ANTICANCER RESEARCH 25: 463-470 (2005)
Role of Tumor-associated Macrophages (TAM) in Advanced Gastric Carcinoma: The Impact on FasL-mediated Counterattack SATOSHI OHNO1,2,4, HIROYUKI INAGAWA3,5, DIPOK KUMAR DHAR2, TOSHIYUKI FUJII2, SHUHEI UEDA2, MITSUO TACHIBANA2, YUMIKO OHNO4, NOBUTAKA SUZUKI1,4, MASAKI INOUE4, GEN-ICHIRO SOMA3,5 and NAOFUMI NAGASUE2 1Department
of Complementary and Alternative Medicine, Kanazawa University, Graduate School of Medical Science, Ishikawa; 2Second Department of Surgery, Shimane Medical University, Shimane; 3Institute for Health Sciences, Tokushima Bunri University, Tokushima; 4Department of Molecular Reproductive Biology, Kanazawa University, Graduate School of Medical Science, Ishikawa; 5Institute for Drug Delivery System, Tokyo University of Science, Chiba, Japan
Abstract. Background: Exactly what role does tumor-derived Fas ligand (FasL) play in cancer: maintaining the immune privilege site or inducing a pro-inflammatory effect? One possible hypothesis is that tumor-associated macrophages (TAM) act as the mediator that enables apoptosis of anti-tumor immune cells without FasLrelated inflammation. We have evaluated the tumor FasL expression and TAM along the tumor margin and/or in cancer stroma, and their impact on the infiltration of immune-competent cells into the tumor nest. Patients and Methods: Tissue specimens from consecutive 84 advanced gastric carcinoma patients, who had undergone a curative resection, were evaluated for TAM (CD68+ cells), tumor FasL expression and immune status (CD8+ T cells). Results: A high number of TAM significantly correlated with lymph node metastasis, intestinal type tumor and FasL expression. Although TAM had a tendency for an inverse correlation with the number of CD8+ T cells within the tumor nest (nest CD8) (p=0.0592), there was no correlation between FasL expression and nest CD8 (p=0.2158). This inverse association was found to be stronger in cases with both FasL-positive and high TAM tumors than in others (p=0.0139). The combination parameter of FasLpositive and high TAM became an independent prognostic factor in Cox’s multivariate analysis, along with the pT status, nest CD8 and tumor cell apoptosis. Conclusion: We suggest that TAM works
Correspondence to: Dr. Satoshi Ohno, Department of Complementary and Alternative Medicine, Graduate School of Medical Science, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 9208640, Japan. Tel: 81-76-265-2147, Fax: 81-76-234-4247, e-mail:
[email protected] Key Words: Gastric cancer, tumor-associated macrophages (TAM), Fas ligand (FasL), cytotoxic T lymphocyte (CTL), immunomodulation, prognosis.
0250-7005/2005 $2.00+.40
harmoniously with tumor-derived FasL and serves as a barrier against the infiltration of CD8+ T cells into the cancer nest. Despite improved tumor diagnosis and various nonsurgical therapies, gastric cancer is the major cause of tumor-related death in Japan (1, 2). Previously, we reported that the number of CD8+ T cells within the cancer cell nest (nest CD8) is a significant prognostic factor in gastric carcinoma patients (3). To develop more effective therapies, particularly immunotherapies, against gastric cancers, elucidation of their local immunological escape mechanisms is an important goal. In neoplastic as well as inflammatory tissue, macrophages play the central role in phagocytosis of cellular debris, angiogenesis and stroma formation (4, 5). Furthermore, recent studies showed that macrophage dysfunction plays a role in tumor-induced immunosuppression. Naama et al. (6) have reported that macrophage production of nitric oxide, superoxide anion and tumor necrosis factor · were significantly decreased, while prostaglandin E2 production was increased in macrophages from melanoma-bearing mice. However, little is known about the immunomodulatory role of macrophages in patients with solid tumor. To date, there is no report describing the role of tumor-associated macrophages (TAM) in terms of immune escape mechanism in vivo. Among the several mechanisms of tumor immune escape, the frequently discussed method describes that tumors expressing Fas ligand (FasL) induce apoptosis of Fas-bearing immune effector cells and help in escape from the host-immune attack (7, 8). The Fas counterattack raised the intriguing hypothesis that cancer might be a site of immune privilege, representing a novel mode of immune evasion. However, despite many assessments of the prognostic impact of FasL expression in cancer patients, the results obtained so far are controversial (9-14). One of the major reasons for this might be
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ANTICANCER RESEARCH 25: 463-470 (2005) Table I. Characterization of the antibodies used in this study. Antibody (Manufacturer)
Species and subclass
Dilution
Incubation time (Temperature)
Epitope retrieval
Antigen
CD68 (DAKO, Copenhagen, Denmark)
Mouse IgG3k
1 : 50
2 hours (RT)
Trypsin
All macrophages (Non cross-reactive with dendritic cells)
CD8 (Novocastra Laboratories, Ltd. Newcastle, UK)
Mouse IgG2b
1 : 30
Overnight (4ÆC)
Microwave
T-cytotoxic/suppressor cells
CD34 (DAKO, Copenhagen, Denmark)
Mouse IgG1k
1 : 50
1.5 hours (RT)
(untreated)
Capillary endothelial cells
Rabbit IgG
1 : 200
Overnight (4ÆC)
Microwave
Fas ligand
FasL (Sant Cruz Biotechnology, Inc., Santa Cruz, USA)
RT: room temperature, Ig:immunoglobulin
a premise that FasL is a double-edged sword that can be used for inducing a pro-inflammatory effect as well as maintaining immune privilege (15). In this paper, we focus attention on the immunomodulatory role of TAM, based on the hypothesis that TAM act as the coexisting mediator that enable apoptosis of anti-tumor immune cells without inducing FasL-related inflammation. The aim of the present work was to investigate the effects of tumor-derived FasL and TAM in advanced gastric carcinoma and to determine their role in tumor immune privilege. This paper also discusses the prognostic significance of FasL, TAM, nest CD8 and tumor cell apoptosis.
