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Clinical Cancer Research Vol. 9, 1826-1836, May 2003
© 2003 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Effect of Interleukin (IL)-4 Cytotoxin on Breast Tumor Growth after in Vivo Gene Transfer of IL-4 Receptor {alpha} Chain1

Koji Kawakami, Mariko Kawakami, Syed R. Husain and Raj K. Puri2

Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although human breast cancer cells express interleukin-4 receptors (IL-4Rs), a recombinant fusion protein, IL-4 cytotoxin, did not mediate desirable antitumor activity in tumor models of breast cancer. Recent studies have identified that a primary IL-4 binding protein, IL-4R{alpha} chain, is internalized after binding to IL-4 in cancer cells. The consequent expression of high-level IL-4R{alpha} in tumor cells sensitizes them to the cytotoxic effect of IL-4 cytotoxin in vitro. To assess whether overexpression of IL-4R{alpha} chain in vivo by plasmid-mediated gene transfer can enhance antitumor activity of IL-4 cytotoxin in mouse models of breast tumor, we injected MDA-MB-231 human breast cancer cells in both flanks of athymic nude mice. Animals then received three intratumoral (i.t.) injections of either IL-4R{alpha} encoding vector (left flank) or vector only (right flank) mixed with liposome followed by IL-4 cytotoxin administration. Both i.p. and i.t. administration of IL-4 cytotoxin profoundly reduced the growth of IL-4R{alpha} plasmid-injected MDA-MB-231 tumors, compared with control. Innate immune cells, including macrophages and neutrophils, were found to infiltrate at the regressing tumor site. This study provides proof of principle that i.t. IL-4R{alpha} plasmid injection followed by systemic or i.t. IL-4 cytotoxin administration may be a useful strategy for the treatment of breast cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breast cancer is the most common cancer in females. In the year 2002 alone, ~205,000 new patients (including 1,500 male) will be diagnosed, and 40,000 are expected to die in the United States (1) . Despite improvements in diagnostic tools and decrease in breast cancer incidence by modification of lifestyle and economic conditions, this disease continues to pose a major public health challenge (2, 3, 4) . Thus, novel approaches are required to overcome this devastating disease. Recent studies have identified a new molecular-targeted approach wherein cell surface or intracellular proteins are being targeted by therapeutic agents for breast cancer therapy (5, 6, 7, 8, 9) . For example, Herceptin, a monoclonal antibody that targets human epidermal growth factor receptor 2 antigen on breast cancer cells, was approved by the Food and Drug Administration for metastatic breast cancer therapy as a single agent or in combination with paclitaxel (5 , 6) . However, only 20–30% of patients were found to express high levels of human epidermal growth factor receptor 2 to benefit from this therapy (7) . Similarly, other molecules that target intracellular signaling molecules are being tested in the clinic for breast cancer therapy (8 , 9) .

Although the monoclonal antibody, cell surface-targeting approach has produced successful results, this method has been additionally improved by the conjugation of whole antibody or antibody fragments to plant bacterial-derived toxins or to chemotherapeutic agents that are otherwise toxic to hosts (10, 11, 12) . Alternatively, specific ligands for overexpressed cell surface receptors have been fused to these toxins or chemicals to create potent-targeted agents (13) . These fusion or conjugated drugs typically bind to cell surface molecules that are subsequently internalized. Once inside the cells, the conjugated toxins or chemicals act as metabolic poisons (10) . The use of these molecules in form of IL3 -2-DT (Ontak) has been realized that targets IL-2 receptors and is approved by the Food and Drug Administration for the treatment of cutaneous T-cell lymphoma (14) . Another agent, Mylotarg, an anti-CD33 antibody conjugated to calicheamicin has been approved for the treatment of relapsed acute myeloid leukemia (15) . Identification of overexpressed plasma membrane protein is critical for this approach (16) .

In that regard, we have identified receptors for IL-4 (IL-4R) that are overexpressed in a variety of human solid tumor cell lines (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) . To target these IL-4Rs on cancer cells, we have developed three generations of potent anticancer therapeutic agents termed IL-4 cytotoxins (24) . The latest molecule is a chimeric protein composed of a circular-permuted IL-4 and a truncated form of a bacterial PE. This recombinant fusion protein is termed IL4 (38–37)-PE38KDEL or IL4-PE (25 , 26) . IL4-PE has been found to be highly efficacious against malignancies of brain (27, 28, 29, 30) , kidney (26 , 31) , head and neck (32) , pancreas (33) , and AIDS-associated Kaposi’s sarcoma (34 , 35) in vitro and in vivo. We also observed that breast cancer cell lines and primary cell cultures were extremely sensitive to the cytotoxic effect of IL4-PE; however, to our disappointment, IL4-PE showed a limited antitumor activity in MDA-MB-231 breast tumor xenografts in nude mice (36) .

