Abstract
In the present study, we studied the expression of T-bet, a key marker for type 1 immune responses, within the tumor microenvironment of gastric cancer, and analyzed its association with clinicopathological parameters. One hundred and fifty-two archival paraffin-embedded gastric tumor tissues were collected, and the expression of T-bet in these cancer tissue specimens was examined by immunohistochemistry. T-bet+ tumor-infiltrating lymphocytes (TILs) in some gastric cancer tissues were further characterized by flow cytometric analysis. The density of T-bet+ TILs in gastric cancer tissues in relation to patient’s clinicopathological parameters and postoperative prognosis has been analyzed. Herein, we have found significant increases in T-bet+ lymphocytes in tumor tissues as compared with normal stomach tissues, gastritis tissues or gastric polyp specimens. T-bet+ cells mainly consisted of CD4+, CD8+ and CD56+ TILs. In addition, lower numbers of T-bet+ TILs were associated with poor clinicopathological parameters such as invasion to muscular layer, larger tumor size and advanced cancer stages. Moreover, patients with higher numbers of T-bet+ TILs have longer disease-free survival and overall survival. Thus, our study supports the idea that tumor growth elicits spontaneous type 1 cellular immune responses and tumor progression is associated with suppression of antitumor immunity. T-bet expression within tumor can serve as a prognostic indicator for gastric cancer and a potential biomarker for immunotherapy.
Keywords: T-bet, Tumor-infiltrating lymphocyte, Gastric cancer, Prognosis
Introduction
Gastric cancer (GC) is a common type of gastrointestinal malignancies and the 10th leading cause of cancer-related death in 2010 worldwide. Surgical tumor resection remains the primary curative treatment for GC, but the overall 5-year survival rate for patients with advanced GC remains poor [1]. Immunotherapy holds great promises as an effective adjuvant therapy for this cancer. However, despite the high levels of anti-tumor immune responses in the blood from patients who have been given tumor vaccines, the objective responses have been limited due to the presence of the immune suppressive tumor microenvironment [2]. Therefore, understanding the molecular and cellular basis of tumor immune microenvironments is important for improving tumor immunotherapy.
Cell-mediated immune responses against tumor are thought to be present spontaneously in human cancer patients as a critical component of tumor immune surveillance [3]. Type 1 immune responses, mediated by Th1 cells, CTLs, γδ T cells and NK cells, are a critical component of cell-mediated immunity against cancer [4]. In humans, the presence of Th1 cells and CTLs within the tumor can be a favorable prognostic indicator [4]. T-bet is a hallmark transcription factor that is essential for differentiation and function of Th1 cells, CD8+ T cells, γδ T cells and NK cells [5]. In animal models, the role of T-bet in anti-cancer immune responses has been attributed to its function in innate cells such as NK cells and DCs [6–9] as well as adaptive immune system such as Th1 and CD8+ T cells [10]. T-bet mRNA is up-regulated in tumor tissues from early-stage colorectal cancer when compared to those from late-stage cancer [11]. Nevertheless, the nature of T-bet expression in tumor tissues and its clinical significance has not been well characterized.
In the present study, we investigated T-bet+ tumor-infiltrating lymphocytes (TILs) in human gastric cancer tissues. The density of T-bet+ TILs in gastric cancer tissues in relation to patient’s clinicopathological parameters and postoperative prognosis has been analyzed. We have found significant increases in T-bet+ lymphocytes in tumor tissues as compared with normal gastric tissues, gastritis tissues and gastric polyp specimens, suggesting T-bet+ lymphocytes play an important role in the process of tumorigenesis. In addition, lower numbers of T-bet+ TILs are associated with poor clinicopathological parameters such as invasion to muscular layer, larger tumor size and advanced cancer stages. Moreover, patients with higher numbers of T-bet+ TILs survive longer than those with lower numbers of such cells. Our study supports the idea that tumor growth elicits spontaneous type 1 cellular immune responses and tumor progression is associated with suppression of antitumor immunity.
