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Keywords:

  • interleukin-13 cytotoxin;
  • pancreatic ductal adenocarcinoma;
  • IL-13Rα2;
  • gemcitabine;
  • orthotopic mice model

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Pancreatic cancer is an aggressive disease with only limited therapeutic options available. We have identified that 71% pancreatic ductal adenocarcinoma (PDA) express high levels of IL-13Rα2, a high-affinity receptor for IL-13. To target IL-13Rα2, we have developed a recombinant immunotoxin, which is a fusion of IL-13 and Pseudomonas exotoxin (IL-13-PE). Since IL-13-PE and a commonly used cytotoxic drug gemcitabine act by a different mechanism, we hypothesized that they synergize in mediating antitumor response. Both IL-13-PE and gemcitabine-mediated cytotoxicity to two pancreatic cancer cell lines and when combined synergistic cytotoxicity was observed. This synergism was also demonstrated in vivo in an orthotopic mouse model of human PDA. IL-13-PE and gemcitabine showed complete eradiation of tumors as assessed by whole body imaging of GFP-transfected tumors in 57% of mice in an early cancer model resulting into prolongation of survival. In contrast, monotherapy with either agent did not produce complete eradiation, but tumor volumes were significantly decreased. In advanced PDA model, combination therapy also produced dramatic reduction in tumor growth and enhanced survival compared to animals treated with either agent alone. When IL-13Rα2 was knocked-down by RNAi prior to tumor implantation, IL-13-PE and gemcitabine did not synergize indicating that IL-13Rα2 is essential. Mechanistically, gemcitabine increased IL-13Rα2 expression in vitro and in vivo, which resulted in a synergism of combination therapy. Interestingly, PDA cancer stem cells were resistant to gemcitabine, but not to IL-13-PE. These results suggest that combination therapy with IL-13-PE and gemcitabine may be a useful strategy for PDA therapy.

Pancreatic cancer is the fourth leading cause of cancer related deaths in the United States. In 2008, an estimated 37,680 people were diagnosed with pancreatic cancer in the United States, and ∼34,290 people died from this disease.1 Although many approaches are used to treat this malignancy, including surgery and chemotherapy, the median survival time (MST) after diagnosis is still 6 months in these patients.

For more than a decade, monotherapy with gemcitabine has remained a standard chemotherapy treatment option for patients with advanced pancreatic adenocarcinoma2 and more than 15 Phase 3 clinical trials have combined novel small molecules, cytotoxic or biologic agents, with gemcitabine to treat this disease. However, only two agents, capecitabine and erlotinib, have been found to prolong the medium overall survival of these patients by less than 1 month.3, 4 Therefore, effective new therapeutic approaches against this aggressive disease are urgently needed.

We have discovered a new therapeutic target in the form of IL-13Rα2 for cancer therapy, which is overexpressed in certain types of human cancers including glioblastoma, head and neck cancer, kidney cancer, ovarian cancer and Kaposi's sarcoma.5–8 IL-13Rα2 is one of the two receptor subunits of IL-13R complex and bind to IL-13. IL-13Rα1 is a low-affinity IL-13R but after binding to IL-13, it recruits IL-4Rα and forms a high-affinity IL-13R complex and activates JAK-STAT6 pathway.9 IL-13Rα2, on the other hand, binds IL-13 with high affinity but it does not activate JAK-STAT6 pathway.10 We have shown that upon binding to IL-13, IL-13Rα2 mediates signal transduction through AP-1 pathway and causes TGF-β production in murine macrophage cell line and human pancreatic ductal adenocarcinoma (PDA) cell lines.11, 12 Most recently, we have reported that IL-13 can enhance pancreatic cancer invasion and metastasis through IL-13Rα2 by increasing matrix metalloproteinase production.13

