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Cancer Research 68, 1154, February 15, 2008. doi: 10.1158/0008-5472.CAN-07-2085
© 2008 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Synergistic Damage of Tumor Vessels with Ultra Low-Dose Endothelial-Monocyte Activating Polypeptide-II and Neovasculature-Targeted Tumor Necrosis Factor-{alpha}

Luca Crippa, Anna Gasparri, Angelina Sacchi, Elisabetta Ferrero, Flavio Curnis and Angelo Corti

Department of Oncology, Cancer Immunotherapy-Gene Therapy Program and IIT Network Research Unit of Molecular Neuroscience, San Raffaele Scientific Institute, Milan, Italy

Requests for reprints: Angelo Corti, DIBIT-Department of Oncology, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milan, Italy. Phone: 39-2-2643-4802; Fax: 39-2-2643-4786; E-mail: corti.angelo{at}hsr.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
High-dose endothelial-monocyte activating polypeptide II (EMAP-II), a tumor-derived antiangiogenic cytokine, can sensitize tumor vasculature to the damaging activity of high-dose tumor necrosis factor (TNF)-{alpha}. However, this combination cannot be used for systemic treatment of patients because of prohibitive toxicity. We have found that this limitation can be overcome by combining a TNF-targeting strategy with the use of ultra low-dose EMAP-II. Coadministration of 0.1 ng of EMAP-II and 0.1 ng of CNGRCG-TNF (NGR-TNF), a peptide-TNF conjugate able to target tumor blood vessels, inhibited lymphoma and melanoma growth in mice, with no evidence of toxicity. This drug combination induced endothelial cell apoptosis in vivo and, at later time points, caused reduction of vessel density and massive apoptosis of tumor cells. Ligand-directed targeting of TNF was critical because the combination of nontargeted TNF with EMAP-II was inactive in these murine models. The synergism was progressively lost when the dose of EMAP-II was increased in the nanogram to microgram range, supporting the concept that the use of low-dose EMAP-II is critical. Studies on the mechanism of this paradoxical behavior showed that EMAP-II doses >1 ng induce the release of soluble TNF receptor 1 in circulation, a strong counter-regulatory inhibitor of TNF. Tumor vascular targeting with extremely low amounts of these cytokines may represent a new strategy for cancer treatment. [Cancer Res 2008;68(4):1154–61]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor cell survival, proliferation, and invasion critically depend on the presence of functional blood vessels in the tumor stroma (1, 2). The use of tumor vascular targeting agents and antiangiogenic molecules capable of damaging existing vessels and inhibiting the formation of new vessels is a promising approach for cancer treatment (1, 3). Among the vascular targeting agents identified thus far, tumor necrosis factor-{alpha} (TNF) is undoubtedly one of the most efficient, due its strong ability to cause selective obliteration and damage of tumor neovasculature (4, 5). For these reasons and for its capability to activate inflammatory-immune mechanisms, this cytokine is currently used in patients for locoregional treatment of tumors of the extremities (6–9). Unfortunately, the clinical use of TNF is limited to locoregional treatments because of prohibitive systemic toxicity.

Recent studies have shown that soluble factors produced by tumors can contribute to sensitize tumor blood vessels to the vascular damaging and procoagulant activity of TNF. For instance, endothelial-monocyte activating polypeptide II (EMAP-II), a soluble tumor-derived cytokine originally discovered in the supernatant of methylcholanthrene A–induced murine fibrosarcoma cells, can alter endothelial functions and sensitize tumor neovasculature to TNF (3, 10–13). Interestingly, complete responses in melanoma patients treated with TNF, by isolated limb perfusion, correlate with increased EMAP-II expression in biopsies (14), suggesting that local production of EMAP-II is critical for the antitumor activity of TNF. Accordingly, transfection of TNF-resistant melanomas with the EMAP-II gene rendered tumors sensitive to systemic TNF therapy (12, 15), and local injection of high-dose EMAP-II (10 µg) into murine mammary adenocarcinomas increased tumor sensitivity to subsequent systemic administration of high dose-TNF, causing local acute thrombohemorrhage (11). Studies on the mechanism of action have shown that EMAP-II can up-regulate the expression of tissue factor, von Willebrand factor, E-selectin, P-selectin, and p55-TNF receptor 1 (TNF-R1) mRNA in tumor vascular endothelial cells (11, 16). EMAP-II can also inhibit endothelial cell adhesion to fibronectin, promote endothelial cell apoptosis, and exert antiangiogenic effects (17, 18). Furthermore, this cytokine can activate monocyte/macrophage and granulocyte chemotaxis and induce the release of TNF and other cytokines by monocytes (11).

