the spheroids were reduced to a single-cell suspension using 0.25% trypsin at 37"C for 10-12 min with continuous agitation.
The stained, disaggregated single-cell suspension was then resuspended in a mixture of growth medium and trysin ( >1 3: 1, v/v) and maintained at 4"C to inhibit stain and drug efflux during the sorting procedure (typically