Three types of IL-4R are expressed in different cell types. Tumor cells express type II IL-4R, whereas immune cells express type I or type III IL-4R (37 , 38) . This classification was based on cross-linking, binding, and molecular signal transduction studies and later confirmed by reconstitution studies (20 , 39, 40, 41, 42, 43) . Type II IL-4R is composed of IL-4R{alpha} (also known as IL-4Rß) and IL-13R{alpha}1 (also known as IL-13R{alpha}') chains (44, 45, 46, 47) , whereas type I IL-4R is composed of IL-4R{alpha} and IL-2R{gamma} ({gamma}c) chains. Either homodimerization of IL-4R{alpha} or heterodimerization of IL-4R{alpha} chain with IL-13R{alpha}1 or {gamma}c chain is required for signal transduction through JAKs/STAT6 pathways (37 , 38 , 43 , 48, 49, 50) . Among the IL-4R components, the IL-4R{alpha} chain plays a premier role in ligand binding (43 , 51, 52, 53) . In addition, a recent study showed that IL-4R{alpha} and {gamma}c chains are responsible for ligand-induced internalization in human T cells (53) . However, our recent studies have demonstrated that IL-4R{alpha} chain is internalized at high levels after binding to IL-4 in Chinese hamster ovary (CHO-K1) cells transfected with IL-4R{alpha} chain (54) . Coexpression of IL-13R{alpha}1 and/or {gamma}c chains with IL-4R{alpha} did not improve the internalization property of the IL-4R{alpha} chain alone. Furthermore, cancer cells transfected with the IL-4R{alpha} chain acquired higher sensitivity to the cytotoxic effect of IL4-PE in vitro (54) .

Because breast cancer cell lines were highly sensitive to IL-4 cytotoxin in vitro but not in vivo in animal models, we hypothesized that IL-4R might have been down-modulated when cells grew as tumors in vivo. Consequently, IL-4 cytotoxin mediated modest antitumor activity. As we later found that gene transfer of IL-4R{alpha} chain in tumor cells increases cytotoxic effect of IL-4 cytotoxin (Ref. 54 ), we further hypothesized that in vivo gene transfer of IL-4R{alpha} chain would enhance the antitumor activity of IL-4 cytotoxin. Therefore, in this study, we explored whether i.t. injections of plasmid vector-encoding IL-4R{alpha} chain mixed with liposome would mediate better antitumor activity against MDA-MB-231 breast tumors s.c. xenografted in immunodeficient mice. In addition, we assessed whether i.t. plasmid administration would result into vector migration and, consequently, organ toxicity when animals receive injections of IL-4 cytotoxin.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Recombinant Cytotoxins.
Human breast cancer cell lines (BT-20, MDA-MB-231, MCF-7, SK-BR-3, and ZR-75-1) were purchased from the American Type Culture Collection (Manassas, VA). Cells were maintained in Eagle minimum essential medium (BT-20 and MCF-7), RPMI 1640 (MDA-MB-231 and ZR-75-1), or McCoy’s 5A medium (SK-BR-3) supplemented with 10% fetal bovine serum, L-glutamine, and antibiotics. For cultures of BT-20 and MCF-7 cells, 0.1 mM nonessential amino acids and 1.0 mM sodium pyruvate were added. Recombinant human IL-4 was produced and purified to homogeneity in our laboratory (55) . Recombinant IL-4 cytotoxin, IL4 (38–37)-PE38KDEL, containing the circularly permuted IL-4 mutant in which amino acids 38–129 were linked to amino acids 1–37 via a GGNGG linker and then fused to truncated toxin PE38KDEL, consisting of amino acids 253–364 and 381–608 of PE, followed by KDEL, was expressed in Escherichia coli and purified by modified procedure as previously described and provided by Neurocrine Biosciences, Inc. (San Diego, CA; Refs. 25 , 26 ). IL-13 cytotoxin (IL13-PE38QQR) was also produced and purified in our laboratory (56 , 57) .

In Vitro Transfection.
cDNA-encoding human IL-4R{alpha} (kindly provided by Dr. Michael Widmer of Immunex Corp., Seattle, WA) and IL-13R{alpha}1 (kindly provided by Dr. Warren Leonard of the NIH, Bethesda, MD) chain was cloned into pME18S mammalian expression vector using XhoI and XbaI sites, and the sequences of flanking regions of junctions were verified by direct sequencing (43) . Plasmid DNA (12 µg/100-mm culture dish) was transfected into semiconfluent cells using GenePORTER transfection reagent (Gene Therapy Systems, San Diego, CA) according to the manufacturer’s instructions (58) .

Western Blot Analysis.
Cells were lysed at 4°C with 1% NP40, 300 mM NaCl, 50 mM Tris (pH 7.4), leupeptin (10 µg/ml), aprotinin (10 µg/ml), phenylmetylsulfonic fluoride (2 mM), 1 mM sodium vanadate, 25 mM sodium fluoride, 10 mM Na PPI, and 1 mM EDTA for 30 min. SDS-PAGE and development of bands was performed as described previously (40) . Polyclonal antibodies to IL-4R (P-6; a kind gift from Immunex Corp.) or actin (Sigma Chemical Co., St. Louis, MO) were used to react with specific bands.

Radioreceptor Binding Assays.
Cells (5 x 105) in 100 µl of binding buffer (RPMI 1640 containing 0.2% human serum albumin and 10 mM HEPES) were incubated with 125I-IL-4 (specific activity, 18.9 µci/µg). Cell-bound 125I-IL-4 was determined with a gamma counter (Wallac, Gaithersburg, MD).

