|DOI : http://dx.doi.org/10.12729/jbr.2012.13.2.157|
1College of Pharmacy
2Medical Research Center, Chungbuk National University, Cheongju 361-763, Korea2Department of Comprehensive Melanoma Research Center (CMRC), H. Lee Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, Florida 33612
|(Received Jun 8, 2012, Revised Jun 21, 2012, Accepted Jun 26, 2012)|
|Human natural killer (NK) cells are major players in innate immune response. The functions of these cells as a scavenger of cancer cells are enhanced by cytokines such as interleukin-2 (IL-2), which play an important role in immune response in both tumors and virally infected cells. Liver cancer has a high incidence rate and is a major cause of death in Korea. We provide evidence that human NK cells inhibit tumor growth of the hepatocellular carcinoma cell line SNU-354. NK cells were cultured with human IL-2 for 14 days, yielding an enriched NK cell population containing 35% CD8+ cells, 6% CD4+ cells, and 51% CD16+ /CD56+ cells. Intravenous injection of NK cells at doses from 2.5 to 10 million cells/mouse was administered once per week in a nude mouse model that retains human liver tumor induced by implantation of SNU-354 cells. The results showed that human NK cells were recruited within tumor tissue and inhibited SNU-354 tumor growth by 32%, 58%, and 65%. The current data suggest the potential for use of NK cell-based immunotherapy for treatment of human liver cancer.|
Hepatocellular carcinoma is the sixth most common neoplasm and the third most frequent cause of cancer death in Korea. Liver cancer mortality rate in Korea is the highest among OECD countries. The primary risk factor is Hepatitis B virus and hepatitis C virus infection of the liver causing chronic liver disease . Initially, lack of specific signs and slow onset of symptoms make liver cancer difficult to diagnose early enough to treat effectively. Depending on the stage, liver cancer is treated surgically, with radiation therapy, and chemotherapy. However, current treatments are problematic in that treatments destroy normal cells near the tumor along with cancer cells. To improve the survival rate of patients, more selective and effective treatments are required. Immunotherapy using immune cells has the advantage of action on cancer cells that is specific and less toxic. For this reason, anti-cancer effects of immune cells have been actively studied, including new research on natural killer cells. Natural killer cells account for about 5% of peripheral blood; they are found in the bone marrow, spleen and lymph nodes as well as in specific organs such as the liver and lungs. They are activated by cytokines and/or by interactions with specific molecules expressed on target cells [2-5]. Human NK cells are broadly defined as CD3- CD56+ lymphocytes. They can be further subdivided into two main functional subsets based on their surface expression of CD56. CD56bright NK cells have potent immunoregulatory properties, and CD56dim NK cells have potent cytotoxic functions. The CD56dim NK cells express high levels of FcγRIIIA (CD16) allowing them to mediate ADCC . NK cells have both activation and inhibitory receptors to recognize cancer or normal cells. Cytotoxicity of NK cells is triggered by tumor cells lacking expression of certain self-MHC class I molecules [7-9]. Thus, immunotherapy using natural killer cells has the advantage overcoming the problems of existing chemotherapy, induction of specific antigens on cancer cells, and possible treatment of metastatic cancer cells. We have reported inhibitory effects of natural killer cells for growth of colon and lung cancers [10-11]. In this experiment, we provide evidence that NK cells exhibit cytotoxic activity on growth of hepatoma tumor and are recruited in the tumor tissue.
Human hepatocellular carcinoma cell line SNU-354 cells were obtained from Korean Cell Line Bank (KCLB, Seoul, Korea) and grown at 37℃ in 5% CO2 in RPMI 1640 medium (Gibco, USA) supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen, USA). NK cells were made from peripheral blood mononuclear cells (PBMC) of healthy volunteers. After obtaining consent, 40 ml of blood was collected with heparin. Buffy coats of PBMC was separated by ficoll-hypaqus density centrifugation and washed with PBS. After washing, cells (1 × 106 cells/ml) were resuspended in lymphomedia containing 5% human serum (Biowhittaker-Cambrex, Walkersville, MD) and cultured with both anti-CD3 antibody (OKT-3 10 ng/ml; BD pharmingen, NJ, USA) and recombinant human IL-2 (Proleukin 500 U/ml, Chiron, Emeryville, USA). After 5 days, the media was changed to one containing rhIL-2 (500 U/ml) and 5% human serum. The activated NK cells NKM were supplied from NKBIO Ltd. (Seongnam 462-807, Korea).
NK cells (1 × 106 cells) were washed once with phosphate-buffered saline (PBS) and then harvested. Cell suspension was treated with anti-CD8-PE/CD4-FITC or anti-CD3-FITC/CD16+56-PE (BD Bioscience, USA) for 20 minutes at 4℃. Labeled cells were washed in PBS to eliminate unbound antibody, fixed in PBS containing 1% paraformaldehyde, and then analyzed on a BD FACS Canto flow cytometer (BD Biosciences, CA USA).