Materials and methods
Patients and tissue samples
We obtained archived formalin-fixed and paraffin-embedded tumor tissues from 152 gastric cancer patients who underwent surgical resection between Jan 1998 and Dec 2009 in the Third Affiliated Hospital of Soochow University. None of the patients received chemotherapy or radiotherapy before surgery. The tumor stages were assigned according to the International Union Against Cancer staging system [12]. Patients’ clinicopathological parameters are shown in Table 1, and patients’ survival intervals are dated toward the end of Dec 2010. Ten cases of gastric polyp tissues, ten cases of chronic atrophic gastritis tissues and five cases of normal gastric tissues obtained from non-malignant portion of stomach resected from surgery were also collected and subjected to the immunohistochemistry assay. The protocol for the present study was approved by the ethics committee of our hospital.
Table 1.
Correlation between patients’ clinicopathological characteristics and infiltration of T-bet+ TILs in gastric cancer tissues
| Clinicopathological features | Cases | Numbers of T-bet+ TILs (mean ± SD) | t/F | p Value |
|---|---|---|---|---|
| Gender | ||||
| Male | 117 | 14.7 ± 9.0 | 1.11 | 0.2683 |
| Female | 35 | 16.7 ± 11.0 | ||
| Age (year) | ||||
| ≤ 45 | 14 | 10.7 ± 8.3 | 1.94 | 0.1471 |
| 45 ~ 60 | 72 | 15.2 ± 9.5 | ||
| >60 | 66 | 16.2 ± 9.5 | ||
| Tumor locations | ||||
| Gastric cardia | ||||
| Yes | 54 | 15.3 ± 9.2 | 0.14 | 0.8853 |
| No | 98 | 15.1 ± 9.7 | ||
| Gastric body | ||||
| Yes | 61 | 14.3 ± 7.9 | 0.95 | 0.3453 |
| No | 91 | 15.8 ± 10.4 | ||
| Gastric antrum | ||||
| Yes | 55 | 15.2 ± 10.4 | 0.03 | 0.9774 |
| No | 97 | 15.2 ± 8.9 | ||
| Tumor size (cm) | ||||
| <5 | 60 | 16.9 ± 11.8 | 2.35 | 0.0202 |
| ≥5 | 70 | 13.0 ± 6.8 | ||
| Histological type | ||||
| Differentiated | 62 | 15.2 ± 10.1 | 0.38 | 0.7046 |
| Poorly differentiated | 83 | 14.6 ± 8.8 | ||
| Invasion to muscular layer | ||||
| Yes | 116 | 14.1 ± 8.3 | 2.50 | 0.0138 |
| No | 17 | 20.2 ± 14.6 | ||
| Nodal metastasis | ||||
| Yes | 98 | 14.2 ± 8.2 | 1.43 | 0.1551 |
| No | 35 | 16.8 ± 12.4 | ||
| Recurrence | ||||
| Yes | 71 | 14.1 ± 7.8 | 1.32 | 0.1873 |
| No | 81 | 16.1 ± 10.6 | ||
| Pathological grade | ||||
| 1–2 | 20 | 17.3 ± 11.7 | 1.19 | 0.3065 |
| 3 | 73 | 15.7 ± 10.1 | ||
| 4 | 59 | 13.9 ± 7.7 | ||
| UICC stage | ||||
| I | 10 | 22.2 ± 14.0 | 3.40 | 0.0196 |
| II | 31 | 15.9 ± 10.1 | ||
| III | 93 | 15.1 ± 9.0 | ||
| IV | 18 | 10.7 ± 5.1 | ||
Values in bold signify p < 0.05
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissues were cut into 3-μm-thick sections and were dewaxed in xylene, rehydrated and graded ethanol solutions. Antigens were retrieved by heating the tissue sections at 100 °C for 30 min in citrate solution (10 mmol/L, pH 6.0). Then sections were cooled and immersed in methanol in the presence of 0.3 % hydrogen peroxide for 15 min to block the endogenous peroxidase activity, subsequently rinsed in PBS for 5 min, and then incubated with primary antibody against T-bet (diluted in 1:120, Santa Cruz, USA) at 4 °C overnight. A negative control was performed by omitting the primary antibody. The sections were then incubated with horseradish peroxidase-labeled goat against mouse/rabbit secondary antibody (ready to use, Maixin Biotechnology Co., Ltd, Fuzhou, China). Diaminobenzene was used as the chromogen and hematoxylin as the nuclear counterstain. Sections were then dehydrated, cleared and mounted.