To target IL-13R, a recombinant fusion IL-13 immunotoxin termed as IL-13-PE, consisting of IL-13 and a mutated form of Pseudomonas exotoxin (PE), has been developed.7, 14, 15 This chimeric protein irreversibly ADP-ribosylates the diphthamide residue of elongation factor 2, using NAD+ as a cofactor, and inhibits protein synthesis and causes cell death.11, 12 IL-13-PE has potent antitumor activity in IL-13R-expressing tumor cells in vitro and in vivo animal models with unique specificity and limited toxicity to normal tissues.7, 14–18 A bispecific cytotoxin that consisted of diphtheria toxin, epidermal growth factor and IL-13 is shown to be effective in inducing tumor cell killing in vitro and in vivo against the MIA-PaCa2 pancreatic cell line.19 In a recent study, we have demonstrated that the IL-13-PE treatment is effective in an orthotopic tumor model of human pancreatic cancer, prolonging the MST more than two times compared to control mice.12

In the current study, to improve antitumor activity of IL-13-PE, we have combined IL-13-PE with gemcitabine in vitro and in vivo in an animal model of human pancreatic cancer. Our results show, in two animal models, that IL-13-PE synergizes with gemcitabine and significantly inhibits the tumor growth, prolongs the survival time and completely eradicates tumors in 57% of mice in the early pancreatic cancer model. Results also show a remarkable antitumor impact in the advanced pancreatic cancer model. We also investigated the effect of IL-13-PE and gemcitabine on cancer stem cells expressing CD24, CD44, and CD133 and markers of tumor aggressiveness [aldehyde dehydrogenase (ALDH1)], angiogenesis (CD31) as well as for apoptosis (caspase 3) and cell proliferation (Ki-67) in tumor specimens.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cell culture, reagents and tissue specimens

Pancreatic cancer cell lines (HS766T and MIA-PaCa2) were obtained from the American Type Culture Collection. IL-13Rα2 positive cell line (PM-RCC) was developed from a renal cell carcinoma in our laboratory. IL-13-PE was produced as described previously.14, 20 Gemcitabine was obtained through the pharmacy of the NIH Clinical Center (Bethesda, MD).

Immunohistochemistry

Immunohistochemistry (IHC) was performed as described previously.21 Deparaffinized tissue sections were incubated for 2 hr at room temperature with anti-human IL-13Rα2 polyclonal antibody at 1 μg/ml (R&D systems, Minneapolis, MN) for overnight at 4°C with anticleaved-caspase 3 antibody (BD, Franklin Lakes, NJ), anti-Ki-67 antibody (Dako, Carpinteria, CA), anti-ALDH1 (BD), anti-CD31 antibody (Santa Cruz, CA), anti-CD44 antibody (Millipore, Billerica, MA), anti-CD24 antibody (Millipore, Billerica, MA) and anti-CD133 antibody (Santa Cruz) or isotype control (IgG) at the same concentration. IL-13Rα2, cleaved-caspase 3 and CD44 are visualized by alexa488 (Invitrogen, Carlsbad, CA). Ki-67 and CD24 are visualized by alexa555 (Invitrogen). The number of cells expressing cleaved caspase 3 and Ki-67 in the total cell number was counted in three fields at 200× magnification. The number of cells expressing CD44 and CD24 in five fields was scored at 400× magnification.

Protein synthesis inhibition assay and assessment of synergism or antagonism

The in vitro cytotoxic activity of IL-13-PE, gemcitabine and their combination was measured by the inhibition of protein synthesis as described previously.12

The synergistic effect of IL-13 cytotoxin and gemcitabine was assessed at a concentration ratio of 1:1, using the combination index (CI), where CI < 1, CI = 1 and CI > 1 indicate synergistic, additive and antagonistic effects, respectively.22 On the basis of the isobologram analysis for mutually exclusive effects, the CI value was calculated as follows:

  • equation image

where (Dx)1 and (Dx)2 are the concentrations of IL-13-PE and gemcitabine, respectively, required to inhibit cell growth by 50%, and (D)1 and (D)2 are the drug concentrations in combination treatments that also inhibit cell growth by 50% (isoeffective as compared with the single drugs).