Materials and Methods Patients and tissue processing. Formalin-fixed paraffin-embedded advanced gastric cancer specimens from 84 patients, who had undergone radical gastrectomy from January 1990 to December 1997 at the Shimane Medical University Hospital, Japan, were obtained for this study. The patients were staged according to the UICC TNM classification (1997) and belonged to either T2 (n=41) or T3 (n=43) stage. These patients did not receive any form of therapy before surgery. No other previous or concomitant primary cancer was present in any patients. All patients had a complete follow-up. Immunohistochemistry. CD68 expression (macrophage), FasL expression, cytotoxic T lymphocytes (CTLs) and microvessel were evaluated in serial sections stained with appropriate antibodies. The sections were cut at 3 Ìm thickness. Staining was performed by the streptavidin-biotin peroxidase complex (SABC) technique, using the
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SAB-PO kit (Nichirei, Tokyo, Japan) as described previously (3, 14). Table I shows the primary antibodies, their sources, dilutions, incubation times, epitope retrieval and specificity/reactivity against antigen. The evaluating procedures for FasL expression and CD8+ T cells have been described in our previously published study (3, 14). Briefly, tumors were considered as positive for FasL when they had stronger expression than the corresponding normal gastric mucosa. Lymphocytes stained by anti-CD8 antibody within the cancer cell nests were considered as nest CD8. For evaluation of the number of CD8+ T cells, three high-power fields (x 400) with the most abundant distribution of CD8+ T cells were selected from each specimen, counted and the results were expressed as the mean number of CD8+ T cells per three high-power fields. Evaluation and counting were done by two observers (S.O. and T.F.) who did not know any details regarding the patients’ background. Evaluation of tumor-associated macrophages. TAM were evaluated on the grounds of their localization by using a modified version of the method described by Naito et al. (16). We expressed CD68positive cells localized to the invasive margin or spatially isolated from cancer cells as margin TAM. For counting margin TAM, the sections were first evaluated at x100, and the five representative areas where TAM accumulated at higher density were identified. TAMs were then counted at x400 in each case, and the average of the five measurements was used for data analysis. Sometimes, numerous macrophages were noted near necrotic areas or in association with large neutrophil clusters; we excluded these macrophages from the evaluation in this study. Evaluation of microvessel density. Quantitative measurement of microvessel density (MVD) in tumor tissue was done by a computer-
Ohno et al: Role of TAM and FasL in Gastric Cancer
Figure 1. Representative serial sections are shown: Tumor with enriched CD68+ cell infiltration (A, x200) and tumor with strong expression of FasL (B, x200).
assisted image analysis system. The most vascular areas were assessed by scanning the entire tumor at low magnification (x40) by using a light microscope (BX50; OLYMPUS, Tokyo, Japan). The selected areas were photographed at a magnification of x200, the images were then visualized on a computer display through a charge-coupled device (CCD) video camera module (HC-300/OL; OLYMPUS, Tokyo, Japan) and color image freezer (Photograbì-300 SH-3; FUJIFILM, Tokyo, Japan). Subsequently, the image files were opened one at a time in the public-domain NIH Image program (developed at the U.S. National Institute of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/) on a Macintosh computer. Under the Analyze menu, measure was selected to calculate the "mean density" of the microvessels at the examined area. For each tumor, the mean of the "mean density" for 10 representative fields was used for statistical analysis in this study. TUNEL methods. Apoptotic cells and bodies were detected using the DeadEnd Colorimetric Apoptosis Detection System Kit (Promega, Madison, USA). To determine the number of apoptotic cells, 15 to 20 representative areas without necrosis and comprising at least 1000 cancer cells were counted from each sample with a light microscope (x400). The results were expressed as apoptotic index (AI), representing the number of apoptotic cells/1000 counted cells and mentioned in percentages. Statistical analysis. The difference in proportions of 2 x 2 tables was evaluated using the Chi-square test and Fisher’s exact test. As the results were similar, only the former is presented. The difference in continuous variables between the groups was compared using the Mann-Whitney U-test. The correlation between two continuous variables was investigated by means of Spearman’s rank correlation coefficient test. The probability of relapse after curative resection was compared with Kaplan-Meier analysis and the log-rank statistic. In the analysis of disease-free survival rates, those who died of causes unrelated to gastric cancer were considered to be disease-free at the end point. Cox proportional hazard analysis was used to determine the relative contribution of various factors to the risk of recurrence. A p value of