Protein Synthesis Inhibition Assay.
The cytotoxic activity of IL-4 cytotoxin was tested as described previously (24) . Typically, 104 cells were used in the assay.

RT-PCR.
To detect mRNA expression of IL-4R{alpha} chain in transfected cancer cells, total RNA was isolated using Trizol reagent (Life Technologies, Inc., Grand Island, NY), then subjected to RT-PCR analysis as described previously (32) . The PCR product (10 µl) was run on 2% agarose gel for UV analysis.

Athymic Mouse Models of Breast Cancer.
Athymic nude mice (4 weeks old, ~20 g in body weight) were obtained from Frederick Cancer Center Animal Facilities (National Cancer Institute, Frederick, MD). Animal care was in accordance with the guidelines of NIH Animal Research Advisory Committee. Human breast tumor models were established in the nude mice by s.c. injection of MDA-MB-231 cells (5 x 106) in 150 µl of PBS into the flank. Palpable tumors developed within 3–4 days.

Preparation of Liposome, in Vivo Gene Transfer, and Treatment.
Animals with established tumors received i.t. injections with 25 µg of IL-4R{alpha} or IL-13R{alpha}1 cDNA-encoding vector mixed with N-(1-[2, 3-dioleoyloxy]propyl)-N,N,N-tri-methylammonium chloride:cholesterol (1:1 molar ratio) liposome (59 , 60) . N-(1-[2, 3-dioleoyloxy]propyl)-N,N,N-trimethylammonium chloride was purchased from Avanti Polar Lipids (Albaster, AL) and cholesterol from Sigma Chemical Co. A mixture of each lipid (20 mM) was dissolved in 10 ml of chloroform and dried in a rotating round-bottomed flask under vacuum. The dried lipid film was hydrated in 5% glucose solution (5 ml) for 30 min and dispersed by vigorous vortexing. The hydrated suspension was sonicated at 4°C for 1 h in a cup-horn-type sonicator (Vibracell; Sonics and Materials, Inc., Danbury, CT) and passed successively through 0.45-, 0.22-, and 0.1-µm sterile syringe filters to yield unilamellar liposomes at a lipid concentration of 30 mg/ml. In s.c. xenografted tumors, DNA/liposome injections (total volume 50 µl/injection) were given from days 4 to 6 after tumor implantation. Mice then received cytotoxin or excipient either i.p. (500 µl/mouse) or i.t. (30 µl/tumor).

Immunohistochemistry.
Tumors were excised from mice 3 days after IL4-PE treatment and immediately fixed in 10% formalin. Sections (5-µm thick) of the paraffin-embedded tissues were prepared and subjected to immunohistochemistry as described previously (33) . Slides were incubated with antibodies against murine macrophage (F4/80; Caltag Laboratories, Burlingame, CA), NK cells (NK1.1; Caltag Laboratories), neutrophils (Gr-1; BD Pharmingen, San Diego, CA), iNOS (M19; Santa Cruz Biotechnology, Santa Cruz, CA; 0.4–1 µg/ml), or isotype control for 18 h at 4°C. The sections were counterstained with hematoxylin. Immunohistochemical assays were performed two to three times independently with similar results, and slides were assessed by two independent investigators.

Immunofluorescence Staining and Fluorescent Microscopy.
Frozen sections prepared from tumors or organs were fixed in acetone at -20°C for 5 min and air dried. Sections were then stained with mouse antihuman IL-4R monoclonal antibody (M57; a kind gift from Immunex Corp.). Nonspecific binding was blocked by treatment with 10% goat serum for 1 h followed by incubation with antibodies or isotype control (IgG1). Sections were subsequently incubated for 1 h with secondary antibodies that had a FITC tag. After three washes with PBS, slides were dried and layered with Vectashield antifluorescence-fading mounting medium (Vector Laboratories) and a coverslip. The slides were viewed in Olympus IX70 fluorescence microscopy using appropriate filters (Olympus Optical Co., Tokyo, Japan). Images were compiled from sets of three consecutive single optical sections using Spot Insight V3.2 software (Diagnostic Instruments, Sterling Heights, MI).

Statistical Analysis.
Tumor size was calculated by multiplying length and width of the tumor on a given day. The statistical significance of tumor regression was calculated by Student t test. All statistical tests were two-sided.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Breast Tumor Cells Acquire Increased Sensitivity to IL-4 Cytotoxin after IL-4R{alpha} Chain Gene Transfer in Vitro.
First, we determined the IL-4R expression level after gene transfer of IL-4R{alpha} chain in breast cancer cell lines. As shown in Fig. 1ACitation Citation , three breast cancer cell lines expressed weak to modest levels of protein for IL-4R. When these cells were transiently transfected with IL-4R{alpha} cDNA, an increased level of IL-4R{alpha} expression could be detected. In another experiment, transfected breast cancer cells were subjected to 125I-IL-4 binding assays. Cells transfected with IL-4R{alpha} cDNA showed increased binding activity to radiolabeled IL-4 (Fig. 1B)Citation Citation . Binding of IL-4R{alpha}-transfected cells to 125I-IL-4 was displaced by excess of unlabeled IL-4, indicating specificity.



