Hepatocellular carcinoma (HCC) is a malignant
tumor arising from the liver’s parenchymal cells (1). It is a
leading cause of cancer-related death worldwide, estimated to be responsible
for around 746000 deaths in 2012 (2, 3). It is currently the second
most common cause of cancer-related mortality (4). The global incidence of HCC is over 600,000
new cases per year, with average survival rates between 6 and 20 months (5,
6).HCC is associated with cirrhosis in >90% of cases (7).
Hepatitis B virus (HBV) infection is the leading risk factor for HCC globally
and accounts for at least 50% cases of HCC. Hepatitis C virus (HCV) is the
second most common risk factor, with an estimated 10%-25% of all cases
attributed to it around the world (8, 9). Long-term persistence of HBV
and HCV in the liver has been suggested to be partially due to the liver
immunosuppressive microenvironment (10). The liver is a tolerogenic
organ with exquisite mechanisms of immune regulation (11). It has to
prevent aberrant immune responses to gut derived antigens that constantly
circulate through the liver (12).
that are detected clinically must have evaded antitumor immune responses to
grow progressively (13). Apart from liver tolerogenic nature, the
loss of tumor-associated antigens (TAAs), decreased major histocompatibility
complex (MHC) antigen expression, inactivation of T cells by reduced TCR
signaling or IL-10 and TGF-?-mediated suppression, cause a scene of immune
tolerance to tumors (14). As the disease progresses from cirrhosis
of the liver to HCC, the functions of various immune cells become dysregulated.
T cells, both helper CD4+ and cytotoxic CD8+, decrease in
numbers with attenuated function and increased expression of inhibitory
receptors. T helper 17 cells increase in number and correlate with angiogenesis
and poor-prognosis (15). Cancer associated fibroblasts (CAFs)
inhibit natural killer cell function (16). Myeloid-derived
suppressor cells (MDSCs) suppress T cell activation, induce other
immune-suppressive cell populations and promote tumor angiogenesis (17).
treatment options available for HCC patient are limited due to the advanced
stage at which most patients are diagnosed. Surgical resection is a good choice
for most early-stage patients (18). Sorafenib is directed therapy and is the standard first-line,
systemic drug for advanced HCC (19). However,
patients with poor performance status or severe hepatic dysfunction do not
derive any survival benefit from HCC-directed therapy (20). Liver
transplant is another perioperative intervention for advanced cases of HCC;
however, there are limitations due to the insufficient number of the matched
donors as well as post-transplant allograft rejection (21). Local
ablative therapies are widely used in HCC for both curative and palliative
treatment in which obstruction of the hepatic artery induces subsequent tumor
necrosis (5). Common ablative procedures are radiofrequency ablation
(RFA), laser ablation, cryoablation, photodynamic therapy, high intensity
frequency ultrasound, and percutaneous ethanol or acetic acid injection (19). HCC is extremely
chemo-resistant as multi-drug resistance (MDR) genes are reported to be highly
expressed in HCC (22).
non-selective effects of conventional treatments, immunotherapy, theoretically,
could selectively target and destroy malignant cells with minimal side effects (22).
It seems to work better in more immunogenic tumors (23). Immunotherapy
acts indirectly and immune responses might take longer to develop, but anti-tumor
effects tend to be more durable than with chemotherapy (24).
Immunotherapy is based on the concept to redirect the patient’s own immune
system against the cancer instead of targeting the cancer itself (e. g., by
chemotherapy) (12). It involves the stimulation the host’s
anti-tumor response by increasing the effector cell number and the production
of soluble mediators and decrease the host’s suppressor mechanisms and by
inducing tumor killing environment (23).
cell therapy (ACT) is one of the main treatment modalities within cancer
immunotherapy (25). It involves expansion and activation of tumour-specific
immune cells in vitro that can then be adoptively transferred back in
large numbers to patients (26). ACT have employed many types of
immune cells, including dendritic cells (DCs), cytotoxic T lymphocytes (CTLs),
lymphokine-activated killer (LAK) cells, natural killer (NK) cells, and cytokine-induced
killer (CIK) cells (27). ACT is a “living” treatment because the
administered cells can proliferate in vivo and maintain their antitumor
effector functions (28). Such immunological stimulation may
counterbalance the strongly immune-suppressive microenvironment in the liver (29).
killer cells (CIK), as the most commonly used cell-based immunotherapy (30),
are a heterogeneous cell population comprising CD3+ CD56+,
CD3+CD56? and CD3?CD56+ cells (31).
The majority of the cytotoxic cells have been shown to be derived from the CD3+?CD56- T cells and not from CD3-CD56+? NK cells (32).
Although PB T cell have 1% to 5% CD3+CD56+ cells (33),
they were readily expanded from the preexisting amount of T cells and
constituted about one third of the total cell number (34). CIK cells have advantage of a higher
proliferation rate (35), not inhibited by immunosuppressive drugs (36)
and particularly important, they have a strong activity against tumors with
minimal toxicity and no graft-vs-host disease (37). CD3+CD56+
subsets are characterized by their MHC-unrestricted antitumor activity (38).
Interferon-gamma (IFN-?) and tumor necrosis factor-alpha (TNF-?) are the main
cytokines produced by CIK cells, which are involved in regulating innate and
adaptive immunities (39). Due to number of advantages of CIK cells, they present a promising
immunotherapy approach that could be used for HCC (35).
the present study evaluates the potential of in vitro expansion of
viable CIK cells from human PBMCs and measures the proportion of the most
effective subset CD3+CD56+ in the culture. In addition,
the study examines TNF-? secretion and the cytotoxicity of expanded CIK cells in
vitro on HCC, HepG2 cell line.