Primary liver cancer is the fifth most common cancer worldwide and the third most common cause of cancer mortality.1-4 Hepatocellular carcinoma (HCC) accounts for between 85% and 90% of primary liver cancers, representing more than 5% of all cancers. The estimated annual number of cases exceeds 500 000, with a mean annual incidence of around 3%-4%.5,6 China alone accounts for more than 50% of the world's HCC cases (age-standardized incidence rate: men, 35.2/100 000; women, 13.3/100 000).5
Spleen, which is the biggest immunity organ, plays an important role in the tumor immunity.7-10 Immune-associated defense against tumor development can be considered a “double-edged sword” by promoting tumor resistance, as well as facilitating tumor escape mechanisms. Thus, the known complexities of tumor immune responses do not lend credence to investigations that may explain these conflicts.11 At present, people think that the anti-tumor effectiveness of the spleen would change in the different stage of tumor. But there is still little direct evidence to support this theory. Macrophage (Mϕ) as the major immunocyte of the spleen, plays a key role in the immune function of the spleen. Assessment of immune function has traditionally been carried out by using cells obtained from either spleen or blood; this apparently is done because cells from these compartments are readily available and/or can be obtained in large number. So we established a spontaneous pulmonary metastasis model of liver cancer in rats by using diethylnitrosamin (DEN), observed the changes in ultrastructure and immune functions of splenic Mϕ from the different stages of the liver cirrhosis and cancer.
Male Sprague-Dawley rats weighing 100-120 g were kept under standardized conditions (light from 6 am to 6 pm, (21±1)°) with food and water supplied ad libitum at the Medical Research Center of School of Medicine, Xi'an Jiaotong University. This research protocol complied with the Guide for the Care and Use of Laboratory Animal (NIH Publication, 1996) and was approved by the Institutional Animal Use and Care Committee of our school.
Induction of spontaneous pulmonary metastasis model of liver cancer
In experiment groups, rats received an intraperitoneal injection of DEN (100 mg/kg; Sigma, St Louis, MO, USA) at first day, and supplied with DEN solution (100 mg/L) to drink at weeks 2-8 and weeks 12-16. The DEN solution was prepared every day, and bottles were covered with tin foil, by which protected from light. The rats were sacrificed at 8, 13, 16 week randomly, and divided into cirrhosis, cancer without metastasis, and metastasis group (10 rats per group) by the pathological examination. Untreated male SD rats were included as control group, which only received an intraperitoneal injection of saline at day 1.
Splenic Mϕ were isolated and purified by anchoring cultivation from all rats as previously described.12 Briefly, the splenic tissue samples were grinded into cell suspension by using a 200-mesh screen and the Mϕ was purified by anchoring cultivation. The cells were harvested prior to use by scraping them from the surface of the culture bottle. The suspension was centrifuged, and the cell pellet was resuspended with RPMI-1640 (Hyclone, Logan, Utah, USA).
Small pieces of splenic tissue samples were fixed in 2.5% glutaraldehyde in 0.2 mol/L phosphate buffer (PH=7.4), and were post-fixed in 1% osmiumtetroxide and embedded in epoxy resin blocks. Ultra-thin sections were stained with uranyl acetate and lead citrate. Samples were subjected to electron microscopic examination using a Hitachi H-600 transmission electron microscope (Hitachi, Japan).
The VybrantTM Phagocytosis Assay Kit (Molecular Probes, Eugene, Oregon, USA) was used according to the protocols supplied by the manufacturer. Briefly, aliquots (100 μl) of splenic Mϕ suspension were then dispensed into a black 96-well culture plate and incubated at 37° in 5% CO2 for 1 hour, allowing the cells to adhere to the bottom of the wells. The culture medium was aspirated, and 100 μl of fluorescent E. coli particles suspended in Hanks’ buffer were added. The plates were further incubated for various time periods. The buffer solution in the wells was removed by aspiration. Extracellular fluorescence was then quenched by adding 100 μl of trypan blue (250 μg/ml, pH 4.4). The dye was removed after 1 minute. The intensity of fluorescence associated with intracellular fluorescent particles was measured directly in the wells using a multifunctional microplate reader at 485 nm excitation and 530 nm emission.
