Molecular Cancer Therapeutics CTRC-AACR San Antonio Breast Cancer Symposium Targeting the PI3-Kinase Pathway in Cancer
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Mol Cancer Ther. 2004;3:137-147
© 2004 American Association for Cancer Research

Specifically targeted killing of interleukin-13 (IL-13) receptor-expressing breast cancer by IL-13 fusion cytotoxin in animal model of human disease

Koji Kawakami, Mariko Kawakami and Raj K. Puri

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

Requests for Reprints:Raj K. Puri, 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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interleukin-13 receptor (IL-13R) {alpha}2 chain binds IL-13 with high affinity and can internalize after binding to ligand. We have exploited this property of IL-13R{alpha}2 chain by receptor-targeted breast cancer therapy. Previous studies have demonstrated that in vivo intratumoral (i.t.) gene transfer of this chain followed by IL-13 cytotoxin [comprised of IL-13 and Pseudomonas exotoxin (IL13-PE38QQR)] therapy causes regression of established human tumors in xenografted models. Breast carcinoma cells do not express IL-13R{alpha}2 chain and are resistant to the antitumor effect of IL-13 cytotoxin. To determine whether IL-13R{alpha}2 chain can render sensitivity of breast cancer to IL-13 cytotoxin, we injected IL-13R{alpha}2 plasmid in s.c. established tumors by i.t. route, followed by systemic or i.t. IL-13 cytotoxin administration. This combination approach showed profound antitumor activity against human breast tumors in xenografted immunodeficient mice. Interestingly, there was dominant infiltration of inflammatory cells in regressing tumors, which were identified to be macrophages producing nitric oxide (NO) and natural killer cells. The partial role of inducible nitric oxide synthase (iNOS)-positive macrophages was confirmed by in vivo macrophage depletion experiments. Serum chemistry, hematology, and organ histology from treated mice did not show any remarkable toxicity resulting from the combination therapy. Taken together, local gene transfer of IL-13R{alpha}2 followed by receptor-targeted IL-13 cytotoxin therapy may be applied safely and effectively to the treatment of localized breast cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Breast cancer is the most common malignancy in females. It is estimated that in the year 2002 alone, 205,000 new patients (including 1,500 male) were diagnosed in the United States and about 40,000 patients died from this disease (1). Although various targeted agents including Herceptin have shown antitumor activity in breast cancer, once metastasized, cure are rare (2, 3). Thus, new approaches are needed to eradicate this disease early.

In recent years, interleukin-13 (IL-13) has received much attention because it has been found to play a major role in malignancies and inflammatory diseases including pulmonary asthma (4–7). Consequently, the plasma membrane receptors for IL-13 (IL-13R) have been vigorously studied (8–12). Similar to other members of type I cytokine receptor superfamily, IL-13R has also been found to exist as multiple different types (13–18). The type 1 IL-13R is comprised of IL-13R{alpha}1 (also known as IL-13R{alpha}'), IL-13R{alpha}2 (also known as IL-13R{alpha}), and IL-4R{alpha} (also known as IL-4Rß) chains, whereas type 2 IL-13R is composed of IL-13R{alpha}1 and IL-4R{alpha} chains. IL-13R{alpha}1 chain forms a complex with IL-4R{alpha} chain to activate a JAK-STAT signaling pathway induced by IL-4 or IL-13 (14, 19–22). Both type 1 and type 2 IL-13R are shown to be expressed on various tumor cell types (11, 13, 15–17, 23–25). The IL-13R{alpha}2 chain binds IL-13 with –50 times higher affinity than IL-13R{alpha}1 chain (26, 27). Although IL-13R{alpha}2 chain has not been shown to mediate any biological activity, it has been shown to play a role in ligand binding and internalization (27–30). In murine systems, IL-13R{alpha}2 chain is shown to function as a decoy receptor because its extracellular domain is cleaved and the protein is detected in serum and urine (31).

To target IL-13R, we have developed a novel anticancer therapeutic agent, recombinant IL-13 cytotoxin (IL13-PE38QQR), which is composed of IL-13 and a mutated short form of Pseudomonas exotoxin (PE) (18, 32). IL13-PE38QQR has been shown to have potent antitumor activity in vitro (18, 23–25, 32) and in vivo (25, 33, 34). Although IL13-PE38QQR is highly cytotoxic to type 1 IL-13R-expressing cancer cells including glioma, renal cell carcinoma, AIDS-associated Kaposi's sarcoma, and head and neck cancer, type 2 IL-13R-expressing tumors have limited sensitivity to IL13-PE38QQR (18, 34). Because the difference between type 1 and type 2 IL-13R is the presence of IL-13R{alpha}2 chain, we hypothesized that if cancer cells acquired this chain, they might become sensitive to IL13-PE38QQR. Our hypothesis was correct because cancer cells transfected with IL-13R{alpha}2 chain ex vivo showed a dramatic response to IL13-PE38QQR in vitro and in vivo (34–36). Furthermore, we have confirmed that in vivo plasmid-mediated gene transfer of IL-13R{alpha}2 sensitized prostate and head and neck tumors to subsequent IL-13 cytotoxin therapy (37). This approach may offer new hope for breast cancer targeting.

