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[Cancer Research 64, 7150-7155, October 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Crucial Role for Interferon {gamma} in the Synergism between Tumor Vasculature-Targeted Tumor Necrosis Factor {alpha} (NGR-TNF) and Doxorubicin

Angelina Sacchi1, Anna Gasparri1, Flavio Curnis1, Matteo Bellone1 and Angelo Corti1

1 Department of Biological and Technological Research and Cancer Immunotherapy and Gene Therapy Program, San Raffaele H Scientific Institute, Milan, Italy


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NGR-TNF is a derivative of TNF-{alpha}, consisting of TNF fused to CNGRCG, a tumor vasculature-targeting peptide. Previous studies showed that NGR-TNF can exert synergistic antitumor effects with doxorubicin and with other chemotherapeutic drugs in murine models. In this study, we have investigated the role of endogenous IFN-{gamma} on the antitumor activity of NGR-TNF in combination with doxorubicin. The study was carried out using murine B16F1 melanoma and TS/A mammary adenocarcinoma implanted subcutaneously in (a) immunocompetent mice, (b) athymic nude mice, and (c) IFN-{gamma}–knockout mice. Synergism between NGR-TNF and doxorubicin was observed in immunocompetent mice but not in nude or IFN-{gamma}–knockout mice. Preadministration of a neutralizing anti-IFN-{gamma} antibody to immunocompetent mice inhibited the NGR-TNF/doxorubicin synergism, whereas administration of IFN-{gamma} to nude and to IFN-{gamma}–knockout mice restored the synergistic activity. The synergism in nude mice was restored also by transfecting tumor cells with the IFN-{gamma} cDNA. Administration of NGR-TNF in combination with IFN-{gamma} to nude mice, but not of NGR-TNF alone, doubled the penetration of doxorubicin in TS/A tumors. These findings point to a crucial role for locally produced IFN-{gamma} in tumor vascular targeting with NGR-TNF and doxorubicin. Finally, addition of IFN-{gamma} to the treatment of immunocompetent mice with NGR-TNF/doxorubicin induced only modest improvement in response, suggesting that exogenous IFN-{gamma} can improve the therapeutic activity of these drugs only in case of suboptimal production of endogenous IFN-{gamma}.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many investigators have shown that systemic or local administration of tumor necrosis factor (TNF) {alpha} to tumor-bearing mice can cause massive tumor necrosis in various tumor models (reviewed in ref. 1 ). Moreover, the results of clinical trials carried out in patients with advanced cancer of the limb have shown that regional administration of high doses of TNF in combination with chemotherapeutic drugs, such as melphalan and doxorubicin, can selectively destroy the tumor vasculature and cause tumor necrosis without affecting normal adjacent tissues (2, 3, 4, 5, 6, 7, 8, 9) . However, despite the impressive results obtained in animal models and patients, the clinical use of TNF as an anticancer drug is limited to locoregional treatments because of systemic toxicity and poor therapeutic index (4) .

We have recently shown that targeted delivery of TNF to aminopeptidase N (CD13) or to {alpha}v integrins, two markers of angiogenic vessels, could be a strategy for improving the therapeutic index of this cytokine (10, 11, 12) . This strategy relies on the rationale that tumor neovasculature is a primary target of TNF (1 , 8 , 13, 14, 15, 16, 17, 18) . Targeted delivery to CD13 or {alpha}v integrins was achieved by fusing the NH2 terminus of TNF with the COOH terminus of peptides containing the NGR or the RGD motives, respectively, previously identified by in vivo panning of peptide-phage libraries (19, 20, 21) . These conjugates (called NGR-TNF and RGD-TNF) induced stronger antitumor effects than TNF, with no evidence of increased toxicity, in animal models (10, 11, 12) . Remarkably, administration of minute amounts of NGR-TNF was sufficient to enhance the antitumor activity of doxorubicin and melphalan in a synergistic manner (11) . Studies on the mechanism of action showed that picogram doses of this conjugate target tumor blood vessels and alter the barriers that limit chemotherapeutic drug penetration in tumors (11) .

