Hepatocellular around the world (8, 9). Long-term

Hepatocellular carcinoma (HCC) is a malignanttumor arising from the liver’s parenchymal cells (1). It is aleading cause of cancer-related death worldwide, estimated to be responsiblefor around 746000 deaths in 2012 (2, 3). It is currently the secondmost common cause of cancer-related mortality (4). The global incidence of HCC is over 600,000new 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 globallyand accounts for at least 50% cases of HCC. Hepatitis C virus (HCV) is thesecond most common risk factor, with an estimated 10%-25% of all cases attributedto it around the world (8, 9). Long-term persistence of HBV and HCV inthe liver has been suggested to be partially due to the liver immunosuppressivemicroenvironment (10).

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The liver is a tolerogenic organ with specialmechanisms of immune regulation (11). It has to prevent aberrantimmune responses to gut derived antigens that constantly circulate through theliver (12).             Cancersthat are detected clinically must have evaded antitumor immune responses togrow progressively (13). Apart from liver tolerogenic nature, theloss of tumor-associated antigens (TAAs), decreased major histocompatibilitycomplex (MHC) antigen expression, inactivation of T cells by reduced TCRsignaling or IL-10 and TGF-?-mediated suppression, cause a scene of immunetolerance to tumors (14). As the disease progresses from cirrhosisof the liver to HCC, the functions of various immune cells become dysregulated.T cells, both helper CD4+ and cytotoxic CD8+, decrease innumbers with attenuated function and increased expression of inhibitoryreceptors. T helper 17 cells increase in number and correlate with angiogenesisand poor-prognosis (15).

Cancer associated fibroblasts (CAFs)inhibit natural killer cell function (16). Myeloid-derivedsuppressor cells (MDSCs) suppress T cell activation, induce otherimmune-suppressive cell populations and promote tumor angiogenesis (17).            Conventionaltreatment options available for HCC patient are limited due to the advancedstage at which most patients are diagnosed. Surgical resection is a good choicefor 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 donot derive any survival benefit from HCC-directed therapy (20). Livertransplant is another perioperative intervention for advanced cases of HCC;however, there are limitations due to the insufficient number of the matcheddonors as well as post-transplant allograft rejection (21). Localablative therapies are widely used in HCC for both curative and palliativetreatment in which obstruction of the hepatic artery induces subsequent tumornecrosis (5). Common ablative procedures are radiofrequency ablation(RFA), laser ablation, cryoablation, photodynamic therapy, high intensityfrequency ultrasound, and percutaneous ethanol or acetic acid injection (19).

HCC is extremely chemo-resistantas multi-drug resistance (MDR) genes are reported to be highly expressed in HCC(22).             Unlike,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).

Immunotherapyacts indirectly and immune responses might take longer to develop, but anti-tumoreffects tend to be more durable than with chemotherapy (24).Immunotherapy is based on the concept to redirect the patient’s own immunesystem against the cancer instead of targeting the cancer itself (e. g., bychemotherapy) (12). It involves the stimulation the host’santi-tumor response by increasing the effector cell number and the productionof soluble mediators and decrease the host’s suppressor mechanisms and byinducing tumor killing environment (23).             Adoptivecell therapy (ACT) is one of the main treatment modalities within cancerimmunotherapy (25). It involves expansion and activation of tumour-specificimmune cells in vitro that can then be adoptively transferred back inlarge numbers to patients (26). ACT have employed many types ofimmune cells, including dendritic cells (DCs), cytotoxic T lymphocytes (CTLs),lymphokine-activated killer (LAK) cells, natural killer (NK) cells, and cytokine-inducedkiller (CIK) cells (27).

ACT is a “living” treatment because theadministered cells can proliferate in vivo and maintain their antitumoreffector functions (28). Such immunological stimulation maycounterbalance the strongly immune-suppressive microenvironment in the liver (29).            Cytokine-inducedkiller 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 andconstituted about one third of the total cell number (34). CIK cells have advantage of a higherproliferation rate (35), not inhibited by immunosuppressive drugs (36)and particularly important, they have a strong activity against tumors withminimal 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 maincytokines produced by CIK cells, which are involved in regulating innate andadaptive immunities (39).

Due to number of advantages of CIK cells, they present a promisingimmunotherapy approach that could be used for HCC (35).            Therefore,the present study evaluates the potential of in vitro expansion ofviable CIK cells from human PBMCs and measures the proportion of the mosteffective subset CD3+CD56+ in the culture. In addition,the study examines TNF-? secretion and the cytotoxicity of expanded CIK cells invitro on HCC, HepG2 cell line.HCC is an aggressive cancer thatoccurs in the setting of chronic liver disease and cirrhosis that frequentlypresents in advanced stages (3).