Real-time RT-PCR

Real-time RT-PCR was performed as described previously.13 Quantification of IL-13Rα2 mRNA expression levels in pancreatic cancer cell lines was determined by real-time RT-PCR using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). Gene expression was normalized to β-actin before the fold change in gene expression was calculated.

Apoptosis detection by TUNEL staining in vitro

HS766T and MIA-PaCa2 cells (0.5 × 106) were plated on chamber slides and incubated with medium only (control), IL-13-PE, gemcitabine or combination of IL-13-PE and gemcitabine for 24 hr. Cells were then washed and fixed with 1% paraformaldehyde in PBS, and stained with the TUNEL-based apoptosis detection kit (Millipore) as per manufacturer's instructions.23 Apoptotic cells were assessed and measured by fluorescent microscopy.

Stable transfection in pancreatic cancer cells

HS766T and MIA-PaCa2 cells expressing GFP were established by culturing with retroviral supernatant derived from HEK293 cells, as described previously.12 For IL-13Rα2 knockdown, retrovirus-mediated RNA interference was performed using the pSuper RNAi system (Oligoengine, Seattle, WA) following the manufacturer's instructions as described previously.13

Animals

Nude nu/nu mice between age 5 and 6 weeks were maintained in a barrier facility on HEPA-filtered racks. All animal studies were conducted under an approved protocol in accordance with the principles and procedures outlined in the NIH Guideline for the Care and Use of Laboratory Animals.

Whole-body imaging

The tumor-bearing mice were periodically examined in a fluorescence light box illuminated by fiberoptic light at 440/20 nm wavelength (Lightools Research, Encinitas, CA). Emitted fluorescence was collected through a long-pass filter GG475 (Chroma Technology, Rockingham, VT) on a Hamamatsu C5810 3-chip cooled color charge coupled device camera (Hamamatsu Photonics Systems, Shizuoka, Japan). Real-time determination of tumor burden was done by quantifying fluorescent surface area as described previously.12

Surgical orthotopic implantation of HS766T-GFP and MIA-PaCa2-GFP tumors

Cells were injected subcutaneously into the right dorsal flank of nude mice. Pancreatic tumors, grown subcutaneously in nude mice, were minced into ∼3 × 3 × 3 mm3 pieces. For orthotopic surgery, the pancreas was carefully exposed, and tumor chunks were transplanted onto the middle of the pancreas with a 6-0 Dexon surgical suture (Tyco Healthcare, Mansfield, MA). The pancreas was then returned to the peritoneal cavity. The abdominal wall and the skin were closed with surgical clips.

Experimental design and treatment

For an early pancreatic cancer model, primary tumor lesions were detected by external whole-body imaging on day 4 after tumor implantation. Once the tumors were visualized, mice were randomized into four groups of seven mice each. Treatment was initiated on day 5 and IL-13-PE was administrated by bolus intraperitoneal infusion (i.p.) twice a day (100 μg/kg/day for 14 days) or continuous infusion at the same dose using a mini-osmotic pump (ALZET, Palo Alto, CA), which was inserted into the mice peritoneal cavity. In an advanced pancreatic cancer model, mice were randomized into six groups of seven mice each and treated from day 29. In an IL-13-PE low dose model, mice were randomized into four groups of seven mice each and treated with lower-dose of IL-13-PE (25 μg/kg/day for 14 days) and gemcitabine from day 5. In an IL-13Rα2-silenced HS766T tumor model and an MIA-PaCa2 early cancer model, mice were randomized into four groups of six mice each and treated similar to HS766T early cancer model.

Statistical analysis

The mean tumor volume in therapeutic and control groups was analyzed by ANOVA. Survival curves were generated by the Kaplan–Meier method and compared by using the log-rank test.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Expression of IL-13Rα2 in pancreatic cancer cell lines

HS766T and MIA-PaCa2 pancreatic cancer cell lines used in this study naturally express IL-13Rα2, which was silenced by RNAi to examine the effect and a relationship between IL-13-PE and IL-13Rα2. The expression of IL-13Rα2 after RNAi knockdown was confirmed by RT-PCR and immunofluorescence assays (Fig. 1a and data not shown). Both analyses showed markedly decreased expression of IL-13Rα2 in HS766T and MIA-PaCa2 cell lines. IL-13Rα2-silenced HS766T and MIA-PaCa2 cells expressed IL-13Rα2 mRNA at only <10% of its original expression (Fig. 1a). In contrast, the mRNA for two other subunits of IL-13Rα1 and IL-4Rα, were unaltered in these cell lines (data not shown and Fig. 1a).

