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OtherCHEMOTHERAPY/GENE THERAPY

The Role of Tumor-Associated Macrophages in the Delivery of Liposomal Doxorubicin to Solid Murine Fibrosarcoma Tumors

Lawrence D. Mayer, Graeme Dougherty, Troy O. Harasym and Marcel B. Bally
Journal of Pharmacology and Experimental Therapeutics March 1997, 280 (3) 1406-1414;
Lawrence D. Mayer
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Graeme Dougherty
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Troy O. Harasym
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Marcel B. Bally
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Abstract

Murine fibrosarcoma tumors arising from subcutaneous inoculation of FSa-N cells exhibit 4-fold higher tumor-associated macrophage (TAM) levels than those from the FSa-R line. These solid tumors were used to assess the role of TAMs in the accumulation of liposomal anticancer drugs. Two liposomal formulations of doxorubicin were investigated: a conventional formulation composed of distearoylphosphatidylcholine (DSPC) and cholesterol and a sterically stabilized liposomal formulation composed of DSPC/cholesterol/poly (ethylene glycol)-modified distearoylphosphatidyethanolamine (PEG-PE). Circulating concentrations of PEG-PE containing liposomes 24 h after i.v. administration were 3-fold greater than those observed after administration of conventional liposomes. No differences were observed in drug retention or tumor (FSa-R or FSa-N) drug and liposomal lipid delivery when comparisons were made between different liposomal formulations. However, tumor doxorubicin concentrations were increased as much as 4-fold for liposomal formulations relative to free drug. Further, there was a 1.5- to 2-fold increase in doxorubicin delivery to TAM-enriched FSa-N tumors compared with FSa-R tumors. Fluorescence microscopy studies revealed a poor correlation between CD11b (Mac-1) positive cells (TAMs) and the appearance of doxorubicin fluorescence. These results suggest that uptake of liposomal drugs by TAMs does not account for the enhanced accumulation of liposomal drugs in solid tumors. Rather, the increased tumor drug delivery may be related to alternative TAM-mediated processes that increase tumor vascular permeability. Therapeutic studies demonstrated that increased tumor drug uptake observed for the liposomal doxorubicin formulations led to marginal improvements in antitumor activity, and it is suggested that much of the drug delivered in liposomal form is not biologically available.

The ability of liposomes to decrease the toxicity of doxorubicin while maintaining or increasing its antitumor potency has been well documented for a variety of liposomal systems in preclinical models (Gabizon et al., 1985; Mayer et al., 1989, 1990a; Huang et al., 1992b) as well as Phase I and Phase II clinical trials (Cowens et al., 1993; Uziely et al., 1995; O’Day et al., 1994). It is generally accepted that the buffering of toxicity observed with liposomal doxorubicin formulations is related to the fact that lipid carriers reduce 1) free drug peak plasma concentration and 2) exposure to susceptible tissues such as the heart (Gabizon et al., 1982, 1989; Mayer et al., 1990b). The latter observation reflects the low uptake of small (100 nm) PC/Chol-based liposomes by these tissues. The mechanism(s) whereby liposomes improve the antitumor activity of doxorubicin, however, are less well understood. This may be caused by the fact that few studies have focused on the processes whereby liposomes gain access to tumor sites and, perhaps more importantly, what factors control exposure of malignant cells to encapsulated drug.

Several potential mechanisms have been postulated for the improved antitumor activity of liposomal doxorubicin compared with unencapsulated drug (Gabizon, 1994). First, because the most efficacious liposomal formulations display increased circulation lifetimes, these systems could act as slow release or systemic infusion delivery vehicles in the blood compartment. Although doxorubicin released from liposomes in the circulation will certainly contribute to therapeutic activity, the fact that liposomes displaying remarkably different plasma drug leakage rates provide similar levels of therapy (Mayer et al., 1994) suggests that release in the blood compartment may not be the most critical factor governing antitumor activity. Second, liposomes may deliver doxorubicin directly to tumors through a passive accumulation process. Subsequent exposure of the tumor cells to the encapsulated drug would therefore rely on drug release from regionally localized liposomes because it seems that nontargeted liposomes are incapable of directly delivering their contents to tumor cells (Working et al., 1994). Third, circulating macrophages may mediate delivery of liposomal drugs and, finally, TAMs may promote liposome delivery to the tumor.

Recent investigations, including elegant imaging studies by Jain and co-workers (Yuan et al., 1994), have demonstrated that intact liposomes are capable of extravasating the tumor vasculature and accumulating in the interstitial space within tumors and areas of tumor growth (Yuan et al., 1994; Mayer et al., 1990a;Huang et al., 1992a; Wu et al., 1993). The processes whereby liposomes cross vascular endothelial barriers as well as the fate of the lipid carriers and their entrapped contents after extravasating into the tumor interstitium, however, are not well understood. In particular, the mechanisms that lead to the release of entrapped anticancer drugs from liposomes, which is presumably required for action on the intracellular tumor target, have not been elucidated. Previous studies have suggested that engulfment and subsequent intracellular degradation of liposomal carriers by macrophages can render encapsulated agents such as doxorubicin bioavailable to surrounding tissue or the central blood compartment (Storm et al., 1988). Given that many human malignancies exhibit significant levels of infiltrated macrophages, studies with two murine fibrosarcoma lines differing primarily in their TAM level were undertaken here to determine whether the presence of phagocytic cells control enhanced tumor drug accumulation observed after i.v. administration of liposomal formulations of doxorubicin.

