Gene Ther Mol Biol Vol 14, 15-23, 2012
IL-15 Gene Therapy inhibited Experimental Liver Metastases of Colon Cancer
Ning Lu1， Dongbing Zhou2， Weidong Li1，Xianghui He1*， Liwei Zhu1
1 Department of General Surgery, Tianjin General Surgery Institute, Tianjin Medical
2 Department of General Surgery II，Nanchong Central Hospital, Sichuan 637000, China
*These authors contributed equally to this work
Correspondence: Dr. Xianghui He, Department of General Surgery, Tianjin General Surgery Institute, Tianjin Medical University General Hospital, No. 154 Anshan Rd. Tianjin 300052, China, Tel：86-22-60363970, Fax:86-22-60362365, Email: firstname.lastname@example.org
Keywords: cancer, gene therapy, interleukin-15
Received: 22 February 2012; Revised: 12 March 2012
Accepted: 14 March 2012; electronically published: 15 March 2012
Liver metastases are common for colon cancer and associate with poor prognosis. Interleukin-15 is considered to be one of the most promising cytokine for cancer gene therapy based on its activity on both innate and adaptive immunity. This study investigated the effect of IL-15 immune gene therapy on metastatic liver disease of colon cancer in murine model. IL-15 over-expression plasmid vector was transfected into CT-26 cells in vitro by lipofection and into mice in vivo by intravenous injection. IL-15 expression was detected by ELISA. The impact of IL-15 on T cell subsets were analyzed by flow cytometry. Murine model of CT-26 colon cancer liver metastases were established and the effect of IL-15 gene therapy on liver metastasis of colon cancer were determined. Results showed that IL-15 was detected in the supernatant of IL-15-overexpression plasmid transfected mouse CT-26 cells. Transgene expression was observed in the liver tissue after intravenous injection of IL-15-overexpression plasmid. IL-15 expression in vivo increased the proportion of CD3+CD8+ cells in peripheral blood of mice. Decreased liver metastases indicated by liver weight were observed in IL-15 gene therapy group. The survival time of mice in IL-15 gene therapy group is also significantly prolonged. In conclusion, IL-15 gene therapy inhibits experimental colon cancer liver metastases, prolongs the survival of tumor-bearing mice. IL-15 gene therapy provides a new approach for the treatment of colorectal liver metastases and is worth further evaluation.
The incidence of colorectal cancer has raised to the third place worldwide and the mortality rates second among cancer related death. In china, the incidence of colorectal cancer has also increased in recent years, and colorectal cancer mortality has been in the fifth place of cancer mortality (Chen and Chen, 2006). Given the lack of specific symptoms, 75% of patients with clinical symptoms are in advanced stage. Although surgery and chemotherapy can significantly improve patient survival, most patients still die of tumor recurrence and metastasis. Liver is the most common site for colon cancer metastasis. Approximately one-half of patients diagnosed with colorectal cancer will develop metastatic liver disease which is the cause of death in the majority of these patients (Aloia et al., 2006). Approach to control tumor cells to form metastases in the liver will improve the survival of colorectal cancer patients. Liver is a specific organ with large number of immune cells of both innate and adaptive immunity. Enhancing the activity of these immune cells is expected to clear the migrated tumor cells and inhibit metastasis formation and development.
Interleukin-15 (IL-15) is a multifunctional cytokine and has an important role in the regulation of innate and acquired immune responses. IL-15 stimulates the activity of neutrophils and macrophages, regulates the function of dendritic cells, and promotes the generation, activation, homing and survival of lymphoid effector cells, especially CD8+ T cells and NK cells. (Obar et al., 2004; Kobayashi et al., 2005; Waldmann, 2006). IL-15 also regulates memory T cells proliferation, maintains their number and is considered the most promising cytokine for cancer immunotherapy (Cheever, 2008). Intratumoral injection of recombinant human IL-15 increases efficacy of cytokine-gene modified tumor cell vaccines in murine model (Basak et al., 2008). IL-15Rα molecule is expressed constitutively by various tissue cells; however, the concentration of IL-15 in the liver tissue is low after intravenous injection, which may limit the therapeutic effect of recombinant IL-15 (Kobayashi et al., 2000). Expression of IL-15 locally by means of gene transfer could be able to enhance the anti-tumor activity of immune cells in the liver. In previous studies we successfully constructed of IL-15 over-expression plasmid vector pHi2-spIL15-CMV-tat (L3). This study further evaluated the effect of IL-15 immunogene therapy on metastatic liver disease of colon cancer using experimental liver metastases model in mice.