It has been reported that coadministration of TNF with IFN-{gamma}, a pleiotropic cytokine mainly produced by T lymphocytes and natural killer cells and in minor amounts by B lymphocytes, macrophages, and dendritic cells (22, 23, 24, 25, 26, 27) , potentiates its antitumor properties in a variety of murine tumors and human xenografts (28, 29, 30) . Moreover, combined treatment of endothelial or tumor cells with TNF and IFN-{gamma} results in synergistic cytotoxic effects (31, 32, 33) . These notions prompted us to investigate the role of endogenous and exogenous IFN-{gamma} on the antitumor activity of NGR-TNF in combination with doxorubicin. We have found that endogenous IFN-{gamma} is indeed critical for the NGR-TNF/doxorubicin synergistic activity and that administration of exogenous IFN-{gamma} can improve the antitumor properties of these drugs in mice with suboptimal production of IFN-{gamma}.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor Cell Lines and Materials.
Mouse B16F1 cells, derived from a spontaneous melanoma of C57BL6 mice (34) , were cultured as described previously (10 , 35) . TS/A cells from a BALB/c spontaneous mammary adenocarcinoma (36) were cultured in RPMI 1640, 10% fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, 2 mmol/L glutamine, and 1% MEM with nonessential amino acids (BioWhittaker Europe, Verviers, Belgium). Doxorubicin (Adriblastina) was purchased from Pharmacia-Upjohn (Milan, Italy). Recombinant murine IFN-{gamma} was purchased from PeproTech (London, United Kingdom; endotoxin content: <1 units/µg). Antimouse IFN-{gamma} monoclonal antibody AN18 (mAb AN18; ref. 37 ) was kindly provided by Paolo Dellabona (Milan, Italy). Biotinylated antimouse IFN-{gamma} mAb XMG1.2 was from BD PharMingen (San Diego, CA).

Preparation of Murine NGR-TNF.
Murine TNF and NGR-TNF (consisting of murine TNF fused with the COOH terminus of CNGRCG) was prepared by recombinant DNA technology and purified from Escherichia coli cell extracts as described previously (10) . All solutions used in the chromatographic steps were prepared with sterile and endotoxin-free water (Salf, Bergamo, Italy). Protein concentration was measured with a commercial protein quantification assay kit (Pierce, Rockford, IL). The cytolytic activity of NGR-TNF was 9.1 x 107 units/mg. The hydrodynamic volume of NGR-TNF was similar to that of TNF, a homotrimeric protein (38) , by gel filtration chromatography on a Superdex 75 high resolution column (Pharmacia, Uppsala, Sweden). Endotoxin content of NGR-TNF was 0.082 units/µg.

IFN-{gamma}–ELISA.
The amount of IFN-{gamma} present in cell supernatants was measured by ELISA as follows: polyvinyl chloride microtiter plates (Becton Dickinson, Oxnard, CA) were incubated with 5 µg/mL mAb AN18 in 0.15 mol/L sodium chloride, 0.05 mol/L sodium phosphate buffer (pH 7.3; assay buffer) for 16 hours at 4°C. The plates were washed three times with assay buffer and blocked with 2% BSA in assay buffer (1 hour at 37°C). The plates were then washed three times with assay buffer and incubated with cell culture supernatants or mouse IFN-{gamma} standard solutions, diluted in assay buffer–BSA. The plates were washed eight times with assay buffer containing 0.05% Tween 20 (Merck) and incubated with biotinylated mAb XMG1.2 (0.2 µg/mL in assay buffer–BSA, 1 hour at 37°C). Plates were washed again with assay buffer–0.05% Tween 20 and incubated for 1 hour at 37°C with streptavidin-horseradish peroxidase (Sigma Chemical Co., St. Louis, MO), diluted 1:3000 in assay buffer–BSA. After washing with assay buffer–0.05% Tween 20, bound peroxidase was detected using o-phenylenediamine dihydrochloride chromogenic substrate (Sigma Chemical Co.). The reaction was blocked after 30 minutes by adding 10% sulfuric acid. The A490 nm was measured with an ELISA microplate reader.

