doxorubicin (DXR) and cisplatin (DDP) of multicellular tumor spheroids (MTS) developed from 2 human lung cancer cell lines; PC-10 squamous cell carcinoma ...
CANCER DRUG DELIVERY Volume 4, Number 4, 1987 Mary Ann Liebert, Inc., Publishers
Effects of Doxorubicin and Cisplatin on Multicellular Tumor Spheroids from Human Lung Cancer SHOICHI INOUE,i2 TAKAO OHNUMA,1 KAZUO TAKAOKA,2 YASUNOSUKE SUZUKI,* MAMORU KANEKO,4 ROBERT SAFIRSTEIN,* and JAMES F. HOLLAND' Departments of Neoplastic Diseases,1 Pathology,4 and Medicine,5 and Environmental Science Laboratory,1 Mount Sinai School of Medicine, New York, N Y 2Department of Medicine, Hokkaido University School of Medicine, Sapporo, Japan
ABSTRACT We tested the sensitivity to doxorubicin (DXR) and cisplatin (DDP) of multicellular tumor spheroids (MTS) developed from 2 human lung cancer cell DDP was lines; PC-10 squamous cell carcinoma and PC-6 small cell carcinoma. able to maintain its efficacy in MTS: PC-10 MTS were only 3-fold more resistant to DDP than in monolayer and in PC-6 cells DDP induced cell lethality was essentially unchanged irrespective of cells being in a monolayer or MTS. Atomic absorption spectrometry revealed that DDP uptake was essentially identical, irrespective of cells being in monolayer or MTS. DDP's efficient cell kill effects in MTS seems to be explained by its good penetration into the MTS core. In contrast to DDP, these 2 types of cells responded differently to DXR. Thus, PC-10 MTS became progressively more resistant to DXR when their size increased, whereas the susceptibility of PC-6 MTS tended to increase when the MTS grew larger. Fluorescent microscopic study revealed that prominent DXR fluorescence was observed only at the outer layer of PC-10 MTS. In PC-6 MTS, however, DXR fluorescence was diffusely seen in the entire MTS at low concentrations; nevertheless, owing to PC-6 cells' high sensitivity DXR was able to exert cell lethality. The differences in distribution of DXR fluorescence between PC-10 and PC-6 MTS were corroborated by flow cytometric analysis.
INTRODUCTION
MTS6 have certain characteristics similar to d_e novo solid tumors (1-7). MTS contain such extracellular matrix as fibronectin, laminin and collagen (3). MTS are composed of chronically hypoxic cell population in the core (4,5) and show heterogenous cell cycle times (6,7). For this reason MTS have been used as an in vitro model to study the effects of radiation (4,5,8) and chemotherapeutic These studies demonstrated limited penetrations of DXR, agents (9-13). methotrexate, bleomycin and vinblastine. 6 Abbreviations used are: MTS, multicellular tumor spheroids; FBS, fetal bovine serum; PBS, Dulbecco's phosphate buffered saline; DXR, doxorubicin; DDP, cisdiamminedichloroplatinum, cisplatin; AU, arbitrary unit.
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In this article we compared DXR and DDP, 2 useful anticancer agents in man, using cells in monolayer and MTS from 2 human lung cancer cell lines. There was a variety of dose response relationships depending on both tumor cell type and chemotherapeutic agent in question. We observed that the MTS from a human small cell carcinoma cell line gave unique DXR penetration patterns. Attempts were made to explain this phenomenon.
MATERIALS AND METHODS
Cell Lines Two human lung cancer cell lines were used. One was PC-10 squamous cell carcinoma line (14) and the other PC-6 small cell carcinoma line (15). Both cell lines were obtained from the Pathology Department of Hokkaido University School of Medicine Cancer Institute, Sapporo, Japan and maintained as a monolayer in RPMI-1640 medium (GIBCO, Grand Island, NY) with 10% FBS (GIBCO) ("complete medium") in a humidified 5% C02/air atmosphere. PC-10 cells were subcultured weekly after trypsinization. PC-6 cells were subcultured once a week after separation of the cells from the flask with a rubber policeman. The rubber policeman has been commonly used for subculture of PC-6 cells because these cells were easily removed from the floor of the culture flask and made to The population doubling single cell suspensions without the aid of trypsin. time of PC-10 cells was approximately 23 hrs and that of PC-6 cells 20 hrs.
