Interleukin-10 gene transfer to peritoneal mesothelial cells suppresses peritoneal dissemination of gastric cancer cells due to a persistently high concentration in the peritoneal cavity

Abstract

Interleukin (IL)-10 has potent biological properties including an inhibitory action on the proliferation and metastasis of various cancer cells. However, it is difficult to maintain a high concentration of this cytokine as it has a short half life. In this study, we evaluated whether peritoneal mesothelial cells (PMCs) could be suitable for maintaining a high concentration of IL-10 using adenoviral gene transfer. We also evaluated the therapeutic effects of an intraperitoneal injection with adenoviral vector containing mouse IL-10 gene (Ad-mIL-10) using a mouse peritoneal dissemination model of MKN45 gastric cancer cells. We demonstrated that in vitro transfection efficiency of a recombinant adenovirus containing the bacterial β-galactosidase gene (Ad-LacZ) was approximately 10-fold higher for primarily isolated PMCs than MKN45. The entire peritoneum was transfected until 3 weeks after an intraperitoneal Ad-LacZ injection. Ad-mIL-10 treatment increased intraperitoneal IL-10 levels until 3 weeks after treatment, and then significantly inhibited peritoneal cancer growth by inhibiting angiogenesis. This treatment also improved cachexia and prolonged mice survival. We thus concluded that IL-10 gene transfer in PMCs could be a new strategy for the prevention of peritoneal dissemination of gastric cancer due to the resulting persistently high IL-10 concentration in the peritoneal cavity.

Introduction

Interleukin (IL)-10 was first described as a cytokine synthesis inhibitor factor secreted by murine Th2 clones of helper T cells that suppressed cytokine production by Th1 clones.1 IL-10 reduces the antigen-presenting capacities of monocytes via downregulation of class II major histocompatibility complex expression, and inhibits the production of proinflammatory cytokines, including IL-1, IL-6, IL-8 and tumor necrosis factor (TNF)-α by monocytes.2, 3, 4 Thus, IL-10 must be one of the key mediators of both the systemic and local immune systems. However, IL-10 also acts in an inhibitory capacity against tumor growth and metastasis. IL-10 inhibits angiogenesis in various cancers, such as melanoma, Burkitt's lymphoma and ovarian cancer.5, 6, 7 Its inhibitory effect is mediated by the stimulation of tissue inhibitors of metalloprotease and inhibition of matrix metalloprotease secretion in prostate tumor cells.8 These antitumor mechanisms have been found to be dependent upon immune cells such as natural killer (NK) cells, cytotoxic T lymphocytes (CTLs) and macrophages.9, 10, 11, 12, 13, 14 Unfortunately, however, the half life of IL-10 is approximately 20 min and it is difficult to maintain a high concentration after administration of IL-10 alone.15, 16, 17

Gastric cancer is one of the most common malignancies in the world, especially in Eastern Asia. Peritoneal dissemination is a common feature of metastasis associated with advanced gastric cancer and is associated with poor prognosis in this disease.18 The peritoneum is composed of a single layer of peritoneal mesothelial cells (PMCs) supported on a connective tissue matrix. When disseminated, it forms the host tissue for cancer cells within the peritoneal cavity. The peritoneum covers the entire interior surface of the abdominal wall and the diaphragmatic, retroperitoneal and pelvic surfaces that comprise the peritoneal cavity. PMCs play a pivotal role in a number of peritoneal events, including transperitoneal transport, peritoneal lubrication and inflammation/host defense.19 PMCs are abundant in the peritoneal cavity and have the potential to produce many cytokines, such as IL-1, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), and stromal cell-derived factor-1 (SDF-1).20, 21, 22, 23, 24, 25 Thus, PMCs may be involved in peritoneal metastasis. However, the precise role of these cells has not been fully investigated. A recent study demonstrated the possibility that PMCs could become target cells of adenovirus-mediated gene transfer for the production of numerous gene products in the peritoneal cavity.26 Therefore, PMCs may be a candidate for the continuous production of IL-10 by gene transfer to maintain a continuously high concentration in the peritoneal cavity.

In the present study, we investigated whether PMCs could be the source for continuous IL-10 production in the peritoneal cavity via adenovirus-mediated IL-10 gene transfer. We also examined the therapeutic effect of IL-10 in terms of the prevention of peritoneal dissemination of gastric cancer cells using PMC-targeting IL-10 gene transfer in mice.