SNU-354 (6 × 106 cells) were injected subcutaneously into female nu/nu mice (SLC Japan, Inc.), For each experiment, mice were randomly distributed into equal groups (10 mice per group) such that NK cells at doses from 2.5, 5 and 10 million cells per mouse were intravenously injected once a week (day 0, 7, and 14) and adriamycin (ADR; Sigma-Aldrich, St.louis, USA) as positive control was intravenously injected once a week (day 0, 7, and 14) at a concentration of 2 mg/kg. Tumor volumes were estimated by the formular: length (mm) × width (mm) × height (mm)/2. On days 21, tumor was separated from mouse and then tumor volume (mean SD; mm3) and the tumor weights were measured. To determine the toxicity of NK cells, body weight changes of the nude mouse were measured.
Mice were sacrificed after the final treatment with PBS or different doses of NK cells. Tumor tissues were isolated and post-fixed for 16 hours with 4% paraformaldehyde-contatining PBS. The fixed tumors were immersed to cryoprotect for overnight in 30% sucrose solution in PBS. Serial sections were cut with a freezing microtome. Immunohistochemical staining was performed using the avidin-biotin peroxidase method. Endogenous peroxidase activity was blocked by treating with 3% H2O2. And also, nonspecific protein binding was blocked with 1% bovine serum albumin (BSA). Initially, tissue sections were incubated at 4℃ with mouse anti CD56 antibody (1:500 dilution, BD Biosciences, USA). After washing with PBS, the sections were incubated in biotinylated goat anti mouse IgG (1:2000dilution, Vector Laboratories, Burlingame, CA USA) for 2 hours at room temperature. The sections were washed and subsequently incubated with streptavidin-conjugated peroxidase complex (ABC kit, 1:200 dilution, Vector Laboratories) for 50 min followed by PBS washing. Peroxidase reaction was carried out using 3,3’-diaminobenzidine tetrahydrochloride (DAB, 0.02%). After washing, the sections were counterstained with a hematoxylin and mounted on polyglycine-coated slides for microscopy.
From In vivo experiment, data on tumor growth were collected and analyzed with 10 mice per group, in vitro data such as FACS analyses were obtained from three independent experiments. The standard deviation (SD) and P-values were calculated using Student’s t-test and ANOVA (GraphPad Prism, GraphPad Software, USA).
NK cells were enriched with PBMC by cultivation in the presence of both IL-2 and anti-CD3 antibody for 14 days. During the cultivation of PBMC, the total immune cells increased by more than 200-fold (data not shown). The cultured NK cells expressed highly IFN-γ, TNF-α, perforin and granzyme B genes involved in cytotoxic function, indicating that NK cells were activated [10, 11]. In order to analyze the population of NK cells, fluorescence flow cytometry was performed. The resulting population comprised 35% CD8+ T cell, 6% CD4+ T cell, and 51% CD16/CD56+ NK cell (Fig. 1). This result indicates that CD3-negative and CD56-positive NK cells were enriched about 10-fold since fresh PBMC usually contains about 5% CD3- CD56+ cells.
Fig. 1. Phenotypic characterization of NK cell. Human PBMCs were cultured in the presence of IL-2 for 14 days and the enriched NK cell populations were stained with monoclonal antibodies, such as anti-CD4-FITC + anti-CD8-PE (A) or anti-CD3-FITC + anti-CD16/CD56-PE (B), and analyzed with FACSCanto flow cytometer as described in Materials and Methods.
We evaluated the inhibition of tumor growth with enriched NK cells in nude mouse xenograft model. In preliminary experiments, it was revealed that three hundred million NK cells did not show any observable adverse effect in nude mice. Mice did not exhibit hair ruffling, lowering morbidity, or weight loss. Thus, we injected NK cells intravenously at doses of less than one hundred million cells. Six million SNU-354 cells were injected subcutaneously into nude mouse and a tumor mass was observed visually after 9 days in nude mouse and showed steady growth. At 21 days, in the control group, tumors had grown an average of 252 ± 102 mm3 (Fig. 2A). When NK cells were injected at 2.5 × 106 cells/mouse, the growth of tumor decreased by 34% as average tumor size was 164 ± 72 mm3. When NK cells were injected 5 × 106 cells/mouse and 10 × 106 cells/mouse, the growth of cancer cells was inhibited by 59% and 66% as mean tumor size was 102 ± 54 mm³ and 85 ± 49 mm3. The positive control group showed inhibition rate of 56% as tumor size was 111 ± 44 mm3.