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Figure 1. IL-13Rα2 expression in pancreatic cancer cell lines and cytotoxicity to gemcitabine and IL-13-PE. (a) RT-PCR shows IL-13Rα2 expression in parent, IL-13Rα2-silenced and mock HS766T and MIA-PaCa2 cells. Renal cell carcinoma cell line, PM-RCC, was used as a positive control. (b) Cytotoxic activity of combination treatment with IL-13-PE and gemcitabine in HS766T cells. Cells (1 × 104) were incubated with various concentrations of IL-13-PE (0–100 ng/ml) and gemcitabine (0–100 nmol/l). (c) Cytotoxic activity of combination treatment with IL-13-PE and gemcitabine in MIA-PaCa2 cells. (d) Cytotoxic activity of IL-13-PE alone in mock and IL-13Rα2-silenced HS766T and MIA-PaCa2 cells. Bars, SD of quadruplicate determinations.

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IL-13 cytotoxin alone and in combination with gemcitabine is cytotoxic to IL-13Rα2 expressing cells

We determined the sensitivity of pancreatic cancer cell lines to IL-13-PE by a protein synthesis inhibition assay. IL-13-PE inhibited protein synthesis of HS766T and MIA-PaCa2 cells in a concentration-dependent manner with IC50 of 1.1 ng/ml in HS766T cells and 51 ng/ml in MIA-PaCa2 cells (Figs. 1b and 1c). Gemcitabine also mediated a dose-dependent inhibition of protein synthesis with IC50 of 19 nmol/l in HS766T cells and 98 nmol/l in MIA-PaCa2 cells. When IL-13-PE was combined with gemcitabine, the protein synthesis inhibition was remarkably enhanced compared to any single agents. IC50 of IL-13-PE in HS766T cells was improved to 0.40, 0.27, 0.14 of 0.014 ng/ml by addition of 0.01, 0.1, 1 and 10 nmol/l gemcitabine, respectively. The IC50 of IL-13-PE in MIA-PaCa2 cells was also improved to 40, 21, 0.7 and 0.01 ng/ml by addition of 0.1, 1, 10 and 100 nmol/l of gemcitabine, respectively. The combination index (CI) at IC50 in HS766T and MIA-PaCa2 cells was <1 at all concentrations of gemcitabine (Table 1). Inhibition of protein synthesis by IL-13-PE was not observed in IL-13Rα2-silenced HS766T and MIA-PaCa2 cells (IC50 >1,000 ng/ml) (Fig. 1d). In our previous studies, we have reported that IL-13-PE has no or very low cytotoxicity to noncancerous normal cell lines such as endothelial cells, pancreatic ductal epithelial cells, fibroblasts and lymphocytes, as they do not express IL-13Rα2.7, 8, 12 The IC50 of IL-13-PE >1,000 ng/ml was observed in these normal cells. In addition, we have observed that the excess of IL-13 neutralizes the cytotoxic activity of IL-13-PE to HS766T and MIA-PaCa2 cell lines (data not shown). These results indicate that IL-13-PE can kill pancreatic cancer cells synergistically with gemcitabine and with highest specificity.