Materials and methods

Isolation and culture of rat PMCs

Rat peritoneum was obtained from 9-week-old male Wistar rats (Charles River Laboratories, Atsugi, Japan). Rat PMCs were isolated from the peritoneal tissue as described previously.27, 28 In brief, the isolated cells were cultured in M199 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 0.5 μg ml−1 insulin, 0.5 μg ml−1 transferrin, 5 ng ml−1 selenine, 2 mM L-glutamine and 0.4 μg ml−1 hydrocortisone (Sigma, St Louis, MO) in an atmosphere of 5% CO2 and 95% O2 at 37 °C. Characteristics of cultured cells were confirmed by immunofluorescence. Monoclonal anti-pan cytokeratin antibody (Abcam, Cambridge, UK), monoclonal anti-vimentin antibody (Dako, Kyoto, Japan), and anti-von Willebrand Factor (vWF) antibody (Dako) were used as the primary antibodies. We used secondary antibodies labeled with Alexa Fluor 488 (Invitrogen, Tokyo, Japan). Samples were examined with a confocal microscope equipped with argon laser sources. Passage 2 cultures were used for experiments.

Gastric cancer cell line

The human gastric adenocarcinoma cell line (MKN45 cells) deposited by Teiichi Motoyama was provided by the Cell Bank, RIKEN Bio Resource Center (Tsukuba, Ibaraki, Japan). Cells were continuously grown as a monolayer in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Sigma) in an atmosphere of 5% CO2 and 95% O2 at 37 °C. The cells were subcultured every 4 days.

Recombinant adenovirus vectors

Recombinant adenovirus vectors expressing the mouse IL-10 gene (AxCAmIL10; Ad-mIL-10) deposited by Hirofumi Hamada were provided by the DNA Bank, RIKEN Bio Resource Center (Tsukuba) with the support of the National Bio-Resources Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT). This vector was constructed by inserting murine IL-10 cDNA into the early region, and expression of cDNA was driven by a cytomegalovirus enhancer and a chicken β-actin promoter, and terminated by the polyadenylation sequence of rabbit β-globulin.29 A recombinant adenovirus containing the bacterial β-galactosidase gene (Ad-LacZ) was constructed as a negative control for Ad-mIL-10. Preparation of the adenoviruses was performed as described previously.30 In brief, all of the viruses were propagated in a package containing 293 cells, purified twice via ultracentrifugation in a cesium chloride gradient, and subjected to dialysis. The titer of the virus was determined by Adeno-X rapid titer kit (Clontech, CA) and expressed in i.f.u.

In vitro transfection efficiency of PMCs and MKN45 cells with an adenovirus vector

Rat PMCs and MKN45 cells were infected with Ad-LacZ at multiplicities of infection (MOI) of 1, 10 and 100 at 37 °C in an atmosphere of 5% CO2 and 95% O2. After 48 h, the cultures were fixed with 1% glutaraldehyde for 10 min. The cultures were rinsed twice with PBS and incubated with a solution of 0.05% 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) in distilled water containing 3 mM potassium ferricyanide, 3 mM potassium ferrocyanide, 1 mM MgCl2, 100 mM KCl, 100 mM sodium phosphate buffer and 0.1% Triton X-100.31 After overnight incubation at 37 °C, the average number of positively stained cells from five randomly chosen high-powered fields (hpf) was counted to determine transfection efficiency.

In situ X-gal staining at the peritoneum in a murine peritoneal dissemination model

Five-week-old female BALB/c nude mice (CLEA Japan Inc., Osaka, Japan) were inoculated with MKN45 cells (3 × 106 per 1 ml PBS) into the peritoneal cavity to generate peritoneal dissemination. Three days later, mice received intraperitoneal injection of Ad-LacZ (1 × 109 i.f.u. per 4 ml PBS) or 4 ml of PBS as a control. After 1 or 3 weeks, mice were killed by cervical dislocation. The entire peritoneal cavity, including the abdominal organs, was fixed with 2% paraformaldehyde/0.2% glutaraldehyde intraperitoneally for 15 min. The peritoneal cavity was washed with PBS 10 times, and then in situ X-gal staining was performed to detect β-galactosidase expression in the peritoneum by intraperitoneal injection of 8 ml of the above-described X-gal solution. After 4 h incubation at 37 °C, the solution was removed and the peritoneal cavity was opened for visual inspection. All animal experimental procedures were approved by the Animal Care Committee of Osaka City University Graduate School of Medicine.