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  		Fig. 1. Expression of IL-4R and effect of IL-4 cytotoxin in breast cancer cell lines. A, cell lysate (20 µg protein/lane) from three breast cancer cell lines transfected with vector or IL-4R{alpha} cDNA were examined for the expression of IL-4R by Western blot analysis using M57 monoclonal antibody. B, IL-4 binding to breast cancer cell lines. Five breast cancer cell lines transfected with vector (control) or IL-4R{alpha} cDNA (5 x 105) were incubated at 4°C for 2 h with 200 pM 125I-labeled IL-4 with or without 40 nM unlabeled IL-4. Results shown are mean of duplicate determinations and shown as percentage of control. C, cytotoxic activity of IL-4 cytotoxin in breast cancer cell lines. After transient transfection, cells were cultured with various concentrations of IL4 (38-37)-PE38KDEL (0–1000 ng/ml). The results are represented as means ± SD of quadruplicate determinations, and the assay was repeated three times. Various chains of IL-4R were transfected in SK-BR-3 cells, and cytotoxic activity of IL4 (38-37)-PE38KDEL (D) or an irrelevant cytotoxin IL13-PE38QQR (E) was examined by protein synthesis inhibition assay.

 


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  		Fig. 1A. Continued

 
We also examined in vitro cytotoxic activity of IL4-PE to breast cancer cell lines transfected with vector or with IL-4R{alpha} gene (Fig. 1C)Citation Citation . Consistent with binding results, the sensitivity to IL4-PE was increased in IL-4R{alpha} chain-transfected breast cancer cells, compared with vector only-transfected control cells. The IC50 (IL4-PE concentration required for the inhibition of protein synthesis by 50%) improved from 2.0 to 0.65 ng/ml in MDA-MB-231 cells, from >1000 to 80 ng/ml in SK-BR-3 cells, from 0.45 to 0.07 ng/ml in MCF-7 cells, from 22 to 2.7 ng/ml in ZR-75–1 cells, and from 100 to 1.6 ng/ml in BT-20 cells. Using SK-BR-3 cell line, we confirmed that the cytototoxic activity of IL4-PE to IL-4R{alpha}-transfected cells was blocked by an excess of IL-4 (2 µg), indicating that cytotoxicity mediated by this molecule is specific to IL-4R (data not shown).

We also examined the cytotoxicity of IL4-PE and an irrelevant cytotoxin, IL13-PE38QQR (56 , 61) , in SK-BR-3 cells transfected with various chains of IL-4R. As shown in Fig. 1DCitation Citation , IL4-PE was cytotoxic to SK-BR-3 cells only when cells were transfected with IL-4R{alpha} chain (IC50 = 50 ng/ml). Cells transfected with {gamma}c or IL-13R{alpha}1 chain showed a limited sensitivity to IL4-PE (IC50 = 750 ng/ml in {gamma}c-transfected cells and IC50 > 1000 ng/ml in IL-13R{alpha}1-transfected cells). On the other hand, IL13-PE38QQR showed no sensitivity for SK-BR-3 cells transfected with any chain of IL-4R system (Fig. 1E)Citation Citation . PM-RCC cells served as positive controls to the IL4-PE and IL13-PE38QQR and were found to be sensitive to both cytotoxins as reported earlier (data not shown; Refs. 26 , 61 ). These data confirm that IL4-PE-mediated cytotoxicity in breast cancer cells is IL-4R{alpha} chain specific, and increased sensitivity of IL-4R{alpha}-transfected breast cancer cells is IL4-PE specific.

IL-4R Expression in Breast Tumors after in Vivo i.t. Injections of IL-4R{alpha} Chain-encoding Plasmid.
To determine the extent and persistence of IL-4R expression in vivo, MDA-MB-231 tumors grown in the flank of the nude mice were i.t. injected with plasmid vector-encoding IL-4R{alpha} cDNA or vector only. Plasmid mixed with liposomes was injected on days 4 through 6 after tumor implantation (total of three injections). Tumors were subsequently excised at various time points and subjected to RT-PCR and immunofluorescence microscopy for IL-4R expression. After IL-4R{alpha}-encoding plasmid injections, we observed augmented levels of IL-4R{alpha} chain mRNA expression until day 17 in MDA-MB-231 tumors (Fig. 2A)Citation . Twenty days after tumor implantation, IL-4R{alpha} expression had decreased to baseline. These results were confirmed by immunofluorescence microscopy using antibody to IL-4R (Fig. 2B)Citation . Increased levels of IL-4R expression was observed on day 7, decreased by day 17, and back to baseline expression level on day 25 (data not shown). In control tumors injected with vector only, no change in IL-4R expression level was observed from days 7 to 25. These results suggest that IL-4R expression in breast tumors i.t. injected by IL-4R{alpha} plasmid was maintained for up to 11–14 days at tumor sites.