Serum levels of IFN-{gamma} was measured using a commercial ELISA kit (R&D Systems, Minneapolis, MN).

Transfection of TS/A Cells with IFN-{gamma} cDNA.
The cDNA coding for murine IFN-{gamma} was obtained by standard reverse transcription-PCR on total RNA purified from the splenocytes recovered from a C57BL/6 mice (Harlan, Udine, Italy). Before RNA extraction, splenocytes were stimulated for 20 hours with 10 µg/mL lipopolysaccharide diluted in RPMI (Euroclone, Milan, Italy) supplemented with 2 mmol/L glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin-B, and 10% fetal bovine serum. Reverse transcription-PCR was set up using the following primers: 5'-AGAATTCATGAACGCTACACACTGCATCTTGGC-3' (forward primer); and 5'-TATATTAAGCTTTCAGCAGCGACTCCTTTTCCGC-3' (reverse primer). Primers were designed to amplify the full-length IFN-{gamma} coding sequence, including the leader sequence. The amplified fragment was cloned into the mammalian expression vector pRS1-neo. Three micrograms of plasmid DNA, called pRS1neo-IFN-{gamma}, was mixed with 100 µL of 0.03 mg/mL Lipofectin Reagent (Life Technologies, Inc., Rockville, MD) in RPMI 1640 and incubated for 20 minutes at room temperature. Then, the mixture was added to TS/A cells plated 1 day before in 24-well microtiter plates (4 x 104 cells per well in 200 µL of culture medium). After incubation at 37°C, 5% CO2 (4 hours) 2 mL of culture medium were added to each well. Two days later, the culture medium was replaced with RPMI 1640 containing 10% fetal bovine serum, 2 mmol/L glutamine, and 1 mg/mL Geneticin. Cells surviving selection were cloned, 1 week later, by limiting dilution in 96-well microtiter plates in the presence of Geneticin. The supernatant of each clone was tested by IFN-{gamma}–ELISA. Ten IFN-{gamma}–secreting clones were obtained. One clone, named TS/A–IFN-{gamma}, able to produce > 1 µg/ml of IFN{gamma} in cell culture supernatants, was selected and used for in vivo experiments.

In vivo Studies.
Studies on animal models were approved by the Ethical Committee of the San Raffaele H Scientific Institute and performed according to the prescribed guidelines. C57BL6 mice and nude BALB/c (nu/nu) mice were purchased from Charles River Laboratories (Calco, Italy); IFN-{gamma}–BALB/c–knockout mice (IFN-{gamma}–/–) were kindly supplied by Fabio Benigni (Bioxell S.p.A., Milan, Italy). Mice were challenged with s.c. injection in the left flank of B16F1 cells (5 x 104) or TS/A cells (105) or TS/A–IFN-{gamma} cells (2 x 105); 5 or 9 days later, mice were treated with NGR-TNF and IFN-{gamma} solutions (100 µL) followed 2 hours later by administration of doxorubicin (100 µL). All drugs were administered i.p. The drugs were diluted with 0.9% sodium chloride, containing 100 µg/mL endotoxin-free human serum albumin (Farma-Biagini, Lucca, Italy), except for doxorubicin, which was diluted with 0.9% sodium chloride alone. Tumor growth was monitored daily by measuring the tumors with calipers as described previously (39) . Animals were sacrificed before the tumors reached 1.0 to 1.5 cm in diameter. Tumor sizes are shown as mean ± SE (five animals per group).