Histological β-galactosidase expression in the peritoneal nodules and main organs

After 3 weeks of Ad-LacZ injection, visible peritoneal nodules larger than 1 mm in diameter, as well as the major organs (liver, kidney, spleen, colon, small intestine, stomach, lung and heart), were harvested and embedded in Tissue-Tek OCT compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan); these were then frozen in dry ice. Serial sections (6-μm thick) were cut with a cryostat and placed on silanized glass slides (Dako) and the sections were fixed in 1% glutaraldehyde at room temperature for 7 min, washed three times with PBS (5 min per wash), and incubated in an X-gal staining solution at 37 °C for 6 h, followed by counterstaining with methyl green (Dako). The average number of positively stained spots from five randomly chosen hpf was used to determine transfection efficiency.

Treatment with intraperitoneal Ad-mIL-10 for peritoneal dissemination of MKN45 cells

On day 3 after peritoneal dissemination generated by the above-described method, mice were randomly assigned to one of three groups in a blinded manner and received intraperitoneal injections as follows: group 1, Ad-mIL-10 (1 × 109 i.f.u. per 4 ml PBS); group 2, Ad-LacZ (1 × 109 i.f.u. per 4 ml PBS); group 3, control vehicle (4 ml PBS). The body weight of each mouse was measured twice per week and after 3 weeks the mice were killed and the whole intestinal tract with peritoneum, omentum and peritoneal disseminated nodules was excised en bloc. The number of peritoneal nodules larger than 1 mm in diameter was counted macroscopically. Using the same protocol, the survival rate of mice was evaluated in each group until day 120.

IL-10 levels in the peritoneal lavage, plasma and peritoneal nodules

In the Ad-mIL-10 group, after 1, 3, 5 or 7 weeks of virus injection, blood samples were collected by transcutaneous heart puncture with a 23-gauge needle. Mice received intraperitoneal injection of 1 ml of PBS and were gently shaken to ensure the solution was spread throughout the peritoneal cavity, and then peritoneal lavage was collected. The lavage was centrifuged at 15 000 rpm, 4 °C for 10 min. Blood samples were stabilized for 60 min, followed by centrifugation at 3000 rpm, 4 °C for 15 min to obtain plasma samples. Excised peritoneal nodules were suspended with 1 ml of PBS, homogenized and then centrifuged at 15 000 rpm, 4 °C for 10 min. These supernatant samples were recovered and stored at −80 °C. IL-10 expression of the sample was quantified using enzyme-linked immunosorbent assay (ELISA) with IL-10 Mouse, Biotrak Easy ELISA (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions. In the Ad-LacZ and control groups, the same procedures were performed after 3 weeks of injections.

Immunohistochemical staining of the peritoneal nodules

Frozen sections obtained from the peritoneal nodules after 1 and 3 weeks of treatment were fixed in acetone at room temperature for 7 min. Immunohistochemical staining was performed using R.T.U. VECTASTAIN universal Quick kit (Vector Laboratories Inc., Burlingame, CA) and DAB substrate according to the manufacturer's instructions. To detect apoptosis, sections were reacted with polyclonal rabbit anti-single-stranded DNA (ssDNA) antibody (Dako) and counterstained by methyl green. To evaluate angiogenesis, sections were reacted with monoclonal rat anti-CD31 (PECAM-1) antibody (BD Pharmingen, CA), the marker for vascular endothelial cells, and counterstained by hematoxylin. The average number of vessels from five randomly chosen hpf was counted to determine microvascular density. Other sections were stained with hematoxylin and eosin (Muto Pure Chemicals, Tokyo, Japan).

Statistical analysis

The values are presented as means±s.e.m. Significant differences were assessed by one-way analysis of variance (ANOVA) followed by the Fisher's protected least significant difference test. Probability values of less than 0.05 were considered to indicate statistical significance. Survival evaluation was carried out using Kaplan–Meier survival analysis.

