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Cancer Therapy: Preclinical |
Authors' Affiliation: Department of Medical Biophysics, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada
Requests for reprints: Andrew I. Minchinton, British Columbia Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3. Phone: 604-675-8032; Fax: 604-675-8049; E-mail: aim{at}bccrc.ca.
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Abstract |
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Experimental Design: We assessed the tissue penetration of paclitaxel and docetaxel in HCT-116 tumor xenografts and in multilayered cell culture (MCC), a three-dimensional cell culture model of the tumor extravascular compartment. In xenografts, taxanes were mapped relative to blood vessels to obtain drug profiles as a function of distance from vasculature. For MCC, cultures were exposed to stirred drug reservoirs and taxanes measured as a function of depth into tissue.
Results: Both taxanes exhibited limited penetration, with little drug reaching further than 100 µm into the tissue. Of the two, paclitaxel exhibited up to 2-fold greater penetration than docetaxel. Mapping tumor cell proliferation following treatment allowed the consequences of limited drug penetration to be assessed. In tumor xenografts where reduced drug exposure to cells far from vasculature is one of several factors influencing response to treatment, up to a 75% reduction in S-phase cells was achieved in cells nearest the vessels, but only 50% reduction was observed in the tissue 150 µm away. In MCC-based data, where the influence of reduced cell proliferation with depth into tissue was circumvented, a 5-fold (paclitaxel) and 10-fold (docetaxel) increase in reservoir drug concentration was required to produce a response in cells 150 µm into the tissue equivalent to that seen in cells directly exposed to the drug.
Conclusion: These results indicate that limited distribution is an important mechanism of tumor resistance to taxanes.
In this study, the tissue penetration of 3H-paclitaxel and 3H-docetaxel was mapped relative to tumor vasculature by combining a film-based dry autoradiography technique with subsequent immunohistochemical detection of tumor vasculature. Superimposition and alignment of tumor vasculature and radiolabeled drug was made possible by capturing high-resolution images of entire tumor cryosections using a robotic tiling microscope. Following automated analysis of whole tumor cryosections, drug distribution was determined as a function of distance from the nearest visible blood vessel. Although mapping radiolabeled taxane distribution relative to tumor vasculature yields information about in vivo taxane distribution, it has the drawback of incorporating the contribution of 3H-labeled liver metabolites, which, being smaller, could diffuse more easily. To minimize this, both taxanes were labeled in the 2-benzoyl ring rather than randomly throughout the molecule. A second drawback of the technique is that drug penetration will seem to be increased by contributions from vessels that just lie outside of the tissue section being analyzed, the occurrence of which will increase in likelihood with increasing distance from an observed vessel. These two factors mean that the in vivo based detection of drug penetration will likely represent the best case scenario and might overpredict taxane tissue penetration.
To validate the tumor-based work, we employed MCC (11, 12), a three-dimensional tissue culture model, in which a disc of tumorlike tissue is grown from single cells attached to a permeable support membrane. The model has been used previously to determine flux of drugs through tumorlike tissue (13–16). Using the MCC model avoided the contributions to apparent taxane distribution from both hidden vessels and 3H-labeled liver metabolites. Although conceptually similar to the tumor histoculture model, the MCC model avoids potential contributions to drug penetration from the edge of the tissue from the existing vascular network in the tumor histocultures.
In addition to comparing the tissue penetration of paclitaxel and docetaxel, a secondary goal of this study was to assess the ability of a second MCC-based assay in which a drug's effect on proliferating cells is used to determine its tissue penetration (17, 18). The assay integrates overall exposure to a drug, rather than determining drug distribution at a discrete time point. Using the distribution of drug effect in tissue as an indicator of drug penetration is generally not possible due to the changes in intrinsic sensitivity of cells that accompanies the reduction in proliferation seen with depth into the tissue (19). The disc geometry of MCCs allowed this problem to be circumvented because there exist two populations of rapidly dividing cells, one on the top and one on bottom of the disc, that are separated by the thickness of the culture itself. Drug penetration was determined by exposing cultures to drug from one side and then comparing the effect on the population of proliferating cells directly exposed to the drug versus the proliferating cells located on the far side. The effect of the taxanes on proliferation was also mapped in the tumor xenografts; however, these experiments did not differentiate between drug penetration and intrinsic changes in cell sensitivity.
