HCC (50). Immunotherapy is a new and promising treatment

HCC is an aggressive cancer that
occurs in the setting of chronic liver disease and cirrhosis that frequently
presents in advanced stages (3). Malignant liver cells often survive
traditional 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 of
cancers. Cell-based immunotherapy is a set of therapeutic strategies based on
manipulating and co-opting a patient’s own immune cells, or donor cells and
using immune cell functions to halt and reverse disease (51).

           In the current work, human PB from
10 healthy volunteers was collected and PBMCs were separated. We found that,
the separated PBMCs count ranged from 30×106 to 34×106 cell/mL
and the lymphocytes account for 60.8 to 67.6% of total separated PBMCs. The
viability range upon separation was 95%-98%.

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         Allogeneic protocols were reported by
Bonanno et al. (52) who have obtained CIK cells using X-VIVO
serum free media with different concentrations of anti-CD3 antibody or with
Thymoglobulin® instead of anti-CD3 antibody. Other
allogeneic protocols were reported by Iudicone et al. (53)
who evaluated expansion and cytotoxicity of CIK cells in combination of IL-15
with IL-2 during a period of four weeks of culture.  Autologous protocols were also reported by
Niam et al. (54), Liu et al. (55) and Chan
WC and Linn YC (56) who have obtained CIK cells from patients
with myeloid leukemia, HCC and polycythemia respectively. According to Zhang et
al. (57), the anti-tumor efficacy of autologous CIK cells
derived from cancer patients was lower because of the immunosuppressive state
of patients, compared with the allogeneic CIK cells obtained from adult healthy
individuals. Protocols involved cord blood MNCs differentiation into CIK cells
were also reported by Zhang et al. (58) and Durrieu et al.
(59).

            Different
culturing protocols for CIK cells were reported but all these protocols share
the main three proliferation pillars of CIK culture, IFN??, IL-2 and monoclonal
anti-CD3 antibody, but with different growth factors’ concentrations or growth
medium used or with different cytokines combinations that may enhance
proliferation or selective tumor toxicity such as IL-6 (60), IL-7 (61),
IL-12 (62), IL-15 (63, 53) and IL-21 (64).

            In
this study, we did not add protein serum to cultured cells, either in the form
of fetal calf serum or as heat inactivated plasma, as the foreign protiens may
lead to sensetization of killer cells to react with them. This finding was in
line with Kerbel and Blakeslee (46) who reported that fetal calf
serum should not be added as it has many drawbacks and can lead to serious
misinterpretations in immunological studies. This hypothesis was followed by
Meng et al. (39) and Li et al. (65) who
used 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 Liu
et al. (55) used 1% heat inactivated plasma during their CIK
culturing protocols.

            In
our study, induced MNCs cells began to grow double in number on day 4 of
culture, which may be due to indirect effect of IFN-? mediated by monocytes
activation, providing soluble proliferating factors (IL-12) and the initial
mitogenic signal provided by the monoclonal anti-CD3 antibody and sustained by
the continuous presence of IL-2 (67). The onset of cells maturation,
cluster-like formation, could be observed on day 7. Fully matured suspended CIK
clusters could be observed on day 14. This goes with the results reported by Li
et al. (45), who cultured MNCs for 14 days and found that the
cells began to proliferate within 3 days and became fully matured within 14
days and he revealed that the cells grew as colonies in suspension. . On the
other hand, Wei et al. (61), Bananno et al. (52)
and Niam et al. (54) cultured human PBMCs for 15, 21 and 28
days respectively to reach the maturation stage. Chan WC and Linn YC (56)
cultured human MNCs for 26 days. They started counting and evaluating CIK cells
on day 10 as they found that it is too early to evaluate CIK cells before day
10.

            In
the present study, cells count, growth rate and viability were reported every 3
days. The result showed a significant increase in cells number along with time,
starting from 10×106 cells/flask till reached 223.600 ± 15.588 x106
cells/flask on day 14. The growth rate (i.e cells density) started on
day zero as 1×106 cells/mL, then reached a peak (3.740 ± 0.124 x106
cells/mL) on day 7, and finally there was a significant decrease till day 14
(2.795 ± 0.190  x106
cells/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 of
starting number at day 11 of culture, after which growth started from about day
14 and approached a plateau by day 28. Unfortunately, the viability of induced
PBMCs in our study decreased significantly with time from 96.901 ± 1.50% to
87.251 ± 2.38% which may be due to increased number of cells, decreased amount
of nutrients and increased wastes formed by the cells in the crowded media.
These results go in agreement with Kim et al. (34) who
reported that the viability range for expanded cells on day 14 was 85-95%.