Results

Efficiency of adenovirus gene transfer targeting PMCs in the in vitro study

Most of the isolated cultured cells were positive for cytokeratin and vimentin, but not for vWF. The findings above, obtained by immunofluorescence, were consistent with previously reported findings for PMCs.27, 28 After confirmation of the characteristics of PMCs, we used these cells in the following experiment. The transfection efficiency of Ad-LacZ at 1 MOI, the infection rate of PMCs, was 0.66±0.58 per hpf (Figure 1a), but no transfection was observed in MKN45 cells (Figure 1d). The transfection rate for PMCs was 6.0±2.65 and 50.7±4.04 per hpf at 10 and 100 MOI, respectively (Figures 1b and c). In contrast, for MKN45 cells, the rate was 0.33±0.58 per hpf at 10 MOI (Figure 1e) and 4.67±0.58 per hpf at 100 MOI (Figure 1f). At 100 MOI, almost all of the cultured PMCs were infected, and the infection rate was about 10.9 times higher than for MKN45 cells. These findings showed that PMCs have a significantly higher potential for successful transfection using the adenovirus than MKN45 cells (P<0.05 at 10 MOI, P<0.01 at 100 MOI).

Figure 1
figure1

Transfection of Ad-LacZ in cultured cells. PMCs (ac) and MKN45 (df) were transfected with Ad-LacZ at 1, 10 and 100 multiplicities of infection (MOI). The average number of X-gal-positive cells from five randomly chosen high-powered fields (hpf) was counted to assess transfection efficiency. Transfection efficacy was found to be significantly higher for PMCs than MKN45 cells. Each value represents the mean±s.e.m. Original magnification: × 200.

β-galactosidase expression on intraperitoneal administration of Ad-LacZ in mice

In the control group, there was no obvious β-galactosidase expression in the peritoneal cavity (Figure 2a). In contrast, after 1 and 3 weeks of intraperitoneal Ad-LacZ administration, the entire peritoneum was strongly and homogenously stained by X-gal solution macroscopically (Figure 2b and c: 1w and 3w, respectively). To clarify the location of the infected cells histologically, most of the organs and peritoneal nodules were sectioned and similarly stained. At first, only the surface of the organs such as the spleen, kidney, colon, small intestine and stomach was positively stained, indicating that there were spots positive for PMCs on the visceral peritoneum but that there were no such spots in the parenchyma (Figure 2d: X-gal staining for the spleen). In the peritoneal nodules, X-gal-positive spots were sparse in the parenchyma, and the number of spots was 9.07±2.41 per hpf (Figure 2e). This stained area was less than 5% of the entire nodule, so the effect of gene transfer into the nodules may be limited. At the parenchyma of the liver, there were faint X-gal-positive spots compared with those in the peritoneal nodules; number of spots was 1.02±0.04 per hpf (P<0.01) (Figure 2f). Obvious histological changes were not found around the blue spot compared with the other areas in the peritoneal nodules and liver. In the lung, heart and spleen, positive staining was not observed at all (data not shown).

Figure 2
figure2

β-Galactosidase expression after intraperitoneal administration of Ad-LacZ in mice (ac). Five-week-old BALB/c mice were intraperitoneally inoculated with PBS (control) or Ad-LacZ (1 × 109 i.f.u. per 4 ml PBS). (a) In controls, the peritoneal surface was not stained by in situ X-gal staining. In contrast, in the Ad-LacZ-treated group, the entire peritoneum showed strong staining at 1 week (b) and also at 3 weeks (c). X-gal staining of peritoneal nodules and the main organs; (d) spleen, (e) peritoneal nodules, (f) liver. After 3 weeks of intraperitoneal Ad-LacZ administration, peritoneal nodules and main organs were excised and X-gal staining was performed. The average number of positive stained spots from five randomly chosen hpf was used to assess transfection efficiency. Each value represents the mean±s.e.m. in three different experiments. Original magnification: (d) × 400, (e, f) × 100.

IL-10 levels in the peritoneal lavage and plasma in vivo

In the Ad-mIL-10-treated group, IL-10 levels in the peritoneal lavage were remarkably elevated at 1 and 3 weeks after the injection (1w: 3431±689, 3w: 4666±862 pg ml−1) (Figure 3). After 5 and 7 weeks, IL-10 levels had decreased considerably compared with at 1 and 3 weeks (5w: 98±88, 7w: 117±73 pg ml−1). In the control and Ad-LacZ groups, IL-10 levels were not detectable at 3 weeks. Although plasma IL-10 levels were detectable at 1 and 3 weeks in the Ad-mIL-10 group (1w: 257±46, 3w: 189±35 pg ml−1), the levels were markedly lower (about 4–7%) than those of the peritoneal lavage (Figure 3). At 5 weeks, plasma IL-10 was not detected.