Malignant liver cells often survivetraditional treatment such as radiation and chemotherapy. Most importantly,small lesions and metastatic cells often remain and cause recurrence of disease(50). Immunotherapy is a new and promising treatment for a number ofcancers. Cell-based immunotherapy is a set of therapeutic strategies based onmanipulating and co-opting a patient’s own immune cells, or donor cells andusing immune cell functions to halt and reverse disease (51).            In the current work, human PB from10 healthy volunteers was collected and PBMCs were separated. We found that,the separated PBMCs count ranged from 30×106 to 34×106 cell/mLand the lymphocytes account for 60.

8 to 67.6% of total separated PBMCs. Theviability range upon separation was 95%-98%.

         Allogeneic protocols were reported byBonanno et al. (52) who have obtained CIK cells using X-VIVOserum free media with different concentrations of anti-CD3 antibody or withThymoglobulin® instead of anti-CD3 antibody. Otherallogeneic protocols were reported by Iudicone et al. (53)who evaluated expansion and cytotoxicity of CIK cells in combination of IL-15with IL-2 during a period of four weeks of culture.  Autologous protocols were also reported byNiam et al.

(54), Liu et al. (55) and ChanWC and Linn YC (56) who have obtained CIK cells from patientswith myeloid leukemia, HCC and polycythemia respectively. According to Zhang etal. (57), the anti-tumor efficacy of autologous CIK cellsderived from cancer patients was lower because of the immunosuppressive stateof patients, compared with the allogeneic CIK cells obtained from adult healthyindividuals. Protocols involved cord blood MNCs differentiation into CIK cellswere also reported by Zhang et al. (58) and Durrieu et al.(59).

            Differentculturing protocols for CIK cells were reported but all these protocols sharethe main three proliferation pillars of CIK culture, IFN??, IL-2 and monoclonalanti-CD3 antibody, but with different growth factors’ concentrations or growthmedium used or with different cytokines combinations that may enhanceproliferation or selective tumor toxicity such as IL-6 (60), IL-7 (61),IL-12 (62), IL-15 (63, 53) and IL-21 (64).            Inthis study, we did not add protein serum to cultured cells, either in the formof fetal calf serum or as heat inactivated plasma, as the foreign protiens maylead to sensetization of killer cells to react with them. This finding was inline with Kerbel and Blakeslee (46) who reported that fetal calfserum should not be added as it has many drawbacks and can lead to seriousmisinterpretations in immunological studies. This hypothesis was followed byMeng et al. (39) and Li et al. (65) whoused X-VIVO serum-free medium and GT-T551 serum-free medium, respectively,during their CIK culturing protocols.

On the contrary, Niam et al. (54)and Wei et al. (66) used 10% defined fetal calf serum and Liuet al. (55) used 1% heat inactivated plasma during their CIKculturing protocols.            Inour study, induced MNCs cells began to grow double in number on day 4 ofculture, which may be due to indirect effect of IFN-? mediated by monocytesactivation, providing soluble proliferating factors (IL-12) and the initialmitogenic signal provided by the monoclonal anti-CD3 antibody and sustained bythe continuous presence of IL-2 (67). The onset of cells maturation,cluster-like formation, could be observed on day 7. Fully matured suspended CIKclusters could be observed on day 14.

This goes with the results reported by Liet al. (45), who cultured MNCs for 14 days and found that thecells began to proliferate within 3 days and became fully matured within 14days and he revealed that the cells grew as colonies in suspension. . On theother hand, Wei et al. (61), Bananno et al. (52)and Niam et al. (54) cultured human PBMCs for 15, 21 and 28days respectively to reach the maturation stage. Chan WC and Linn YC (56)cultured human MNCs for 26 days.

They started counting and evaluating CIK cellson day 10 as they found that it is too early to evaluate CIK cells before day10.            Inthe present study, cells count, growth rate and viability were reported every 3days. The result showed a significant increase in cells number along with time,starting from 10×106 cells/flask till reached 223.600 ± 15.588 x106cells/flask on day 14. The growth rate (i.e cells density) started onday zero as 1×106 cells/mL, then reached a peak (3.740 ± 0.

124 x106cells/mL) on day 7, and finally there was a significant decrease till day 14(2.795 ± 0.190  x106cells/mL). The present findings were not in line with Niam et al.

(54)who reported that the number of CIK cells dropped to a median of 0.44 fold ofstarting number at day 11 of culture, after which growth started from about day14 and approached a plateau by day 28. Unfortunately, the viability of inducedPBMCs in our study decreased significantly with time from 96.901 ± 1.50% to87.251 ± 2.38% which may be due to increased number of cells, decreased amountof nutrients and increased wastes formed by the cells in the crowded media.These results go in agreement with Kim et al.

(34) whoreported that the viability range for expanded cells on day 14 was 85-95%.            Ourstudy aims at measuring the proportion of the most effective subset CD3+CD56+in the culture. Flow cytometric analysis for CIK cells’ specific markers CD3and CD56 were performed at different time points on day 0, 7 and 14. Theresults showed that cells with positive CD3 phenotype upon seeding on day zerowere 6.