Intracellular staining and flow cytometry analysis
The TILs were harvested from fresh surgical resected gastric cancer tissues according to the method described in our previous study [10]. FITC- or ECD-labeled mouse anti-human CD45, mouse anti-human CD4-PE-Cy7, mouse anti-human CD8α-PE-Cy5, mouse anti-human CD56- FITC, mouse anti-human T-bet-PE, fluorescence-labeled mouse or rat IgG Isotypes and the FixPerm buffer were purchased from eBioscience (San Diego, CA). The TILs were isolated from gastric cancer tissues as well as adjacent normal tissues using the standard ficoll-hypaque density gradient centrifugation. For intracellular staining of T-bet, the TILs were surface stained, then permeabilized with the eBioscience FixPerm buffer. Cells were then washed and stained with mouse anti-human T-bet-PE or mouse IgG1-PE Isotype Control. Flow cytometric analysis was performed using a FACS flow cytometer (Beckman-Coulter, USA).
Density evaluation of T-bet+ TILs in gastric cancer tissues
All slides were examined independently by two pathologists who were blinded to patients’ clinical data. The method of assessing the density of T-bet+ TILs in tumor nest was as described by our previous studies [13, 14]. In brief, numbers of T-bet+ TILs in tumor nest were determined according to the immunohistochemical staining and were counted as follows: five areas in tumor nest with the most intense TILs were selected at low magnification (40×), then the T-bet+ TILs were counted and recorded at high power field (HPF, 200× magnification).
Statistical analysis
All data were imported into Epidata3.0 database with double-check and were analyzed by the SAS 9.13 software package (version 9.13; SAS Institute, Cary, NC USA). Quantitative data were expressed as mean ± SD. ANOVA/Dunnett-test was used to compare means of two or more groups. Chi-square test was used to compare the difference in the rates or proportions between different groups. Survival data were analyzed by Kaplan–Meier method, and log-rank test Cox model was performed to estimate the association between the T-bet expression and hazard risk of gastric cancer patients. A p value < 0.05 was deemed statistically significant.
Results
Immunolocalisation of T-bet+ lymphocytes in normal gastric tissues, gastric polyp, gastritis and gastric cancer tissues
To study type 1 immune responses in tumor nest of gastric cancer, we examined T-bet+ cells by IHC. The positive T-bet staining was readily observed on lymphoid cells within tumor nests and predominantly localized within the cell nuclei (Fig. 1a, b). We also found T-bet+ lymphoid cells in gastritis (Fig. 1c, d), gastric polyp (Fig. 1e, f) and normal gastric tissue specimens (Fig. 1g, h). The densities of infiltrating T-bet+ lymphoid cells in gastric cancer tissues, polyp tissues, gastritis tissues and normal gastric tissues were 15.2 ± 9.5, 2.4 ± 1.9, 2.6 ± 1.3 and 6.6 ± 1.2 (number/HPF, mean ± SD), respectively. As shown in Fig. 2, the density of infiltrating T-bet+ lymphoid cells in gastric cancer tissues was significantly higher than that in gastric polyp tissues, gastritis tissues or normal gastric tissues (p < 0.0001, < 0.0001 and < 0.0001 respectively). These findings are consistent with the nature of immune infiltration in these lesions. In gastritis cases, the stomach mucosa showed predominant lymphoplasmacytic infiltrates within the lamina propria. In contrast, the cancer tissues are characterized by lymphocytic infiltration. Thus, gastric cancer development is associated with an increase in T-bet+ lymphoid cells, consistent with immunosurveillance of cancer (Tables 2 and 3).
