Gene
Ther Mol Biol Vol 14, 15-23, 2012
IL-15 Gene Therapy inhibited
Experimental Liver Metastases of Colon Cancer
Research Article
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: humphreyhe@163.com
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.
III.
Results
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.
Acknowledgments
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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