Figure 3
figure3

IL-10 levels in the peritoneal lavage, plasma and peritoneal nodules of the Ad-mIL-10-treated mice. Five-week-old BALB/c mice received intraperitoneal vehicle, Ad-LacZ, or Ad-mIL-10. After administration, IL-10 levels in the peritoneal lavage, plasma and peritoneal nodules were evaluated using enzyme-linked immunosorbent assay (ELISA). IL-10 levels in the peritoneal lavage increased remarkably up to 3 weeks but no such increase was seen in either the plasma or in the peritoneal nodules. Each value represents the mean±s.e.m. (N=4–12).

IL-10 levels in the disseminated tumors

IL-10 levels in the tumors were elevated at 1 and 3 weeks in the Ad-mIL-10 group (1w: 85±32, 3w: 115±46 pg ml−1) but these levels were very low compared with those from the peritoneal lavage as well as from the plasma samples (Figure 3). All these findings indicated that intraperitoneal IL-10 was mainly produced from PMCs rather than from the peritoneal nodules.

Therapeutic efficacy of intraperitoneal Ad-mIL-10 administration in peritoneal dissemination models

Three weeks after the administration of PBS or Ad-LacZ, large peritoneal nodules were observed on the peritoneum and omentum macroscopically (Figure 4a and b: control and Ad-LacZ, respectively). In contrast, in the Ad-mIL-10 group, the peritoneal nodules were obviously smaller than in the other groups and were barely detectable macroscopically (Figure 4c). The total number of peritoneal metastatic nodules was 59±5.4 and 37±5.8 in the control and Ad-LacZ-treated groups, respectively (Figure 4d). In contrast, Ad-mIL-10 treatment significantly decreased the number of peritoneal nodules compared with those in the control or Ad-LacZ-treated group (8.7±1.4, P<0.01) (Figure 4d). The number of peritoneal nodules did not increase until 5 weeks after Ad-mIL-10 injection (8.8±3.0). In the peritoneal nodules treated with Ad-mIL-10, some regions where there were cancerous cells showed cytoplasmic aggregation and nuclear condensation. These regions gradually expanded from 1 to 3 weeks after injection (Figure 5a and b: 1w and 3w, respectively). These cells stained positive for ssDNA, which indicated that they proceeded through the apoptotic cell death process (Figure 5e). Such changes were not observed in controls (Figure 5c) or in the Ad-LacZ-treated group (Figure 5d).

Figure 4
figure4

Treatment with intraperitoneal Ad-mIL-10 for peritoneal dissemination of MKN45 cells. Five-week-old female BALB/c nude mice were inoculated with MKN45 cells (3 × 106 per 1 ml) in the peritoneal cavity to generate peritoneal dissemination. Three days later, mice were randomly assigned to one of three groups in a blinded manner and were treated with PBS (control), Ad-LacZ (1 × 109 i.f.u. per 4 ml) or Ad-mIL-10 (1 × 109 i.f.u. per 4 ml). After 3 weeks, mice were killed and the peritoneal cavity was opened. Macroscopic findings relating to the peritoneal nodules of the control (a), Ad-LacZ (b) and Ad-mIL-10 mice (c). In the Ad-mIL-10-treated group, the peritoneal nodules (arrows) were obviously smaller and rarely seen macroscopically compared with the other groups. The number of peritoneal nodules with a diameter larger than 1 mm (d). Bars show the mean±s.e.m. (N=5–9 for each group). Ad-mIL-10 treatment was associated with a significantly lower number of peritoneal nodules. **P<0.01 vs control or Ad-LacZ.

Figure 5
figure5

Histological findings of peritoneal nodules after intraperitoneal Ad-mIL-10 injection; after 1 week (a), and 3 weeks (b) following Ad-mIL-10, and after 3 weeks in the control group (c) and Ad-LacZ group (d). In the peritoneal nodules from mice treated with Ad-mIL-10, a region of the cancer cells showed cytoplasmic aggregation (arrow) and nuclear condensation, and this region gradually expanded from 1 to 3 weeks. Such changes were not observed in the control and Ad-LacZ groups. Original magnification: × 200, (inset) × 400. Immunohistochemical staining for single-stranded DNA (ssDNA) in the peritoneal nodules after 1 week of Ad-mIL-10 treatment (e). Cancer cells with cytoplasmic aggregation stained positive for ssDNA (arrow). Original magnification: × 200, (inset) × 400. Immunohistochemical staining for CD31 in the peritoneal nodules after 3 week of treatment; (f) control, (g) Ad-LacZ, (h) Ad-mIL-10. The average number of vessels in the peritoneal tumors (i). Ad-mIL-10 treatment significantly suppressed neovascularization. **P<0.01 vs control or Ad-LacZ. Original magnification: × 400.