Fig. 1.
Immunolocalisation of T-bet+ lymphocytes in gastric cancer, gastritis, gastric polyp and normal tissues. a, b infiltrating T-bet+ lymphocytes in gastric cancer tissue. c, d T-bet staining in gastritis tissue. e, f T-bet staining in gastric polyp tissue. g, h T-bet staining in normal gastric tissue
Fig. 2.
Comparison of the numbers of the infiltrating T-bet+ lymphocytes in gastric tissues. The number of infiltrating T-bet+ lymphocytes in gastric cancer tissues was significantly higher than that in gastric polyp tissues (p < 0.0001), gastritis tissues (p < 0.0001) and normal gastric tissues (p < 0.0001) using unpaired t test
Table 2.
Cox model disease-free survival analysis of patients’ clinicopathological parameters and density of T-bet+ TILs in gastric cancer
| Clinicopathological parameters | Comparison/reference | HR (95 % CI) | p Value |
|---|---|---|---|
| Density of T-bet+ TILs | Higher/lower | 0.56 (0.32–0.98) | 0.043 |
| Gender | Female/male | 1.04 (0.63–1.73) | 0.867 |
| Age | >45/≤45 | 1.58 (0.72–3.46) | 0.253 |
| Tumor location | Gastric cardia/others | 1.36 (0.86–2.13) | 0.186 |
| Invasion to muscular layer | Yes/No | 1.48 (0.62–3.54) | 0.375 |
| Tumor size (cm) | ≥5/<5 | 2.39 (1.48–3.84) | 0.0003 |
| UICC stage | IV + III + II/I | 1.93 (1.07–3.47) | 0.029 |
Values in bold signify p < 0.05
Table 3.
Cox model survival analysis of patients’ clinicopathological parameters and density of T-bet+ TILs in gastric cancer
| Clinicopathological parameters | Comparison/reference | HR (95 % CI) | p Value |
|---|---|---|---|
| Density of T-bet+ TILs | Higher/lower | 0.55(0.30–0.99) | 0.047 |
| Gender | Female/male | 1.18(0.69–2.02) | 0.536 |
| Age | >45/≤45 | 1.56(0.68–3.57) | 0.293 |
| Tumor location | Gastric cardia/others | 1.37(0.86–2.20) | 0.189 |
| Invasion to muscular layer | Yes/no | 1.26(0.52–3.04) | 0.613 |
| Tumor size (cm) | ≥5/<5 | 2.52(1.53–4.17) | 0.0003 |
| UICC stage | IV + III + II/I | 1.91(1.04–3.53) | 0.039 |
Values in bold signify p < 0.05
Flow cytometry analysis of T-bet+ TILs subgroups in gastric cancer tissues
In order to study the nature of T-bet+ TILs in gastric cancer, we used flow cytometry to characterize TILs isolated from fresh surgical resected gastric cancer tissues as well as adjacent normal tissues. T-bet was detected in large portions of CD4 T cells, CD8 T cells and NK cells in the peripheral blood (Fig. 3a, Tables 4, 5). In both tumor and adjacent normal tissues, T-bet was detected in CD4, CD8 T cells and NK cells (Fig. 3a). Interestingly, although not statistically significant, the percentage of T-bet+ CD8+ T cells in tumor seemed to trend higher than adjacent normal tissues (Table 5). CD4, CD8 T cells and NK cells made up the majority of T-bet+ lymphocytes in peripheral blood, tumor and tumor adjacent normal tissues (Fig. 3b). It was noted that NK cells were the largest group of cells among T-bet+ TILs (Fig. 3b).
Fig. 3.
Flow cytometry analysis of subgroups of T-bet+ TILs in gastric cancer tissues. Peripheral blood cell, TILs and mononuclear cells from adjacent normal tissues were stained with CD45, CD4, CD8, CD56 and T-bet and analyzed by flow cytometry. a Events were gated on CD45 and further gated on CD4, CD8 or CD56. Percentage of T-bet+ cells was shown. b Events were first gated on T-bet and CD45. Percentage of CD4, CD8 or CD56 within T-bet+CD45+ cells was shown. PBL, tumor and normal tissues shown were from the same patient. Data are representative of independent experiments with three patients
Table 4.