The remarkable synergistic effects of EMAP-II and TNF observed in animal models have continued to nourish hopes with regard to the possibility of exploiting the powerful antivascular effects of TNF for systemic treatment of cancer patients. Unfortunately, also the systemic administration of high-dose EMAP-II/TNF induces toxic reactions and the efficacy of nontoxic doses of this combination is very low.

In the attempt to overcome this limitation, we have investigated in murine models the antitumor properties of EMAP-II in combination with low doses of NGR-TNF, a TNF derivative currently tested in phase I and II clinical studies. NGR-TNF consists of TNF fused with the COOH terminus of the CNGRCG peptide, a ligand of aminopeptidase N (CD13) expressed by endothelial cells in tumor vessels. Previous studies showed that targeted delivery of low doses of NGR-TNF to the tumor vasculature is a valuable strategy for overcoming major TNF counter-regulatory mechanisms and for increasing the penetration of chemotherapeutic drugs in tumors (19–21). We have unexpectedly found that EMAP-II completely suppresses, rather than increases, the antitumor properties of low-dose NGR-TNF, either alone or in combination with chemotherapy. However, we have also found that administration of low doses of EMAP-II, various orders of magnitude lower than the dose currently used in animal models, induces strong synergistic antitumor effects with NGR-TNF, leading to marked apoptosis of tumor cells and inhibition of tumor growth, with no evidence of toxicity. We also provide evidence to suggest that the mechanism underlying this paradoxical behavior is related to dose-dependent activation of TNF counter-regulatory mechanisms by EMAP-II, such as soluble TNF receptor shedding.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents. Human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical cord vein by collagenase treatment as described (22), following ethical approval, and cultured in 1% gelatin–coated flasks (Falcon, Becton Dickinson) using endotoxin-free Medium 199 (BioWhittaker, Cambrex Bio Science Verviers) containing 20% heat-inactivated fetal bovine serum (Hyclone), 1% bovine retinal-derived growth factor, 90 µg/mL heparin, 100 IU/mL penicillin, and 100 µg/mL streptomycin (Biochrom; M199 complete medium). All experiments were carried out with HUVEC at passages 1 to 4. Human microvasculature endothelial cells, HMEC-1, were provided by Dr. A. Manfredi (San Raffaele Scientific Institute, Milan, Italy). Mouse B16F1 melanoma cells and RMA lymphoma cells were cultured as previously described (19, 23).

4',6-Diamidino-2-phenylindole (DAPI) was from Invitrogen and normal goat serum was from Vector Laboratories. Affinity-purified rabbit polyclonal anti–EMAP-II antibody was from Sigma-Aldrich; rat anti-mouse CD31 monoclonal antibody (mAb) MEC 13.3 was from BD PharMingen; rabbit anti–cleaved caspase-3 polyclonal antibody, purified by protein A and peptide affinity chromatography, was from Cell Signaling Technology. AlexaFluor 488 donkey anti-rabbit IgG (H + L) and AlexaFluor 546 goat anti-rat IgG (H + L) were from Invitrogen.