II. Material and Methods
A. Animals and cell lines
BALB/c mice (aged 6-10 weeks) were purchased from the animal center of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (Tianjin, China) and maintained in the animal facility at the Tianjin Medical University General Hospital. Murine colon carcinoma CT-26 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (Invitrogen, Beijing, China), 2 mM glutamine, 1 mM pyruvate, 50 mM 2-mercaptoethanol, penicillin (200 units/ml), and streptomycin (200 mg /ml) at 37 ¡C in a 5% CO2/95% air atmosphere.
B. Genetic Constructs
The IL-15 expression plasmid vector used in this study was pHi2-spIL15-CMV-tat (L3). The construction and characterization of these vectors has been described previously (He et al., 2008). Briefly, IL-15 expression was driven by the HIV2 promoter in this vector, and the activity of the HIV2 promoter was amplified by the expression of tat driven by the CMV promoter. In addition, high levels of IL-15 expression were achieved by replacing the IL-15 signal sequence with the IL-2 signal sequence. Green fluorescence expression vector, pHi2-EGFP-CMV-tat （L6）vector, in which the gene encoding enhanced green fluorescence protein (EGFP) was cloned into the same backbone plasmid as L3 behind the HIV2 promoter, were used to evaluate transfection efficiency.
C. Cell transfection
Tumor cells were transfected with plasmid DNA using the cationic lipid, GenePORTER 3000 (Genlantis, CA, USA) according to the manufacturerÕs protocol. Briefly, cells were plated at a concentration of 1.5 X106 cells/well in 2 ml medium in 6-well plates and incubated for 30 min. For each well, plasmid DNA (2mg) were diluted with 200ml GP3K diluent, and then mixed with 28ml GenePORTER 3000 reagent diluted by 112ml serum-free medium OptiMEM (GIBCO, Rockville, MD) medium. After 5 min incubation, the mixture was added to the well. Cells were incubated with lipid/DNA complexes for 4 hours. Equal volume completed medium was then added to the well. Cells were incubated for 24 hours at 37 ¡C in a 5% CO2/95% air atmosphere. Transfection efficiency was determined by simultaneous transfection of an EGFP expression plasmid. IL-15 expression was tested by ELISA.
D. ELISA detection of IL-15 expression Human IL-15DuoSet ELISA Development kit (R & D Systems China, Shanghai, PRC) was used to detect the IL-15 expression in the cell culture supernatant of transfected cells according to the manufactureÕs protocol. Briefly, a 96-well plate was coated with anti-mouse IL-15 antibody and blocked with bovine serum albumin-containing buffer. After washing, a standardized IL-15 solution and the cell culture supernatants were added to the wells. Following 2 hours of incubation, and the plate was washed, the biotin-labeled detection antibody and avidin-horseradish peroxidase (HRP) were added. After another 1 hour incubation and washing, the substrate solution was added and the plate read at 450nm.
For in vivo transfection, the green fluorescent protein expression plasmid pHi2-EGFP-CMV-tat (L6) was diluted with 0.5ml PBS (50mg/ml) and injected rapidly into the penile dorsal vein of mice. After 24 hours, mice were sacrificed and liver tissues were harvested for frozen sections. Green fluorescent protein expression was determined using fluorescent microscopy. Similarly, IL-15 expression plasmid pHi2-spIL15-CMV-tat (L3) was injected into mice and blood samples were collected 24 hours later for determining serum IL-15 expression by ELISA.
For establishing mice model of colon cancer metastasis, BALB/c mice were anesthetized with chloral hydrate and the abdomen was opened through the midline abdominal incision. Spleen was located and 100 ml CT-26 cell suspension (1×107 cells/mL) were injected into the lower pole of the spleen. After hemostasis with pressure, the abdominal cavity was closed and mice were monitored for tumor formation in the liver. To investigate the therapeutic effect of IL-15, 18 mice with tumor inoculation were randomly divided into three groups. Mice in negative control group were injected with PBS through penile dorsal vein at day 3, 5 and 7 after the inoculation of tumor. Mice in fluorescent protein control group were injected with 25 μg plasmid L6, whereas mice in IL-15 gene therapy group were injected with 25 μg plasmid L3 for each mouse. Two weeks later the mice were sacrificed and the liver weight, liver tumor metastasis and blood T cell subsets were monitored. In addition, another 18 mice with tumor inoculation were randomly divided into three groups and treated with the PBS, plasmid L6 or L3. The survivals of each group of mice were monitored.
H. Statistical analyses
Values are represented as the means ± standard deviation (SD). Results were compared using the Student's t-test. p value < 0.05 were considered to be statistically significance.
A. IL-15 was expressed by murine cells after in vitro transfection
Murine colon carcinoma CT-26 cells were transfected with green fluorescent expression plasmid pHi2-EGFP-CMV-tat (L6) or IL-15 expression plasmid pHi2-spIL15-CMV-tat (L3) by liposome mediated transfection. Cells were observed under inverted microscope 24 hours after transfection and GFP expression were seen within L6 transfected cells (Figure 1A). Cell culture supernatants were collected 24 hours and 48 hours after transfection. ELISA results showed that IL-15 was expressed by L3 transfected CT-26 cells: the concentration of IL-15 was 69.76 ± 29.91 pg / mL at 24h and 246.69 ± 55.13 pg / mL at 48h (Figure 1B).