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  		Fig. 2. IL-4R{alpha} chain expression in MDA-MB-231 tumors and vital organ tissues after plasmid-mediated gene transfer of IL-4R{alpha} chain. s.c. xenografted MDA-MB-231 tumors i.t. injected with plasmid vector mixed with liposome (on days 4, 5, and 6 after tumor implantation) were collected at indicated time. A, RT-PCR using total RNA from tumors and PM-RCC cell line for a positive control, glyceraldehyde-3-phosphate dehydrogenase served as internal control. B, immunofluorescence assay using monoclonal antibody against human IL-4R (M57). Original magnification, x100. C, IL-4R{alpha} chain transgene expression in vital organs after i.t. plasmid injections. Tumors and various vital organs were harvested at indicated time, and total RNA extracted from tissue sections was subjected to RT-PCR analysis for IL-4R{alpha} mRNA expression. RNA from PM-RCC cells served as positive control.

 
In vivo i.t. gene transfer of IL-4R{alpha}-encoding plasmid may migrate to distant vital organs, resulting in organ toxicity when these mice are systemically treated with IL4-PE. To examine this possibility, animals bearing MDA-MB-231 tumors i.t. injected with IL-4R{alpha}-encoding plasmid on days 4, 5, and 6 after tumor implantation were subjected to harvest of vital organs, including liver, lung, kidney, spleen, heart, and blood, as well as tumors from days 4 to 30. Total RNA extracted from samples was analyzed for IL-4R{alpha} mRNA expression. On day 7 (1 day after third plasmid injection), IL-4R{alpha} mRNA was detected in all of the organs, except for blood, as assessed by RT-PCR (Fig. 2C)Citation . IL-4R{alpha} gene expression levels appeared to decrease in all organs until day 11, and on day 13, no detectable transgene expression was observed.

In addition to RT-PCR analysis, we also performed immunohistochemical staining of frozen sections obtained from same samples, using antibody to IL-4R. Interestingly, no detectable protein expression was observed in IL-UR any organs examined, except for IL-4R{alpha} chain expression in IL-4R plasmid vector-injected tumors (data not shown). These observations indicate that although vital organs were unexpectedly transfected with IL-4R{alpha} transgene because of in vivo i.t. plasmid injections, detectable levels of IL-4R protein were not translated in these organs.

Effect of IL-4 Cytotoxin on MDA-MB-231 Tumor Growth after i.t. IL-4R{alpha} Chain Gene Transfer.
We next examined whether overexpression of IL-4R{alpha} chain in breast tumor cells enhanced antitumor effect of IL4-PE in s.c. xenografted MDA-MB-231 tumors in nude mice. We developed tumors in both right and left flanks of nude mice. Animals received either a vector only injection (right flank) or an injection of IL-4R{alpha} chain plasmid (left flank) i.t. on days 4, 5, and 6. Subsequently, these animals were treated from days 8 to 12 with IL4-PE by either an i.p. (50 or 100 µg/kg b.i.d. for 5 days) or i.t. (250 µg/kg qd for 5 days) routes. Drug dosages were chosen based on our previous studies on breast tumor-xenografted nude mice (36) .

i.p. IL-4 Cytotoxin Treatment.
Tumors injected with vector only (right flank) minimally responded to i.p. IL4-PE treatment (Fig. 3A)Citation . By day 40, tumor growth in control (mean tumor size, 215 mm2) and 50 µg/kg dose IL4-PE-treated animals showed no significant difference in tumor size (mean tumor size, 176 mm2). A small but statistically significant antitumor effect was observed in 100 µg/kg dose animals (mean tumor size, 143 mm2; P < 0.001), and tumors reduced by 34% compared to control. On the other hand, tumors injected with IL-4R{alpha} encoding plasmid (left flank) started to regress during i.p. IL4-PE administration in both 50 and 100 µg/kg dose groups. Although tumors in treated mice gradually grew after the treatment period, mean tumor size at the end of experiment (day 40) was significantly smaller (106 mm2 in 50 µg/kg dose; 67 mm2 in 100 µg/kg dose) compared with mean tumor size in control mice (211 mm2; P < 0.001 for both doses). Tumor regression rate was 50% in 50 µg/kg dose and 68% in 100 µg/kg dose group compared with control tumor.



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  		Fig. 3. Effect of IL-4R{alpha} chain gene transfer and IL-4 cytotoxin therapy on MDA-MB-231 breast tumor xenografted in athymic mice. MDA-MB-231 cells (5 x 106) were injected in both flanks of nude mice (day 0). Tumors in the left flank were injected with IL-4R{alpha} plasmid, whereas the right flank was i.t. injected with vector only on days 4, 5, and 6, followed by i.p. (50 or 100 µg/kg (b.i.d.) for 5 days) (IP; A) or i.t. (250 µg/kg qd for 5 days) (IT; B) IL4 (38-37)-PE38KDEL administration on days as indicated by arrows. Mean tumor size was determined at indicated days by calculating the cross-sectional area (mm2); each group had at least five mice.

 
i.t. IL-4 Cytotoxin Treatment.
Effect of i.t. IL4-PE administration was also evaluated in tumors injected with either vector or IL-4R{alpha} chain-encoding plasmid. After treatment with IL-4PE, vector only-injected tumors showed significant regression in tumor size (Fig. 3B)Citation . Mean size of control excipient-treated tumor was 215 mm2, compared with 102 mm2 in IL4-PE-treated tumors on day 40 (P < 0.001). In contrast, after i.t. administration of IL4-PE, growth of IL-4R{alpha} chain gene-transferred tumors was profoundly arrested. By day 13, three of five mice showed complete disappearance of their left flank tumors. Although tumors recurred in one mouse, mean tumor size in treated group remained significantly smaller (17 mm2; P < 0.001) than control (211 mm2) by day 40: 92% reduction in tumor size compared with control.