Because localized breast tumor mass can be injected through skin, new approaches can be tested wherein tumor cells can be sensitized by intratumoral (i.t.) administration of appropriate gene followed by targeted agent therapy by systemic or local routes. Therefore, in this study, we examined the effect of IL-13R{alpha}2 gene transfer on the cytotoxicity of IL13-PE38QQR in breast cancer cells in vitro and in vivo. Established human breast tumors were injected with vector encoding IL-13R{alpha}2 cDNA to determine in vivo gene expression, vector migration to other organs, and examine antitumor activity of combination approach of gene transfer and IL-13 cytotoxin therapy. Our results demonstrate that this approach of in vivo forced expression of IL-13R{alpha}2 followed by IL13-PE38QQR therapy can mediate dramatic antitumor activity in animal models of localized human breast cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant Cytokine, Cytotoxin, and Cell Cultures
Recombinant human IL-13 and IL-13 cytotoxin (IL13-PE38QQR) were produced and purified to homogeneity in our laboratory (18, 32, 38). 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) and maintained in culture according to the supplier's specifications.

In Vitro Transfection
cDNA encoding human IL-13R{alpha}2 (16, 26) or IL-13R{alpha}1 (19) 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 (39). The resulting construct was expanded in Escherichia coli and purified using endotoxin-free EndoFree Mega kit (QIAGEN Inc., Valencia, CA). pME18S plasmid vector is driven by an SV40 promoter. 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. Briefly, cells (2 x 106/100-mm dish) were incubated with the DNA-GenePORTER mixture for 5 h in DMEM followed by 20 h incubation in fresh DMEM containing 20% fetal bovine serum and additional 24 h in medium with 10% fetal bovine serum.

RT-PCR
To detect the mRNA expression in cells or tumors developed in mice, total RNA was isolated using TRIZOL reagent (Life Technologies, Inc., Grand Island, NY), then RT-PCR was performed using specific primers as described (13). PCR mixture was first incubated for 10 min at 94°C and amplified 35 cycles at 94°C for 45 s, 54°C for 30 s, and elongated at 72°C for 55 s.

Protein Synthesis Inhibition Assay
The protein synthesis inhibition caused by IL-13 cytotoxin was tested as previously described (40). Typically, 104 cells were cultured in quadruplicate in leucine-free medium with or without various concentrations of IL13-PE38QQR for 20–22 h at 37°C. Then, 1 µCi of [3H]leucine (NEN Research Products, Boston, MA) was added to each well and incubated for an additional 4 h. Cells were harvested and radioactivity incorporated into cells was measured by a ß plate counter (Wallac, Gaithersburg, MD).

Human Breast Cancer Xenografts
Athymic nude mice 4 weeks old (about 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 or MCF-7 tumor cells (5 x 106) in 150 µl of PBS into the flank. Palpable tumors developed within 3–4 days. Two perpendicular diameters of tumors were carefully measured by Vernier calipers. In general, five or six mice were used for each group.

In Vivo IL-13R{alpha}2 Gene Transfer and Treatment
Immunodeficient mice with established tumors were injected i.t. with 25 µg of IL-13R{alpha}2 cDNA encoding vector mixed with 20 mM N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTAP):cholesterol (1:1 molar ratio) liposome (41, 42). DOTAP was purchased from Avanti Polar Lipids (Albaster, AL) and cholesterol from Sigma-Aldrich (St. Louis, MO). A mixture of each lipid (20 mM) was dissolved in 10 ml chloroform and dried in a rotating round-bottom 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 & Materials Inc., Danbury, CT) and extruded 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, vector injections (total volume 50 µl/injection) were performed from day 4 through 6 after tumor implantation. Typically injections were made in upper and lower half of tumor. Mice were then given injections of IL13-PE38QQR or excipient either i.p. (500 µl/mouse) or i.t. (30 µl/tumor) 2 days after last vector administration.

Immunohistochemistry
Immunohistochemistry was performed using the Vector ABC peroxidase kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. S.c. tumor samples were taken at 3 days after the IL13-PE treatment (day 15) and fixed with 10% formalin or snap frozen with OCT compound. Paraffin-embedded sections were deparaffinized by xylene treatment and washed with alcohol (100–50%) and PBS. Slides were incubated with antibodies (at a concentration of 0.4–1 µg/ml) against murine macrophage (F4/80; Caltag Laboratories, Burlingame, CA), NK cells (NK1.1; Caltag Laboratories), or inducible nitric oxide synthase (iNOS) (M19; Santa Cruz Biotechnology, Santa Cruz, CA) or isotype control (0.4–1 µg/ml) for 18 h at 4°C. Slides were then developed using DAB substrate biotinylated peroxidase reagent (Vector Laboratories) and counterstained with hematoxylin. Immunohistochemical assays were performed 2–3 times independently with similar results and slides were assessed by two authors (K.K. and M.K.).

For immnofluorescent assays, frozen sections were stained with anti-human monoclonal antibody for IL-13R{alpha}2 (Diaclone, Besancon, France) or co-stained with F4/80 and M19. Slides were fixed in acetone at –20°C for 5 min and air-dried. Nonspecific binding was blocked by treatment with 10% serum for 1 h followed by incubation with antibodies or isotype control. Sections were subsequently incubated for 1 h with secondary antibodies that had either tetramethylrhodamine isothiocyanate or 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 V 3.2 software (Diagnostic Instruments, Sterling Heights, MI).