Preparation of NGR-TNF and murine EMAP-II. Murine and human NGR-TNF (consisting of murine or human TNF fused with the COOH terminus of CNGRCG) were prepared by recombinant DNA technology and purified from E. coli cell extracts as previously described (19). Mature EMAP-II was prepared as follows: the cDNA coding for murine EMAP-II (residues 145–310) was amplified by PCR on the cDNA obtained by reverse transcriptase-PCR on total RNA of NIH-3T3 fibroblasts using the following primers: 5'-CACCATGTCCAAGCCTATCGACGCATC-3' (forward primer) and 5'-TCATTTAATTCCACTATTGGCCATGG-3' (reverse primer). The forward primer, including the CACC sequence, was designed to enable directional cloning of the EMAP-II gene into the pET101/D-TOPO vector of the Champion pET Directional TOPO Expression Kit (Invitrogen). E. coli BL21(DE3) cells were transformed with this plasmid and left to grow overnight in 10 mL of Luria-Bertani/Miller broth containing 100 µg/mL ampicillin. The culture was diluted 1:50 with fresh medium and left to grow at 37°C until the absorbance at 600 nm reached 0.7 units. Isopropyl-D-thiogalactopyranoside (1 mmol/L) was then added to the culture to induce EMAP-II expression. After 3 h at 37°C, cells were harvested by centrifugation (4,000 x g, 20 min, 4°C) and resuspended in 20 mmol/L Tris-HCl buffer (pH 7.4) containing 1 mmol/L benzamidine, 2 mmol/L EDTA, 0.02% sodium azide, 0.075% octyl-β-D-glucoside, and 4 units/mL benzonase. The resuspended material was sonicated and dialyzed overnight against 20 mmol/L Tris-HCl (pH 7.4) containing 0.075% octyl-β-glucoside. The product was filtered through a 0.45-µm filter (Nalgene), loaded onto an FPLC Mono Q HR 5/5 column (Pharmacia), and eluted with sodium chloride (gradient 0–1.0 mol/L). Fractions were analyzed by SDS-PAGE. The flow-through fraction, containing EMAP-II, was loaded onto a Source 15 reverse-phase column (Pharmacia) and eluted with acetonitrile (gradient 5–95%) containing 0.1% trifluoroacetic acid. Fractions containing EMAP-II were neutralized by adding 5 N sodium hydroxide, pooled, and partially dried with a centrifugal vacuum concentrator. The product was then loaded onto a Shodex gel-filtration column (Phenomenex) and eluted with 20 mmol/L Tris-HCl (pH 7.4) containing 0.075% octyl-β-glucoside. Fractions containing EMAP-II were pooled and filtered through a 0.22-µm filter (Nalgene). The final product was aliquoted and stored at –80°C. All solutions used in the chromatographic steps were prepared with sterile and endotoxin-free water (SALF Laboratorio Farmacologico SpA). Protein concentration was measured with the BCA Protein Assay Reagent (Pierce Chemical Co.). The endotoxin content of EMAP-II, measured using the quantitative chromogenic Limulus amoebocyte lysate test (BioWhittaker, Inc.), was 0.002 units/µg.

In vivo studies. Studies on animal models were approved by the Ethical Committee of the San Raffaele Scientific Institute and done according to the prescribed guidelines.

C57BL6/N or C57BL6/J mice (Charles River Laboratories) weighing 18 to 20 g were challenged with s.c. injection in the left flank of 7 x 104 RMA wt cells or 200 x 104 B16F1 cells, respectively; 9 to 11 days later, mice were treated with EMAP-II or NGR-TNF solutions, alone or in combination, at various doses. All drugs were diluted with 0.9% sodium chloride containing 100 µg/mL endotoxin-free human serum albumin (Farma-Biagini SpA) and administered i.p. Tumor growth was monitored daily by measuring the tumors with calipers. Animals were sacrificed before the tumors reached 1.5 cm in diameter. Tumor sizes are shown as mean ± SE (five animals per group).

Detection of apoptotic cells by immunofluorescence. Cell apoptosis in tumor tissue sections was analyzed by immunofluorescence as follows: 16 days after tumor cell implantation, tumor-bearing mice were treated i.p. with EMAP-II and NGR-TNF, alone or in combination. Two, eight, or twenty-four hours later, mice were sacrificed and tumors were embedded in Killik frozen section medium (Bio-Optica) for quick freezing. Cryostatic sections, 6 µm thick, were prepared, adsorbed on polylysine-coated slides, fixed for 30 min with PBS containing 4% paraformaldehyde, and frozen at –80°C. Detection of apoptotic and endothelial cells was done as follows: tissue sections were incubated with 150 µL of PBS containing 1% bovine serum albumin, 0.1% Triton X-100 (PBS-BT), and 5% normal goat serum for 1 h at room temperature. The solution was then removed and replaced with PBS-BT containing anti-CD31 mAb MEC-13.3 (1:100), anti–cleaved caspase-3 polyclonal antibody (1:100), and 2% normal goat serum. After incubation for 1 h at room temperature, each tissue section was rinsed for 15 min with PBS containing 0.1% Triton X-100 and further incubated for 1 h with AlexaFluor 488 donkey anti-rabbit IgG (H + L) antibody and AlexaFluor 546 goat anti-rat IgG (H + L) antibody (Invitrogen), both diluted 1:500 in PBS-BT. The slides were rinsed again and incubated for 5 min with PBS containing 0.1 µg/mL DAPI (Sigma) to stain cell nuclei. Sections were analyzed with an Axioplan2 fluorescence microscope (Zeiss, equipped with 40x/0.75 and 63x/1.4 objective lenses and AxioVision acquisition software).