Effects of intraperitoneal Ad-mIL-10 administration on microvascular density in the peritoneal nodules

In controls and in the Ad-LacZ-treated group, many vessels, for which localization was confirmed by CD31 staining, were found at the periphery and at the center of the tumors (Figure 5f and g: control and Ad-LacZ, respectively). However, in the Ad-mIL-10-treated group, few vessels were found in the tumors (Figure 5h). The average number of vessels in the peritoneal tumors of the control, Ad-LacZ and Ad-mIL-10-treated groups were 9.56±1.7, 9.27±0.97 and 1.67±0.18, respectively (Figure 5i).

Effects of intraperitoneal Ad-mIL-10 administration on the survival of mice with disseminated tumors

Ad-mIL-10 treatment significantly inhibited weight loss in the mice with disseminated tumors (Figure 6a). Moreover, although all mice in the control and Ad-LacZ-treated groups were dead within 80 days, half of the Ad-mIL-10-treated mice were still alive at this time. Additionally, the survival rate was significantly prolonged in the Ad-mIL-10-treated group (P<0.01 vs control or Ad-LacZ group) (Figure 6b).

Figure 6
figure6

Relative body weight of treated mice (a). MKN45 cells (3 × 106 cells) were intraperitoneally inoculated, and on day 3, mice were treated with PBS (control), Ad-LacZ (1 × 109 i.f.u. per 4 ml), or Ad-mIL-10 (1 × 109 i.f.u. per 4 ml). Ad-mIL-10 treatment significantly inhibited weight loss of the mice with disseminated MKN45 cells compared with the control or Ad-LacZ groups. Each value represents the mean±s.e.m. (N=6 for each group). *P<0.05; **P<0.01 vs control or Ad-LacZ. Survival rate of control, Ad-LacZ- or Ad-mIL-10-treated groups (b). Although all mice in the control and Ad-LacZ groups were dead by day 80, half of the Ad-mIL-10 treated mice were still alive on day 80. The survival rate was significantly prolonged in the Ad-mIL-10 group (N=10 for each group). P<0.01 vs control or Ad-LacZ.

Discussion

In the present study, we demonstrated that IL-10 gene transfer to PMCs suppressed peritoneal dissemination of gastric cancer cells, which is correlated with transiently high levels of IL-10 in the peritoneal cavity, and prolonged survival in inoculated mice.

IL-10 is an anti-inflammatory cytokine that inhibits the production of proinflammatory cytokines including IL-1, IL-6, IL-8 and TNF-α originating from monocytes.2, 3, 4 As well as having anti-inflammatory properties, IL-10 also inhibits proliferation and metastasis of cancer cells.5, 6, 7 This action is mediated by various mechanisms.8, 9, 10, 11, 12, 13, 14 IL-10 also improves cancer-associated cachexia through suppression of the production of TNF-α and other cytokines.3, 32 Thus, IL-10 may be an important factor for improving conditions associated with malignant tumors. However, it is difficult to maintain a high concentration of IL-10 as it has a very short half life.15, 16, 17 Furthermore, the types of adverse effects that could be expected with IL-10 remain unknown when IL-10 levels are systemically increased in the presence of malignant tumors. Therefore, before determining the therapeutic possibility of IL-10 for in vivo cancer cells, we needed to establish a system that was continuous but limited to focal circumstances. Therefore, we focused on the PMCs, since PMCs are abundant in the closed peritoneal cavity and have a potent capacity to produce various cytokines such as IL-1, IL-6, IL-8, G-CSF, VEGF and SDF-1.20, 21, 22, 23, 24, 25 We demonstrated that IL-10 levels were markedly high in the peritoneal lavage but low in plasma and not detectable in the nodules at 1 and 3 weeks after Ad-mIL-10 administration. Considering the transfection efficacy for PMCs in the in vitro study, these findings suggest that our aim in the present study of developing a system for continuous, focal production of IL-10 may have been established. Thus, PMCs could be the appropriate target for gene transfer of IL-10 using the adenovirus vector.