Macrophage Depletion
In vivo macrophage depletion was performed as described previously (37). Carrageenan (type II; Sigma-Aldrich, Inc.) was dissolved in sterile PBS at 5 mg/ml. The solution was heated to 56°C to ensure complete solubilization. Mice (n = 7/group) were treated by i.p. injection of 200 µl (1 mg) of carrageenan on days 3, 7, and 14 after the tumor implantation. Control mice received 200 µl of sterile PBS.

Statistical Analysis
Tumor sizes were calculated by multiplying length and width of tumor on a given day. The statistical significance of tumor regression was calculated by Student's t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Breast Cancer Cells Acquire Increased Sensitivity to IL-13 Cytotoxin after IL-13R{alpha}2 Chain Gene Transfer in Vitro
First we determined IL-13R{alpha}2 mRNA expression before and after gene transfer of IL-13R{alpha}2 chain in breast cancer cell lines. As shown in Fig. 1A, four breast cancer cell lines (BT-20, MDA-MB-231, SK-BR-3, and ZR-75-1) do not constitutively express mRNA for IL-13R{alpha}2 chain. MCF-7 cell line expresses mRNA for IL-13R{alpha}2 chain; however, the band intensity was weaker compared to positive control (PM-RCC cell line). PM-RCC is renal cell carcinoma cell line that has been shown to express IL-13R{alpha}2 chain (13). When breast cancer cell lines were transiently transfected (for 2 days) with IL-13R{alpha}2 cDNA, abundant levels of IL-13R{alpha}2 mRNA expression could be detected. Using control vector or IL-13R{alpha}2-transfected cell lines, protein synthesis inhibition activity of IL13-PE38QQR was examined in vitro. As shown in Fig. 1B, IL13-PE38QQR caused modest inhibition of protein synthesis in four breast cancer cell lines transfected with vector only at the highest concentration (1000 ng/ml) of the drug except that MCF-7 cell line showed moderate sensitivity to IL13-PE38QQR because these cells expressed low levels of IL-13R{alpha}2 mRNA. In contrast, when cells were transfected with IL-13R{alpha}2 cDNA, increased sensitivity to IL13-PE38QQR was observed in four of five cell lines. In ZR-75-1 cells, the effect of IL-13R{alpha}2 gene transfer was minimal, because IL13-PE38QQR caused higher inhibition of protein synthesis only at the highest concentration (1000 ng/ml) of the drug. The IC50 (the protein concentration required for the inhibition of protein synthesis by 50%) improved from >1000 to 100 ng/ml in MDA-MB-231, from 130 to 8.5 ng/ml in MCF-7, from >1000 to 52 ng/ml in SK-BR-3, and from 1000 to 320 ng/ml in ZR-75-1 cell line, respectively. Using MCF-7 cell line, we also confirmed that the protein synthesis inhibition activity of IL13-PE38QQR in IL-13R{alpha}2-transfected cells was blocked by an excess of IL-13 (2 µg), indicating that the activity mediated by this molecule is specific (data not shown).



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  		Figure 1. Breast cancer cell lines transfected with IL-13R{alpha}2 show increased sensitivity to protein synthesis inhibition activity of IL-13 cytotoxin in vitro. A, RT-PCR analysis for IL-13R{alpha}2 chain transcripts in breast cancer cell lines. Total RNA (2 µg) from five cell lines transfected with vector alone (Control) or IL-13R{alpha}2 cDNA was examined for the expression of IL-13R{alpha}2 mRNA by RT-PCR analysis. An equivalent amount of total RNA from renal cell carcinoma cell line (PM-RCC) served as a positive control (13). B, After transient transfection, cells were cultured with various concentrations of IL13-PE38QQR (0–1000 ng/ml). Points, means of quadruplicate determinations; bars, SD. The assay was repeated three times.

 
An Irrelevant Cytotoxin (IL4-PE) Does Not Mediate Protein Synthesis Inhibition in SK-BR-3 Breast Cancer Cells Transfected with IL-13R{alpha}2 Chain
We also examined the protein synthesis inhibition activity of an irrelevant cytotoxin, IL4(38-37)-PE38KDEL, which is comprised of circular permuted IL-4 and a mutated form of PE (43), in SK-BR-3 cells transfected with vector only or IL-13R{alpha}2. Previous studies have shown that SK-BR-3 cells are insensitive to this IL-4 cytotoxin (44). As shown in Fig. 2, IL4(38-37)-PE38KDEL only moderately mediated protein synthesis inhibition in cells transfected with vector or IL-13R{alpha}2 (right panel; IC50 > 1000 ng/ml), while IL13-PE38QQR showed profound protein synthesis inhibition activity in IL-13R{alpha}2-transfected cells compared to vector-transfected cells (left panel; IC50 = 52 ng/ml). PM-RCC cells were used as positive control (data not shown) and were found to be sensitive to both IL13-PE38QQR and IL4(38-37)-PE38KDEL as previously reported (23, 45). These data confirm that IL13-PE-mediated protein synthesis inhibition activity in breast cancer cells is IL-13R{alpha}2 specific.



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  		Figure 2. IL-4 cytotoxin did not mediate protein synthesis inhibition in SK-BR-3 cells transfected with IL-13R{alpha}2 cDNA. SK-BR-3 cells were cultured with various concentrations (0–1000 ng/ml) of IL13-PE38QQR (left panel) or IL4(38-37)-PE38KDEL (right panel). Points, means of quadruplicate determinations; bars, SD. The assay was repeated twice.