Three sections per tumor (obtained from three different regions) were analyzed. The quantification of apoptotic tumor cells in each section was done as follows: a score ranging from 0 to 4 was given to 10 fields (random) of each section, depending on the percentage of apoptotic tumor cells: 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%).

Similarly, quantification of apoptotic endothelial cells was done on three sections per tumor and by inspecting the entire sections; a score ranging from 0 to 4 was given to each section depending on the number of vessels containing apoptotic endothelial cells (CD31/caspase-3 double positive): 0 (0 vessels), 1 (1–10 vessels), 2 (11–20 vessels), 3 (21–30 vessels), and 4 (>30 vessels).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production and characterization of murine recombinant EMAP-II. Mature murine EMAP-II is a 166-residue-long protein derived from a 310-residue precursor (proEMAP; ref. 11). Recombinant murine EMAP-II was produced by expressing the cDNA coding for residues 145 to 310 of proEMAP in E. coli cells and purified from cell extracts by ion-exchange, reverse-phase, and gel-filtration chromatography. The final product was homogeneous by analytical gel-filtration chromatography, reverse-phase HPLC, SDS-PAGE under reducing and nonreducing conditions, and Western blot analysis with an anti–EMAP-II antibody, the latter showing single bands of ~18 kDa (Supplementary Fig. S1A–D). The molecular mass of the purified product was 17,988.5 Da by electrospray mass spectrometry. This value corresponds very well to the mass expected for mature murine EMAP-II (17,989.94 Da).

Low-dose, but not high-dose, EMAP-II increases the antitumor activity of NGR-TNF in murine lymphoma and melanoma models. We examined, first, the antitumor activity of EMAP-II and NGR-TNF, alone and in combination, against murine RMA lymphomas transplanted in immunocompetent C57BL6 syngeneic mice. A single administration of EMAP-II or NGR-TNF (0.1 ng, i.p., alone) induced a modest delay in RMA tumor growth (Fig. 1A, left ). No antitumor effects were observed when the dose of EMAP-II was increased to 5,000 ng (Fig. 1B, middle). When mice were treated with a combination of EMAP-II (0.01 or 0.1 ng) and NGR-TNF (0.1 ng, 0.5 h later), we observed significant antitumor effects, pointing to synergistic mechanisms (Fig. 1A, middle and right). The synergism was progressively lost when the dose of EMAP-II was increased to 1, 10, 100, and 1,000 ng, the latter dose being completely inactive (Fig. 1A, right). The plot of tumor volume versus dose of EMAP-II showed a bell-shaped dose-response curve, maximal antitumor effects being achieved with 0.1 ng of EMAP-II in combination with 0.1 ng of NGR-TNF (Fig. 1C). Thus, EMAP-II and NGR-TNF exerted synergistic effects only when very low doses of both cytokines were used. No synergistic effects were observed when EMAP-II was digested with trypsin (data not shown). Furthermore, no loss of body weight was observed after administration of EMAP-II/NGR-TNF, suggesting that anticancer effects were induced without causing major toxicity (not shown).


Figure 1
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  		Figure 1. Low-dose, but not high-dose, EMAP-II increases the antitumor activity of NGR-TNF against RMA lymphoma tumors. A to D, C57BL6/N mice (n = 5 per group) were treated i.p. at day 9, 10, or 11 after RMA tumor implantation (arrows) with the indicated combinations of drugs. TNF and NGR-TNF were administered 0.5, 2, or 16 h after EMAP-II, as indicated. C, tumor volume before and 7 d after treatment with different doses of EMAP-II followed 0.5 h later by 0.1 ng of NGR-TNF. Columns, mean tumor volume; bars, SE. A, right, P < 0.01, open circle versus reversed triangle (two-tailed t test) at day 19.

 
In the attempt to optimize the administration schedule, we treated mice with both cytokines with intervals of 0.5, 2, and 16 h between drug administrations. Optimal effects were obtained when NGR-TNF was administered 0.5 h after low-dose EMAP-II (Fig. 1B, left) or when the two drugs were given together (not shown). In contrast, lack of synergism was observed when NGR-TNF was given 16 h after low-dose or high-dose EMAP-II (Fig. 1B, left and middle). Noteworthy, in the latter case, we observed complete inhibition of NGR-TNF effects (Fig. 1B, middle). This pretreatment inhibited also the NGR-TNF/cisplatin synergism (Fig. 1B, right), further supporting the concept that high-dose EMAP-II inhibits, rather than potentiates, NGR-TNF.