Materials and Methods

Materials.

1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] (PEG2000-DSPE) were purchased from Avanti Polar Lipids (Alabaster, AL). Chol was obtained from Sigma Chemical Co. (St. Louis, MO). Radiolabeled 3H-CHDE was purchased from Amersham (Oakville, ON) and DOX from Adria Laboratories (Mississauga, ON). FITC-labeled rat antimouse CD11b (MAC-1) antibody was purchased from Pharmingen (San Diego, CA) and Tissue-Tek O.C.T. compound from Miles (Etobicoke, ON). Dulbecco’s Modified Eagle’s Medium and Hanks’ Balanced Salt Solution were purchased from StemCell Technologies, Inc. (Vancouver, Canada). Tissue culture grade fetal bovine serum, trypsin and glucose were obtained from ICN Biomedicals (Costa Nesa, CA). Solvable was obtained from NEN (Mississauga, ON), Pico-fluor 40 scintillation cocktail from Packard and microtainer EDTA-coated tubes from Becton Dickinson (Mississauga, ON). Female C3H/HeJ mice were obtained from the Joint Animal Care Facility breeding colony at the BC Cancer Research Centre. Parental stock were obtained from Jackson Laboratories (Bar Harbor, ME).

Preparation of liposomes.

Large unilamellar vesicles (100 nm) were prepared as described previously (Mayer et al., 1986). Lipid mixtures consisting of DSPC, Chol and PEG2000-DSPE (50:45:5, mole %) were prepared in chloroform and subsequently concentrated to a homogeneous lipid film under a stream of nitrogen gas. The lipid film was then placed under high vacuum for at least 4 h before hydration at 65°C with 300 mM citrate, pH 4.0. The resulting multilamellar vesicle preparation was frozen and thawed five times (Mayer et al., 1985) before extruding the sample 10 times through stacked 100-nm polycarbonate filters (Poretics) with an extrusion device (Lipex Biomembranes, Inc., Vancouver, Canada) at 65°C. The resulting liposomes were sized by QELS with a Nicomp 270 submicron particle sizer operating at 632.8 nm.

DOX encapsulation.

DOX was encapsulated in liposome preparations with the transmembrane pH gradients as described previously (Mayer et al., 1989). The liposome preparation (prepared at pH 4.0) was heated to 65°C for 10 min before addition to a preheated (65°C for 10 min) solution of DOX (5–6 mM in saline). A final drug-to-lipid ratio of 0.2 (w/w) was typically employed. This mixture was incubated with periodic mixing for 10 min at 65°C. Unencapsulated DOX was removed by passing the sample through a Sephadex G-50 column and the DOX-to-lipid ratio was measured by fluorescence detection at 550 nm as described previously (Bally et al., 1994).

Murine tumor models.

The cell line designated as FSa-R is a highly immunogenic, methylcholanthrene-induced fibrosarcoma and the FSa-N cell line is a “spontaneous” fibrosarcoma arising in a nonirradiated part of an irradiated mouse (Howie et al., 1982). Cell lines were maintained in culture with Dulbecco’s Modified Eagle’s Medium supplemented with 4500 mg/l glucose and 10% fetal bovine serum. Cells were passaged (1:10) every 3 to 4 days after removal of adherent cells with a 2.5% trypsin solution. For tumor initiation, harvested cells were centrifuged at 250 ×g for 5 min and resuspended in Hanks’ Balanced Salt Solution at a concentration of 2 × 106 cells/ml. Female C3H/HeJ mice weighing 18 to 22 g were then injected in both the left and right flank with 1 × 106 cells (50 μl). When tumors grew to approximately 200 mg (12 and 18 days for FSa-R and FSa-N, respectively) tumor-loading studies were initiated. Large unilamellar vesicles were injected via the lateral tail vein at a lipid and drug dose of 100 and 20 mg/kg, respectively (0.2 drug/lipid). For tumor efficacy studies, tumor weights were determined by converting caliper measurements to grams with the formula; (length × width2)/2 (Mayer et al., 1990a) which was confirmed by comparing the actual weights of excised tumors with those estimated from caliper measurements. Treatment was given i.v. when tumors reached a size of 200 to 500 mg. Statistical significance in tumor growth was determined by analysis of variance analysis with the statistical software program Statistica.

Preparation of plasma and tissues.

Mice were anesthetized with an intraperitoneal injection (100 μl) of a ketamine/xylazine mixture (160 and 20 mg/kg, respectively). Whole blood was collectedvia cardiac puncture and immediately placed in EDTA-coated Microtainer tubes. Plasma was subsequently prepared by centrifugation at 1500 × g for 10 min. One tumor from each mouse was prepared for histology (see below), the second tumor as well as lung, liver, spleen, kidney and heart were collected, weighed and homogenized to a 10% homogenate (w/v in H20) with a Polytron tissue homogenizer.