Antigen processing and presenting
DQTM Ovalbumin Kit (Molecular Probes) was used to evaluate the antigen processing and presenting of splenic Mϕ according to the protocols supplied by the manufacturer. Briefly, cells were plated at 1×105 cells/well in 24-well plates with RPMI-1640. After incubating with DQTM ovalbumin at 1 mg/ml for 3 hours, the cells were washed with PBS and were tested immediately for fluorescence using a FACS Caliber (Becton Dickinson, Mountain View, CA, USA), at the excitation wavelength of 505 nm and emission wavelength of 515 nm. The data were depicted by the percentage of positive cells.
ELISpot kits for the detection of rat tumor necrosis factor (TNF)-α (Diaclone, France) were used according to the protocols supplied by the manufacturer. In short, the cell suspension and controls were added into the PVDF-backed microplates coated with polyclonal antibody specific for rat TNF-α, and were incubated at 37° in 5% CO2 for 2 hours. Thereafter, the detection antibody was added and incubated at 2-8° overnight. After which, the diluted Streptavidin-AP was added into each well and incubated for 30 minutes at room temperature, followed by the addition of BCIP/NBT Chromogen for 1 hour at room temperature. Between incubations, the plates were washed with Wash Buffer. The developed microplates were analyzed by counting spots using the AID EliSpot reader system (Strassberg, Germany).
Cells were plated in 96-well plates with a density of 5×105 per well. After incubating for 2 hours to allow adhesion to the microplate surface, 3-[4,5-dimethylthiozol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) (Sigma) at 5 mg/ml was added to each well for 4 hours. Subsequently, the culture medium was removed. Thereafter, 150 ml of dimethyl sulfoxide (DMSO) (Sigma) was added to each well. The absorbance was measured at 570 nm using a multifunctional microplate reader (POLARstar, OPTIMA, Germany).
Results are presented as mean and standard error of the mean (SEM). One-way repeated-measures analysis of variance (ANOVA) was used for the analysis of differences between the experimental and control groups. The threshold for significance was set at P <0.05.
Changes in ultrastructure of Mϕ
In the normal spleen, the Mϕ had few microvillus or pseudopod-like protrusions on cell surface. There were mitochondria in shape of sphere or ellipse, few rough endoplasmic reticulums and free ribosomes, and some lysosomes in cytoplasm. Few Mϕ contained phagocytized erythrocytes. In cirrhosis group, splenic Mϕ had more pseudopod-like protrusions, lysosomes, mitochondria in shape of sphere or ellipse, and rough endoplasmic reticulums. The count of Mϕ with cell debris in the cytoplasm also increased than that of control group. In cancer without metastasis group, splenic Mϕ had many villi and pseudopodium-like protrusion on the cell surface, with many mitochondria and lysosomes, dead cells and cell debris in the cytoplasm. The electron density in rough endoplasmic reticulum also increased. In metastasis group, the integrity of cytomembrane was broken in Mϕ, pseudopod-like protrusions reduced. There were only few organelle and secondary lysosome. The endocytoplasmic reticulum intumesced into shape of vacuole, and the electron density decreased. Part of cytoplasm became into blank. Cell debris was seldom seen in the cytoplasm of Mϕ (Figure 1).
Changes in immune function of Mϕ
As compared to control group, the phagocytosis rate, viability, spot number of positive cells for TNF-α, and positive rates of antigen processing and presenting in cirrhosis and caner without metastasis group were all increased (P <0.05, P <0.01). There were no significant differences between cirrhosis and cancer without metastasis group in the phagocytosis rate, viability (P >0.05). But the spot number of positive cells for TNF-α, and positive rates of antigen processing and presenting in caner without metastasis group were significantly higher than that of cirrhosis group (P <0.01). In metastasis group, the phagocytosis rate, viability, spot number of positive cells for TNF-α, and positive rates of antigen processing and presenting were all decreased significantly than that of other groups (P <0.05, P <0.01. Table, Figures 2 and 3).
As the largest secondary lymphoid organ, the spleen has a number of important roles in immune response, including antitumor immunity. It generally accepted that spleen played a complex role in the tumor immunity, which would change in the different periods of cancer, i.e. the characteristics of “two-way” and “phase”. In the early stage of tumor, spleen had a positive effect in tumor immunity, but this effect was insufficient. With the development of tumor and the inhibitory state of tumor immunity, the antitumor effect of spleen decreased, even reversed in the advanced stage of malignant tumor. If the primary lesion of cancer had been removed, spleen would be benefit for the clearance of cancer cell because the inhibitory effect of cancer bearing on antitumor immunity had diminished. Until now, the research about effects of spleen on tumor immunity mainly focused on the impact of splenectomy on tumor growth, and changes in subgroup of peripheral blood lymphocyte. It still need to be cleared that the changes in structure and function of spleen.