To investigate the role of the NGR domain in the "low-dose EMAP-II/NGR-TNF" synergism, we carried out similar studies using EMAP-II in combination with nontargeted TNF. In this case, no antitumor effects were observed (Fig. 1D), suggesting that the NGR domain is crucial for activity, likely for targeting TNF to the tumor vasculature.

Similar experiments were done using the syngeneic B16F1 melanoma model. Also in this case, high-dose EMAP-II (1,000 ng) was unable to potentiate the antitumor activity of NGR-TNF against B16F1 melanomas. In contrast, 40% to 50% reduction of tumor growth rate was observed when low-dose EMAP-II (0.1 ng) was given before NGR-TNF (not shown). Immunohistochemical analysis of several tumor tissue sections with anti–cleaved caspase-3 antibodies showed marked increase of tumor cell apoptosis 24 h after treatment (Fig. 2A and B ). Similar effects on tumor cell apoptosis were observed also with the RMA model (not shown).


Figure 2
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  		Figure 2. Low-dose EMAP-II in combination with low-dose NGR-TNF induces B16F1 tumor cell apoptosis in vivo. A and B, effect of treatment with NGR-TNF and EMAP-II on tumor cell apoptosis. Tumor-bearing mice (n = 3 per group) were treated at day 15 with EMAP-II (0.1 ng) or NGR-TNF (0.1 ng; administered 0.5 h later), alone or in combination. The number of apoptotic cells was analyzed 24 h later in tumor tissue sections (3 tumors, 3 sections per tumor, 10 fields per section, total of 90 fields per treatment) after immunostaining of cleaved caspase-3 (green); cell nuclei were stained with DAPI (blue); a score ranging from 0 to 4 was given to 10 random fields of each section, depending on the amount of apoptotic cells (green; see Materials and Methods). A, columns, mean apoptotic score (90 fields per treatment); bars, SE. ***, P < 0.0001, versus controls (two-tailed t test). B, microscopy photographs showing CD31 (red) and activated caspase-3 (green) in tumor sections 24 h after treatment. Bar, 20 µm.

 
Low-dose EMAP-II/NGR-TNF combination induces endothelial cell apoptosis and decreases tumor vascular density. The mechanism of action underlying the massive apoptosis of tumor cells after treatment with low-dose EMAP-II/NGR-TNF was then investigated. Cytotoxicity assays with cultured RMA cells showed that this cell line is resistant in vitro to EMAP-II/NGR-TNF treatment over a wide range of concentrations and ratios (Supplementary Fig. S2A). Similar results were obtained also with B16F1 melanoma cell cultures (Supplementary Fig. S2B), suggesting that the in vivo apoptotic effect was indirect. To assess the original hypothesis of vascular damage as a primary antitumor mechanism, we measured the number of apoptotic endothelial cells in RMA tumors by immunofluorescence microscopy with anti-CD31 (an endothelial cell marker) and anti–activated caspase-3 (a marker of apoptosis) antibodies. We observed a significant increase of apoptotic endothelial cells 8 h after treatment with EMAP-II/NGR-TNF (low-dose) and, apparently, a decrease after 24 h (Fig. 3A, top ). At variance, the number of apoptotic tumor cells (CD31 negative/caspase-3 positive cells) was maximal after 24 h [Fig. 3A (bottom) and B], suggesting that endothelial cell apoptosis preceded tumor cell apoptosis. Of note, control treatments with cytokines alone showed that NGR-TNF, but not EMAP-II, could induce endothelial cell apoptosis, although to a lower extent compared with the combined treatment, at least after 8 h (Fig. 3A and B). The lower number of double-positive cells observed 24 h after the treatment with the drug combination, compared with NGR-TNF alone, likely reflected a marked loss of CD31 in late apoptotic cells, due to stronger antivascular effects. A significant reduction of CD31-positive vessels, 24 h after treatment with EMAP-II/NGR-TNF, was observed also in the B16F1 melanoma model, compared with single agents (Fig. 4 ). These results strongly suggest that the combined treatment led to reduction of vascular density more efficiently than single agents and support the original hypothesis of vascular targeting and damage.