Quantitation of liposomal lipid and doxorubicin.

Liposomal lipid was quantified by use of the nonexchangeable and nonmetabolizable lipid marker 3H-CHDE (Bally et al., 1994). Solvable (0.5 ml) was added to 200 μl of the 10% tissue homogenates. Samples were then incubated in a water bath at 50°C for 3 h. After cooling to room temperature, 50 μl of EDTA (200 mM), 200 μl H2O2 (30%) and 25 μl HCl (10 N) were added. After 1 h at room temperature 5 ml of Pico-fluor 40 scintillation cocktail was added and samples were analyzed in a Packard Tri-carb 1900 TR liquid scintillation analyzer. For plasma samples, 100 μl of plasma was added directly to the Pico-fluor 40 and analyzed.

DOX was quantified by a solvent extraction and fluorescence detection assay as outlined previously (Bally et al., 1994). Fifty microliters of plasma or 200 μl of 10% tissue homogenate were adjusted to 0.8 ml with H2O. One hundred microliters of 10% sodium dodecyl sulfate and 100 μl H2SO4(10 mM) were then added. Subsequently, DOX extraction was achieved by additions of 2 ml of chloroform/isopropyl alcohol (1:1, v/v) followed by vigorously vortexing and freezing at −20°C overnight. After thawing and further vortexing the samples were centrifuged at 1500 × g for 10 min and the organic phase (lower phase) collected. The fluorescence of this phase was determined (excitation wavelength, 500 nm; emission wavelength, 550 nm) with a Perkin Elmer LS50 B Luminescence Spectrometer. A standard DOX curve was prepared with tissue and plasma derived from control (untreated) animals by a similar extraction procedure.

Tumor fixation and staining.

Isolated tumors (approximately 200 mg) were collected in PBS at 4°C. Tumors were subsequently fixed with a 3% paraformaldehyde solution in PBS at 4°C for 30 min (sufficient to fix through the tumor), washed with PBS and immersed in increasing sucrose gradients for 20 min each: 10% sucrose-PBS, 15% sucrose-PBS and 15% sucrose-PBS containing O.C.T. compound (1:1, v/v). The processed tissue was then embedded in O.C.T. compound and frozen in liquid nitrogen. Five-micrometer sections were then prepared on a Frigocut 2800E microtome from Leica.

For antibody staining of sections, the sections were washed three times in PBS. Nonspecific antigens were blocked with 0.02% bovine serum albumin (30 min) and an antibody specific for macrophages (FITC-labeled anti-CD 11b) was added and incubated (30 min) in a wet box at room temperature. The sections were then washed with H2O to remove salt and mounted on a microscope slide. A Leitz Dialux microscope was used to evaluate FITC fluorescence of the sections (430–490 nm bandpass filter block with a 520-nm suppression filter) and DOX fluorescence (530–550 nm bandpass filter block with a 580-nm suppression filter) with fluorescent photomicrographs obtained with a Orthomat microscope camera under 400× magnification with a 40× objective lens. Fluorescent micrographs were obtained under constant exposure time once appropriate exposure was identified by use of manual settings for the most intensely fluorescent sample. Phase contrast photomicrographs of the sections were also obtained. All images were recorded on Fuji color ASA400 negative film.

Results

Characterization of TAM levels in FSa-R and FSa-N tumors.

The murine fibrosarcoma lines used here for characterizing the tumor-accumulating properties of liposomes were selected on the basis of the different levels of infiltrating macrophages displayed in vivo (Howie et al., 1982). Cellular isolates from FSa-N and Fsa-R tumor digests have been characterized for macrophage content by immunohistochemistry (G. Dougherty, unpublished observations). These studies revealed that the percent macrophage content in cells obtained from cytospins of FSa-N homogenates were 2.3-fold higher than observed for cytospins from FSA-R tumors. The higher TAM level in FSa-N tumors occur throughout tumor progression when growing as subcutaneous solid tumors in C3H/HeJ mice (Howie et al., 1982). Macrophage content comparisons were also determined here with frozen thin sections of FSa-R and FSa-N tumors that were stained with FITC-labeled anti-CD 11b antibody and visualized for positive cells by fluorescence microscopy. Comparison of three fields in three different tumors of each cell type revealed that the FSa-N tumor sections contained 28.8 ± 4.4 CD 11b positive cells per 400× magnification field, whereas FSa-R tumor sections contained 7.3 ± 2.4 CD 11b positive cells per field. This reflected a 3.9-fold increase in macrophage content in the FSa-N tumors relative to the FSa-R line. It should be noted that other studies (not presented here) demonstrated that both FSa tumor types had similar vascular plasma volumes (8 μl/g tumor).In vitro cytotoxicity studies demonstrated that the FSa-N line was 3-fold less sensitive to DOX than the FSa-R line (24 h continuous exposure DOX IC50 values of 1 μM and 3 μM, respectively, data not shown).