 
In Vivo Intratumoral Plasmid Injections Resulted in IL-13R{alpha}2 Chain Expression in Breast Tumors Subcutaneously Xenografted in Nude Mice
To assess the persistence of IL-13R{alpha}2 chain gene expression in vivo, MDA-MB-231 tumors growing in the flank of the nude mice were i.t. injected with plasmid vector encoding IL-13R{alpha}2 cDNA or vector only. Plasmids mixed with liposomes were injected on days 4 through 6 after tumor implantation (total of three injections). Tumors were subsequently resected at various time points after plasmid injection and subjected to RT-PCR and immunofluorescence microscopy analyses for IL-13R{alpha}2 gene expression. As shown in Fig. 3A, an optimal level of IL-13R{alpha}2 mRNA expression was evident until day 17, which then disappeared by day 21. These results were confirmed by immunofluorescence microscopy using monoclonal antibody to IL-13R{alpha}2 chain (Fig. 3B). IL-13R{alpha}2 chain protein expression was observed on day 7 (positive cells in entire section were 20–30%), decreased by day 17 (positive cells less than 10%), and undetectable on day 25. In control tumors injected with vector only, no positive cells were detected from day 7 through 25. These results suggest that gene expression of IL-13R{alpha}2 chain in breast tumors was maintained for up to 11–14 days at tumor site after three i.t. plasmid injections.



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  		Figure 3. IL-13R{alpha}2 mRNA and protein expression in MDA-MB-231 tumor tissues after plasmid-mediated gene transfer of IL-13R{alpha}2 chain. Subcutaneously xenografted 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 analysis using total RNA from tumors and PM-RCC cell line for a positive control. B, immunofluorescence assay using monoclonal antibody to IL-13R{alpha}2 chain (100x). Both RT-PCR and immunofluorescence assays were performed twice using duplicate sets of animals.

 
IL-13R{alpha}2 Transgene Expression in Vital Organs after Intratumoral Injections of Plasmid Vector
In vivo i.t. injection of IL-13R{alpha}2 encoding plasmid may cause migration and expression of transgene in distant vital organs resulting in organ toxicity when these mice are systemically treated with IL13-PE. To examine this possibility, animals bearing MDA-MB-231 tumors were i.t. injected with IL-13R{alpha}2 encoding plasmid on day 4, 5, and 6 after tumor implantation and vital organs including liver, lung, kidney, spleen, heart, and blood as well as tumors at various days were harvested and analyzed. Total RNA extracted from these samples was analyzed for IL-13R{alpha}2 mRNA expression. As shown in Fig. 4, IL-13R{alpha}2 mRNA was detected in all the organs except in blood cells on day 7, as assessed by sensitive RT-PCR. Although RT-PCR is not a quantitative assay, the IL-13R{alpha}2 mRNA level appears to decrease on day 9, disappearing by day 11, and on day 13 and beyond, no detectable transgene expression was observed in any organ.



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  		Figure 4. IL-13R{alpha}2 chain transgene expression in vital organs after i.t. plasmid injections. Nude mice bearing MDA-MB-231 tumors were given injections of IL-13R{alpha}2 cDNA on days 4, 5, and 6 after tumor implantation. 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-13R{alpha}2 mRNA expression. RNA from PM-RCC cells served as positive control for IL-13R{alpha}2.

 
Breast Tumors Xenografted in Animals Are Susceptible to in Vivo IL-13R{alpha}2 Gene Transfer Followed by Systemic IL-13 Cytotoxin Therapy
Because breast tumors s.c. growing in nude mice were able to express IL-13R{alpha}2 chain by i.t. plasmid injection, we evaluated the antitumor activity of IL13-PE in two breast tumor models. We developed MDA-MB-231 and MCF-7 tumors in both right and left flanks of nude mice. Animals then received i.t. injections of either vector only in the right flank tumor or IL-13R{alpha}2 plasmid in the left flank tumors on days 4, 5, and 6. MDA-MB-231 tumor-bearing animals were subsequently treated with i.p. IL13-PE38QQR [25 or 50 µg/kg twice a day (b.i.d.) for 5 days] from day 8 through day 12. Tumors injected with vector only (right flank) showed only limited response to IL13-PE38QQR treatment. As shown in Fig. 5A, by day 39, tumor growth in control (excipient only injected; 182 mm2) and IL13-PE38QQR-treated animals did not show a significant difference in tumor size in 25 µg/kg i.p. treated group (162 mm2, P = 0.09). However, statistically significant modest tumor regression was observed in 50 µg/kg treated mice (136 mm2, P < 0.0006 at day 39). Tumors injected with IL-13R{alpha}2 chain plasmid (left flank) began to regress during the treatment period in IL13-PE38QQR-treated animals. In mice treated with 25 µg/kg dose of IL13-PE38QQR, complete regression of tumors was not observed; however, the mean size of tumors measured on day 39 (88 mm2) was 49% reduced compared to control tumors (174 mm2) (P < 0.001). Animals receiving 50 µg/kg dose of IL13-PE38QQR showed better tumor response including complete disappearance of tumors in two of five mice by day 39 (mean tumor size 14 mm2; P < 0.0005 compared to control).