Figure 3
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  		Figure 3. Low-dose EMAP-II/NGR-TNF combination induces endothelial cell apoptosis in tumors, followed by tumor cell apoptosis. A and B, C57BL6/N mice bearing RMA tumors (n = 3 per group) were treated at day 16 after tumor implantation with EMAP-II (0.1 ng) or NGR-TNF (0.1 ng, 0.5 h later), alone or in combination. Tumors were excised 2, 8, or 24 h later and tumor sections were immunostained with anti-CD31 mAb (red) and anti–cleaved caspase-3 polyclonal antibody (green) to identify endothelial apoptotic cells (CD31/caspase-3 double-positive cells) and tumor apoptotic cells (CD31-negative/caspase-3–positive cells); cell nuclei were stained with DAPI (blue). A, columns, mean of endothelial apoptotic cells (top) and tumor apoptotic cells (bottom) observed in 3 tumors, 3 sections per tumor, 10 fields per section (total of 90 fields per treatment; see Methods for protocol and score determination); bars, SE. *, P < 0.01; **, P < 0.001; ***, P < 0.0001, versus controls (two-tailed t test). B, microscopy photographs of tissue sections, taken 8 or 24 h after treatment, as indicated. Arrows, apoptotic endothelial cells. Bar, 20 µm.

 

Figure 4
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  		Figure 4. Low-dose EMAP-II/NGR-TNF combination decreases vascular density in B16F1 tumors. A, C57BL/J mice bearing B16F1 tumors (n = 3/group) were treated at day 15 with EMAP-II (0.1 ng) or NGR-TNF (0.1 ng, 0.5 h later), alone or in combination. Vessel density was evaluated 24 h later in tumor tissue sections (3 slices per tumor) after immunostaining with anti-CD31 antibody. The number of vessels (CD31-positive) was counted in 10 random fields of each section. Columns, mean of vessels (90 fields per treatment); bars, SE. ***, P < 0.0001, versus controls (two-tailed t test). B, microscopy photographs showing CD31 (red) in tumor sections 24 h after treatment (as indicated). Bar, 20 µm.

 
To investigate the mechanism of endothelial cell apoptosis observed in vivo and to assess whether other factors, besides EMAP-II/NGR-TNF, are necessary for cell killing, we carried out in vitro cytotoxicity assays with cultured HUVEC and HMEC-1 endothelial cells. These cells were incubated with various concentrations of EMAP-II and NGR-TNF in the range of picogram to microgram per milliliter. After 60 h, viable cells were quantified by staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. These drugs, either alone or in combination, could not induce cytotoxic effects (Supplementary Fig. S2C and D), suggesting that other factors present in the tumor microenvironment were likely critical for the in vivo activity.

NGR-TNF/TNF-R1 interactions are sufficient for the synergism with EMAP-II in vivo. It is well known that TNF exerts its biological functions by interacting with two membrane receptors of 55 kDa (TNF-R1) and 75 kDa (TNF-R2). To assess the role of TNF receptors in the synergistic effect of EMAP-II/NGR-TNF on cell apoptosis, we next examined the effect of this combination in the RMA model using human NGR-TNF in place of murine NGR-TNF. This experiment takes advantage of the fact that human TNF binds only TNF-R1 because binding to murine TNF-R2 is species specific (24).

Microscopic analysis of several tumor tissue sections showed that the combination of EMAP-II (0.1 ng)/human NGR-TNF (0.3 ng) is sufficient to induce marked apoptosis of tumor cells (Fig. 5 ). These results suggest that TNF-R1 is primarily involved in the proapoptotic mechanism of EMAP-II/NGR-TNF combination.


Figure 5
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  		Figure 5. Human NGR-TNF in combination with low-dose EMAP-II induces RMA tumor cell apoptosis in vivo. Tumor-bearing mice (n = 3/group) were treated at day 15 with EMAP-II (0.1 or 1 ng) or human NGR-TNF (0.3 ng; administered 0.5 h later), alone or in combination. Tumor cell apoptosis was analyzed 24 h later in tumor tissue sections by immunostaining of cleaved caspase-3. Microscopy photographs show CD31 (red) and activated caspase-3 (green) in tumor sections 24 h after treatment. Cell nuclei were stained with DAPI (blue). Bar, 20 µm.

 
Of note, a lower number of apoptotic cells was observed when the same study was done with a 10-fold higher amount of EMAP-II (1 ng; Fig. 5).

