The role of tumor cells in the modification of T lymphocytes activity — the expression of the early CD69+, CD71+ and the late CD25+, CD26+, HLA/DR+ activation markers on T CD4+ and CD8+ cells in squamous cell laryngeal carcinoma. Part I

The role of interactions between tumor cells and autologous immunocompetent cells, the impact on the modulation of the activity of T CD4+ and CD8+ lymphocytes, as well as the influence on the regulation and determination of antitumor cellular immune response in patients with head and neck squamous cell carcinomas (HNSCC) is not completely clear. The aim of this study was to analyze early and late activation antigens expression on T cells subpopulations modified under the influence of the presence of cancer cells to investigate the regulatory mechanisms of the local cellular immune response in carcinoma of the larynx. Cytofluorymetric analysis of the early (CD69+, CD71+) and late activation markers (CD25+ high, CD26+, HLA/DR+) expression on T CD3+CD4+ and CD3+CD8+ cells subpopulations in mixed cellular cultures of freshly isolated tumor cells (MLTMC) and non-cancerous normal epithelial cells (MLNCC) with immunocompetent cells was performed in 55 cases of squamous cell laryngeal carcinoma. The whole peripheral blood concentrations of IL-10 and IFN-γ in 21 h and 72 h of experiments were also measured by ELISA. The relationships between the activation markers expression depending on the type of cells used in co-cultures, as well as the level of secreted cytokines, were investigated. Our work has revealed a statistically significant dependence of cytofluorymetric results on the presence of TMC or NCC in mixed cellular cultures. Increased expression of CD69+, CD71+ and CD25+ high, CD26+, HLA/DR+ antigens on T CD3+CD4+ and CD3+CD8+ cells was higher in MLTMC cultures, in comparison with MLNCC. We demonstrated negative significant relationships of IFN-γ and IL-10 secretion with regard to CD4+CD69+, CD8+CD69+, CD4+CD71+, CD8+CD71+ antigens expression in 21 h of experiments without mitogenic stimulation. Furthermore, this study revealed negative significant relationships of IFN-g secretion with regard to CD4+HLA/DR+ and CD8+HLA/DR+ as well as between IL-10 concentration and CD4+HLA/DR+ in trials without PHA stimulation. Our findings have confirmed a key role for tumor cells in determining the function of T cells involved in the immunological processes and impact of neoplastic cells on modulating the activity of T CD4+ and CD8+ lymphocytes in laryngeal carcinoma. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 579–592

Prognostic value of the immunological phenomena and relationship with clinicopathological characteristics of the tumor — the expression of the early CD69+, CD71+and the late CD25+, CD26+, HLA/DR + activation markers on T CD4+ and CD8+ lymphocytes in squamous cell laryngeal carcinoma. Part II

One of the most important challenges in contemporary oncology is to find objective biomarkers of tumor aggressiveness, which help to identify more invasive phenotypes of the carcinoma. The purpose of this study was to investigate the relationships between the early and the late activation markers expression on T CD4+ and CD8+ cells subpopulations and certain clinicopathological characteristics of the neoplastic infiltration in order to determine their role as biomarkers for tumor behavior in squamous cell laryngeal carcinoma. Analysis of the early (CD69+, CD71+) and the late activation antigens (CD25+ high, CD26+, HLA/DR+) expression on T CD4+ and CD8+ lymphocytes by cytofluorymetry in 55 patients treated for squamous cell laryngeal carcinoma was performed. Clinicomorphological analysis on the basis of TNM criteria and tumor front grading, which included tumor-related features and adjacent stroma-related characteristics of the peripheral edge of infiltration was carried out. The relationships between the activation markers expression and parameters of tumor aggressiveness were investigated. Our work revealed statistically significant differences in the expression of the studied activation markers on T cells with regard to certain clinicomorphological fetaures. The expressions of CD69+ and CD71+ antigens on T CD3+CD4+ and CD3+CD8+ cells as well as CD4+HLA/DR+ markers were higher for pT3 and pT4 tumors, in comparison with pT2 carcinomas. Moreover, tumors with the smallest number of TFG points were characterized by significantly lower values of the average expression of CD3+CD69+ and CD3+CD71+ as well as CD4+HLA/DR+ markers on T lymphocytes. In addition, more aggressive and deeply infiltrating laryngeal carcinomas were most often characterized by significantly higher values of the average expression of CD69+ and CD71+ antigens on CD8+ as well as HLA/DR+ markers on CD4+. Our study confirmed the implication of the early and the late activation antigens expression on CD4+ and CD8+ T lymphocytes in clinicomorphological parameters of the tumor, especially TFG total score and depth of invasion, and their importance as indicators of the invasive phenotype of laryngeal carcinoma. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 593–603

The expression of SOCS1 and TLR4-NFkappaB pathway molecules in neoplastic cells as potential biomarker for the aggressive tumor phenotype in laryngeal carcinoma.