After developing a satisfactory in vivo transfection system, we went on to investigate the therapeutic efficacy of intraperitoneal IL-10 treatment against peritoneal dissemination of gastric cancer cells. We found that intraperitoneal IL-10 treatment was able to reduce peritoneal metastasis and increase survival rate, as well as decrease the occurrence of cachexia. Numerous previous reports have demonstrated the antitumor effects of IL-10. IL-10 suppresses tumor growth and metastasis by acting as an angiogenesis inhibitor.5, 6, 7, 8 Huang et al.5 reported that the in vivo decrease in neovascularization found in IL-10-secreting tumors is most likely due to the ability of IL-10 to downregulate angiogenetic factors such as VEGF in tumor-associated macrophages. Thus, IL-10 is associated with angiogenesis in tumors via certain chemical mediators. Our present finding that Ad-mIL-10 treatment significantly decreased microvascular density may be supported by these previous data. These findings may also suggest that Ad-mIL-10 treatment might be involved in the antitumor effects on the disseminated nodules mediated by angiogenesis. In contrast, previous studies have shown that the antitumor mechanisms of IL-10 are dependent upon immune cells such as NK cells, CTLs and macrophages.9, 10, 11, 12, 13 Giovarelli et al.33 reported the local release of IL-10 following transfection of mouse mammary adenocarcinoma cells and noted that this was associated with an enhanced antitumor reaction and also elicited a strong CTL and antibody-dependent immune memory. It may be that it is induction of these parts of the immune system that is the underlying mechanism(s) of action of Ad-mIL-10 in this study. IL-10 has the potential to suppress Th1 type immune responses that might be expected to mediate antitumor activity. IL-10 might suppress the production of proinflammatory cytokines from PMCs and have anti-inflammatory effects. Progressive peritoneal inflammation induced by bacterial infection and cancer cell dissemination produces massive quantities of peritoneal fluid containing IL-1, IL-6, IL-8 or VEGF.34, 35 However, in the initial condition with mild peritoneal inflammation, as in this model, peritoneal fluid and proinflammatory cytokines in the peritoneal cavity are not increased.

Our observed histological findings, including cytoplasmic aggregation and nuclear condensation, following treatment are thought to be consistent with the characteristic features of apoptotic cell death associated with ischemia due to the decrease in tumor vascularization. To date, we have examined the presence of leukocytes, macrophages and NK cells in the peritoneal nodules by immunohistochemical staining. However, no significant localization of any of these inflammatory cells was detected in nodules in the Ad-mIL-10-treated group or in the control groups (data not shown). In fact, a tumor nodule is composed primarily of tumor cells. All of the findings suggested that these changes demonstrated a series of features of the tumor cells as they proceeded through the cell death process that might be mainly mediated by antiangiogenesis.

In the present study, the effect of a single intraperitoneal administration of Ad-mIL-10 on inhibition of metastatic nodules was maintained until 5 weeks without any significant adverse effects. At 5 weeks, the survival rate in the control group began to decrease although the rate was not changed in the Ad-mIL-10-treated group. In addition, IL-10-associated gene therapy does not directly depend on the cancer cells species. Thus, this treatment may be useful and applicable to other types of cancer such as pancreatic, colon or ovarian cancer where there is the possibility of peritoneal metastasis associated with a poor prognosis.

In conclusion, we demonstrated that IL-10 gene transfer to PMCs effectively suppressed peritoneal dissemination of gastric cancer cells, which is correlated with transiently high levels of IL-10 in the peritoneal cavity. This gene therapy has the potential to become a novel treatment with promising clinical applications in advanced gastric cancer with peritoneal dissemination.

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Acknowledgements

This study was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Culture, Sports Science and Technology of Japan and the Yasuda Medical Foundation.

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Correspondence to K Tominaga.

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Tanaka, F., Tominaga, K., Shiota, M. et al. Interleukin-10 gene transfer to peritoneal mesothelial cells suppresses peritoneal dissemination of gastric cancer cells due to a persistently high concentration in the peritoneal cavity. Cancer Gene Ther 15, 51–59 (2008). https://doi.org/10.1038/sj.cgt.7701104

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Keywords

  • interleukin-10
  • peritoneal mesothelial cells
  • peritoneal dissemination
  • gastric cancer

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