Plasma elimination characteristics of DSPC/Chol and PEG-PE/DSPC/Chol liposomal systems.

Plasma levels of DOX and liposomal lipid were determined after i.v. injection of 100 nm drug-loaded DSPC/Chol and PEG-PE/DSPC/Chol liposomes to C3H/HeJ mice. Comparisons were also made between tumor-bearing and tumor-free mice to fully characterize the pharmacokinetic and tumor accumulation properties of the liposomal systems. Figure 1A presents the plasma liposomal lipid levels in tumor-free mice after injection of 100 mg lipid/kg (equivalent to 20 mg/kg DOX dose). As indicated elsewhere (Parr et al., 1993) liposomes containing DOX (entrapped by the pH gradient loading technique) exhibited extended circulation lifetimes compared with empty liposome counterparts (results not shown). DOX-loaded liposomes containing PEG-PE exhibited circulation half-lives that were increased significantly over those observed for DSPC/Chol-formulated liposomal doxorubicin in the C3H/HeJ mice (18 and 3.5 h, respectively). Plasma concentrations of3H-CHDE indicated that 13% and 37% of the injected dose of DSPC/Chol and PEG-PE/DSPC/Chol liposomes, respectively, were present in the circulation 24 h after i.v. injection (fig.1A).

Figure 1
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Figure 1

Plasma clearance of DOX-loaded DSPC/Chol (▪) and PEG-PE/DSPC/Chol (•) 100 nm liposomes in normal (A) and Fsa-N tumor-bearing (B) C3H/HeJ mice (three per time point). DOX was encapsulated in the liposomes at a drug-to-lipid ratio of 0.2:1 (w/w). Liposomes were injected i.v. at a dose of 100 mg lipid/kg and DOX at a dose of 20 mg/kg.

Plasma clearance properties of the two liposomal DOX formulations and free drug were also completed in Fsa-N tumor-bearing C3H/HeJ mice (fig.1B). Initial rates of liposomal drug and lipid removal from the circulation in tumor-bearing mice are increased (fig. 1B) compared with tumor-free mice (see fig. 1A). This is reflected primarily in the 1-h time point at which lipid levels decrease from 98% of the injected dose to 73% for PEG-PE/DSPC/Chol liposomal DOX and from 77% to 63% for DSPC/Chol liposomal DOX (fig.1A). The 24-h liposomal lipid levels in FSa-N tumor-bearing mice were similar to those obtained in tumor-free animals. It should also be noted that similar results were obtained in mice bearing the FSa-R tumor (data not shown).

Regarding the drug clearance properties, the amount of doxorubicin retained in the plasma compartment is at least two orders of magnitude greater for the liposomal drugs than for the free drug (fig. 2). Greater than 99% of the administered dose of free DOX was cleared from the plasma within 1 h after injection in mice with FSa-N tumors, whereas greater than 20% of the injected DOX dose remained in the plasma 24 h after injection for the liposomal formulations. Consistent with the results shown in figure 1, PEG-PE-containing liposomes displayed elevated plasma drug levels compared with DSPC/Chol liposomes during the entire time course. Specifically, 24-h plasma DOX concentrations were 1.8- to 2.3-fold higher for PEG-PE/DSPC/Chol than for DSCP/Chol liposomal systems in FSa-N tumor-bearing mice. As indicated above, the results obtained in animals bearing FSa-R tumors were not statistically different from those bearing FSa-N tumors.

Figure 2
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Figure 2

Plasma clearance of DOX in C3H/HeJ mice (three per time point) bearing FSa-N tumors after i.v. injection of free and liposomal DOX at a drug dose of 20 mg/kg (100 mg lipid/kg for liposomal formulations). Mice were administered free drug (▾) as well as DOX encapsulated in 100 nm DSPC/Chol (▪) or PEG-PE/DSPC/Chol (•) liposomes.

Tumor lipid and drug accumulation properties.

DOX and liposomal lipid levels in FSa-R and macrophage-enriched FSa-N tumors after i.v. injection were determined for DSPC/Chol and PEG-PE/DSPC/Chol formulations as well as free (unencapsulated) drug. The rapid distribution of free DOX from plasma is accompanied by maximum FSa-N tumor drug uptake levels of 12.8 μg/g 1 h after administration (fig. 3A). Tumor drug levels subsequently decreased gradually from this value to 11.5 μg/g and 7.6 μg/g at 4 h and 24 h, respectively. In contrast, DOX accumulates in FSa-N tumors at a slower rate but for a prolonged period of time when administered in liposome-encapsulated form. FSa-N tumor-associated DOX levels after injection of drug entrapped in DSCP/Chol or PEG-PE/DSPC/Chol liposomes were approximately 2.5-fold lower than those obtained for free DOX at 1 h, equivalent to free drug levels at 4 h and roughly 4-fold higher at 24 h (fig. 3A). This pattern of drug accumulation was similar to that observed for liposomal lipid accumulation in FSa-N tumors for both liposomal doxorubicin formulations (fig. 3B). Tumor-associated lipid levels of approximately 16 μg/g were observed at 1 h and increased to 45 to 60 μg/g and 130 to 140 μg/g at 4 h and 24 h post-injection, respectively. It is important to note that FSa-N tumor levels of liposomal lipid and DOX were not statistically (P < 0.05) different between DSPC/Chol and PEG-PE/DSPC/Chol systems throughout the entire time course.

