ONCOLOGY repOrts 26: 215-221, 2011
Abstract. the aim of this study was to investigate the role of
specific cytotoxic T lymphocytes (CTLs) activated by dendritic
cells (DCs) presenting cationic nanoparticles with the K-ras
(12-Val) mutant peptide in the killing of different pancreatic
cancer cell lines in vivo and in vitro. Peripheral blood DCs
were induced by rhGM-CSF and IL-4 and cultured. DCs were
sensitized by whole antigen of PANC-1 with expression of K-ras
mutant, K-ras mutant peptide (K-ras+peptide) and cationic
nanoparticles with K-ras mutant peptide (K-ras+peptide-CNP),
respectively. Cell surface markers were measured by flow cyto-
metry. Lymphocyte proliferation was detected by the 3H-tdr
test, and IL-12 and IFN-γ secretion was detected by ELISA.
125I-UdR was used to measure the killing effect of CTLs. The
antitumor activity of CTLs in tumor-bearing nude mouse
models prepared with PANC-1 and SW1990 cells was evaluated.
Results showed that, compared with K-ras+peptide, low concen-
trations of K-ras+peptide-CNP were effectively presented by
DCs (P<0.05). CTLs induced by DCs pulsed with whole tumor
antigen had a significantly greater killing effect (P<0.05) on
PANC-1 and SW1990 pancreatic cancer cells compared with
K-ras+peptide- and K-ras+peptide-CNP-induced CTLs. CTLs
induced by DCs pulsed with K-ras+peptide and K-ras+peptide-
CNP had a specific killing effect (P<0.05) on PANC-1 cells and
no effect (P>0.05) on SW1990 cells. In conclusion, cationic
nanoparticles with the K-ras (12-Val) mutant peptide can be
effectively presented by DCs at a low concentration. CTLs
induced by K-ras+peptide-CNP had specific killing activity
for the pancreatic cancer cell line with the K-ras mutant and
significantly inhibited tumor growth and increased the survival
time of tumor-bearing nude mice. Although this study
confirmed that whole cell antigen induced a good antitumor
immune response, the possibility of immune tolerance and
autoimmunity which has been previously proven contribute to
the difficulty in the application of this DC vaccine.
pancreatic cancer accounts for 1-5% of all malignant tumors
(1). Its incidence has shown a clear upward trend in recent
years with 230,000 new cases of pancreatic cancer each year
worldwide (2). Pancreatic cancer lacks specific clinical mani-
festations. the surgical resection rate of pancreatic cancer is
less than 20%, and it is not sensitive to radiotherapy or chemo-
therapy. the 5-year survival rate is less than 5%. the prognosis
of pancreatic cancer has not improved in the last 20 years
(3,4). Immunotherapy for pancreatic cancer mainly includes
specific active immunotherapy, monoclonal antibody-directed
therapy, cytokine therapy and adoptive cellular immunotherapy
(5). Dendritic cells (DCs) are known to be the most powerful
antigen presenting cells (APCs) in the human body (6,7).
Studies have found that the K-ras gene mutation rate in
pancreatic cancer patients was approximately 90% and that
the gene mutation site was the ideal target for DC immuno-
therapy. In this study, we investigated the effects of DCs
sensitized with different antigens and the killing effect of
induced specific CtLs on different pancreatic cancer cell
lines in vivo and in vitro.
Materials and methods
Materials. PANC-1 and SW1990 cell lines were purchased
from ATCC. The cationic polymer β (1,4)-2-amino-2-deoxy-
D-glucan was purchased from Sigma (USA). Human
recombinant GM-CSF and IL-4 were purchased from R&D
Systems (USA), and mouse anti-human CD80-PE, CD83-PE,
CD86-PE, CD40-FITC, CD1a-FITC monoclonal antibodies
were obtained from Immunotech (France). Ficoll (lymphocyte
separation medium) was purchased from Second Plant of
Induction of CTLs by DCs pulsed with K-ras mutant
peptide on the surface of nanoparticles in
the treatment of pancreatic cancer
GUANG TAN, ZHoNGyU WANG, XIN ZHANG, ZHENGANG CAI and JUNKAI ZHANG
Department of General Surgery, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, P.R. China
Received November 16, 2010; Accepted December 21, 2010
Correspondence to: Dr Zhongyu Wang, Department of General
Surgery, The First Affiliated Hospital of Dalian Medical University,
No. 222, Zhongshan Road, Dalian 116011, P.R. China
Abbreviations: CTLs, cytotoxic T lymphocytes; DCs, dendritic
cells; mDC, mature dendritic cell; GM-CSF, granulocyte-macrophage
colony-stimulating factor; IL, interleukin; CNP, cationic nano-
particles; APCs, antigen-presenting cells; SI, stimulating index;
TNF-α, tumor necrosis factor; PBS, phosphate-buffered saline;
PBMCs, peripheral mononuclear cells; IFN-γ, interferon-γ; FCS,
fetal calf serum; ELISA, enzyme-linked immunosorbent assay
Key words: pancreatic cancer, dendritic cells, K-ras peptide, antigen,
cationic nanoparticles, cytotoxic T lymphocytes, mutation
ONCOLOGY repOrts 26: 215-221, 2011
the present study, we found that CTLs induced by DCs loaded
with PANC-1 cell antigen demonstrated strong cytotoxic
effects not only on PANC-1 cells but also on SW1990 cells.
Many effective antigens in PANC-1 cells, including K-ras
protein, were phagocytized and presented by DCs in this
experiment; one or a variety of antigens was the effective
recognition component of SW1990 cells by CTLs. Thus, DCs
sensitized by PANC-1 cell antigen have a greater therapeutic
effect on SW1990 pancreatic cancer cells in vivo and in vitro.
Although this study confirmed that whole cell antigen induced
an ideal antitumor immune response, it contained the normal
cellular components, which make immune tolerance and auto-
immunity possible. Additionally, the tumor antigen content
cannot be easily controlled, which causes difficulty in the
application of DC-sensitized antigen (27-29).
In the present study, whole tumor cell antigen-, K-ras+peptide-
and K-ras+peptide-CNP-sensitized DCs promoted the
expression of surface molecules of mature DCs and enhanced
the cytotoxic activity of CTLs on target cells, IL-12 secretion
by DCs and IFN-γ secretion by T cells. All CTLs induced by
the three types of antigen were able to kill target cells effectively.
K-ras+peptide-CNP- and K-ras+peptide-sensitized DCs
exhibited more specificity for the target cells. Sensitization by
K-ras+peptide-CNP was more effective than K-ras+peptide.
In vivo experiments also support these results, which lay the
foundations for the future development of DC vaccines for
The study was sponsored by the Nature Science Foundation of
China (NSFC) (30670624, 30870719).
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