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  		Figure 5. Effect of IL-13R{alpha}2 chain gene transfer and systemic IL-13 cytotoxin therapy in breast cancer xenograft models. MDA-MB-231 (A) or MCF-7 (B) tumors were xenografted in both flanks of nude mice. Tumors in the left flank were injected with IL-13R{alpha}2 plasmid while those in the right flank with vector only by i.t. injection on days 4, 5, and 6 followed by i.p. IL13-PE38QQR administration on days as indicated by arrows. Each group had six animals and experiments were performed twice. Bars, SD.

 
MCF-7 tumor-bearing mice were treated with lower doses of i.p. IL13-PE38QQR administration [5, 15, or 25 µg/kg twice a day (b.i.d.) for 5 days] from day 8 through 12 and 15 through 19, after vector only injection (right flank) or IL-13R{alpha}2 encoding plasmid (left flank) injection i.t. on days 4, 5, and 6. Vector only-injected control tumors (right flank) grew to 86 mm2 by day 37, and 5 or 15 µg/kg doses of IL13-PE38QQR treatment did not affect tumor growth (Fig. 5B). Twenty-five micrograms per kilogram dose IL13-PE38QQR treatment demonstrated a modest effect on arresting tumor growth. In contrast, tumors receiving in vivo gene transfer of IL-13R{alpha}2 chain by plasmid injection (left flank) responded well to the antitumor effect of IL13-PE38QQR. Five micrograms per kilogram IL13-PE38QQR dose did not show antitumor effect; however, after 15 or 25 µg/kg doses (b.i.d. x 10 days) of i.p. IL13-PE38QQR administration, the growth of IL-13R{alpha}2-targeted tumors was profoundly inhibited in all mice. By the last day of the experiment (day 37), mean size of tumors was 42% (15 µg/kg dose; 49 mm2) or 75% (25 µg/kg dose; 21 mm2) smaller compared to control tumors (84 mm2) (P < 0.0005 at both doses). These results suggest that IL13-PE38QQR can show significant antitumor activity in breast tumors after in vivo IL-13R{alpha}2 gene transfer.

In Vivo IL-13R{alpha}2 Gene Transfer Followed by Intratumoral IL-13 Cytotoxin Treatment Demonstrates Complete Regression of Breast Cancer Xenografts in Nude Mice
We then assessed the antitumor effect of i.t. administration of IL13-PE38QQR after in vivo IL-13R{alpha}2 encoding plasmid injections. We again developed tumors in both right and left flanks of nude mice. Similar to experimental procedures in Fig. 5, animals received either vector only injections (right flank) or IL-13R{alpha}2 encoding plasmid injections (left flank) i.t. on days 4, 5, and 6. MDA-MB-231 or MCF-7 tumor-bearing animals were subsequently treated with i.t. IL13-PE38QQR [100 or 250 µg/kg a day (q.d.) for 5 days] from day 8 through day 12. The doses selected for these experiments were based on our previous findings that higher doses of IL13-PE38QQR can be administrated i.t. without organ toxicities and show better antitumor responses (25, 33, 34, 36–38). As shown in Fig. 6A, i.t. administration of IL-13PE38QQR in vector only-injected MDA-MB-231 tumors (right flank) showed a modest antitumor activity in both 100 and 250 µg/kg dose groups. Mean tumor size of 250 µg/kg dose group at day 39 was 88 mm2, which is 52% smaller than excipient only injected control tumors (182 mm2) (P < 0.0005). On the other hand, IL-13R{alpha}2 encoding plasmid injected tumors (left flank) showed highly improved sensitivity to the antitumor effect of IL13-PE38QQR. During the i.t. treatment with IL13-PE38QQR, all the tumors drastically regressed. By day 12, all the 12 tumors treated by either 100 or 250 µg/kg dose were completely invisible and nonpalpable. Some of the tumors appeared and slowly grew again; however, by day 39, mean tumor size was 86% (24 mm2; 100 µg/kg dose) or 93% (12 mm2; 250 µg/kg dose) smaller compared to control (P < 0.0005 in both doses). One of five animals in 100 µg/kg dose and three of five mice in 250 µg/kg dose showed complete regression of their established tumors.



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  		Figure 6. Effect of IL-13R{alpha}2 chain gene transfer and i.t. IL-13 cytotoxin therapy in breast cancer xenograft models. MDA-MB-231 (A) or MCF-7 (B) tumors were xenografted in both flanks of nude mice. Tumors in the left flank were injected with IL-13R{alpha}2 plasmid while those in the right flank with vector only by i.t. injection on days 4, 5, and 6. Mice then received i.t. administration of IL13-PE38QQR from day 8 to day 12. Each group had six animals and experiments were performed twice. Bars, SD; CR, complete regression.

 
We also treated MCF-7 tumor xenografted mice with a similar protocol of IL-13R{alpha}2 gene transfer followed by i.t. IL13-PE38QQR administration [100 µg/kg a day (q.d.) for 5 days]. As shown in Fig. 6B, IL13-PE38QQR showed some antitumor effect in vector only-injected tumors (right flank). Mean tumor size in treated group was 55 mm2 by day 37, which is 36% less compared to control (86 mm2; P < 0.0005). Similar to MDA-MB-231 tumors, MCF-7 tumors injected with IL-13R{alpha}2 encoding plasmid (left flank) were extremely sensitive to IL13-PE38QQR treatment. As a result of IL13-PE38QQR treatment, five of six tumors were completely disappeared by day 14. Although two tumors recurred, three of six animals remained tumor-free in their left flank by the end of the experiment (day 37). Mean tumor size at day 37 was 8 mm2, which is 93% smaller compared to control (P < 0.0005). These data indicate that IL-13R{alpha}2 i.t. injections followed by i.t. IL13-PE38QQR therapy causes dose-dependent regression of human breast tumors xenografted in nude mice.