The amount of 25 mg and 50 mg pHi2-EGFP-CMV-tat (L6) or pHi2-spIL15-CMV-tat (L3) diluted with 100 ml PBS was injected rapidly into the penile dorsal vein of mice. Mice were sacrificed 24 hours later and frozen sections of liver tissue were prepared. Different levels of GFP expression were seen in liver tissue of mice injected with L6, but not L3. In the group of mice injected with IL-15 expression plasmid pHi2-spIL15-CMV-tat (L3), IL-15 expression was detected in the serum of transfected mice: the concentration of IL-15 was 10.60±1.21 pg/ml when injecting 25 mg L3 and 13.31±1.82 pg/ml when injecting 100ug of L3 (Figure 2). The results indicated that hepatic transgene expression can be achieved in vivo through intravenous plasmid injection.
Mice were injected with three doses of PBS, 25 mg or 50 mg L3 respectively at day 1, day 3 and day 7. Two weeks later, mice were sacrificed and blood samples were collected for flow cytometry analysis of T cell subsets. The results showed that the proportion of CD3+CD8+ T cells in IL-15 gene transfer groups were significantly higher than that of PBS control group (p<0.05), whereas there was no significant difference for CD3+CD4+ T cells and CD4+CD25+ T cells (Figure 3).
D. IL-15 gene transfer inhibited experimental liver metastasis of colon cancer in mice
Colon cancer CT-26 cells were injected into mice spleen to induce liver metastasis. Mice were randomly divided into three groups and intravenously administered with PBS, fluorescent protein expression plasmid L6 or IL-15 expression plasmid L3 at day 3, day 5 and day 7 after tumor cells inoculation. Two weeks later, the mice were sacrificed and liver metastases were determined. Metastatic tumor nodules increased the weight of murine liver in our experiment. There were liver tumors formation in all groups, but gross and microscopic observation showed that liver metastasis in IL-15 plasmid treated mice was less severe compared to L6 and PBS treated mice. Average weight of normal mice liver were 1.49 ± 0.07 g, mean liver weight of tumor inoculated PBS control mice were 4.06 ± 0.76 g, L6 control mice were 4.41 ± 1.01 g, L3 treated mice were 2.84 ± 0.61 g, (L3 vs PBS, L3 vs L6, p <0.05) (Figure 4).
E. IL-15 gene therapy extended survival time of mice with colon cancer liver metastasis
Figure 1. Transgene expression after in vitro transfection. A. EGFP expression was observed in CT-26 cells transfected with L6 (200X). B. IL-15 expression were detected in the supernatant of CT-26 cells transfected with pHi2-spIL15-CMV-tat, 15 69.76 ± 29.91 pg / mL (24h after transfection), 246.69 ± 55.13 pg / mL (24h after transfection).
Figure 2. Transgene expression after in vivo gene transfer. EGFP expression was observed in liver tissue 24 hours after intravenous injection of plasmid pHi2-EGFP-CMV-tat (L6) 25ug (B) or 50ug (C). No EGFP expression was seen in the liver tissue of mice injected with pHi2-spIL15-CMV-tat (L3) (A). However, IL-15 expression were detected in the serum of mice intravenous injection of plasmid L3, but not L6 (D).
Figure 3. The effect of IL-15 expression on the subsets of T cells after in vivo gene transfer. The proportion of CD3+ CD8+ cells were significantly increased in the peripherial blood of mice injected with IL-15 expression plasmid (* p <0.05).
Figure 4. IL-15 gene transfer inhibited experimental colon cancer liver metastases in mice. Mice was intravenously injected with PBS 100 ml (B); plasmid pHi2-EGFP-CMV-tat (L6) 25 mg (C); plasmid pHi2-spIL15-CMV-tat (L3) 25 mg (D) by day 3, 5 and 7 after splenic inoculation of CT-26 cells, two weeks later, mice were sacrificed and liver metastasis were determined. A. normal mouse liver; E. The average weight of the liver in each group (L3 vs PBS, L3 vs L6, p <0.05); F. Pathology of metastatic tumor (HE staining).
Figure 5. IL-15 gene therapy increased the survival of mice with colon cancer liver metastasis. Each mouse were intravenously injected with PBS 100 μl; plasmid pHi2-EGFP-CMV-tat (L6) 25 μg; or plasmid pHi2-spIL15-CMV-tat (L3) 25ug at day 3, 5 and 7 after splenic inoculation of CT-26 cells. The survival of mice in each group was monitored. Results showed IL-15 treatment significantly increased the survival of mice compared to control (P <0.05).