Detection of Doxorubicin in Tumors.
Nude mice bearing TS/A tumors (diameter, 0.5 to 1 cm) were treated (i.p.) with or without NGR-TNF (0.1 ng) and IFN-{gamma} (300 ng), followed 2 hours later by doxorubicin (500 µg, i.p.). After 2 hours, the animals were sacrificed, and the tumors were excised. Each tumor was weighed, disaggregated, resuspended in cold PBS, and filtered through 70-µm filters. The cells were resuspended with cold PBS (50 mL), centrifuged (1500 rpm, 10 minutes, 4°C), resuspended in cold PBS (2.5 g/mL tumor tissue) and mixed with freshly prepared PBS containing 8% formaldehyde (2.5 g/mL tissue). The cells were stored in the dark at 4°C overnight and then analyzed by fluorescence-activated cell sorting. The FACScan (Becton-Dickinson) was calibrated with cells recovered from untreated tumors. Each sample was then analyzed using the FL-3 filter and Cell Quest software.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Endogenous IFN-{gamma} Is Critical for NGR-TNF/Doxorubicin Therapeutic Activity.
We have shown previously that administration of 0.1 ng of murine NGR-TNF to B16F1 melanoma-bearing C57BL6-mice increases the antitumor activity of doxorubicin (11) . These drugs act synergistically because NGR-TNF alone is poorly active when used at low dosage (0.1 ng) in this model (11) . To investigate the role of endogenous IFN-{gamma} on this synergism, we evaluated, first, the effect of a neutralizing anti-IFN-{gamma} antibody (mAb AN18) on the antitumor activity of 0.1 ng of NGR-TNF in combination with 80 µg of doxorubicin against s.c. B16F1 melanomas in C57BL6-immunocompetent mice. As expected, the antitumor effect induced by the combination of these drugs was stronger than that obtained with doxorubicin alone (Fig. 1)Citation . However, when we administered 250 µg of mAb AN18 24 hours before NGR-TNF/doxorubicin, the response to the combined therapy was similar to that of doxorubicin alone. No inhibitory effect was observed with an irrelevant monoclonal antibody (9E10; data not shown). These results suggest that endogenous IFN-{gamma} is an important player in the overall control of tumor growth after therapy with these drugs.



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  		Fig. 1. Effect of a neutralizing anti-IFN-{gamma} mAb (AN18) on the antitumor activity of NGR-TNF and doxorubicin against B16/F1 melanoma tumors growing (s.c.) in immunocompetent C57BL6 mice. Animals were treated (i.p.) at day 5 after tumor implantation (arrow) with the indicated doses of NGR-TNF and, 2 hours later, with doxorubicin. mAb AN18 was administered at day 4. Tumor volume: mean ± SE (five mice per group). {triangledown} versus {bullet} (P < 0.05); {circ} versus {bullet} (P < 0.05; two-tailed t test, at day 11).

 
To demonstrate that IFN-{gamma} is indeed critical for the antitumor activity of NGR-TNF/doxorubicin, we evaluated the efficacy of these drugs in BALB/c IFN-{gamma}–knockout (IFN-{gamma}–/–) and wild-type (IFN-{gamma}+/+) mice. Because B16F1 melanoma cells are not syngeneic with BALB/c mice, this experiment was carried out with mice challenged with TS/A cells, a spontaneous BALB/c mammary adenocarcinoma (36) . Treatment with NGR-TNF (0.1 ng)/doxorubicin (40 µg) significantly reduced the tumor growth in IFN-{gamma}+/+ mice (Fig. 2Citation , black bars) but not in IFN-{gamma}–/– mice (white bars). Notably, administration of exogenous IFN-{gamma} (300 ng) to IFN-{gamma}–/– mice restored the synergy between NGR-TNF and doxorubicin (Fig. 2Citation , white bars). These results support the hypothesis that IFN-{gamma} is necessary for the NGR-TNF/doxorubicin synergistic activity. Of note, coadministration of IFN-{gamma} and doxorubicin without NGR-TNF did not induce significant antitumor effects in BALB/c IFN-{gamma}–/– mice (Fig. 2Citation , white bars), indicating that IFN-{gamma} acts synergistically with NGR-TNF and minimally or not at all with doxorubicin.



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  		Fig. 2. Antitumor activity of various combinations of IFN-{gamma}, NGR-TNF, and doxorubicin against TS/A mammary adenocarcinoma implanted (s.c.) in BALB/c mice (IFN{gamma}+/+) and in IFN-{gamma}–knockout mice (IFN{gamma}–/–). Animals (n = 5 per group) were treated (i.p.) at day 9 after tumor implantation with the indicated combinations of NGR-TNF (0.1 ng), IFN-{gamma} (300 ng), and doxorubicin (2 hours later; 40 µg). Tumor volume: mean ± SE (five mice per group). P < 0.05 (*), by two-tailed t test.