T-bet+ cells in peripheral blood mononuclear cell subsets
| CD8+Tbet+/CD8+ | CD4+Tbet+/CD4+ | CD56+Tbet+/CD56+ | |
|---|---|---|---|
| Mean ± SD (%) | 54.46 ± 18.31 | 28.35 ± 12.91 | 41.70 ± 10.73 |
| Cases (n) | 5 | 5 | 4 |
Table 5.
T-bet+ cells in infiltrating lymphocyte subsets in adjacent normal tissues and gastric cancer tissues
| Adjacent normal tissues | Cancer tissues | |||||
|---|---|---|---|---|---|---|
| CD8+Tbet+/CD8+ | CD4+Tbet+/CD4+ | CD56+Tbet+/CD56+ | CD8+Tbet+/CD8+ | CD4+Tbet+/CD4+ | CD56+Tbet+/CD56+ | |
| Mean ± SD (%) | 7.48 ± 1.84 | 12.68 ± 4.53 | 18.10 ± 10.94 | 17.00 ± 5.31a | 16.03 ± 7.74b | 29.01 ± 18.85c |
| Cases (n) | 4 | 4 | 4 | 6 | 5 | 4 |
a p = 0.0667 cancer tissues versus adjacent normal tissues
b p = 0.7302 cancer tissues versus adjacent normal tissues
c p = 0.4857 cancer tissues versus adjacent normal tissues
Correlation between the density of T-bet+ TILs and patients’ clinicopathological parameters
In order to determine the clinical significance of T-bet+ TILs, we correlated the density of T-bet+ TILs with patients’ clinicopathological parameters (Table 1). We found that the density of T-bet+ TILs in human gastric tumor nests was significantly correlated with tumor size (p = 0.0202), invasion depth (p = 0.0138) and UICC stage (p = 0.0196), whereas it is not correlated with patient’s gender, age, tumor location, nodal metastasis, recurrence or pathological grade. Thus, our data demonstrate that the lower density of T-bet+ TILs in human gastric tumor nests correlates with more advanced gastric cancer. This is consistent with the idea that the type 1 lymphocyte-mediated tumor immunosurveillance is inhibited during cancer progression.
Prognostic value of the density of T-bet+ TILs in gastric cancer tissues
We next examined relationship between T-bet expression and cancer patient survival. Based on the minimum p value seek, we selected a cutoff value 19.05/HPF to separate patients into two groups with higher or lower densities of T-bet+ TILs respectively. Our univariate analysis demonstrated that the disease-free survival (DFS) for the group with the higher density of T-bet+ TILs was significantly better than the group with lower T-bet+ TILs. The median DFS for the higher and lower groups were 41 m (95 % CI: 16–55 m) and 26 m (95 % CI: 17–32 m), respectively (χ 2 = 3.905, p = 0.0481, Fig. 4a). In addition, the overall survival (OS) for the group with higher density of T-bet+ TILs was also significantly better than the group with lower T-bet+ TILs. The median survival time for the higher and lower groups were 55 m (95 % CI: 31–69 m) and 32 m (95 % CI: 25–39 m), respectively (χ 2 = 4.002, p = 0.0455, Fig. 4b).
Fig. 4.
The density of T-bet+ TILs in gastric cancer tissues correlates with patient’s survival. Based on the minimum p value seek, patients were divided into two groups with higher or lower densities of T-bet+ TILs respectively. a The result of patient’s DFS comparing T-bet high and T-bet low groups of patients by Kaplan–Meier survival analysis. b OS comparing T-bet high and T-bet low groups of patients by Kaplan–Meier survival analysis
As shown in Tables 2 and 3, our multivariate analysis demonstrates that the higher density of T-bet+ TILs was significantly associated with an about twofold decreased hazard risk of recurrence or death compared with the lower group, and the HR and 95 % CI for recurrence or death of gastric patients were 0.56 (0.32–0.98) and 0.55 (0.30–0.99) respectively. As expected, the higher UICC stage or larger tumor size was significantly associated with elevated hazard risk of recurrence or death for gastric patients. These data show that higher numbers of T-bet+ TILs are associated with better outcome for gastric cancer patients.