We compared paclitaxel penetration with that of the analogue docetaxel and also examined the effect of paclitaxel vehicle, Cremophor EL, on tissue penetration. Both taxanes exert their antimitotic effects through binding and stabilization of microtubules leading to cell cycle arrest (20). In the human colorectal carcinoma cell line used here, HCT-116, both drugs exhibit a similar IC50 (21). However, there are numerous differences between them which might affect their tissue penetration. Docetaxel has been reported to have a higher binding affinity to microtubules as well as faster accumulation and longer retention than paclitaxel (22, 23), which might limit its ability to distribute within the tissue. In addition, in vivo, both drugs are highly plasma bound, with paclitaxel 88% to 98% and docetaxel 
94% (24–26). The extent and speed at which this binding occurs and the degree of reversibility for each drug could play an important role in determining the amount of free drug available for diffusion into the tissue. Paclitaxel has the added factor of its formulation vehicle, Cremophor EL, which, through micelle formation, is thought to reduce the free drug available for diffusion and increase the duration of exposure (27, 28). The tissue distribution of the two drugs and the effect of vehicle Cremophor EL were compared in HCT-116 tumor xenografts. Then, in isolation of plasma pharmacokinetics, tissue distributions were determined using MCCs under comparable exposures.
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Materials and Methods |
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Cell culture. HCT-116 human colorectal carcinoma cells were purchased from American Type Culture Collection. Cells were maintained as monolayers using minimum essential media (Life Technologies/BRL) supplemented with 5% bovine growth serum (HyClone) and passaged every 3 to 4 days. Multilayered cell cultures were grown using the apparatus shown in Fig. 1A as previously described (17).
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/6(abc). The mean weight (±SD) of the excised tumors was 60 ± 24 mg. For radiolabel-based work, drugs were given i.v. For BrdUrd-based work, paclitaxel in Cremophor EL and docetaxel in Tween 80 were given i.p., and mice were given 1,500 mg/kg BrdUrd 2 h before sacrifice, as previously described (18, 30). MCC drug penetration assays and control assay. MCC drug penetration was done using the apparatus shown in Fig. 1B as previously described (17), with the following modifications. Reservoir volumes were 2.5 mL per MCC instead of the original 7.5 mL. For radiolabeled experiments, cultures were frozen immediately following the 1- or 2-h drug exposures. For the BrdUrd-based assay, following the 1-h drug exposure, MCCs were incubated for two 30-min washout periods before removal from the penetration apparatus. They were incubated for 2 days before BrdUrd labeling via exposure of both sides of MCCs to 100 µmol/L BrdUrd for 4 h and subsequent freezing. The MCC control assay was done using the apparatus shown in Fig. 1C as previously described (17). Control experiments exposed both sides of MCC to 0.1 µmol/L drug with normal conditions (20% O2, 1 g/L glucose) on one side of cultures versus deprived conditions (0% O2, 0.1 g/L glucose, 1 mmol/L lactate) on the other side. Three MCCs were used for each set of experimental conditions.
Autoradiography. Tumor xenograft (10 µm thick) and MCCs cryosections were placed in triplicate on glass slides, allowed to dry overnight, and then clamped against tritium-sensitive film (Hyperfilm-3H; GE Healthcare) under GBX filter safelight conditions (Kodak) and stored in darkness for a period of 80 days. The film was developed for 1 min using a 1:1 dilution of D-19 developer (Kodak). Calibration of film was done using a tritium standard slide with range of 0 to 489 µCi/g (American Radiolabeled Chemicals inc.).