In addition to efficacy studies, major organs (heart, liver, lung, kidney, and spleen) from mice receiving i.t. IL-4R{alpha} plasmid injections followed by IL4-PE either by i.p. (50 µg/kg dose) or i.t. (250 µg/kg dose) routes were evaluated for toxicity. By histological examination, we found no evidence of toxicity in any organs when these organs were evaluated on day 15 after tumor implantation (data not shown). All of the treated mice tolerated the therapy very well without any sign of visible toxicity. These results suggest that animals receiving in vivo i.t. gene transfer of IL-4R{alpha} chain are highly susceptible to the antitumor effect of IL-4 cytotoxin when given i.p. or i.t. routes of administration with no undesirable side effects.

Effect of IL-4 Cytotoxin on Large Breast Tumors Injected with IL-4R{alpha} Plasmid.
We also assessed antitumor activity of IL4-PE in animals bearing large, established MDA-MB-231 tumors. Eighteen days after the tumor implantation (both flank) when mean tumor size had reached 85 mm2, mice received either a vector injection (right flank) or an injection of IL-4R{alpha} chain plasmid (left flank) i.t. on days 18, 19, and 20. Subsequently, these animals were treated from days 21 to 28 with IL4-PE by either an i.p. (200 µg/kg b.i.d. for 8 days) or i.t. (500 µg/kg qd for 8 days) route of administration. Tumors receiving i.p. administration of excipient only grew linearly, regardless of vector (right flank) or IL-4R{alpha} chain (left flank) gene plasmid injection (Fig. 4)Citation . By termination of the experiment (day 40), tumors grew to 275 mm2 in vector only group and to 271 mm2 in IL-4R{alpha} plasmid-injected group, respectively. Because of both routes of IL4-PE injection, tumor growth was slightly inhibited in vector-injected tumors. Mean tumor size at day 40 was 186 mm2 (P < 0.0073 versus control) in the i.p.-treated group and 167 mm2 (P < 0.0034 versus control) in the i.t.-treated group. There was no significant difference between i.p. and i.t. treatment groups on day 40 (P = 0.49). In contrast, tumors injected with IL-4R{alpha} cDNA-encoding plasmid mediated rapid tumor regression during the IL4-PE treatment period. Two of 6 mice appeared to be tumor free from day 33 in i.t. treatment group. By the end of the experiment (day 40), mean size tumor was 97 mm2 in i.p. treatment group and 24 mm2 in i.t. treatment group. In both groups, tumor size was significantly smaller compared with control tumors: 64% reduction in i.p. treatment group (P < 0.001) and 91% reduction in i.t. treatment group (P < 0.001), respectively. These results indicate that even larger breast tumors can be successfully treated by IL-4R{alpha} gene transfer followed by IL-4 cytotoxin therapy.



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  		Fig. 4. Effect of IL-4R{alpha} chain gene transfer and IL-4 cytotoxin therapy in large MDA-MB-231 breast tumor models. Cells (5 x 106) were injected in both flanks of nude mice (day 0). When tumors reached 85 mm2 in size, animals received i.t. injections with IL-4R{alpha} chain plasmid (left flank) or vector only (right flank) on days 18, 19 and 20 and treated with IL4 (38-37)-PE38KDEL either i.p. (IP; 50 or 100 µg/kg b.i.d. for 8 days) or i.t. [IT; 250 µg/kg qd for 8 days) on days indicated by arrows. Mean tumor size was determined on indicated days. Each group had at least six mice; results shown are mean tumor size (mm2).

 
Effect of IL-4 Cytotoxin Is Specific to IL-4R{alpha}-transfected Breast Tumors.
To demonstrate whether the effect of IL4-PE is specific to breast tumors receiving in vivo gene transfer of IL-4R{alpha} chain, we i.t. injected plasmid encoding either IL-4R{alpha} cDNA or IL-13R{alpha}1 chain on days 4, 5, and 6 after tumor implantation. Mice then received i.p. administration of IL4-PE (50 µg/kg b.i.d. for 5 days) from day 8 to 12. IL4-PE treatment caused growth inhibition of tumors injected with IL-4R{alpha} cDNA, whereas only modest tumor regression was observed in mice with IL-13R{alpha}1 cDNA injected tumors during the treatment period (Fig. 5A)Citation . By last day of the experiment (day 40), mean size in IL-13R{alpha}1-injected tumors (154 mm2) was significantly smaller than control tumors (212 mm2; P < 0.001), however, reduction in tumor size was only 27%. On the other hand, mean tumor size on day 40 in IL-4R{alpha}-plasmid injected mice group (81 mm2) was 62% smaller compared with control tumors (P < 0.001).