Table 1. Cytotoxicity of IL-13-PE, gemcitabine and their combination in pancreatic cancer cell lines
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Complete eradication of tumors by combination therapy with IL-13-PE and gemcitabine in an early pancreatic cancer model

The effect of IL-13-PE and gemcitabine combination therapy in an orthotopic pancreatic cancer mouse model was assessed by real-time whole-body imaging (Fig. 2a). In an early cancer model, single therapy with IL-13-PE or gemcitabine decreased tumor size, but no tumor was eliminated by these treatments (Fig. 2b). IL-13-PE was administrated by bolus intraperitoneal infusion or continuous infusion using an osmotic pump. Mice receiving continuous IL-13-PE exhibited better tumor response compared to bolus administration (p = 0.0074 on day 98). However, they did not show statistically significant improvement of survival compared to mice receiving bolus intraperitoneal IL-13-PE administration (Figs. 2b and 2c). Combination therapy with IL-13-PE and gemcitabine strongly suppressed tumor growth, and tumor lesions were undetectable in four of seven mice treated with gemcitabine and IL-13-PE by bolus infusion and in six of seven mice treated with gemcitabine and IL-13-PE pump infusion on day 21. Four of seven mice treated with gemcitabine and IL-13-PE pump infusion remained tumor free throughout the course of the study.

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Figure 2. Sequential whole-body imaging and real-time analysis of pancreatic tumor growth in an early cancer model using HS766T cells. (a) Sequential in vivo imaging of tumor progression over time in early pancreatic cancer mice model using HS766T cells is shown. Selective tumor GFP fluorescence facilitated real-time visualization of tumor burden in the live animal. Panels depict a representative mouse from each six groups. Treatment plan and imaging strategy are described in “Material and methods” section. (b) Quantification of pancreatic tumor GFP fluorescence enabled real-time determination and comparison of tumor load during the course of each treatment and, therefore, permitted real-time comparison of treatment efficacy between groups. Points, mean area of GFP fluorescence in each treatment group (n = 7 mice); bars, SD. (c) Kaplan–Meier survival curves of HS766T early cancer model mice after tumor implantation.

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Synergistic increase in survival of mice treated with combination of IL-13-PE and gemcitabine in an early cancer model

As shown in Figure 2c, MST of animals was 156 days in the no treatment group, whereas it was significantly increased to 212, 218, 232, 274 and >300 days in IL-13-PE bolus group (p = 0.0068), IL-13-PE pump group (p = 0.0013), gemcitabine group (p = 0.0002) and combination with gemcitabine and IL-13-PE group (p < 0.0001), respectively. Increase in significant survival difference correlated with tumor area as detected by whole-body imaging. We monitored the body weight of mice and their general conditions throughout the experimental period to detect any adverse effects caused by the treatment. However, no loss of body weight or change in general conditions were observed (data not shown). In addition, we observed no organ toxicity in vital organs such as the liver, heart, lung, kidney and spleen of IL-13-PE and gemcitabine-treated mice evaluated by histological examination (Supporting Information Fig. 1).

Combination therapy with gemcitabine and IL-13-PE strongly suppresses tumor growth even in an advanced cancer model

To mimic the clinical situation, we examined the antitumor effect of combination therapy with gemcitabine and IL-13-PE in animals with HS766T pancreatic tumors. Mice began receiving therapy starting day 29 when the average fluorescent area of tumor was 41 mm2. In all treatment groups, tumors were significantly regressed compared to the no treatment group (Fig. 3a). However, no tumor was completely eradiated even in combination therapy with the gemcitabine and IL-13-PE continuous treatment group.

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Figure 3. Real-time analysis of pancreatic tumor growth in an advanced cancer model, and IL-13Rα2-silenced tumor model using HS766T cells. (a) Quantification of pancreatic tumor GFP fluorescence in an advanced cancer model using HS766T cells is shown. Mice were randomly divided into six groups, which were same as the early cancer model. Treatment was initiated on day 29 after tumor implantation. Tumor sizes were measured every 14 or 21 days by scanning fluorescence area. (b) Quantification of pancreatic tumor GFP fluorescence in an IL-13-PE low-dose model using HS766T cells. (c) Quantification of pancreatic tumor GFP fluorescence in an IL-13Rα2-silenced tumor model using HS766T cells. IL-13Rα2-silenced HS766T tumor expressing GFP protein was implanted in pancreas of nude mice. Mice were randomly divided into four groups, which were same as an IL-13-PE low-dose model. Each treatment was initiated on day 5. Points, mean area of GFP fluorescence (n = 7 mice); bars, SD. Statistical significances are shown by *p < 0.05 and p < 0.001.