Figure 3
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Figure 3

DOX (A) and liposomal lipid (B) accumulation in s.c. FSa-N tumors after i.v. injection of free DOX (▾) or DOX encapsulated in 100 nm DSPC/Chol (▪) or PEG-PE/DSPC/Chol (•) liposomes. Mice (four per time point) were injected with a DOX dose of 20 mg/kg and a lipid dose of 100 mg/kg.

Although the time courses for drug and liposomal lipid accumulation in FSa-N tumors were similar to those for FSa-R tumors (results not shown), absolute levels of drug were significantly increased for the TAM-enriched FSa-N tumors. This is shown in table 1where the tumor DOX levels as well as the FSa-N/FSa-R drug uptake ratio is presented for the drug formulations studied. DOX levels in the FSa-N tumors were consistently elevated 1.4- to 2.7-fold compared with FSa-R tumors. It should be noted that the increase in tumor-associated drug levels observed for FSa-N tumors did not appear to be related to differences in the extent of tumor vascularization, because both FSa-N and FSa-R tumors exhibited similar blood and plasma volumes (results not shown). Further, no correlation could be made between the FSa-N/FSa-R ratio and whether DOX was in free or liposome-encapsulated form.

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Table 1

Comparison of DOX uptake levels in FSa-R and TAM-enriched FSa-N tumors after i.v. injection of free and liposomal drug1-a

Tumor and plasma DOX levels for DSPC/Chol and PEG-PE/DSPC/Chol formulations were also compared to assess the efficiency of liposomal DOX extravasation into the solid tumor. This correlation can be made because of the fact that plasma drug-to-lipid ratios for the liposomes used here remained unchanged (0.2:1, w/w) over 24 h, which indicated negligible release of entrapped DOX in the central blood compartment (data not shown). Consequently, tumor uptake of DOX for these systems is related primarily to liposomes that have delivered drug directly to the extravascular tumor site. Figure 4presents the tumor (μg/g) to plasma (μg/ml) ratios for DSPC/Chol and PEG-PE/DSPC/Chol liposomal DOX preparations in FSa-R and FSa-N tumor-bearing mice 24 h after injection. Approximately 2-fold increased tumor/plasma values are observed in mice bearing TAM-enriched FSa-N tumors compared with FSa-R tumors for both liposomal formulations studied. This increase reflects an increased efficiency in the extravasation of DSPC/Chol and PEG-PE/DSPC/Chol liposomes across the vasculature of FSa-N tumors. Further, the extravasation efficiency of DSPC/Chol liposomes was greater than that for PEG-PE/DSPC/Chol liposomes by 1.8- and 2.4-fold in FSa-N and FSa-R tumors, respectively (fig. 4). This was caused by the fact that similar tumor levels of drug were obtained for both liposomal formulations under conditions in which plasma levels of DSPC/Chol liposomes were 2- to 3-fold lower than those observed for the PEG-PE-containing liposomes. It should also be noted that similar relationships are observed at earlier time points (data not shown).

Figure 4
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Figure 4

Tumor-to-plasma DOX ratios observed in FSa-R and FSa-N tumors 24 h after i.v. injection of 100 nm DSPC/Chol (open bars) and PEG-PE/DSPC/Chol (solid bars) liposomes containing DOX at a drug-to-lipid ratio of 0.2:1 (w/w). Drug and liposomal lipid doses were 20 mg/kg and 100 mg/kg, respectively. Tumor-to-plasma ratios were calculated as the tumor DOX content (μg/g tumor) divided by the plasma concentration of DOX (μg/ml).

Studies on the mechanism of tumor drug and liposome accumulation.