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  		Fig. 5. Effect of IL-4R{alpha} or IL-13R{alpha}1 chain gene transfer and IL-4 cytotoxin or IL-13 cytotoxin therapy in MDA-MB-231 breast tumor models. Cells (5 x 106) were injected s.c. in nude mice on day 0. Tumors were injected with IL-4R{alpha} or IL-13R{alpha}1-encoding plasmid on days 4, 5, and 6. Animals then received i.p. (IP) administration of either IL4 (38-37)-PE38KDEL (50 µg/kg b.i.d. for 5 days; A) or IL13-PE38QQR (50 µg/kg b.i.d. for 5 days; B) on days as indicated by arrows. Mean tumor size was determined at indicated days by calculating the cross-sectional area (mm2). Each group had at least five mice; results shown are mean tumor size (mm2).

 
To additionally show the specificity, another group of animals i.t. injected with IL-4R{alpha}-encoding plasmid was treated with an irrelevant cytotoxin, IL-13 cytotoxin (IL13-PE or IL13-PE38QQR) by i.p. route (50 µg/kg b.i.d. for 5 days) from days 8 to 12 (Fig. 5B)Citation . During treatment period, IL13-PE did not induce tumor regression in IL-4R{alpha} gene-transferred tumors. However, by day 40, mean tumor size of IL13-PE treatment mice group was slightly smaller (161 mm2) compared with excipient-treated control tumor (212 mm2), which was statistically significant (P < 0.003). On the other hand, upon IL4-PE treatment, profound antitumor effect was observed (P < 0.001, compared with control). These results suggest that the effect of IL-4 cytotoxin is specific to tumors expressing IL-4R{alpha} chain.

Histological Changes in IL-4R{alpha}-transfected and IL4-PE-treated Breast Tumors.
MDA-MB-231 tumors were s.c. established by injection of cells on day 0. These mice received i.t. injection of IL-4R{alpha} chain plasmid on days 4, 5, and 6 and, subsequently, treated from days 8 to 12 with IL4-PE (50 µg/kg b.i.d. for 5 days) or excipent by i.p. route. Tumors were excised on day 15 after the tumor implantation, and tumor sections were prepared. In tumors treated with excipient only, no tumor cell death was observed (Fig. 6A)Citation . On the other hand, tumor necrosis was observed after IL4-PE treatment in tumors that were injected with IL-4R{alpha} chain gene (Fig. 6, B and C)Citation . To determine whether inflammatory cells had infiltrated at the area between remaining tumor cells and necrotic region (Fig. 6C)Citation , we performed immunohistochemical staining of sections from these tumors. Interestingly, we found moderate levels of macrophage marker (F4/80)-positive cells (Fig. 6D)Citation , neutrophil marker (Gr-1)-positive cells (Fig. 6E)Citation , and iNOS marker (M19)-positive cells (Fig. 6F)Citation . It seemed that iNOS-positive cells were mostly macrophages. Because iNOS is an inducible enzyme that produces NO, these data suggest that NO-producing macrophages and neutrophils may also participate in antitumor activity of IL4-PE in IL-4R{alpha} gene-transferred breast cancer.



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  		Fig. 6. Histological examination of MDA-MB-231 tumors receiving IL-4R{alpha} gene followed by IL-4 cytotoxin therapy. Tumor cells (5 x 106) were injected s.c. in nude mice. Tumors were then injected with IL-4R{alpha} plasmid by i.t. injections on days 4, 5, and 6, followed by i.p. IL4 (38-37)-PE38KDEL administration (50 µg/kg b.i.d.) from days 8 to 12. Tumors were excised 3 days after the completion of IL-4 cytotoxin treatment (15 days after tumor implantation). H&E sections show tumors treated with excipient only (A) or IL4-PE (B and C) injections, after i.t. IL-4R{alpha} chain cDNA injections. Tumor sections from IL4-PE-treated animals were subjected to immunohistochemistry using antibodies to macrophages (F4/80; D), neutrophils (Gr-1; E), and iNOS (M19; F). Original magnification, A and B, x100; C–F, x600.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We demonstrate that in vitro transfection of IL-4R{alpha} chain in breast cancer cell lines enhances the number of IL-4 binding sites and sensitivity to IL4-PE in an IL-4R-specific manner. IL4-PE mediated significant inhibition of breast tumor growth in vivo when these tumors were injected with plasmid for IL-4R{alpha} chain. Antitumor activity of IL4-PE was specific to IL-4R{alpha} chain in breast tumor cells because gene transfer of IL-13R{alpha}1 chain did not enhance the cytotoxic activity of IL4-PE in both in vitro and in vivo experiments. Moreover, an irrelevant cytotoxin, IL13-PE did not show enhanced antitumor activity in IL-4R{alpha} gene-transferred breast tumors in vitro and in vivo. These results indicate that (a) IL-4R{alpha} chain is a primary binding/internalization component in breast cancer cells, and (b) IL-4R{alpha} chain by itself does not mediate strong binding/internalization of IL-13 in breast cancer cells, although this chain is one of the binding components of the IL-13R complex (Refs. 36 , 42 ). These observations are similar to our previous findings, which demonstrated that IL-4R{alpha} chain forms a signaling complex with IL-13R{alpha}1 chain and that IL-4R{alpha} chain by itself binds and internalizes after binding to IL-4 (43 , 54) .