Mϕ, as the major immunocyte of the spleen, plays a key role in the immune function of the spleen.13,15 Mϕ also contributed to the antitumor immunity by phagocytois, tumor antigen processing and presenting, antibody dependent cell mediated cytotoxicity, and secreting cytokines like TNF and NO. An investigation of the functional change in splenic Mϕs would therefore provide a good platform to evaluate the function of the spleen in antitumor immunity.
Ultrastructure could be regarded as an indicator of the changes in cell function. After stimulated with interferon (INF)-γ and lipopolysaccharide, the Mϕ would have more pseudopod-like protrusions and organelle, by which the enhancement of Mϕ function could be reflected. It generally accepted that TEM would be an ideal method to observe the changes in ultrastructure. Cell viability, which could be detected by MTT, is also an important indicator for cell function.
Phagocytosis is one of the most important functions of Mϕ in antitumor immunity.16 The process of phagocytosis can be observed and quantitated in Mϕ by following the internalization of a foreign particle, such as fluorescently labeled immune complexes and bacterial particles. The VybrantTM Phagocytosis Assay Kit provides a model system for quantifying the phagocytic function. This technique takes advantage of the detectability of the intracellular fluorescence emitted by the engulfed particles, as well as the effective fluorescence quenching of the extracellular probe by trypan blue. This kit can be used in Mϕ and other adherent cell lines.17,18
In addition activated Mϕ contributes to the production of multiple cytokines and inflammatory mediators synthesize, which have been considered to be cytotoxic towards certain tumor targets.19,20 TNF-α is a critical molecule that promotes tumor immune surveillance in vivo. TNF-α signaling may be required only for immune responses in conditions of limited immunostimulatory capacity, such as tumor surveillance.21,22 ELISPOT assay is a common method for monitoring immune responses in humans and animals. The ELISPOT assay is based on, and was developed from a modified version of the ELISA immunoassay. ELISPOT assays have been adopted for the identification and enumeration of cytokine-producing cells at the single cell level. Simply put, at appropriate conditions the ELISPOT assay allows visualization of the secretory product of individual activated or responding cells. Each spot that develops in the assay represents a single reactive cell. Thus, the ELISPOT assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information.23-25
Mϕ plays a central role in the immune response by presenting antigen to lymphocytes during the development of specific immunity. DQTM ovalbumin is a self-quenched conjugate of ovalbumin that exhibits bright green fluorescence upon proteolytic degradation. This substrate, which is labeled with our pH insensitive BODIPY FL dye, is designed especially for the study of antigen processing and presentation.
Thus, we detected the changes in structure and function of splenic Mϕ listed above in different stages of liver cancer model induced by DEN. The results showed that the functions of phagocytosis, secretion, and antigen processing and presenting of splenic Mϕ were increased in groups of cirrhosis and cancer without metastasis, especially the cancer group, as compared to the control group. But these functions were all decreased significantly in the metastasis group. There has been some similar tendency in the changes in ultrastructure. These results further supported the viewpoint that the characteristics of “two-way” and “phase” of spleen in tumor immunity.
It is difficult to figure out how splenic Mϕ performed their function in the tumor immunity, and it may be a complicated procedure, involving multiple factors. So the mechanism of changes in structure and function of splenic Mϕ in the tumor immunity is still need to be clarified.
1. Laurent-Puig P, Zucman-Rossi J. Genetics of hepatocellular tumors. Oncogene 2006; 25: 3778-3786.
2. Kirk GD, Bah E, Montesano R. Molecular epidemiology of human liver cancer: insights into etiology, pathogenesis and prevention from The Gambia, West Africa. Carcinogenesis 2006; 27: 2070-2082.
3. Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 2006; 6: 674-687.
4. Chen YB, Yan ML, Gong JP, Xia RP, Liu LX, Li N, et al. Establishment of hepatocellular carcinoma multidrug resistant monoclone cell line HepG2/mdr1. Chin Med J 2007; 120: 703-707.
5. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007; 132: 2557-2576.
6. Llovet JM, Beaugrand M. Hepatocellular carcinoma: present status and future prospects. J Hepatol 2003; 38 Suppl 1: S136-S149.
7. Wakeman CJ, Dobbs BR, Frizelle FA, Bissett IP, Dennett ER, Hill AG, et al. The impact of splenectomy on outcome after resection for colorectal cancer: a multicenter, nested, paired cohort study. Dis Colon Rectum 2008; 51: 213-217.
8. Renukaradhya GJ, Khan MA, Vieira M, Du W, Gervay-Hague J, Brutkiewicz RR. Type I NKT cells protect (and type II NKT cells suppress) the host's innate antitumor immune response to a B-cell lymphoma. Blood 2008; 111: 5637-5645.
9. Blishchenko EY, Sazonova OV, Yatskin ON, Kalinina OA, Tolmazova AG, Philippova MM, et al. beta-Actin-derived peptides isolated from acidic extract of rat spleen suppress tumor cell growth. J Pept Sci 2008; 14: 811-818.
10. McGory ML, Zingmond DS, Sekeris E, Ko CY. The significance of inadvertent splenectomy during colorectal cancer resection. Arch Surg 2007; 142: 668-674.
11. Jones HP, Wang YC, Aldridge B, Weiss JM. Lung and splenic B cells facilitate diverse effects on in vitro measures of antitumor immune responses. Cancer Immun 2008; 8: 4.
12. Yan F, Li Z, Zhang S, Yang J, Li A, Liu X. Isolation and purification of macrophages from human spleen. J Xi'an Jiaotong Univ (Med Sci) 2004; 25: 513-516.
13. Cesta MF. Normal structure, function, and histology of the spleen. Toxicol Pathol 2006; 34: 455-465.
14. Mebius RE, Kraal G. Structure and function of the spleen. Nat Rev Immunol 2005; 5: 606-616.
15. Li ZF, Zhang S, Huang Y, Xia XM, Li AM, Pan D, et al. Morphological changes of blood spleen barrier in portal hypertensive spleen. Morphological changes of blood spleen barrier in portal hypertensive spleen. Chin Med J 2008; 121: 561-565.
16. Oflazoglu E, Stone IJ, Gordon KA, Grewal IS, van Rooijen N, Law CL, et al. Macrophages contribute to the antitumor activity of the anti-CD30 antibody SGN-30. Blood 2007; 110: 4370-4372.
17. Foukas LC, Katsoulas HL, Paraskevopoulou N, Metheniti A, Lambropoulou M, Marmaras VJ. Phagocytosis of Escherichia coli by insect hemocytes requires both activation of the Ras/mitogen-activated protein kinase signal transduction pathway for attachment and beta3 integrin for internalization. J Biol Chem 1998; 273: 14813-14818.
18. Wan CP, Park CS, Lau BH. A rapid and simple microfluorometric phagocytosis assay. J Immunol Methods 1993; 162: 1-7.
19. Kim KR, Son EW, Rhee DK, Pyo S. The immunomodulatory effects of the herbicide simazine on murine macrophage functions in vitro
. Toxicol In Vitro 2002; 16: 517-523.
20. Keller R, Keist R, Frei K. Lymphokines and bacteria, that induce tumoricidal activity, trigger a different secretory response in macrophages. Eur J Immunol 1990; 20: 695-698.
21. Calzascia T, Pellegrini M, Hall H, Sabbagh L, Ono N, Elford AR, et al. TNF-alpha is critical for antitumor but not antiviral T cell immunity in mice. J Clin Invest 2007; 117: 3833-3845.
22. Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci U S A 1975; 72: 3666-3670.
23. Khalil G, El-Sabban M, Al-Ghadban S, Azzi S, Shamra S, Khalife S, et al. Cytokine expression profile of sensitized human T lymphocytes following in vitro stimulation with amoxicillin. Eur Cytokine Netw 2008; 19: 131-141.
24. Biglino A, Crivelli P, Concialdi E, Bolla C, Montrucchio G. Clinical usefulness of ELISPOT assay on pericardial fluid in a case of suspected tuberculous pericarditis. Infection 2008; 36: 601-604.
25. Yoon JC, Rehermann B. Determination of HCV-specific T-cell activity. Methods Mol Biol 2009; 510: 403-413.