Immunohistochemistry. Cryosections were fixed in acetone-methanol for 10 min at room temperature. Vasculature was stained using an antibody to PECAM/CD31 (BD PharMingen) and fluorescent Alexa 546 anti-rat secondary antibody (Invitrogen). Slides stained for incorporated BrdUrd were placed in distilled water for 10 min and then treated with 2 mol/L HCl at room temperature for 1 h, followed by neutralization for 5 min in 0.1 mol/L sodium borate. Slides were then washed and transferred to a PBS bath. Incorporated BrdUrd was detected using a monoclonal mouse anti-BrdUrd (clone BU33, Sigma-Aldrich), followed by an anti-mouse peroxidase-conjugated antibody (Sigma-Aldrich) and a metal-enhanced 3,3'-diaminobenzidine substrate (Pierce). Slides were then counterstained with hematoxylin, dehydrated, and mounted using Permount (Fisher Scientific) before imaging.
Image acquisition. The imaging system consisted of a robotic fluorescence microscope (Zeiss Imager Z1), a cooled, monochrome CCD camera (Retiga 4000R, QImaging), a motorized slide loader and x-y stage (Ludl Electronic Products), and customized NIH-ImageJ software1 (public domain program developed at the NIH) running on a G5 Macintosh computer (Apple). The system allowed for tiling of adjacent microscope fields of view. Using this system, images of entire tumor cryosections up to 1 cm2 and MCC sections, 0.8 cm in length, were captured at a resolution of 0.75 µm per pixel.
Image analysis. Using the NIH-Image software application and user-supplied algorithms, digital images of 3H-taxane distributions were analyzed in the following manner. For tumors, digital images of tritium-sensitive film and CD31 immunostained tumor cryosections were superimposed and aligned. Then, each pixel making up the film image was sorted based on its distance relative to CD31-positive vasculature. Finally, the fraction of pixels from the film that were positive was tabulated as a function of distance from vasculature. Data were scaled using results for fraction-positive pixels versus radioactivity (µCi/g) using a tritium standard slide. For MCCs, digital images of tritium-sensitive film were aligned with bright-field images of unstained tissue cryosections. Pixels making up the tissue were then sorted based on distance from each edge of the section. For BrdUrd-based work, analysis was carried out as previously described (17, 18, 30).
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Results |
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100 µm of tissue, with little drug reaching 100 to 150 µm away from vessels for both drugs. The vehicle Cremophor EL, which was expected to reduce plasma levels of free paclitaxel and increase its duration of exposure, reduced tissue drug levels at the 2-h time point relative to Tween 80 by more than 2-fold (Fig. 2A and B). At the 8-h time point, paclitaxel in vehicle Tween 80 is seen to have undergone substantial washout, whereas for the Cremophor EL, vehicle data paclitaxel is still accumulating in the tissue. The drug profiles at 8 h are found to be similar. Docetaxel at the 2-h time point (Fig. 2C) is seen to fall off more sharply relative to paclitaxel. In contrast to paclitaxel in Tween 80, docetaxel showed increased accumulation at 8 h.
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3-fold increase in peak tissue levels, indicating at least partial saturation of sites. At the higher concentration, both drugs show improved tissue penetration, indicating that consumption of the drugs by tissue plays an important role in limiting their penetration. The longer exposure times are seen to improve tissue distributions. Peak radioactivity levels were observed at a depth of 20 µm into tissue. This was likely due to averaging over gaps in the tissue on the edge of the cultures and alignment of autoradiography images, which blurred slightly on the edges. These factors would not apply to the tumor-based work, where the peaks in radioactivity 30 µm into tissue at the 8-h paclitaxel time point is likely caused by drug washout due to falling plasma levels.
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3-fold lower for paclitaxel and 10-fold lower for docetaxel than the 2-h, 0.3-µmol/L MCC results. In MCCs, peak levels (Fig. 4A) did not increase when going from a 1- to a 2-h exposure (P > 0.05 ANOVA except for the 0.3 µmol/L docetaxel); however, total tissue radioactivity (Fig. 4B) did increase moderately (significant differences, P < 0.005 ANOVA, for 1-h versus 2-h exposures in all cases except for 0.3 µmol/L paclitaxel). As observed from the tissue distribution profiles (Figs. 2 and 3), the depth at which the drug levels fell to half-maximum is seen to increase with concentration (Fig. 4C). The xenografts were seen to exhibit surprisingly large penetration half-depths in comparison with the MCC-based data given their low-peak tissue levels.