High-dose EMAP-II induces soluble TNF-R1 shedding in circulation. Shedding of soluble TNF receptors is an efficient counter-regulatory mechanism of TNF (21). To assess whether this mechanism was responsible for the loss of synergism with high-dose EMAP-II, we have analyzed the circulating levels of soluble TNF-R1 (sTNF-R1) and soluble TNF-R2 (sTNF-R2) in tumor-bearing mice before and 30 min after treatment with 0.1, 1, and 1,000 ng of EMAP-II. Whereas 0.1 ng of EMAP-II could not affect the circulating levels of soluble receptors, a significant increase, particularly of sTNF-R1, was induced by higher doses (Fig. 6A ). This mechanism could contribute to NGR-TNF inhibition observed after administration of high-dose EMAP-II. Of note, whereas 1-ng EMAP-II could induce a significative increase in the circulating levels of sTNF-R1, the same dose could still induce significant antitumor effects, although to a lower extent (Fig. 1A, right). This suggests that other mechanisms contribute, together with soluble TNF receptor shedding, to the complete inhibition of NGR-TNF when this cytokine was combined with 1 µg of EMAP-II.


Figure 6
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  		Figure 6. Induction of sTNF-R1 and sTNF-R2 shedding in the blood of RMA tumor–bearing mice and in the supernatants of primary and immortalized endothelial cells after treatment with EMAP-II and NGR-TNF. A, animals were treated 11 d after tumor implantation with 0.1, 1, or 1,000 ng of EMAP-II (n = 3). Blood samples were taken from the tail vein before and 30 min after the treatment. B and C, HUVEC and HMEC-1 were seeded in microtiter plates (1.87 x 105/cm2 and 2.5 x 105/cm2, respectively). After overnight incubation, the cells were cultured for 48 h in the absence or presence of EMAP-II and NGR-TNF. sTNF-R1 and sTNF-R2 in sera and cell supernatants were measured by ELISA using the Mouse sTNF RI/TNFRSF1A and sTNF RII/TNFRSF1B Quantikine ELISA Kits (R&D Systems). *, P < 0.05 (two-tailed t test).

 
To assess whether endothelial cell activation by EMAP-II can directly cause the release of soluble TNF receptors, we carried out in vitro studies with cultured HUVEC cells. Neither NGR-TNF nor EMAP-II, alone or in combination, could affect soluble TNF receptor shedding in the supernatant of these cells over a wide range of concentrations (Fig. 6B). Furthermore, EMAP-II could not affect the shedding of soluble TNF receptors induced by NGR-TNF in the SV40 immortalized HMEC-1 cells (Fig. 6C). These results suggest that sTNF-R1 shedding induced by EMAP-II in mice is unlikely related to direct effects of EMAP-II on endothelial cells. More likely, other cells were activated by high-dose EMAP-II in vivo to release sTNF-R1 in circulation, or other factors possibly present in the tumor microenvironment were necessary.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Despite TNF sensitization of tumor blood vessels by EMAP-II being a well-known phenomenon, systemic administration of high, biologically relevant doses of EMAP-II and TNF cannot be used for cancer treatment because of toxicity. The main finding of this work is that this limitation can be overcome by combining a TNF targeting strategy (NGR-TNF) with the use of ultra low-dose EMAP-II. In particular, we have found that 0.1 ng of EMAP-II and 0.1 ng of NGR-TNF can exert synergistic antitumor effects, with no evidence of toxicity, in two murine tumor models poorly sensitive to TNF. Maximal synergism, leading to endothelial cell damage, tumor cell apoptosis, and tumor growth inhibition, was achieved when EMAP-II was given to mice 0.5 h before NGR-TNF.

Remarkably, nontargeted TNF could not synergize with low-dose EMAP-II. The simplest explanation for this observation is that low-dose TNF could not reach local concentrations in tumor vessels to induce antivascular effects, whereas NGR-TNF could reach bioactive concentrations and activate membrane receptors by virtue of a targeting mechanism (21). However, no synergism was observed even when high-dose TNF (5 µg) was combined with 0.1 ng of EMAP-II (data not shown). One possibility is that the NGR domain of NGR-TNF plays additional roles besides targeting TNF to CD13-positive endothelial cells, such as signaling. However, whether the NGR domain contributes to the synergism by activating membrane receptors still remains to be clarified.