            Our
study aims at measuring the proportion of the most effective subset CD3+CD56+
in the culture. Flow cytometric analysis for CIK cells’ specific markers CD3
and CD56 were performed at different time points on day 0, 7 and 14. The
results showed that cells with positive CD3 phenotype upon seeding on day zero
were 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% on
day 7 and ranged from 27.70% to 34.70% with mean value 31.20 ± 3.50%.

            According
to pervious results, there was a consecutive significant increase in all CIK
cells 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+ ranged
from 21.37% to 28.67% with mean value 25.137 ± 3.656% of all samples. The
present results of expanded CD3+CD56+ subset were
supported by Kim et al. (34) who reported that cells bearing
the characteristic of the CD3+CD56+ phenotype were
readily expanded and constituted about one third of the total cell number. Guo et
al. (68) who cultured CIK cells from healthy volunteers’ blood
donors for 14 day reported CD3+CD56+ proportion on day 14
as 25.31 ± 7.42%. Li et al. (65) cultured CIK cells
from 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.

            In
the present work, TNF-? production and secretion by induced MNCs was measured
in culture supernatant at the beginning of the study, day zero, and at the end
of it, day 14. The results showed that TNF-? was Nil on day zero. On day
14, the TNF-? concentration reached up to 14.538 ± 6.672 pg/mL. The present
results were supported by those demonstrated by Zhang et al. (58)
who investigated the secretion of TNF-? from expanded umbilical cord – CIK
cells. The result reported was much less than that of our study (6 ± 5.5
pg/mL), which may be due to different CIK cells origin or the usage of fetal
calf serum that should not be used in immunological studies (46).

            The
CIK cells’ cytotoxic effect on cancerous cells showed variable degrees of
efficacy in several tumors including malignant lymphoma either Hodgkin’s
disease or non-Hodgkin’s lymphoma (NHL) (69), hematological malignancies
as acute myeloid and acute lymphocytic leukemia (70) and chronic
lymphocytic leukemia (CLL) (71). Recent in vitro studies has further
showed the potential activity of CIK cells differentiated from blood of healthy
or patient volunteers against breast cancer (68),   pancreatic cancer (72), ovarian
cancer (73), sarcomas (74), metastatic melanoma (75),
glioblastoma or brain cancer (76) solid tumors (67) and
gallbladder cancer (7).

            Over
the past few years, CIK cell has entered clinical trials as adjuvant therapy
with promising efficacy for colorectal cancer (78), endometrial
cancer (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).

            In
the current study, we examined the cytotoxic effect of mature CIK cells on day
14 on HCC cells in vitro. Our results showed that there was a
significant increase in HepG2 cytotoxicity with the increase in CIK: HepG2
ratio. The cytotoxic effect of CIK cells was maximal at 40:1 ratio, where the
tumor cell killing ability was 58.889 ± 1.104%. This finding was in line with
Wang et al. (30) and Yu et al. (35), who
reported the strong cytolytic activities of CIK cells and their ability to
recognize a number of tumors.

CONCLUSION

            Collectively,
the present study has provided data to support the ongoing practice of
generating CIK cells from human PB, which is an important and promising
strategy for future work involving HCC immunotherapy. PB-derived MNCs can
differentiate into CIK cells in vitro when cultured in complete nutrient
media containing IFN-?, anti-CD3 antibody and IL-2. The CIK culture showed
different proportions of effector and cytotoxic subsets T cells, NK cells and
NK-T cells. CIK cells showed high functional capacity as evidenced by secretion
of cytokine TNF-? and cytotoxicity against HCC cell line, HepG2.

            This
study provides a simple and cheap strategy for in vitro differentiation
of human PB-derived MNCs into CIK cells. Further studies are also needed to
address whether CIK cells may be integrated into current HCC treatment
protocols as well as CIK cells’ in vivo toxicity to normal and cancerous
cells. Several ongoing clinical trials were performed on the efficacy of CIK
cells on HCC patients with no results available yet (86-92).