Antitumor Activity of IL-13 Cytotoxin in MCF-7 Tumors Was Specific to in Vivo Gene-Transferred IL-13R{alpha}2 Chain
To assess whether improved antitumor activity of IL13-PE38QQR is due to gene transfer of IL-13R{alpha}2 chain, we also injected IL-13R{alpha}1 chain encoding plasmid into the MCF-7 tumor, instead of IL-13R{alpha}2 chain encoding plasmid. Plasmid vector encoding IL-13R{alpha}1, IL-13R{alpha}2, or vector only i.t. injected mice received i.p. administration of IL13-PE38QQR (25 µg/kg twice a day for 10 days). As shown in Fig. 7, tumors injected with IL-13R{alpha}2 cDNA responded very well to IL13-PE38QQR. By day 37, mean tumor size in these mice was significantly smaller (19 mm2) than control (P < 0.0005). On the other hand, IL-13R{alpha}1 cDNA-injected tumors did not show significant regression when compared to vector only-injected tumors. Mean tumor size in these animals (76 mm2) was similar to control by day 37 (P = 0.9). These results indicate that IL13-PE38QQR-mediated tumor killing in this therapeutic approach is strictly IL-13R{alpha}2 chain specific.



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  		Figure 7. IL-13 cytotoxin did not show antitumor activity in MCF-7 tumors injected with IL-13R{alpha}1 cDNA in vivo. Tumors implanted in nude mice were injected with plasmid vector encoding IL-13R{alpha}1 or IL-13R{alpha}2 chains by i.t. injection on days 4, 5, and 6 followed by i.p. IL13-PE38QQR administration on days as indicated by arrows. Each group had six animals and experiments were performed twice. Bars, SD.

 
Cellular Infiltration into Tumor Site Was Observed after IL-13R Targeted Therapy of Breast Cancer in Nude Mice
Although 20–30% of tumor cells were transfected with IL-13R{alpha}2 chain after in vivo i.t. injections, breast tumor growth was profoundly arrested by systemic or i.t. IL13-PE38QQR treatment. These findings indicate that other mechanism(s) may exist for bystander antitumor effect to untransfected tumor. To investigate this, s.c. xenografted MDA-MB-231 tumor-bearing mice were treated under the same protocol as shown in Fig. 5A. Tumors were resected on day 15, sections were prepared, and subjected to H&E staining and immunohistochemistry for infiltrating cells. Control tumors injected with vector alone followed by IL13-PE38QQR (i.p. 50 µg/kg dose) injection neither showed tumor regression nor tumor necrosis as assessed by H&E (Fig. 8A). On the other hand, central tumor necrosis was clearly observed after the same dose (50 µg/kg) of i.p. IL13-PE38QQR injection after IL-13R{alpha}2 gene transfer (Fig. 8B). Under higher magnification (Fig. 8C), infiltration of inflammatory cells was observed at the junction between necrosis and remaining tumor region. No infiltration of inflammatory cells was observed in control tumors injected with vector alone. To characterize these cells, we performed immunohistochemical staining from the same tissue block as shown in Fig. 8, B and C. Through these experiments, we observed moderate number of macrophage marker (F4/80) positive cells (Fig. 8D). Some macrophages were also stained with the maker for iNOS (M19) (Fig. 8E). Weakly positive NK1.1 cells were also found at tumor site (data not shown). To confirm whether iNOS was produced by macrophages, we co-stained the frozen sections from same tumor tissue block with F4/80 and M19 antibodies and assessed by fluorescence microscopy. Similar to the results shown in Fig. 8, D and E, tumor sections were stained with antibodies to macrophages (red; Fig. 8F) and iNOS (green; Fig. 8G). These two markers were found to co-localize in the same cells, as assessed by image merger (Fig. 8H). Because iNOS is an inducible enzyme necessary to produce nitric oxide (NO), these data indicate that NO-producing macrophages may, in part, participate in in vivo IL-13R{alpha}2 gene transfer and IL-13 cytotoxin therapy for breast cancer.



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  		Figure 8. Cellular infiltration in breast tumors injected with IL-13R{alpha}2 cDNA followed by IL-13 cytotoxin therapy. MDA-MB-231 breast tumors were established and treated as mentioned in legend to Fig. 5A. Tumors were resected 3 days after the completion of IL-13 cytotoxin treatment (day 15). H&E sections show tumors treated with i.p. IL13-PE38QQR (50 µg/kg dose) administration following either vector only (A) or IL-13R{alpha}2 encoding plasmid (B and C) injections. Same tumor specimens as B and C were subjected to immunohistochemistry using antibodies for macrophages (D) and iNOS (E). Arrows indicate stained cells. Results of immunohisochemistry experiments were confirmed by immunofluorescent microscopy using same tumor specimens double-stained with antibodies to macrophages (F) and iNOS (G). H, merged image of F and G. Original magnification: A and B, 100x; C–H, 600x. Experiments were performed using duplicate sets of animals with similar data.