 
Role of T Cells and Locally Produced IFN-{gamma} in NGR-TNF/Doxorubicin Therapeutic Activity.
Activated T and natural killer cells are the primary source of IFN-{gamma} in immunocompetent mice (22 , 23) . To investigate the importance of T cells as a source of IFN-{gamma}, critical for the NGR-TNF/doxorubicin-combined therapy, we investigated the effect of these drugs against B16F1 and TS/A tumors implanted in nude mice lacking functional T cells. No synergism between NGR-TNF (0.1 ng) and doxorubicin was observed in the B16F1 model (Fig. 3A–C)Citation . However, when we administered these drugs in combination with exogenous IFN-{gamma}, the synergistic effect was restored (Fig. 3D)Citation . Similarly, no synergism between NGR-TNF and doxorubicin was observed in the TS/A model, even when mice were treated with 200 ng of NGR-TNF (Fig. 4Citation , white bars).



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  		Fig. 3. Antitumor activity of various combinations of IFN-{gamma}, NGR-TNF, and doxorubicin against B16F1 melanomas implanted (s.c.) in nude mice. Animals (n = 5 per group) were treated (i.p.) at day 5 after tumor implantation (arrows), with the indicated drug combinations and doses. IFN-{gamma} and NGR-TNF were administered at the same time, whereas doxorubicin was administered 2 hours later. Tumor area: mean ± SE (five mice per group). {permzspch197}, None; {permzspch154}, Doxorubicin (80 µg); {permzspch159}, IFN{gamma} (300 ng) + Doxorubicin (80 µg); {permzspch157}, NGR-TNF (0.1 ng) + Doxorubicin (80 µg); {permzspch158}, IFN{gamma} (300 ng) + NGR-TNF (0.1 ng) + Doxorubicin (80 µg).

 


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  		Fig. 4. Antitumor activity of various combinations and doses NGR-TNF and doxorubicin against TS/A ({square}) and TS/A–IFN-{gamma} ({blacksquare}) tumors implanted (s.c.) in nude mice. Animals (n = 5 per group) were treated (i.p.) at day 9 after tumor implantation, with the indicated drug combinations and doses. IFN-{gamma} and NGR-TNF were administered at the same time, whereas doxorubicin was administered 2 hours later. Tumor volume: mean ± SE (five mice per group); P < 0.05 (*) and P < 0.01 (**), by two-tailed t test.

 
To assess whether local production of endogenous IFN-{gamma} within the tumor microenvironment is sufficient to induce the synergism between NGR-TNF and doxorubicin in nude mice, we transfected TS/A cells with murine IFN-{gamma} cDNA. One clone able to secrete IFN-{gamma} in culture medium was selected and named TS/A–IFN-{gamma}. TS/A–IFN-{gamma} cells were then implanted, s.c., in nude mice. As expected, the combination of 50 µg of doxorubicin with 0.1 or 200 ng of NGR-TNF exerted significant antitumor effects against TSA–IFN-{gamma} (Fig. 4Citation , black bars). These results are in line with the hypothesis that local production of IFN-{gamma}, likely by T cells in immunocompetent mice, is critical for the NGR-TNF/doxorubicin synergism.