Unexpectedly, the synergism was completely lost, and even reverted to inhibition of NGR-TNF activity, when higher doses of EMAP-II (1–5 µg) were administered to tumor-bearing mice. In addition, the NGR-TNF/chemotherapy synergism was inhibited by high-dose EMAP-II. What is the mechanism of this paradoxical behavior? In previous work, we have shown that sTNF-R2 shedding is an efficient counter-regulatory mechanism of NGR-TNF, either alone or in combination with chemotherapy (21). When we measured serum soluble TNF receptors after administration of increasing doses of EMAP-II, we observed a significant increase of sTNF-R1. Given that small changes in soluble TNF receptor levels are sufficient to significantly inhibit TNF (25–27), this mechanism could have contributed to NGR-TNF inhibition when we used high-dose EMAP-II.

The results of previous studies showing that local injection of high-dose EMAP-II (10 µg) into murine mammary adenocarcinoma can increase tumor sensitivity to high-dose TNF (5 µg; ref. 11) are not necessarily in contrast with this view because, in this case, circulating levels of TNF were likely to exceed the buffering capacity of soluble TNF receptors, leaving a fraction of molecules able to interact with membrane receptors on vessels. However, for the same reason, one might expect that also the undesired systemic effects of TNF are not blocked as well, leading to systemic toxicity.

The results of in vitro experiments with cultured human primary and immortalized endothelial cells showed that EMAP-II does not induce soluble TNF receptor shedding from these cells over a wide range of concentrations. No EMAP-II–dependent induction of soluble TNF receptors was observed even when cells were cultured with EMAP-II/NGR-TNF. These observations argue against the hypothesis that the endothelial lining of vessels is the source of EMAP-II–induced sTNF-R1 in vivo, and suggest that other cells in the body produce sTNF-R1 after EMAP-II administration. However, although previous work showed that murine EMAP-II can affect human endothelial cells (11), we cannot exclude that species differences could also account for the lack of soluble TNF receptor induction or that other factors present in the tumor microenvironment are necessary.

With regard to the mechanism of tumor cell apoptosis, it is unlikely that the massive apoptotic effect observed after treatment with low-dose EMAP-II/NGR-TNF was due to direct interaction of these cytokines with tumor cells. More likely, it was a consequence of vascular damage. Indeed, considering that the total number of NGR-TNF or EMAP-II molecules injected in mice was ~109 and that only a fraction of them could reach the tumor (thus a number of molecules lower than the total number of cells present in ~1-cm3 tumors), the effect was likely indirect (e.g., involving the endothelial compartments of the tumor, which represents a small fraction of the tumor mass). Accordingly, the results of studies on antitumor mechanisms have shown that the combination of low-dose EMAP-II and NGR-TNF can induce apoptosis of endothelial cells in vivo apparently before that of tumor cells. This suggests that tumor cell apoptosis is a consequence of vascular damage.

We also observed that human NGR-TNF could efficiently induce apoptotic effects in tumors when combined with EMAP-II, similarly to murine NGR-TNF. Given that human TNF can bind murine TNF-R1, but not murine TNF-R2 (24), this observation suggests that NGR-TNF interaction with TNF-R1 is sufficient for the synergism. Interestingly, previous work has shown that EMAP-II up-regulates TNF-R1 mRNA expression in endothelial cells (28). Furthermore, a recent study showed that EMAP-II induces TNF-R1 redistribution from Golgi storage pools to cell membrane and facilitates TNF-R1 apoptotic signaling in endothelial cells via TNF receptor 1–associated death domain mobilization (29). However, EMAP-II can exert a variety of other effects on endothelial cells and monocytes potentially leading to activation and secretion of various factors (11). It is therefore possible that tumor cell apoptosis is the result not only of vascular damage and deprivation of oxygen and nutrients but also of the release of a cascade of secondary mediators that in turn affect tumor cell viability.

In conclusion, our results suggest that the combination of low-dose NGR-TNF with low-dose EMAP-II is a novel strategy for avoiding counter-regulatory as well as toxic reactions. This concept is probably more general and provides a new rationale for ligand-directed targeted delivery of low-dose cytokines to tumors. Administration of ultra-low doses of cytokines that affect tumor blood vessels could represent a novel concept in cancer therapy.


   Acknowledgments
 
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.

Conflict-of-interest disclosure: Angelo Corti, Luca Crippa, and Flavio Curnis are the inventors of a patent on NGR-TNF/EMAP-II combination that has been licensed to Molmed SpA.

We thank Barbara Colombo for technical assistance in animal studies and Angela Bachi for mass spectrometry analysis of EMAP-II.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 6/ 5/07. Revised 12/11/07. Accepted 12/19/07.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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