Development
of MTS
MTS were produced by the method described by Yuhas et al (1) with minor One x 105 viable PC-10 or PC-6 cells in 10 ml of the complete modifications. medium were placed in 100 mm plastic Petri dishes (No. 3003, Falcon, Cockysville, MD) previously coated with 0.5% agar (Noble, Difco Laboratories, Detroit, MI). They were cultured in humidified 5% C02/air at 37°C. The dishes were inspected daily for changes in medium color and fed with fresh medium before the medium became acidic. When MTS were developed, MTS of a uniform size were transferred into a new medium placed on agar plates using 1 ml plastic
pipettes (No. 7520, Falcon).
Drug Exposure In this study we used DXR (Adria laboratories, Columbus, OH) and DDP DXR and DDP are the most widely used (Bristol laboratories, Syracuse, NY).
anticancer agents in man. Their differences in the molecular dimension and the diversity of their structures were considered worthy for comparison in MTS. Ten of 4 week old MTS, 15 of 3 week old MTS and 24-26 of 2 week old MTS were preincubated in 4.9 ml of fresh medium placed in agar-coated 60 mm Petri dish (No. 25011, Corning Glass Work, Corning, NY) for 24 hrs. MTS were then treated by the addition of 0.1 ml of graded concentrations of DXR or DDP for 1 hr in a The MTS were then washed once by humidified 5% C02/air atmosphere at 37°C. suctioning the medium from the plate and replacing it with calcium- and magnesium-free PBS (GIBCO), transferred into culture tubes (No. 3033, Falcon) and gently washed twice more with PBS. A single cell suspension was obtained by trypsinization: washed PC-10 MTS were mixed with 0.1% trypsin (type III from bovine pancreas, Sigma, St. Louis, M0) containing 0.01% EDTA, and for PC-6 MTS with 0.025% trypsin containing 0.01% EDTA. The different trypsin concentrations were used for these 2 cell lines, so that it required approximately 10 min for both monolayer cells to become viable single cell suspensions. Pilot studies showed that differences in the concentration of trypsin used for PC-10 and PC-6 cells did not influence the results of subsequent clonogenic assays. Trypsin digestion was terminated by addition of the same volume (2 ml) of the complete
214
The cells were then washed by centrifugation, and the viable cells were the Trypan Blue dye exclusion method. The viable cell number was adjusted to 5 x 10^ cells/ml of fresh medium for PC-10 and 1 x 10^ cells/ml for PC-6 cells. Aliquots of 0.1 ml cell suspensions were seeded on 5 ml of 0.5% Noble agar containing complete medium in 60 mm Petri dishes for clonogenic assay (16). After 7 days of incubation in a humidified 5% C02/air atmosphere at 37°C, the colonies (defined as more than 50 cell aggregrates) were counted under an inverted microscope and expressed as the surviving fraction. Fifty cell aggregates were arbitrarily chosen because it corresponded to a MTS with a diameter of 8 cells in row which was easily recognizable under the microscope. As a control, sensitivities of cells in monolayer cultures were tested as follows. Single cell suspensions from a monolayer culture were adjusted to 1 x 105 cells/ ml and prelncubated for 24 hrs in 60 mm Petri dishes. After confirmation that the cells had attached to the floor of the flask as a monolayer, they were exposed to graded concentrations of DXR or DDP for 1 hr, washed thrice with PBS and trypsinized with identical trypsin solutions used in MTS treatment The numbers of PC-10 and PC-6 cells were adjusted to 5 x for each cell line. 104 and 1 x 1CP cells/ml, respectively, and subjected to clonogenic assay. Plating efficiency for PC-10 and PC-6 cells were 40 and 20%, respectively. medium. counted
using
Observation of Cell Surface Structure and Intercellular Contact
Aliquots of single cell suspensions were seeded drop-wise on a sterile filplaced in Petri dishes and incubated for various periods of time up to 48 hrs. The filter papers were carefully removed from the Petri dish and doubly After dehydration in 100% ethanol, fixed with gluteraldehyde and osmic acid. the specimen were dried by the critical point method (17), mounted on SEM specimen mounts using silver conducting paint and coated with gold palladium alloy. ter paper