In our previous study, IL4-PE did not mediate significant antitumor activity in MDA-MB-231 xenografts when injected by i.p. route at a dosage of 100 µg/kg (b.i.d. 5 days; Ref. 36 ). MDA-MB-231 tumor cells expressed a moderate number of IL-4R, and IL4-PE was highly cytotoxic to these cells in vitro. Lack of antitumor activity of IL4-PE in vivo was considered to be attributable to most probably inefficient drug delivery to the tumor bed. Alternatively, IL-4 binding sites may be down-regulated in vivo as cells formed tumors in nude mice (36) . However, in this study, when we used in vivo gene transfer of IL-4R{alpha} chain into breast tumors followed by IL4-PE therapy by same route and same dose (i.p. 50 or 100 µg/kg doses, b.i.d. 5 days), remarkable antitumor activity was observed. Thus, when more target (IL-4R{alpha} chain) was inserted in breast tumor cells, better target-specific antitumor activity was observed. Regardless of percentage of breast tumor cells transfected in vivo with IL-4R{alpha} chain by i.t. plasmid injections, we observed significant MDA-MB-231 tumor regression after the treatment of tumor-bearing animals with IL4-PE. Thus, additional mechanism(s) may be operational along with direct IL4-PE-mediated cytotoxicity to cause significant regression of established tumors.

The mechanism of tumor regression was additionally investigated by histological examination of tumors. Pronounced cellular infiltration was observed at tumor sites because of IL-4R{alpha} gene transfer and IL4-PE administration. The infiltrating cells were identified to be macrophages and neutrophils by immunohistochemistry analysis, and macrophages were found to produce NO by iNOS expression. Because macrophages and neutrophils and NO released from macrophages have been implicated in mediating antitumor activity, it is possible that these cells and NO play an important role in IL4-PE-induced breast tumor regression (62, 63, 64) . Thus, breast cancer therapeutic approach through IL-4R{alpha} gene transfer and IL4-PE therapy causes not only a direct killing of breast tumor cells but also activates innate immunity at local tumor site, which is one of the key mechanisms in tumor immunotherapy (65) . Alternatively, dying tumor cells attracted macrophages and neutrophils, and these cells may have little to do with tumor regression. Additional studies are ongoing to unravel these mechanisms and explore the significance of IL-4R expression in breast cancer.

We observed IL-4R{alpha} transgene mRNA expression in vital organs after three i.t. plasmid vector injections, which persisted for 5 days after completion of injections, as assessed by RT-PCR analysis. However, transgene mRNA expression level was not sufficient for translation of enough IL-4R{alpha} protein in various organs to be detectable by immunohistochemical analysis. In addition, we did not observe any histological organ toxicity in mice receiving three i.t. IL-4R{alpha} cDNA injections followed by systemic or i.t. IL4-PE administration. These observations suggest that a breast cancer therapeutic approach that uses in vivo IL-4R{alpha} chain gene transfer and IL4-PE administration may be performed safely in humans.

Advanced breast cancer frequently metastasizes to bone (2 , 66) . Despite many efforts, only a limited number of chemotherapeutic agents have shown to be effective (67) . To treat both local and metastasized breast tumor legions, one may need to administer a particular drug by both systemic and localized routes. The combination of i.t. and systemic IL4-PE administration after IL-4R{alpha} chain gene transfer may eliminate breast cancer locally. However, for metastatic disease, IL-4R{alpha} gene expression in secondary legions would be needed. Therefore, our ongoing efforts have focused on breast tumor-specific selective gene expression by systemic administration of vector. Our therapeutic approach may form a basis for consideration for a novel approach for breast cancer therapy.


   ACKNOWLEDGMENTS
 
We thank Pamela Dover for the procurement of reagents and laboratory supplies and Dr. Takashi Murata of Graduate School of Biomedical Science Nagasaki University, (Nagasaki, Japan) for providing pME18S plasmids. We also thank Drs. Melanie Hartsough and Ralph Bernstein at CBER/Food and Drug Administration for critical reading of this manuscript.


   FOOTNOTES
 
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.

1 These studies were conducted as part of a collaboration between the Food and Drug Administration and Neurocrine Biosciences, Inc., under Cooperative Research and Development Agreement (CRADA). Back

2 To whom requests for reprints should be addressed, at Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, NIH Building 29B, Room 2NN10, 29 Lincoln Drive MSC 4555, Bethesda, MD 20892. Phone: (301) 827-0471; Fax: (301) 827-0449; E-mail: puri{at}cber.fda.gov Back

3 The abbreviations used are: IL, interleukin; IL-4R, IL-4 receptor; IL-13R, IL-13 receptor; PE, Pseudomonas exotoxin A; RT-PCR, reverse transcription-PCR; i.t., intratumoral; iNOS, inducible nitric oxide synthase; qd, once daily; b.i.d., twice a day; NO, nitric oxide. Back

Received 11/18/02; revised 12/18/02; accepted 12/18/02.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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