Fig. 2. Antitumor effect of activated NK cells on SNU-354-induced liver tumor. SNU-354 cells were implanted subcutaneously into nude mice (n=10) on day 0. Activated NK cells NKM at doses from 2.5 to 10 million cells/mouse were injected intravenously once in a week (day 0, 7 and 14). Adriamycin (ADR) was injected intravenously at 2 mg/kg. (A) Tumor volumes were estimated by the formula: length (mm) × width (mm) × height (mm)/2. Statistical significance was determined using the Student’s t-test versus PBS-treated control group (*P<0.05, ***P<0.001). (B) On day 21, the mice were sacrificed and the tumor weights were measured. (C) Representative photographs of tumor masses are shown. (D) The body weights of the tumor-bearing nude mice were measured to evaluate overall toxicity. Statistical significance was determined using Student’s t-test versus PBS-treated control group (**P<0.01).
On 21 days, the tumor masses were isolated from the nude mice for measuring tumor weight. In the control group, tumor weight was 527 ± 231 mg. When NK cells were injected at 2.5 × 106 cells/mouse, the growth of cancer cells was inhibited by 32%, a mean tumor weight of 355 ± 141 mg. When NK cells were injected at 5 × 106 cells/mouse and 10 × 106 cells/mouse, growth of cancer cells was inhibited by 58% and 65%. Mean values of tumor weight were 220 ± 139 mg and 184 ± 113 mg, respectively (Figs. 2B and 2C). The adriamycin-treated group as a positive control showed the growth inhibition of 54% as tumor size was 241 ± 124 mg.
To observe the toxicity by treatment of NK cells, the body weight of nude mouse was measured for 21 days and behavior changes verified visually. The body weight of the control group was 123% compared to baseline, While the NK cell-treated groups were 121~125%. The ADR group was likewise 123% of baseline (Fig. 2D). So no abnormal weight changes were observed, nor was there any strange behavior. This result indicates that treatment of NK cells does not induce any adverse effect in our experimental system.
We determined accumulation of human NK cells within the hepatoma tumor implanted in the nude mice model, since NK cells exhibit anticancer activity mainly by direct contact to target cells. Because CD56 surface molecules are specifically expressed on NK cells, we decided to observe distribution of NK cells by immunohistochemical staining using anti CD56 antibody. We found that the number of brown colored (CD56-labeled) cells was observed only in tumor sections from 5 million NK cell-treated mice, but not in control tumor. CD56-positive NK cells were located in tumor and around periphery of tumor mass (Fig. 3). Moreover, density of NK cells in the hepatoma tumors was dependent on the doses of the treated NK cells (data not shown). This data indicates that NK cells are sufficiently recruited to inhibit the growth of tumor
Fig. 3. Recruitment of NK cells in hapatoma tumor. Human NK cells in hepatoma tumors of SNU-354-implanted mice were detected by using anti CD56 antibody followed by treatments of biotin-conjugated goat anti mouse IgG and streptavidin-HRP complex as described in Materials and Methods. Thick-tumor sections (5 μm) were prepared from mice treated with PBS (A) or 5 × 106 NK cells (B). Arrows indicate positively stained NK cells inside tumor and around periphery of tumor mass. Original magnifications, ×400.
Previous studies have used a dose of NK cell in animal experiments varying from 1 × 105 cells/mouse to 1 × 108 cells/mouse, depending upon kinds of cancer cells, implantation site, density of immune cells, type of experimental animals, and the experimental period. While such differences exist, these assays show similar antitumor activity.
To verify in vivo antitumor effect, the cultured hepatocellular cancer cell SNU-354 was transplanted 6 × 106 cells/ mouse into nude mouse and formed a solid tumor of statistically similar size. When NK cells were injected intravenously at a dose of 2.5 ~ 10 × 106 cells/mouse, tumor growth was strongly inhibited. When administered once a week, we observed weak antitumor activity at a concentration of 2.5 × 106 cells/mouse compared to the concentration of 5 × 106 cells/mouse and 10 × 106 cells/mouse. In other words, increased concentration of NK cell produces an increase of antitumor activity, indicating the dose dependency of the anticancer effects.
From immunohistochemical staining with anti-CD56 antibody, we observed that NK cells are recruited in and around the tumor tissue, suggesting that NK cells and SNU-354 cancer cells make a direct contact to inhibit the growth of the hepatoma tumor in the animal model. The NK cell dose treated in the nude mouse has proven to be non-toxic. During the test period, both weight changes and abnormal behavior of nude mouse were not observed. In preliminary experiments, it was proven that an injection of 3 × 108 NK cells/mouse (30 times of nomal dosage) was likewise observed to be non-toxic.
In this study, the anti-tumor effects of NK cells were identified in human hepatoma cells using nude mouse xenograft model. In addition, we found that NK cells are possible to use as a therapeutic agent for the treatment of liver cancer.
This work was supported by the research grant of the Chungbuk National University in 2010.
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