Observations of the cell surface structures were made with a Hitachi S450 scanning electron microscope. For transmission electron microscopy MTS grown for 3 weeks were fixed doubly with paraformaldehyde solution (Karnovsky) and 1% phosphate buffered osmic acid each for 30 minutes. After ethanol dehydration the specimens were embeded in Epon, ultrathin-sectioned with a LKB ultramicrotome and stained with uranyl acetate and lead. Observations were made with a Hitachi 11ES type electron
microscope. Histological Studies under Fluorescent Microscopy Three week old MTS were treated with 1 x 10~4M of DXR for 1 hr at 37°C in a humidified 5% C02/air atmosphere. They were washed with ice-cold PBS once, embedded in 0CT compound (Miles Scientific, IL) and frozen rapidly. Frozen sections of 5 um thickness were prepared using Cryomicrotome (Lipshaw Elect. MI). Observations of DXR fluorescence were, made with a fluorescence microscope equipped with epi-illumination (E. Leitz, Wetzlar, W. Germany) using dichroic mirror TK580 and suppression filter K580. Flow
Cytometric
Measurement of DXR
Uptake
Monolayer culture or 3 week old MTS were exposed to 1 x 10-¿,M of DXR for 1 They were washed twice with ice cold PBS and made to a single cell suspension by trypsinization. Relative DXR content per cell was measured by detecting red fluorescence at 630 nm wave lengths emitted by an argon laser Model 164-05 at a wave length of 488 nm (Spectro-Physics, NJ).
hr at 37°C.
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215
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Survival curves for PC-10 cells (Panel A) and PC-6 cells (Panel B) monolayers (open circles) or spheroids (filled circles, frown for 2 weeks; filled squares, for 3 weeks; filled triangles, for 4 weeks) following Error bars, mean exposure to graded concentrations of doxorubicin for 1 hr. + SD from 3-4 experiments. Each experiment was done in triplicate. FIGURE
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Briefly, cells in monolayer or in MTS were exposed to 4 x 10~*M of DDP for 1 hr, washed twice with ice cold PBS and trypsinized. Cell numbers were adjusted to 1 x 10* cells/ml. One ml aliquots of cells were partially digested by heating with 60% HNO3 and digestion was completed with the addition of a mixture of 3 parts of 70% HCIO4 and 2 parts of 60% HNO3 (v/v). When the samples were almost dry the residue was mixed in 0.5 ml of 0.1 N HC1, evaporated and resolubilized in a known volume of 0.3 N NaOH. Aliquots of the digest were then analyzed by flameless atomic absorption spectrometer (Instrumentation laboratory, Edison, NJ) and values were calculated using standards prepared by dilution of a 1000 ppm tetrachloroplatiniç acid. RESULTS Mean Diameter of MTS
Mean diameters (AxB) H, where A is the longest diameter and B is the widest perpendicular width, of PC-10 MTS were 120 + 20 urn (mean + SD) after 1 week, 290 + 30 um after 2 weeks, 790 + 100 um after .3 weeks, and 970 + 180 p.m after 4 weeks. For PC-6 MTS they were 120 + 10 urn, 310 + 30 um, 990 + 180 yim and 1790 + 210 um, respectively. Effects of DXR
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Cell
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With increasing DXR concentrations, the surviving fractions of PC-10 and PC-6 cells both in monolayer and in MTS decreased progressively (Figure 1A and B). As expected, both PC-10 and PC-6 carcinoma cells in monolayer were more In PC-10 cells MTS were progressively more sensitive to DXR than those in MTS. resistant when their size increased. The dose-effect curves of 3 and 4 week old MTS have virtually flattened at high DXR concentrations indicating that maximally no more than 70% and 50%, respectively, of cells can be killed. The DXR dose-effect relationship for PC-6 MTS were different from PC-10 MTS, in that MTS were only 10-20 fold more resistant than monolayer and the size of MTS appeared to have little influence on the efficacy of DXR cell lethality or if any, the
216
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FIGURE 2. Survival curves for PC-10 (Panel A) and PC-6 cells (Panel B) grown as monolayers (open circles) or spheroids (filled circles, grown for 2 weeks; filled squares for 3 weeks; filled triangles, for 4 weeks) following exposure to graded concentrations of cisplatin. Error bars, mean + SD from 3-4 experiments.
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Scanning electron micrograph showing PC-10 (Panel A) and PC-6 (Panel Length of the bar in the bottom of the figure indicates 5 pm. 217
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