Given that TAM-enriched FSa-N tumors take up significantly more drug and lipid than FSa-R tumors and that PEG-PE-containing liposomes are less efficient in accumulating in these tumors, studies were undertaken to determine whether TAMs mediate tumor delivery of liposomal DOX, particularly for PEG-free liposomes. Figure5 shows fluorescence micrographs of FSa-N tumor frozen sections of tissues derived 24 h after i.v. injection of DOX (20 mg/kg) encapsulated in DSPC/Chol (fig. 5, A and C) and PEG-PE/DSPC/Chol (fig. 5, B and D) liposomes. DOX fluorescence was monitored directly (fig. 5, A and B) and TAMs were visualized after staining with FITC-labeled anti-CD 11b antibody (fig. 5, C and D). The fluorescence micrographs presented reflect representative fields from tumor sections exhibiting DOX fluorescence; however, it should be noted that areas within the tumors could be found that exhibited negligible DOX fluorescence. Such drug-poor areas were not associated with any specific tumor ultrastructure and could be detected near the tumor periphery or the tumor interior. The pattern and intensity of DOX fluorescence was similar in drug-containing sections for DSPC/Chol- and PEG-PE/DSPC/Chol-based liposomal DOX formulations. Evidence of diffuse nuclear-associated DOX fluorescence as well as concentrated foci of drug were present (fig. 5, A and B). Direct coordinate comparisons (by caliper measurements) of DOX fluorescence and CD 11b positive cells revealed that no intense foci of drug fluorescence occurred in these cells identified as macrophages (fig. 5). In fact, CD 11b positive cells were often observed in positions devoid of fluorescence when visualized with optical filters specific for DOX fluorescence. Similar observations were made with frozen sections of FSa-R tumors (data not shown). It should be noted that the amount and distribution of DOX fluorescence was unaffected by the immunohistochemistry processing steps; DOX fluorescence appeared similar for sections visualized immediately after sectioning as well as after antibody-staining procedures. Further, liposomes containing DOX remained intact and fluorescent when processed through the cryofixation procedure, and isolated peritoneal macrophages that had phagocytosed liposomal DOX exhibited strong fluorescent images when exposed to the cryofixation conditions (data not shown). These results in conjunction with previous studies demonstrating fluorescent imaging of liposomal DOX accumulated in peritoneal macrophages (Bally et al., 1994) indicate that liposomal DOX is not actively taken up by macrophages residing in the fibrosarcoma tumors studied here.

Figure 5
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Figure 5

Fluorescence micrographs of FSa-N tumor frozen sections obtained from C3H/HeJ mice 24 h after i.v. injection of DOX (20 mg/kg) encapsulated in 100 nm DSPC/Chol (A and C) or PEG-PE/DSPC/Chol (B and D) liposomes. Frozen sections were stained with FITC-labeled anti-CD 11b antibody and visualized by optical filters specific for DOX (A and B) or FITC (C and D) fluorescence with a 40× objective lens.

Whereas the microscopic evidence above does not establish a direct correlation between TAMs and tumor uptake of liposomal DOX, careful examination of tumor drug and lipid levels reveals interesting differences between the two fibrosarcoma lines. Monitoring the drug-to-lipid ratio of the liposomal DOX formulations provides a measure of the extent of drug leakage from liposomes. Plasma drug-to-lipid ratios for both liposome formulations studied here remained unchanged over the entire 24-h time course for both FSa-R and FSa-N tumors and were within 5% of the drug-to-lipid ratios of the formulations before injection, which indicates negligible drug leakage from the liposomes in the circulation (fig. 6). In contrast, FSa-N tumor-associated liposomal lipid and DOX content reflect drug-to-lipid ratios that are as much as 1.7-fold higher than the original formulated liposomal DOX preparations (fig. 6). The increased drug-to-lipid ratios observed in FSa-N tumors (compared with the corresponding plasma drug-to-lipid ratios in the same mice) are maximal at 1 h (P < .05), but remain elevated 1.5- (P < .05) and 1.3-fold (P < .05) in the tumor at 4 h and 24 h post-injection, respectively. Given that the liposomal lipid label used is nonexchangeable and nonmetabolizable and that liposome tumor accumulation increases over the 24-h time course, these data indicate that the drug uptake in FSa-N tumors after liposomal DOX administration arises from free drug that has been released from the liposomes in the tumor vasculature in addition to drug carried into the tumor interstitium via the liposomes. In contrast, all of the drug accumulated in Fsa-R tumors can be accounted for by direct uptake of DOX contained in the liposomes.

Figure 6
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Figure 6

Plasma and tumor drug-to-lipid ratios in C3H/HeJ mice (four per experimental point) bearing FSa-R (solid bars) and FSa-N (open bars) tumors 1 h after i.v. injection of DOX (20 mg/kg) encapsulated in 100 nm DSPC/Chol and PEG-PE/DSPC/Chol liposomes. Quantitation of DOX and liposomal lipid was accomplished as described under “Materials and Methods.”

Therapeutic activity of free and liposomal DOX formulations against FSa-R and FSa-N solid tumors.

The data shown to this point suggest that DOX delivery to both FSa-R and FSa-N tumors is enhanced when the drug is given in liposomal form. The amount of drug obtained in the tumor is similar for conventional liposomes (DSPC/Chol) and sterically stabilized liposomes (PEG/DSPC/Chol) that exhibit enhanced circulation lifetimes. In addition, the macrophage-enriched FSa-N tumors accumulate approximately 2-fold more drug than the FSa-R tumor. Clearly, it is important to determine whether formulation and/or tumor-induced differences in drug delivery result in a therapeutic advantage. Therefore, the efficacy of free DOX as well as DOX encapsulated in DSPC/Chol and PEG-PE/DSPC/Chol liposomes against FSa-R and FSa-N solid tumors growing s.c. in C3H/HeJ mice was evaluated. Mice bearing FSa-R and FSa-N tumors (50–250 mg) were treated with a single i.v. dose of 20 mg DOX/kg (100 mg lipid/kg for liposomal formulations) or saline for control animals. FSa-R tumors in control mice grew rapidly and reached sizes requiring termination of the animals within 12 days after initiation of therapy. Administration of free or liposomal DOX resulted in a significant delay in tumor growth (fig. 7A). The antitumor effects induced by free DOX and DOX entrapped in DSPC/Chol liposomes were similar throughout the course of the study, and the decreased tumor growth rates observed for the PEG-PE/DSPC/Chol liposomal DOX preparation are statistically significant (P < 0.05) compared with free and DSPC/Chol formulations only on days 14 and 16 post-treatment.