Discussion
Our present study has shown that many T-bet+ lymphoid cells were present in gastric cancer tissues and the density of intratumoral T-bet+ lymphoid cells was significantly higher than that in gastric polyp, gastritis or normal gastric tissues. In addition, the number of T-bet+ TILs inversely correlated with tumor size, invasion depth and UICC stage. Most importantly, the patients with higher numbers of T-bet+ TILs survived longer than those with lower numbers of T-bet+ lymphoid cells. Collectively, these data support the idea that the density of T-bet+ intratumoral lymphoid cells is associated with effective anti-tumor immune responses and an important prognostic indicator for gastric cancer.
The previous studies using various mouse tumor models have supported a role of T-bet in antitumor immune responses. T-bet was shown to exert a significant inhibitory function in the development of metastatic disease, but not primary tumor in a mouse autochthonous prostate cancer model [15]. We have shown that T-bet and Eomes are required for adaptive immune responses against transplantable melanoma [10]. Surprisingly, T-bet deficiency prevented Helicobacter felis-induced gastric cancer [16]. T-bet-deficient mice responded to H. felis infection with a blunted Th1 response and a greatly reduced IL-1beta and TNF-alpha but increased IL-10 levels. This result is, however, not contradictory to the idea that T-bet promotes antitumor immune responses. It is because in this case, the main role of T-bet is to facilitate H. felis-driven inflammation which precedes and is likely critical for gastric cancer development. Therefore, T-bet-driven immune responses against malignant tumor cells should be distinguished from T-bet-mediated inflammation in response to infection. For gastric cancer prevention, strategies aimed at blockade of T-bet function should be used to prevent chronic gastritis from developing into gastric cancer. Once cancer has developed, T-bet-mediated antitumor immune responses should be boosted with various immunotherapeutic approaches.
T-bet is important for the proper function of Th1, CD8 T cells and NK cells [17]. Indeed, we confirmed that T-bet+ TILs consisted of mainly CD4, CD8 and NK cells. The primary gene regulated by T-bet is IFN-γ [18] which is important for tumor immunosurveillance [19]. In addition, T-bet promotes trafficking of T cells to tumor sites by up-regulating CXCR3 [10]. T-bet also controls the type of adaptive immune responses within the tumor site because it is important not only for promoting Th1 fate but also critical for suppressing alternative Th2 and Th17 differentiation [10]. The lack of T-bet resulted in elevated Th17 responses within tumor site [10]. Th17 cells were recently found associated with tumor progression in human gastric cancer [20]. Similarly, it has been shown that the ratio of GATA-3+/T-bet+ lymphoid cells is an indicator of poor survival in pancreatic cancer [21]. Thus, a decrease in T-bet-mediated immune responses and a corresponding increase in deviation to other types of inflammation may coordinately promote cancer progression, and such possibility should be further examined in the setting of gastric cancer.
Acknowledgments
We thank senior pathologists Chang-qing Lu, Wei Zhao, Jun Xie and Yuan-dong Chen (Department of Pathology, The Third Affiliated Hospital of Soochow University, Jiangsu Changzhou, China) for their expert suggestions and technical assistances. This work was supported in part by grants from the National Natural Science Foundation of China (No. 81171653, 31170866, 30950022 and 30972703), Jiangsu Province Science and Technology Innovation Fund (2012-85), and the Innovative Talents Training Project of Changzhou Health Bureau.
Conflict of interest
The authors declare that they have no competing interests to this paper.
Footnotes
L. Chen and X. Zheng contributed equally to this work.
Contributor Information
Bin-feng Lu, Email: binfeng@pitt.edu.
Jing-ting Jiang, Email: jjtnew@163.com.
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