Mechanism of Action of the Triple Combination (IFN-{gamma}, NGR-TNF, and Doxorubicin).
We have shown previously that an important mechanism for the NGR-TNF/doxorubicin synergism is related to alteration of endothelial barrier function by NGR-TNF and to increased penetration of doxorubicin in tumors (11) . Thus, we investigated whether T cells and IFN-{gamma} are critical for this effect. To this aim, we measured the penetration of doxorubicin in TS/A tumors, implanted s.c. in nude mice, by measuring the fluorescence intensity of tumor cells recovered from animals 2 hours after treatment. This experiment takes advantage from the fact that doxorubicin is a fluorescent compound that remains bound to cells after fixation with formaldehyde (11) . Pretreatment of tumor-bearing nude mice with NGR-TNF did not increase doxorubicin uptake by tumor cells (Table 1)Citation . The lack of activity of NGR-TNF in this model suggests that T cells are important for NGR-TNF–induced barrier alteration. When we added exogenous IFN-{gamma} to the treatment we observed a 2-fold increase of doxorubicin uptake by tumor cells (Table 1)Citation . Statistical analysis of data showed that the difference between the groups treated with IFN-{gamma}/NGR-TNF/doxorubicin or doxorubicin alone did not reach statistical significance (P = 0.08). However, a statistically significant difference (P = 0.03) was observed when the data of animal treated without IFN-{gamma} were cumulated (n = 10; Table 1Citation ). This suggests that IFN-{gamma} can indeed affect doxorubicin penetration in tumors.


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  		Table 1 Effect of NGR-TNF and IFN-{gamma} on doxorubicin uptake by TS/A tumor cells in nude mice

 
Effect of IFN-{gamma} on the Therapeutic Activity of NGR-TNF/Doxorubicin in Immunocompetent Mice.
The therapeutic activity of NGR-TNF and doxorubicin, alone or in combination with exogenous IFN-{gamma}, was then evaluated in B16F1 and TS/A tumor-bearing mice (C57BL6 and BALB/c, respectively). The combination of NGR-TNF and doxorubicin induced significant (P < 0.05) antitumor effects in the B16F1 model, even in the absence of exogenous IFN-{gamma} (Fig. 5A)Citation . Addition of exogenous IFN-{gamma} to the treatment with NGR-TNF and doxorubicin induced a modest (nonsignificant) increase in response. Similar results were obtained in the TS/A (BALB/c) model (Fig. 5B)Citation . These findings suggest that endogenous IFN-{gamma} in these models is sufficient to act synergistically with NGR-TNF/doxorubicin and that addition of exogenous IFN-{gamma} can only induce modest increase in the overall antitumor effects.



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  		Fig. 5. Antitumor activity of various combinations of IFN-{gamma}, NGR-TNF, and doxorubicin against B16F1 and TS/A tumors implanted (s.c.) in immunocompetent C57BL6 (A) and BALB/c (B) mice. Animals (n = 5 per group) were treated (i.p.) at day 5 or day 12 after tumor implantation (arrows), with the indicated drug combinations and doses. A. NGR-TNF and doxorubicin were administered 2 and 4 hours after IFN-{gamma}, respectively; B. IFN-{gamma} and NGR-TNF were administered at the same time, whereas doxorubicin was administered 2 hours later. Tumor volume and animal weight: mean ± SE (five mice per group). A, {triangledown} versus {bullet} (P < 0.05); {triangledown} versus {blacksquare} (P < 0.005); {bullet} versus {blacksquare} (non significant; two-tailed t test at day 13).

 
Endogenous IFN-{gamma} was then measured by ELISA in the serum of B16F1 tumor-bearing mice 24 hours after treatment with NGR-TNF/doxorubicin. The circulating levels of IFN-{gamma} were below the detection limit of our assay (<20 pg/mL) in both treated and untreated mice. On the basis of these data, it is difficult to speculate whether local production of IFN-{gamma} was induced by NGR-TNF/doxorubicin or preceded treatment.

Role of the NGR-Targeting Domain of NGR-TNF.
To investigate whether the NGR-targeting domain is critical for the antitumor effects of low doses of NGR-TNF and doxorubicin and for the synergism with IFN-{gamma}, we treated TS/A tumor-bearing mice (BALB/c) with NGR-TNF/doxorubicin or with TNF/doxorubicin at days 5, 12, and 15. Significant antitumor effects were induced by NGR-TNF/doxorubicin but not by TNF/doxorubicin (Fig. 6)Citation . No effect was observed with TNF/doxorubicin, even when IFN-{gamma} was added to the treatment on days 19 and 25. These results suggest that the targeting mechanism is critical for the synergism with endogenous IFN-{gamma}, at least when very low doses of TNF are used.