Figure 7
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Figure 7

Antitumor activity of DOX (20 mg/kg) administered as a single i.v. dose (day 0) to C3H/HeJ mice (five per group) bearing bilateral 50- to 250-mg s.c. FSa-R (A) or FSa-N (B) tumors. DOX was administered in free form (▾) as well as encapsulated in 100 nm DSPC/Chol (▪) or PEG-PE/DSPC/Chol (•) liposomes. Control mice (▴) were administered sterile saline. Tumor growth was monitored by caliper measurements and conversion to tumor weights as described under “Materials and Methods.” Error bars represent the standard error of the mean resulting from determinations on 10 tumors per group.

Administration of DOX to mice bearing FSa-N tumors also resulted in a delay in tumor growth compared with control tumors (fig. 7B). In this tumor line, however, free drug appeared most effective and was significantly better than both liposomal formulations in retarding tumor growth beyond day 16 (P < 0.05). The antitumor activity of the PEG-PE/DSPC/Chol and DSPC/Chol liposomal systems against the FSa-N tumors were similar and statistically significant differences in tumor growth were only observed beyond day 20.

Discussion

The studies described here were conducted to determine whether TAMs play a role in the accumulation and antitumor activity of DOX administered in liposomal form. The rationale for comparing the pharmacokinetic, tumor accumulation and therapeutic properties of free and liposomal DOX formulations in fibrosarcoma tumors differing in TAM content was based on three primary considerations. First, the use of liposomes exhibiting extended circulation lifetimes and minimized plasma drug leakage properties will require processing events at the tumor site to render the DOX bioavailable to tumor cells. Second, previous studies have demonstrated the ability of macrophages to engulf, process and re-release free DOX after phagocytosis of liposome-entrapped drug (Storm et al., 1988). Third, the use of RES avoiding liposomes containing steric stabilizing lipids may compromise the beneficial effects of potential TAM-based liposome-processing phenomena. The results from these studies suggest that TAMs do not have a direct role in liposomal drug accumulation in tumors. Specifically, there was no histological correlation observed between DOX and macrophages within the tumors studied. The results, however, do suggest that TAMs may play a secondary role in the accumulation of liposomal drugs. The discussion provided here focuses on how this information can be used to enhance the antitumor potency of liposomal DOX as well as other liposome-based anticancer drug formulations.

Pharmacokinetic studies with liposomal DOX in the FSa-R and FSa-N tumor models suggest that, in tumor-bearing mice, a faster plasma liposome distribution phase is observed. This can account for approximately 20% of the injected drug and liposome dose within 1 h after administration and could not be attributed to accumulation in the tumor or to tumor-induced increases in blood volume. This effect, therefore, maybe caused by tumor-induced nonspecific activation of alternative particulate clearance mechanisms. Regardless, the results obtained here are consistent with previous reports (Gabizon et al., 1989;Huang et al., 1992b) which indicate that incorporation of PEG-PE into liposomal DOX formulations results in significantly enhanced circulation lifetimes. In accordance with our original hypothesis, the TAM-enriched FSa-N tumors exhibited significantly elevated levels of drug and liposome uptake for encapsulated DOX compared with FSa-R tumors. This phenomenon was observed at all time points studied and peaked at approximately 4 h after drug administration. The direct role of TAMs in mediating this increased uptake, however, was not consistent with several experimental observations: 1) increases in drug accumulation in the FSa-N tumor were observed for free drug as well as both liposomal formulations, and 2) time-dependent increased uptake by FSa-N tumors did not correlate with plasma pharmacokinetics or tumor-specific changes in kinetics of accumulation compared with FSa-R tumors. This behavior was unexpected in view of the dramatic differences in plasma drug levels observed between free and liposomal DOX (fig. 2) as well as the fact that peak free drug accumulation in tumors occurs shortly after injection compared with the extended accumulation kinetics obtained with both liposomal preparations (fig 3A).

Although drug and liposome accumulation is increased for TAM-enriched FSa-N tumors, the lack of specificity of this effect for liposome-encapsulated DOX formulations compared with free drug suggests that direct phagocytosis of the lipid carriers is not intricately involved. This interpretation is supported by histological studies which demonstrate that tumor DOX fluorescence did not correlate with TAM location and often was completely absent in CD IIb positive cells after injection of liposomal DOX. Because the role of TAMs within tumors will be partly to remove cellular debris and dead cells, it may be expected that these mature phagocytic cells will already be saturated in terms of phagocytic capacity and unable to accumulate additional foreign particulates, such as liposomes. Further, unlike the condition in ascitic tumor models (Bally et al., 1994), the contact between extravasated liposomes and macrophages within the tumor may be restricted because of the tumor matrix itself.