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We also examined the antitumor effect of combination therapy in an early pancreatic cancer model (therapy starting on day 14) with a suboptimal dose of IL-13-PE (25 μg/kg/day for 14 days) and an optimal dose of gemcitabine. Low-dose IL-13-PE alone, by continuous intraperitoneal administration decreased tumor size compared to the no treatment group (Fig. 3b). In comparison, gemcitabine at the optimal dose showed better tumor response. However, best tumor response was observed using combination therapy with optimal dose of gemcitabine and low dose of IL-13-PE by pump.

We confirmed these results by developing tumors and treating them with IL-13-PE using IL-13Rα2-silenced HS766T pancreatic cancer cells. An IL-13Rα2-silenced HS766T tumor was also implanted onto a pancreas and antitumor effects were monitored as above (Fig. 3c). Even though IL-13-PE showed no effect against the IL-13Rα2-silenced tumor, gemcitabine alone and a combination of gemcitabine and IL-13-PE significantly decreased tumor size. Interestingly, combination therapy with gemcitabine and IL-13-PE showed slightly better antitumor effect than gemcitabine alone (p = 0.0077 on day 105).

We further confirmed the antitumor effect of combination therapy using gemcitabine and IL-13-PE using another pancreatic cancer model developed by MIA-PaCa2 cells, which expressed IL-13Rα2, but its expression was weaker than HS766T cells. In this model, even though single therapy with IL-13-PE or gemcitabine significantly decreased MIA-PaCa2 tumor size compared to the no treatment group, combination therapy with gemcitabine and IL-13-PE more strongly inhibited tumor growth compared to monotherapy with either agent (Figs. 4a and 4b). Monotherapy with IL-13-PE and gemcitabine prolonged MST from 60 to 78 days and 84 days, respectively. However, combination therapy with gemcitabine and IL-13-PE prolonged MST (105 days) more than each single therapy (p = 0.005 to IL-13-PE and 0.012 to gemcitabine) (Fig. 4c).

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Figure 4. Sequential whole-body imaging and real-time analysis of pancreatic tumor growth in an early cancer model using MIA-PaCa2 cells. (a) Sequential in vivo imaging of tumor progression over time in early pancreatic cancer mice model using MIA-PaCa2 cells. Panels depict a representative mouse from each of the four groups. MIA-PaCa2 tumor expressing GFP protein was implanted in pancreas of nude mice. Treatment was initiated on day 5. Images are captured every 10 days beginning on day 4. (b) Quantification of pancreatic tumor GFP fluorescence in the early cancer model using MIA-PaCa2 cells. Points, mean area of GFP fluorescence (n = 6 mice); bars, SD. (c) Kaplan–Meier survival curves of MIA-PaCa2 early cancer model mice after tumor implantation.

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Gemcitabine increases IL-13Rα2 expression in pancreatic cancer cells in vitro and in vivo

In the advanced cancer model, primary tumors were resected from each group after treatment (day 70) and IL-13Rα2 expression was determined by real-time RT-PCR (Fig. 5a). IL-13-PE pump infusion significantly decreased IL-13Rα2 mRNA expression in primary tumors (p < 0.001). However, in sharp contrast, gemcitabine treatment increased IL-13Rα2 expression in primary tumors (p < 0.05). Tumors from mice treated with both gemcitabine and IL-13-PE showed a decrease in mRNA expression compared to gemcitabine alone (p < 0.001). To confirm in vivo observations, HS766T cells were cultured in vitro with gemcitabine and IL-13Rα2 expression was examined at various time points (Supporting Information Fig. 2). Gemcitabine significantly increased IL-13Rα2 mRNA expression in a time-dependent manner plateauing at 24 hr.