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  		Fig. 6. Antitumor activity of TNF and NGR-TNF in combination with doxorubicin against TS/A tumors implanted (s.c.) in immunocompetent BALB/c mice. Animals (n = 5 per group) were treated (i.p.) at days 5, 12, and 15 after tumor implantation (arrows), with the indicated drug combinations and doses. At day 19 and 25, IFN-{gamma} (300 ng) was added to the treatment with TNF/doxorubicin and with NGR-TNF/doxorubicin. {triangledown} versus {bullet} (P < 0.01; two-tailed t test at day 26). {permzspch197}, None; —{diamond}—, TNF (0.3 ng) + Doxorubicin (40 µg); {permzspch157}, NGR-TNF (0.3 ng) + Doxorubicin (40 µg).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study suggest that endogenous IFN-{gamma} is a critical component in the synergistic antitumor activity of NGR-TNF and doxorubicin. This view is supported by the observation that (a) no synergism occurs when these drugs are coadministered with a neutralizing anti-IFN-{gamma} antibody to tumor-bearing mice or when they are administered to mice lacking the IFN-{gamma} gene, (b) coadministration of IFN-{gamma} with NGR-TNF and doxorubicin induces significant antitumor responses in IFN-{gamma}–knockout mice, (c) no antitumor effects are induced by NGR-TNF/doxorubicin in nude mice, except when these drugs are coadministered with exogenous IFN-{gamma}, (d) endogenous production by tumor cells transfected with IFN-{gamma} cDNA restores the synergistic activity also in nude mice. Collectively, these findings point to a crucial role for locally produced IFN-{gamma} in tumor vascular targeting with NGR-TNF and doxorubicin.

Considering that activated T and natural killer cells are the primary sources of IFN-{gamma} in immunocompetent mice and that athymic nude mice lack functionally mature T cells but not natural killer cells (22 , 23 , 40) , the results obtained in nude mice suggest that T lymphocytes play a relevant role. Interestingly, previous studies showed that also nontargeted TNF, alone or in combination with melphalan, does not induce antitumor effects in nude mice bearing human melanoma xenografts (41) . This suggests that the requirement of a functional immune system, probably necessary for the production of IFN-{gamma}, is not a peculiarity of targeted TNF and doxorubicin.

The finding that coadministration of doxorubicin with IFN-{gamma} alone does not induce significant antitumor response in nude or IFN-{gamma}–knockout mice indicates that IFN-{gamma} synergizes minimally or not at all with doxorubicin and that both IFN-{gamma} and NGR-TNF are necessary to produce the synergistic effects.