Although the data discussed above do not support mechanisms for enhanced tumor accumulation of liposomal DOX based on direct uptake by TAMs, macrophages may indirectly increase drug and liposome tumor levels. Factors secreted by TAMs may alter tumor properties such as vascular permeability or intratumor pH, both of which could contribute to increased tumor drug accumulation. Such phenomena would be consistent with the observation that the drug-to-lipid ratios for both liposomal DOX formulations are significantly elevated compared with plasma drug-to-lipid ratios in FSa-N tumors, which indicates tumor site-specific increases in drug release from the liposomes (see fig.6). The fact that tumor drug accumulation is increased in FSa-N tumors for both free and liposomal DOX is also consistent with an indirect TAM effect on characteristics such as vascular permeability.

Two interesting observations in the studies presented here concern the efficiency of liposome accumulation in tumors as well as the relative efficacy of free and liposomal DOX formulations against FSa-R and FSa-N tumors. Empty and DOX-containing liposomes composed of PEG-PE/DSPC/Chol exhibited significantly extended circulation lifetimes compared with DSPC/Chol systems. However, this increase in plasma area under the curve liposome exposure did not result in a substantial improvement in the uptake of liposomal DOX by FSa-R and FSa-N tumors. These results are in contrast to some previous reports with similar systems (Huanget al., 1992b; Wu et al., 1993; Gabizon and Papahadjopoulos, 1988) and indicate that optimization of liposomal drug carriers based solely on extended plasma pharmacokinetic properties may not necessarily reflect improvements in tumor drug delivery. We have observed similar tumor uptake efficiency relationships for DSPC/Chol and PEG-PE/DSPC/Chol liposomes in various other solid tumor models including murine Lewis lung and human xenograft A431 squamous cell carcinoma (L. Mayer and M. Bally, unpublished observations). This suggests that particular attention should be given to the nature of the tumor model used for the evaluation of liposome-based drug formulations. It is important to note that tumor accumulation characteristics of liposomes containing the steric stabilizing lipid PEG-PE were not adversely affected by the presence of elevated TAM levels. This suggests that the RES-avoiding properties of these systems do not compromise their biological activity in macrophage-enriched tumors and is consistent with our conclusion that TAMs do not play a significant direct role in tumor accumulation.

The therapeutic studies performed here indicate that the 3.4-fold increases in tumor DOX concentrations achieved after 24 h with the liposomal formulations (compared with free DOX) provide only marginal improvements in antitumor activity against FSa-R tumors and are actually less effective than free drug in treating the TAM-enriched FSa-N tumors (see fig. 7). We would suggest that these surprising results are a consequence of poor DOX bioavailability in tumors in which liposomal drug has accumulated compared with free doxorubicin. These results also argue against the ability of macrophages to engulf liposomes and release DOX after intracellular processing because this should result in increased bioavailability and improved therapeutic activity for DOX administered in liposomal form compared with free drug. This indicates that the full therapeutic potential of liposomal anticancer drugs, such as DOX, will require increased sophistication in controlling tumor site-specific release of encapsulated agents to enhance activity. The advantage of such an approach has been demonstrated recently with thermosensitive liposomal DOX formulations (Huang et al., 1994).

Acknowledgments

The authors thank Guoyang Zhang and Dana Masin for their excellent technical assistance.

Footnotes

  • Send reprint requests to: L.D. Mayer, Division of Medical Oncology, BC Cancer Agency, 600 W. 10th Ave., Vancouver, BC V5Z 4E6.

  • ↵1 This work was supported through a research grant from the Cancer Research Society, Inc. (L.D.M.) and the Medical Research Council (M.B.B.) of Canada.

  • Abbreviations:
    PC
    phosphatidylcholine
    DSPC
    distearoylphosphatidylcholine
    PEG-PE
    polyethyleneglycol-distearoylphosphatidylethanolamine
    DOX
    doxorubicin
    TAM
    tumor-associated macrophage
    RES
    reticuloendothelial system
    QELS
    quasielastic light scattering
    CHDE
    cholesterylhexadecyl ether
    EDTA
    ethylenediamine tetraacetic acid
    Chol
    cholesterol
    i.v.
    intravenous
    PBS
    phosphate-buffered saline
    FITC
    fluorescein isothiocyanate
    • Received February 12, 1996.
    • Accepted November 13, 1996.
  • The American Society for Pharmacology and Experimental Therapeutics

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The Role of Tumor-Associated Macrophages in the Delivery of Liposomal Doxorubicin to Solid Murine Fibrosarcoma Tumors

Lawrence D. Mayer, Graeme Dougherty, Troy O. Harasym and Marcel B. Bally
Journal of Pharmacology and Experimental Therapeutics March 1, 1997, 280 (3) 1406-1414;

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The Role of Tumor-Associated Macrophages in the Delivery of Liposomal Doxorubicin to Solid Murine Fibrosarcoma Tumors

Lawrence D. Mayer, Graeme Dougherty, Troy O. Harasym and Marcel B. Bally
Journal of Pharmacology and Experimental Therapeutics March 1, 1997, 280 (3) 1406-1414;
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