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Figure 5. Analysis of IL-13Rα2 expression, apoptosis, cell proliferation and cancer stem cell markers in the primary tumors derived from murine tumor model and cultured pancreatic cancer cells after treatment with IL-13-PE and gemcitabine. (a) Real-time RT-PCR showed IL-13Rα2 expression in mice with HS766T pancreatic tumor after IL-13-PE and gemcitabine treatment. In advanced cancer model, primary tumor and lymph node metastasis were resected from each group after any treatment (day 70). Data showed the ratio of IL-13Rα2/β-actin expression. Bars, SD of quadruplicate determinations. Statistical significances are shown by *p < 0.05, and p < 0.001. (b) TUNEL assay in HS766T and MIA-PaCa2 cells treated with IL-13-PE, gemcitabine and their combination. Cells were seeded on the chamber slide and treated with IL-13-PE, gemcitabine and their combination for 24 hr. After fixation, cells were stained with ApopTag Fluorescein. Percentage of apoptotic cells in HS766T and MIA-PaCa2. Bars, mean ± SD of quadruplicate determination. (c) IHC analysis of cleaved caspase 3 and Ki-67 as markers of apoptosis and cell proliferation. Cleaved caspase 3 (green) and Ki-67 (red) were stained in the tumor tissues obtained from no treatment, pump IL-13-PE, gemcitabine and pump combination group. The percent of positive cells were counted in these specimens. (d) IHC of CD44 and CD24 as markers of cancer stem cells. CD44 and CD24 were stained by alexa 488 and alexa 555, respectively; in the same tumors as in (c). The number of positive cells in five fields was counted.

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Combination therapy with gemcitabine and IL-13-PE induces apoptosis and suppresses cell proliferation

HS766T and MIA-PaCa2 cells were cultured with IL-13-PE (0.1 ng/ml in HS766T and 1 ng/ml in MIA-PaCa2), gemcitabine (0.1 and 1 mmol/l, respectively), and combination. Then apoptosis was assessed by TUNEL assay (Fig. 5b). IL-13-PE and gemcitabine both induced modest apoptosis in both pancreatic cancer cell lines. However, when both IL-13-PE and gemcitabine were combined, a marked increased in apoptosis was observed indicating synergistic effect. Cell apoptosis and proliferation were also examined in the primary tumor tissues obtained from the advanced cancer model by IHC. This was done by staining for cleaved-caspase 3 and Ki-67 in tumor sections derived from no treatment, pump IL-13-PE, gemcitabine and pump combination therapy groups. Similar to in vitro results, IL-13-PE and gemcitabine both induced modest apoptosis in the primary tumor. However, combination therapy showed marked increased in apoptosis compared to the individual therapy. Moreover, combination therapy drastically decreased the positivity of cell proliferation index, Ki-67 marker, compared to individual therapy (Fig. 5c).

IL-13-PE but not gemcitabine treatment inhibits cancer stem cell population

Next, we examined the effect of IL-13-PE and gemcitabine to eliminate cancer stem cells by performing IHC assay. As shown in Figure 5d, IL-13-PE did not change the number of CD44+/CD24 cells but in gemcitabine group, CD44+/CD24 cells increased. In addition, CD44+/CD24 cells increased in combination therapy of IL-13-PE and gemcitabine. We did not detect CD44+/CD24+ double positive cells in these specimens and the number of CD44/CD24+ was not changed by any of these treatments. We also performed double immunostaining for CD44 and CD133 in these tumor specimens and did not find appreciable double positive staining cells (data not shown). In addition, no definite staining was observed when tumor tissue sections were stained with CD31 and ALDH1 antibodies (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In our study, we demonstrate antitumor effect of IL-13-PE in pancreatic cancer models and its synergistic antitumor effect with gemcitabine, an approved drug for pancreatic cancer therapy. We demonstrate the antitumor effect of combination therapy in vitro as well as in two different animal models of human pancreatic tumors that were implanted onto the animal pancreas. In the early disease model, gemcitabine along with continuous infusion of IL-13-PE produced almost complete eradication of established tumors. Similarly, in the advanced disease model, a combination of both produced longest survival of animals. Gemcitabine as a monotherapy has been shown to prolong the survival of pancreatic cancer patients by 1 month.2 In addition, gemcitabine has also been tested in combination with chemotherapeutic drugs and small molecules, cisplatin, fluvastatin and gefitinib, in animal models.24–26 However, in most cases these approaches have not made any significant impact on this disease in clinical trials.27–30 Various chemotherapeutic drugs have been combined. For example, doxorubicin has been combined with anti-B4-blocked ricin, Ara-C with granulocyte macrophage colony-stimulating factor fused to truncated diphtheria toxin, and fludarabine with rituximab/saporin-S6 conjugated protein for leukemia and lymphoma.31–33 In addition, we have reported synergistic antitumor effects of gemcitabine and IL-4-PE, which is an immunotoxin consisting of IL-4 and a mutated form of PE.22 Although IL-4-PE strongly inhibited pancreatic cancer growth, combination therapy with gemcitabine and IL-4-PE synergistically suppressed tumor growth and prolonged survival more than each single therapy in an orthotopic mice model. These studies support our observation of gemcitabine synergizing with IL-13-PE.