 
Macrophage-Depleted Mice Exhibited Less Sensitivity to IL-13R{alpha}2 Gene Transfer Followed by IL-13 Cytotoxin Therapy
To assess whether NO-producing macrophages had an impact on the tumor regression mechanism, macrophage-depleted nude mice were used to evaluate the antitumor activity of IL-13R{alpha}2 gene transfer followed by IL-13 cytotoxin treatment. Mice given injections of MDA-MB-231 breast cancer cells s.c. (day 0) were given injections of carrageenan on days 3, 7, and 14. Three IL-13R{alpha}2 cDNA injections (on days 4, 5, and 6) and i.t. IL-13 cytotoxin treatment (50 µg/kg, once a day for 5 days from day 7 through 11) were performed. As shown in Fig. 9, IL-13 cytotoxin treatment caused tumor regression in both control and macrophage-depleted groups; however, the degree of tumor regression in macrophage-depleted mice was slightly less compared to control mice. By the termination of the experiment (day 42), mean tumor size in macrophage-depleted mice was larger (115 ± 34 mm2) compared with undepleted mice (79 ± 33 mm2) (P < 0.065). These results confirm that macrophages infiltrating into regressing tumors during IL-13R{alpha}2 gene transfer followed by IL-13 cytotoxin therapy play some role in the antitumor mechanism.



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  		Figure 9. Macrophage-depleted mice showed less sensitivity to the antitumor effect of IL-13R{alpha}2 gene transfer followed by IL-13 cytotoxin administration. Mice (n = 7/group) given s.c. injections of MDA-MB-231 breast cancer cells were then given i.p. injections of carrageenan (1 mg/injection) on days 3, 7, and 14. Established tumors received injections with IL-13R{alpha}2 cDNA followed by IL-13 cytotoxin administration from day 7 through 11 (50 µg/kg dose) as shown by arrows. Asterisk indicates P value of <0.065, comparing tumor sizes in macrophage-depleted versus not depleted mice on day 40.

 
Assessment of Toxicity in Mice Treated with Intratumoral IL-13R{alpha}2-Encoding Plasmid Followed by Local or Systemic IL13-PE Administration
We collected blood and major organs (heart, liver, lung, kidney, and spleen) from mice receiving i.t. IL-13R{alpha}2 plasmid followed by IL13-PE38QQR treatment either i.p. (50 µg/kg twice a day for 5 days in MDA-MB-231 xenografted mice) or i.t. (250 µg/kg a day for 5 days in MDA-MB-231 xenografted mice) on days 14 and 22 after tumor implantation. As shown in Table 1, blood serum chemistry analysis showed no remarkable changes in any parameters in all the mice tested except minor elevation of lactate dehydrogenase in some groups. Similarly, no hematological alterations were observed (data not shown). Organ toxicities were assessed by histological examination. A slight focal tubular necrosis and cell hypertrophy was observed in kidney from mice treated with i.t. IL13-PE38QQR, but, all other organs were free from any evidence of toxicity (data not shown). Organs from untreated control mice did not show any evidence of toxicities. All the treated mice tolerated therapy very well without visible toxicity.


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  		Table 1. Changes in blood serum chemistry after i.t. IL-13R{alpha}2 gene transfer followed by IL13-PE therapya

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IL-13R{alpha}2 chain has been shown to play a unique role in tumor biology. It is overexpressed in a variety of primary tumor cell cultures and tumor cell lines (13, 15–18, 25, 34), whereas normal cells including lymphoid cells, endothelial cells, and astrocytes (13, 15, 17, 26) do not express or express low levels of this cytokine receptor chain. Recent studies have demonstrated that overexpression of IL-13R{alpha}2 chain in certain breast and pancreatic cancer cell lines resulted into loss of tumorigenicity, whereas unmodified control tumor cells formed enlarging tumor nodules when injected in immunodeficient mice (46). In another study, Terabe et al. (47) have demonstrated that the soluble form of IL-13R{alpha}2 chain (5) can modulate immune environment and shift Th2 phenotype to a dominant Th1 phenotype. This shift resulted into resistance of 15-12RM cell-derived tumor recurrence. Thus, further investigation of the role of IL-13R{alpha}2 chain in tumor immunology and targeting has become extremely important.

In the current study, we investigated whether IL-13R{alpha}2 chain can be targeted in vivo by an IL-13 fusion protein for breast cancer theraphy. As IL-13R{alpha}2 chain binds IL-13 with high affinity and after binding the ligand-receptor complex is internalized inside the cell, we hypothesized that IL-13 cytotoxin will have potent antitumor activity against IL-13R{alpha}2-expressing cancer cells in vivo (28, 35). I.t. administration of a plasmid encoding IL-13R{alpha}2 chain resulted into expression of IL-13R{alpha}2 gene and protein for a prolonged period (>day 17 of tumor implantation) as determined by RT-PCR and immunofluorescence studies (Fig. 3). Although immunofluorescence studies are not quantitative, we chose this method because commercially available antibody to IL-13R{alpha}2 does not work in Western blot analysis. When tumor-bearing mice were given injections of IL-13R{alpha}2 plasmid i.t. followed by IL-13 cytotoxin administration by i.p. or i.t. routes, dramatic reduction of established tumors was observed. However, tumors injected with control plasmid vectors continued to grow vigorously without any sign of tumor response.