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5-fold decrease in drug exposure on the far side of the culture for paclitaxel versus 10-fold decrease for docetaxel. Despite paclitaxel exerting a more uniform effect within the cultures, both drugs were similar in their ability to control MCC thickness (Fig. 5B and C, bottom). Control experiments were carried out to determine the effect that closing off the culture during drug exposure had on the response of cells on the far side. Conditions encountered on the far side of the cultures during drug exposure were simulated using the apparatus shown in Fig. 1C, in which both sides of the cultures were exposed to equal drugs, 0.1 µmol/L, but under normal conditions on the near side versus oxygen/nutrient-deprived conditions on the far side. Results, also shown in Fig. 5B and C (top), indicate that temporary deprivation of oxygen and nutrients on the far side of the cultures during drug exposure did not affect the response to the drugs.
Effect of taxanes on S-phase cell distribution in tumor xenografts. Before conducting the tumor-based radiolabeled drug experiments, we mapped the drug effect on S-phase cell distribution in the tumor xenografts using the same technique applied to MCCs. In these experiments, S-phase cells were mapped relative to tumor vasculature as a function of taxane dose and time following treatment, and the results are summarized in Fig. 6
. Results for the time course of paclitaxel effect on S-phase cells 1, 3, and 6 days following treatment are shown in Fig. 6A, with raw data in the top and data normalized to the untreated controls in the bottom. Results show that there is a decrease in S-phase cells over the first 3 days following treatment. From the normalized data, paclitaxel is seen to produce up to a 75% reduction in proliferation near vessels, but only a 50% reduction in cells 150 µm away. Further work looking into the drug effect at the 3-day time point as a function of dose is shown in Fig. 6B for paclitaxel and Fig. 6C for docetaxel. Drug doses were chosen to span the range typically given to mice and produce equitoxic effects for the two drugs (31). From the normalized data in Fig. 6B and C (bottom), a preferential effect is seen on the cells in the first 50 µm of tissue away from vessels, where proliferation and drug exposures are highest.
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Discussion |
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The formulation vehicle Cremophor EL had the expected effect of reducing initial peak levels of paclitaxel in tumors, but increasing its duration of exposure. It was interesting that at the 8-h time point, both paclitaxel formulations produced similar drug profiles; however, the tissue area under the curve for 0 to 8 h would still be higher for the Tween 80 data. Later time points might eventually result in greater accumulation for the Cremophor EL data.
The MCC-based work was intended to serve as a comparison between the drugs in isolation of their in vivo pharmacokinetic differences rather than to reproduce blood pharmacokinetic profiles. Hence, drug exposures were chosen to bracket the expected tumor exposures. However, peak tumor levels for paclitaxel and docetaxel were, respectively, 3- and 10-fold lower than those seen in MCC following the 0.3 µmol/L exposure. This is despite reported plasma areas under the curve and peak levels in mice in the range of 35 and 5 µmol/Lxh and peak levels of 40 and 10 µmol/L for paclitaxel and docetaxel, respectively (25, 29). These results indicate that blood binding of the drugs plays a significant role in limiting their tissue distribution, and that only the free form of the drug in plasma is likely to contribute to tissue penetration. MCC work was carried out in 5% serum, which likely results in 50% unbound drug (24) as compared with 2% to 10% unbound drug expected in vivo (25, 26). Based on these findings, future work could be carried out to look at taxane penetration in MCCs at lower drug concentrations, matching the levels detected from the tumor-based work. Under these conditions, the taxanes might exhibit an even greater reduction in penetration or perhaps behave in a similar way to the 0.3 µmol/L data. It would also be interesting to use the assay to examine the relation between exposure duration and drug concentration on overall drug buildup at lower drug concentrations and longer exposures.