25 ± 3.36%. On day 7, their proportion was nearly triplicated (21.05 ±2.91%) and ranged on day 14 from 77.83% to 62.

2% with mean value 72.22 ± 8.70%.CD56 phenotype peering cells were 1.

74 ± 0.27% on day zero, 12.12 ± 1.63% onday 7 and ranged from 27.70% to 34.70% with mean value 31.

20 ± 3.50%.            Accordingto pervious results, there was a consecutive significant increase in all CIKcells subsets at each time point. The proportion of CD3+CD56+subset on day zero was 0.387 ± 0.091%, then it reached 6.457 ± 1.046% on day 7.

At the end of culture, the proportion of CD3+CD56+ rangedfrom 21.37% to 28.67% with mean value 25.137 ± 3.656% of all samples. Thepresent results of expanded CD3+CD56+ subset weresupported by Kim et al.

(34) who reported that cells bearingthe characteristic of the CD3+CD56+ phenotype werereadily expanded and constituted about one third of the total cell number. Guo etal. (68) who cultured CIK cells from healthy volunteers’ blooddonors for 14 day reported CD3+CD56+ proportion on day 14as 25.

31 ± 7.42%. Li et al. (65) cultured CIK cellsfrom patients with early stage melanoma blood for 14 days; reported CD3+CD56+proportion on day 14 as 21.8 ± 8%.

On the other hand, Bananno et al. (52)who cultured PBMCs for 21 days with different concentrations of Anti-CD3 antibody(50, 250, and 500 ng/mL) reported that the proportion of CD3+CD56+subsets on day 21 were 69.6% , 47.9%, and 29.3% respectively.             Inthe present work, TNF-? production and secretion by induced MNCs was measuredin culture supernatant at the beginning of the study, day zero, and at the endof it, day 14. The results showed that TNF-? was Nil on day zero. On day14, the TNF-? concentration reached up to 14.

538 ± 6.672 pg/mL. The presentresults were supported by those demonstrated by Zhang et al. (58)who investigated the secretion of TNF-? from expanded umbilical cord – CIKcells. The result reported was much less than that of our study (6 ± 5.5pg/mL), which may be due to different CIK cells origin or the usage of fetalcalf serum that should not be used in immunological studies (46).            TheCIK cells’ cytotoxic effect on cancerous cells showed variable degrees ofefficacy in several tumors including malignant lymphoma either Hodgkin’sdisease or non-Hodgkin’s lymphoma (NHL) (69), hematological malignanciesas acute myeloid and acute lymphocytic leukemia (70) and chroniclymphocytic leukemia (CLL) (71). Recent in vitro studies has furthershowed the potential activity of CIK cells differentiated from blood of healthyor patient volunteers against breast cancer (68),   pancreatic cancer (72), ovariancancer (73), sarcomas (74), metastatic melanoma (75),glioblastoma or brain cancer (76) solid tumors (67) andgallbladder cancer (7).

            Overthe past few years, CIK cell has entered clinical trials as adjuvant therapywith promising efficacy for colorectal cancer (78), endometrialcancer (79), pancreatic cancer (80), gastric cancer (81),non small cell lung cancer (82), metastatic nasopharyngeal carcinoma(83), Metastatic renal carcinoma (84), hematological malignancies(69, 70) and solid tumors (85).            Inthe current study, we examined the cytotoxic effect of mature CIK cells on day14 on HCC cells in vitro. Our results showed that there was asignificant increase in HepG2 cytotoxicity with the increase in CIK: HepG2ratio. The cytotoxic effect of CIK cells was maximal at 40:1 ratio, where thetumor cell killing ability was 58.

889 ± 1.104%. This finding was in line withWang et al. (30) and Yu et al. (35), whoreported the strong cytolytic activities of CIK cells and their ability torecognize a number of tumors.CONCLUSION            Collectively,the present study has provided data to support the ongoing practice ofgenerating CIK cells from human PB, which is an important and promisingstrategy for future work involving HCC immunotherapy.

PB-derived MNCs candifferentiate into CIK cells in vitro when cultured in complete nutrientmedia containing IFN-?, anti-CD3 antibody and IL-2. The CIK culture showeddifferent proportions of effector and cytotoxic subsets T cells, NK cells andNK-T cells. CIK cells showed high functional capacity as evidenced by secretionof cytokine TNF-? and cytotoxicity against HCC cell line, HepG2.            Thisstudy provides a simple and cheap strategy for in vitro differentiationof human PB-derived MNCs into CIK cells. Further studies are also needed toaddress whether CIK cells may be integrated into current HCC treatmentprotocols as well as CIK cells’ in vivo toxicity to normal and cancerouscells. Several ongoing clinical trials were performed on the efficacy of CIKcells on HCC patients with no results available yet (86-92).


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