In previous work, we showed that one mechanism underlying the synergism between 0.1 ng of NGR-TNF and doxorubicin, in immunocompetent mice, is related to vascular barrier alteration and increased penetration of this chemotherapeutic drug in tumors (11) . Thus, one attractive hypothesis is that endogenous IFN-{gamma} is a critical cofactor for these effects on endothelial cells and vascular leakage. The importance of IFN-{gamma} in TNF-induced reduction of endothelial cell-cell/cell-extracellular matrix adhesion and barrier alteration has been previously documented by other investigators using different experimental models (42, 43, 44) . For instance, experiments carried out with the rat mesentery microvascular networks showed that perfusion with TNF in combination with IFN-{gamma}, but not with TNF alone, increases the number of sites of albumin leakage in postcapillary venules (42) . In this model, increased permeability was dose dependent and correlated with focal loss of cadherin-5 intercellular adhesion (42) . Another work showed that the paracellular transport of FITC-dextran through endothelial cell monolayers is increased by IFN-{gamma}/TNF mixtures but little or not at all by TNF alone (43) . It would appear, therefore, that the presence of IFN-{gamma} is critical for efficient alteration of endothelial barrier by TNF. This may suggest that IFN-{gamma} is also critical for NGR-TNF–induced penetration of doxorubicin in tumors. In line with this hypothesis, we have found that pretreatment of nude mice with NGR-TNF (0.1 ng) in combination with IFN-{gamma} induced a 2-fold increase of doxorubicin penetration in tumors, whereas NGR-TNF alone was not effective. This finding could offer an explanation for the poor activity of NGR-TNF/doxorubicin in nude mice and for the increased activity when combined with IFN-{gamma}. However, many other mechanisms could contribute to the synergism of the IFN-{gamma}/NGR-TNF/doxorubicin combination considering that TNF and IFN-{gamma} can exert many other effects on both endothelial and tumor cells. For instance, TNF can induce on endothelial cells leukocyte adhesion molecules (45 , 46) , proinflammatory cytokines (47 , 48) , fibrin deposition (13 , 49) , nitric oxide production (50 , 51) , and apoptosis (31) . On the other hand, IFN-{gamma} can induce MHC I/II and intercellular adhesion molecule-1 expression on endothelial cells and cytokine and chemokine secretion (52) . IFN-{gamma} can also inhibit tumor angiogenesis, activate macrophages to nonspecifically kill tumor cell targets, and induce antiproliferative and proapoptotic effects on many tumor cell types (52) . Remarkably, at least some of these biological responses are triggered by TNF and IFN-{gamma} in a synergistic manner. For instance, it has been shown that treatment of endothelial cells with a combination of TNF and IFN-{gamma} results in reduced activation of {alpha}vß3 integrin and, consequently, in detachment and apoptosis of angiogenic endothelium (31) . Moreover, synergistic cytotoxic effects have been observed after treatment of transformed cell lines with TNF and IFN-{gamma} (32 , 33) . It is likely that the pleiotropic and synergistic activity of these cytokines could lead not only to increased vascular leakage and drug penetration in tumors but also to apoptosis of endothelial and tumor cells and to activation of an inflammatory-immune response. All of these mechanisms are not mutually exclusive and could contribute to the antitumor effects observed with the triple combination.

Our findings may also have other important implications. Several studies in animal models and in patients showed that TNF can selectively affect and damage tumor vessels but not vessels associated with normal tissues. Accordingly, the vasculature of tumors, but not of normal tissues, was extensively damaged after patients were given isolated limb perfusion with high doses of TNF in combination with IFN-{gamma} and melphalan (5) . The molecular basis of this selectivity is unclear. It has been hypothesized that structural differences within tumor vessels and/or the presence of tumor-derived sensitizing factors could be responsible for the TNF vascular selectivity (1) . Our results suggest that local production of IFN-{gamma} could be one of these sensitizing factors.

Finally, it has been reported that addition of IFN-{gamma} to isolated limb perfusion with TNF and melphalan can enhance the overall response rate of patients with melanoma from 91 to 100% (53) . Small difference (not statistically significant) in complete response rate was observed also in the BN175 rat sarcoma limb perfusion model (54 , 55) . Similarly, we have found that addition of IFN-{gamma} to the treatment of immunocompetent mice with low doses of NGR-TNF and doxorubicin can only induce modest (nonsignificant) increases in the antitumor response. This suggests that in the isolated limb perfusion setting and in our models, endogenous IFN-{gamma} is expressed in a sufficient amount to act synergistically with TNF and chemotherapy, even in the absence of exogenous IFN-{gamma}. However, the results of our study also suggest that when endogenous IFN-{gamma} is not expressed in sufficient amounts, the response rate to targeted TNF could be significantly improved by exogenous IFN-{gamma}. This is an interesting possibility that deserves to be investigated.


   ACKNOWLEDGMENTS
 
We thank Cristina Longoni for TS/A cell transfection and Anna Mondino for helpful discussion.


   FOOTNOTES
 
Grant support: Associazione Italiana per la Ricerca sul Cancro.

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.

Note: A. Sacchi and A. Gasparri contributed equally to this work.

Requests for reprints: Angelo Corti, Department of Biological and Technological Research, San Raffaele H Scientific Institute, via Olgettina 58, 20132 Milan, Italy. Phone: 39-02-26434802; Fax: 39-02-26434786; E-mail: corti.angelo{at}hsr.it

Received 4/23/04. Revised 7/ 1/04. Accepted 7/27/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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