Interestingly, only limited number of breast tumor cells (20–30%) seem to be transfected with IL-13R{alpha}2 chain by plasmid-mediated gene transfer; however, a dramatic tumor regression was observed. The mechanism of dramatic tumor response could be attributed to bystander effect of dying tumor cells to adjoining viable tumors, or infiltration of "effector cells" at the tumor site as a result of IL-13R{alpha}2 gene transfer and IL-13 cytotoxin therapy. These "effector cells" may mediate direct or indirect cytotoxicity of remainder tumor cells. Although we did not observe direct bystander effect in vitro (data not shown), infiltration of cells including macrophages was observed in vivo. These macrophages were speculated to produce NO as they were positive for iNOS expression. These observations suggest that IL-13R{alpha}2 gene transfer in vivo followed by IL-13 cytotoxin administration results in direct cell death of IL-13R{alpha}2-transfected tumor cells, and in part recruitment of innate immune cells that eliminate "non-transfected" tumor cells. The role of NO-producing macrophages was further investigated by in vivo depletion of macrophages by carrageenan administration. Our results demonstrated that IL-13 cytotoxin treatment caused tumor regression in both control and macrophage-depleted IL-13R{alpha}2 plasmid-injected groups; however, the extent of tumor regression in macrophage-depleted mice was slightly less compared to control mice (Fig. 9). Thus, it is suggested that this approach can mediate tumor regression by two independent mechanisms: direct cell death and in part cell death mediated by innate immune activation. Innate immune response is one of the key mechanism of rejection or elimination of cancers in vivo (48, 49), and additional studies are currently ongoing in our laboratory to clearly understand the mechanisms involved in this therapeutic approach.

The safety of our approach of i.t. plasmid injection followed by local or systemic IL-13 cytotoxin administration was also investigated. The i.t. plasmid injection may lead to vector uptake by blood circulation and migration to vital organs resulting into undesirable toxicity. In fact, we observed IL-13R{alpha}2 mRNA expression in vital organs (liver, kidney, and spleen) after three i.t. vector injections. mRNA expression persisted in kidney and spleen for up to 5 days after plasmid injection (day 11 from tumor implantation). On the other hand, mRNA expression in tumors persisted at least 7 days after plasmid injection (day 13). As immunofluorescence assays of vital organs for IL-13R{alpha}2 expression could not be reliably investigated, we performed extensive toxicity studies to examine whether i.t. IL-13R{alpha}2 gene transfer followed by systemic or i.t. IL-13 cytotoxin injection mediated vital organ toxicities. Histological examination of vital organs did not show any detectable changes except in kidney from mice receiving three i.t. IL-13R{alpha}2 cDNA injections followed by i.t. IL-13 cytotoxin administration. Kidney samples showed slight focal necrosis and cell hypertrophy. However, all the animals remained healthy throughout the experimental period. Hematology and blood serum chemistry results also showed no remarkable parameter changes including CPK, AST, ALT, total bilirubin, and creatinine levels. These observations suggest that i.t. injection of plasmid may migrate to kidney and result into some IL-13R{alpha}2 protein expression, inducing susceptibility to IL-13 cytotoxin-mediated cytotoxicity but overall other major organs remained intact without any detectable damage. These results are in agreement with our previous study in which IL-13 cytotoxin at 100 µg kg–1 day–1 dose for 7 days did not cause changes in blood serum chemistry or organ toxicity (50). Thus, it is possible that our approach may be applied safely for breast cancer therapy in the clinic.

Because early breast cancer may be a localized disease, it is possible that IL-13R{alpha}2 gene may be injected under ultrasound guidance resulting in enhanced sensitivity to local or systemic IL-13 cytotoxin administration. However, in advanced breast cancer, tumor nodules are interspersed in abundant connective tissue, and it is not clear how transfection of IL-13R{alpha}2 followed by cytotoxin therapy would generate significant bystander effect by infiltrating macrophages or by other mechanisms. Therefore, additional strategies need to be devised for gene transfer in infiltrating tumor. One possible approach could be careful injection of IL-13R{alpha}2 plasmid in multiple lesions followed by local or systemic IL-13 cytotoxin therapy. This strategy may not only eliminate breast cancer cells burden and lessen the risk of "spilt-over" of cancer cells to other organs during necessary surgical mastectomy or lumpectomy but may also provide an additional curative impact on breast cancer therapy. Nevertheless, because we hereby show the proof-of principle that our approach may be a useful strategy for effective breast cancer therapy, further preclinical studies should be carefully planned. Finally, this treatment strategy will be useful for not only breast cancer but also for other localized and "accessible" cancers such as ovarian, prostate, head and neck, and malignant glioma.


   Acknowledgments
 
We thank Dr. Syed Rafat Husain for preparing liposomes, Dr. Takashi Murata for providing pME18s plasmids, and Pamela Dover for the procurement of reagents and laboratory supplies. We also thank Dr. Lee Pai-Scherf for critical reading of this manuscript.


   Footnotes
 
These studies were conducted as part of a collaboration between the FDA and NeoPharm Inc. under Cooperative Research and Development Agreement (CRADA).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3/12/03; revised 10/23/03; accepted 11/20/03.


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 Introduction
 Materials and Methods
 Results
 Discussion
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