Liver is the most common site of distant metastasis for colon cancer. Approximately 15% of colon cancer patients have liver metastases at the time of diagnosis, and 20% of patients developed metachronous liver metastasis within five years following diagnosis (Manfredi et al., 2006). Approaches to control liver metastasis will definitely improve the prognosis of colon cancer and other gastrointestinal tumor. Cytokine gene therapy as a means of biological therapy for cancer has been widely studied in animal models. IL-2, GM-CSF and IL-12 all showed certain therapeutic effects (Cross and Burmester, 2006). The effect of gene therapy determined by the function of therapeutic genes, the efficiency of gene transfer approach and the status of host immune system. Targeted gene expression to the liver can be achieved by non-viral and viral vector-mediated gene transfer (Ferry and Heard, 1998; Maruyama et al., 2002). Emerging evidences suggest that liver is an immunological organ. Therefore, enhancement of liver immunity through genetic approach is expected to control malignant liver metastasis to a certain degree.
Interleukin -15 (Interleukin-15) is a cytokine found by Grabstein et al. (Grabstein et al., 1994) with important role in homeostasis and activation of both innate and adaptive immunity. IL-15 and IL-2 share βand γc subunits of their receptors on T/NK-cells. However, compared to IL-2, IL-15 is less toxic, inhibits activation-induced cell death and stimulates the persistence of memory CD8+ T cells. The role of IL-15 in tumor immunotherapy has attracted much attention in recent years (Roychowdhury et al., 2004; Jakobisiak et al., 2011). Expressing IL-15 by genetic engineering methods in tumor cells resulted in slow tumor formation and reduced ability to metastasis (Meazza et al., 2000; Araki et al., 2004). In previous studies, we constructed IL-15-overexpression vector to achieve efficient IL-15 expression of human tumor cells (He et al., 2008). We also demonstrated that intraperitoneal IL-15 gene transfer alleviated peritoneal inoculation and growth of colon cancer cells in mice (He et al., 2012).
In this study, we further demonstrated that intravenous injection of IL-15-overexpression vector achieved IL-15 expression in vivo, especially in the liver, inhibited the experimental colon cancer liver metastases and prolonged the survival of tumor-bearing mice.
The liver is enriched in innate and adaptive immune cells. The average human liver contains a population of approximately 1010 lymphocytes, which include conventional and unconventional lymphocyte subpopulations of the innate (NKT and NK cells) and adaptive immune systems (T and B cells), respectively (Racanelli and Rehermann, 2006). Kuffer cells and liver dendritic cells (DCs), as well as multiple populations of nonhematopoietic liver cells, including sinusoidal endothelial cells, stellate cells, serve as antigen-presenting cells to induce immunity or tolerance to specific antigen. (Crispe, 2009). In normal condition, NK cells and NKT cells in the liver are ready to be activated to eradicate pathogen, however, T cells primed by the liver APCs in the context of immunosuppressive cytokines and inhibitory cell surface ligands usually end in tolerance. The liver is the predominant organ for the metastasis of tumor cells originated in the digestive tract. Approaches to boost liver immunity rather than tolerance could inhibit metastatic tumor formation.
On the other hand, the liver is the major target organ of intravenous gene transfer. In mice, injection of naked plasmid DNA encoding interferon through tail vein resulted in significant amount of interferon expression in the liver and decreased tumor metastasis (Kobayashi et al., 2002). Although hydrodynamic tail vein injection was thought to be an effective approach for liver gene transfer, the large injection volume renders it an unsuitable approach for clinical application. In this study, we expressed IL-15 in the liver through normal intravenous plasmid injection. IL-15 expressed locally in the liver should not only enhance the function of NK and NKT cells, but also restore the immunity of T cells. In fact, there were data showed that IL-15 promoted the survival of adoptively transferred tumor-specific T-cell and enhanced tumor rejection (Klebanoff et al., 2004). Teague et al. showed that IL-15 induced the CD8+ T cells which was in a tolerant state to proliferation and restored the immune function of CD8+ T cells (Teague et al., 2006). Accordingly, the increased peripheral blood CD8/CD4 ratio of mice transfected with IL-15 expression plasmid may result from the proliferation of CD8+ cells in our study.
In conclusion, we investigated the potential of IL-15-overexpression plasmid vector as gene medicine for the control of colon cancer liver metastasis. Results showed that intravenous IL-15 gene therapy inhibited experimental colon cancer liver metastasis and prolonged the survival of tumor-bearing mice. IL-15 gene therapy provides a novel approach for the treatment of liver metastasis of colon cancer and is worth further exploration.
The project was sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (to X. H.), Research Foundation of Tianjin Medical University (to X. H.).
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