The antitumor effect of IL-13-PE was mediated through the expression of IL-13Rα2, as in vitro cytotoxicity assay, cytotoxicity was abrogated when IL-13Rα2 was knocked down by RNAi. The mechanism of synergistic effect of gemcitabine and IL-13-PE was also investigated. Gemcitabine was found to increase IL-13Rα2 expression in vitro as well as in vivo in pancreatic cancer samples. These results suggest that gemcitabine can enhance the antitumor effect of IL-13-PE, which may be one of the reasons why gemcitabine showed synergistic antitumor effect with IL-13-PE even in pancreatic cancer cell lines expressing low levels of IL-13Rα2. Both IL-13-PE and gemcitabine caused apoptosis and inhibited cell proliferation in tumors derived from animals with orthotopic model. However, a significant increase in apoptosis and less proliferation was observed in tumors derived from mice treated with a combination therapy compared to mice treated with either gemcitabine or IL-13-PE alone.

Cancer stem cells are hypothesized to be resistant to conventional chemotherapy and thought to cause metastasis and cancer recurrence after clinical remission. In pancreatic cancer, CD44 and CD24 are well-known cell surface markers, which are expressed on pancreatic cancer stem cells.34 In addition, several studies have reported that a small subpopulation of cells bearing CD44+/CD24 show high tumorigenicity.35 In our study, CD44+/CD24 population, but not CD44/CD24+ population continue to proliferate in gemcitabine group. Our findings confirm previously published results by Hong et al. that CD44+ cells have a resistance to gemcitabine treatment.36 Interestingly, CD44+ population did not increase in IL-13-PE group, which suggests that IL-13-PE killed the CD44+ cells similar to the CD44 cells. Thus, this study demonstrates a novel role of IL-13-PE in eradicating cancer stem cells.

IL-13-PE has been administrated intracranially to patients with glioblastoma, with no extracranial toxicity.37 In addition, IL-13-PE has been administrated to patients with advanced renal cell carcinoma.38 A maximum tolerated dosage of 2 μg/kg IL-13-PE given intravenously every alternate day, has been determined. Our current study provides a proof of concept for the clinical study. The combination of IL-13-PE administration by two different routes of drug delivery, along with gemcitabine was well tolerated and no visible evidence of toxicity observed in animals. Hence, it is possible that this approach will produce a similar safety profile in the clinical trial. Therefore, a combination of gemcitabine with IL-13-PE may be tested in patients with PDA.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Drs. Robert Aksamit and Brenton McCright for reviewing the manuscript and members of Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research for their suggestions.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Cartwright T, Richards DA, Boehm KA. Cancer of the pancreas: are we making progress? A review of studies in the US Oncology Research Network. Cancer Control 2008; 15: 30813.
  • 2
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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IJC_25437_sm_SuppFig1.tif13869KSupporting Information Figure 1.

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