| Objective: To observe the indoleamine 2,3-dioxygenase(IDO) expression and its association with lymphocyte subset distribution in tumor draining lymph nodes(TDLNs) of esophageal squamous cell cancer(ESCC) patients. By detecting the expression of IDO and bridging integrator-1(Bin1) in tumor microenvironments(TMEs) and TDLNs at protein and gene level, the relationships between IDO and Bin1 expressions were aimed to be analyzed. Furthermore, to investigate the clinical significances by classifying the associations of IDO and Bin1 expressions with clinical parameters of ESCC patients.Methods:1.Tumor tissue, para-carcinoma tissue and TDLN specimens were collected from 66 cases of ESCC patients. Each specimen was divided to 3 piece and treated as follows for different uses: grinded sterilely to prepare single cell suspension for flow cytometry, put in liquid nitrogen for extracting RNA and fixed in 4% formaldehyde for making paraffin embedded blocks.2.Flow cytometry was used to detect the distribution of lymphocyte subsets(CD3+ T cell, CD3+CD4+ T cell, CD3+CD8+ T cell, CD3-CD16+CD56+ NK cell, CD3-CD19+ B cell and CD4+CD25+ Treg cell).3.Immunohistochemistry was used to detect the protein expression of IDO and Bin1 in tumor tissues, para-carcinoma tissues and TDLNs. Then the association of IDO expression with lymphocyte subset distribution was analyzed. In addition, the relationship between IDO and Bin1 protein expression was analyzed.4.RT-PCR was used to detect the m RNA expression of IDO and Bin1 in tumor tissues, para-carcinoma tissues and TDLNs. The relationship between IDO and Bin1 m RNA expression was analyzed. In addition, for IDO and Bin1, the relationships between gene expression and protein expression were analyzed.5.The associations of IDO and Bin1 expression in ESCC tumor tissue with clinical parameters were analyzed.Results:1.Among the 66 TDLNs, 23 cases were metastasis positive while 43 cases were metastasis negative. Compared with metastatic lymph nodes, the proportions of CD3+ T cell(45.57 ± 10.17% vs. 60.65 ± 7.06%, P < 0.001), CD3+CD4+ T cell(30.37 ± 7.20% vs. 34.54 ± 6.44%, P = 0.019), CD3+CD8+ T cell(15.07 ± 3.99% vs. 21.18 ± 4.29%, P < 0.001) and CD3-CD16+CD56+ NK cell(4.30 ± 1.56% vs. 5.78 ± 1.14%, P < 0.001) in non-metastatic lymph nodes were lower while proportions of CD3-CD19+ B cell(34.58 ± 12.47% vs. 21.63 ± 5.87%, P < 0.001) and CD4+CD25+ Treg cell(4.42 ± 1.31% vs. 3.17 ± 0.92%, P < 0.001) were higher, significantly. Among the 23 metastatic and 43 non-metastatic lymph nodes, respectively 20(86.96%) and 25(58.14%) cases were with high IDO expression, which meant the IDO expressions were higher in metastasis positive lymph nodes than those in negative ones(P = 0.017).2.High expression of IDO was observed in 45 case(68.18%) of TDLNs. Compared to TDLNs with low IDO expression, the proportions of CD3+ T cell(51.65 ± 10.54% vs. 63.43 ± 6.71%, P < 0.001), CD3+CD4+ T cell(31.34 ± 6.60% vs. 36.83 ± 6.29%, P = 0.002), CD3+CD8+ T cell(18.03 ± 5.02% vs. 21.25 ± 4.63%, P = 0.029) and CD3-CD16+CD56+ NK cell(4.74 ± 1.31% vs. 6.40 ± 1.15%, P < 0.001) in TDLNs with high IDO expression were lower while proportions of CD3-CD19+ B cell(29.33 ± 10.84% vs. 19.33 ± 6.23%, P < 0.001) and CD4+CD25+ Treg cell(4.08 ± 0.99% vs. 2.58 ± 0.88%, P < 0.001) were higher, significantly.3.Among the 66 tumor tissues, totally 38 cases showed IHC staining of high IDO expression while no case of para-carcinoma tissues showed it, and the difference was of statistical significance(P < 0.001). Compared to TMEs with low IDO expression, the proportions of CD3+ T cell(45.67 ± 10.98% vs. 57.69 ± 11.40%, P < 0.001), CD3+CD4+ T cell(48.35 ± 6.34% vs. 41.54 ± 6.71%, P = 0.002), CD3+CD8+ T cell(18.26 ± 4.05% vs. 20.17 ± 4.57%, P = 0.019) and CD3-CD16+CD56+ NK cell(4.97 ± 1.25% vs. 5.87 ± 1.09%, P < 0.001) in TMEs with high IDO expression were lower while proportions of CD3-CD19+ B cell(24.43 ± 7.13% vs. 18.34 ± 6.58%, P = 0.027) and CD4+CD25+ Treg cell(5.08 ± 1.09% vs. 2.78 ± 0.84%, P < 0.001) were higher, significantly.4.37 TDLN and 42 TME specimens showed IHC staining of low Bin1 expression.The IDO expression was negatively correlated with Bin1 expressi- on in TDLNs and TMEs at protein level(P < 0.001).5.Compared to the TMEs and TDLNs with low IDO protein expression, the expressions of IDO m RNA were higher in those with high protein expression(P < 0.001). Compared to the TMEs and TDLNs with high Bin1 protein expression, the expressions of Bin1 m RNA were lower in those with low protein expression(P < 0.001). These results indicated that the expressions of IDO and Bin1 were consistent at gene and protein level.6.The IDO expression in tumor tissue was correlated with tumor differentiation grade, patient TNM stage and lymph node metastasis(P < 0.001; P = 0.018; P = 0.004) while not correlated with gender, age and invation range(P = 0.902; P = 0.793; P = 0.159). Likewise, the Bin1 expression in tumor tissue was correlated with tumor differentiation grade, patient TNM stage and lymph node metastasis(P = 0.010; P = 0.045; P = 0.015) while not correlated with gender, age and invasion range(P = 0.914; P = 0.797; P = 0.103).Conclusions:1.Compared to the non-metastatic TDLNs, the IDO expressions were higher in metastatic TDLNs. Likewise, the expressions of IDO were higher in tumor tissues than those in para-carcinoma tissues. The disordered distributions of lymphocyte subsets in TDLNs and TMEs were correlated with high expression of IDO, indicating that IDO could induce tumor immune escape.2.In TMEs and TDLNs, the IDO expression was negatively correlated with Bin1 expression at protein level and gene level. The expressions of IDO and Bin1 were consistent at gene and protein level, indicating that there might be no post-transcriptional control mechanisms.3.The expressions of IDO and Bin1 in ESCC tumor tissues were correlated with tumor differentiation grade, patient TNM stage and lymph node metastasis while not correlated with gender, age and invasion range. |
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