The apparent depth of penetration of the drugs in tumors as compared with MCCs was greater than expected given the marked concentration dependence of tissue penetration seen here. The radial geometry seen in the HCT-116 tumor type, in which cords of tumor cells surround individual vessels (30), should pose a greater barrier to penetration than does the planar geometry of MCC discs. Two factors might explain the increased penetration seen in the tumor data. First, there will exist unobserved vessels that lie just above or below the 10-µm tissue sections cut from the tumors. The likelihood of occurrence of unobserved vessels will increase with distance from observed ones, and hence, they will increase the apparent penetration half-depth of a drug. Another factor that might increase the apparent penetration of the taxanes in vivo is the contribution of 3H-labeled liver metabolites, which might diffuse more easily than the parent drug.
A unique property of the disc geometry of MCCs is that it presents a barrier to penetration in which there exist rapidly proliferating cells at two positions in the tissue (either edge). This symmetry makes it possible to use the effect of the drugs as an indicator of their penetration, in effect, using the cells themselves as the drug measurement end point. Results from the effect-based assay were interesting in that the differential in drug effect on the two edges was not as great as might be expected from the steep drug gradients observed in the autoradiography-based data. However, the radiolabeled data produced discrete snapshots of drug distribution at two time points during drug buildup, whereas the effect-based assay integrated exposure over the entire process of buildup and washout. Redistribution of the drugs during washout and differences in cellular retention of the drugs would affect results. It was also interesting that both taxanes controlled MCC thickness similarly despite their having different profiles of effect, with paclitaxel exerting a more uniform effect on both sides of the tissue, whereas docetaxel had a greater effect on the near side and less on the far side.
The proliferation-based tumor xenograft data showed that both drugs were less effective on the cells farthest from tumor vasculature. However, it was difficult to interpret these data on their own in terms of tissue penetration of the drugs because, unlike the MCC-based BrdUrd work, the cells farthest into the tissue exhibited only a fraction of the proliferation seen in those cells near vessels. The tissue farthest from vasculature will not only exhibit reduced sensitivity to the drugs but also a different lag time between drug exposure and progression through the cell cycle to G2-M block (32). The fact that at the lowest doses, both drugs seemed to exert a uniform effect within the tumors, but at higher doses, a greater effect is observed near vessels (Fig. 6B and C, bottom) might be explained by indirect host toxicity, which could conceivably reduce proliferation throughout the tumor.
Based on the results of this study, the effect-based MCC assay could be used as a high-throughput method for screening new taxane analogues for tissue penetration. In the development of new taxanes with improved antitumor capabilities, tissue penetration would be one of many parameters that could be modified. However, for new analogues that exhibited in vitro efficacy and desirable in vivo pharmacokinetics and tolerance, knowledge of tissue penetration could provide additional criteria for selecting potential analogues to further develop. Because a drug that penetrates poorly will require a higher driving concentration from the blood to reach and kill the cells situated farthest from blood vessels, improving drug penetration could reduce system toxicity by reducing the blood drug levels required to achieve maximum response. Also, in addition to increasing the concentration of drug seen by the cells farthest from blood vessels, improved drug penetration might also increase the window of exposure of these cells to therapeutic levels of the drug, which could counter the effects of the lower S-phase fraction observed in this population.
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8/ 4/06; revised 10/13/06; accepted 2/12/07.
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) and 8 h (
) following tail vein injection; 10 mg/kg paclitaxel, vehicle Tween 80 (A), vehicle Cremophor EL (B); and 5 mg/kg docetaxel, vehicle Tween 80 (C). Data were obtained from tritium-sensitive film clamped against 10-µm cryosections for 80 d followed by immunohistochemical staining of CD31-positive tumor vasculature. Points, average; bars, SE; n = 9.
), docetaxel (
), and for tumor data, paclitaxel in Cremophor EL (
).
) side of MCCs for paclitaxel (B) and docetaxel (C). Data show BrdUrd immunostaining expressed as the fraction of levels seen in untreated MCCs. Results from control experiments under conditions of normal (
) versus low oxygen and glucose and high lactate (
) are also shown. Bottom, MCC thickness 2 d after treatment plotted relative to thickness at time of treatment. Points, average; bars, SE; n = 4-6. *, P < 0.005 (ANOVA), data points where the drug effect on the far side was significantly less than on the near side. The effect of paclitaxel versus docetaxel on thickness were comparable for all concentrations, P > 0.05 (ANOVA).