Suppressor of cytokine signaling 1 (SOCS1) is the key regulator of cytokine-mediated innate and adaptive immunity. One of the molecular mechanisms of SOCS1 is connected with inhibition of TLR4-NFkappaB pathway. The relationships among these molecules in laryngeal carcinoma are not exactly known. In this preliminary study we focused on their special activity and role in regulation of development and progression of laryngeal carcinoma. To investigate NFkappaB (p65 subunit) nuclear and cytoplasmic expression in 45 tumor samples of advanced laryngeal carcinoma IHC staining was performed. To determine the mRNA expression levels of TLR4, IRAK1, TRAF6 and SOCS1 in isolated neoplasm cells and non-cancerous adjacent mucosa epithelial cells RT-PCR was used. The invasiveness of laryngeal carcinomas was evaluated according to tumor front grading, TFG, which included tumor-related features (cytoplasmic differentiation, nuclear polymorphism, number of mitoses) and adjacent stroma-related characteristics of the peripheral edge of tumor infiltration (mode of infiltration, depth of invasion and plasmalymphocytic infiltration). The relationships between pT, pN status, the histological G grade, certain clinicopathological characteristics as well as postoperative observation time and the mRNA expression of the molecules mentioned earlier were investigated. Significant differences of TLR4-NFkappaB pathway molecules and SOCS1 mRNA expression in laryngeal tumor cells and normal adjacent mucosa cells as well as significant interconnections of TLR4, SOCS1 and NFkappaB(p65) in isolated tumor cells were obtained. This preliminary study demonstrated that the expression of SOCS1 and TLR4-NFkappaB pathway molecules had a strong association with the aggressiveness of laryngeal carcinoma. Positive relationships of TRAF6 in tumor margin cells with the histological grade and the mode of tumor invasion as well as the TFG total score were highlighted. Significant positive correlations were found between the TLR4 in tumor central cells and the TFG total score. Negative relationships of SOCS1 in tumor central cells with the histological grade were also noted. Significant positive correlations were found between the cytoplasmic NFkappaB(p65) and the mode of invasion as well as TFG total score. Our findings confirmed the importance of SOCS1 and TLR4-NFkappaB pathway molecules as potential biomarkers for assessment of the aggressive tumor phenotype in laryngeal carcinoma

Gene and protein expression of glucose transporter 1 and glucose transporter 3 in human laryngeal cancer—the relationship with regulatory hypoxia-inducible factor-1α expression, tumor invasiveness, and patient prognosis

Increased glucose uptake mediated by glucose transporters and reliance on glycolysis are common features of malignant cells. Hypoxia-inducible factor-1α supports the adaptation of hypoxic cells by inducing genes related to glucose metabolism. The contribution of glucose transporter (GLUT) and hypoxia-inducible factor-1α (HIF-1α) activity to tumor behavior and their prognostic value in head and neck cancers remains unclear. The aim of this study was to examine the predictive value of GLUT1, GLUT3, and HIF-1α messenger RNA (mRNA)/protein expression as markers of tumor aggressiveness and prognosis in laryngeal cancer. The level of hypoxia/metabolic marker genes was determined in 106 squamous cell laryngeal cancer (SCC) and 73 noncancerous matched mucosa (NCM) controls using quantitative realtime PCR. The related protein levels were analyzed by Western blot. Positive expression of SLC2A1, SLC2A3, and HIF-1α genes was noted in 83.9, 82.1, and 71.7 % of SCC specimens and in 34.4, 59.4, and 62.5 % of laryngeal cancer samples. Higher levels of mRNA/protein for GLUT1 and HIF-1α were noted in SCC compared to NCM (p<0.05). SLC2A1 was found to have a positive relationship with grade, tumor front grading (TFG) score, and depth and mode of invasion (p<0.05). SLC2A3 was related to grade and invasion type (p<0.05). There were also relationships of HIF-1α with pTNM, TFG scale, invasion depth and mode, tumor recurrences, and overall survival (p<0.05). In addition, more advanced tumors were found to be more likely to demonstrate positive expression of these proteins. In conclusion, the hypoxia/metabolic markers studied could be used as molecular markers of tumor invasiveness in laryngeal cancer.This work was supported, in part, by the statutory fund of the Department of Cytobiochemistry, University of Łódź, Poland (506/811), and by grant fromtheNational Science Council, Poland (N403 043 32/2326)

Gene and protein expression of O-GlcNAc-cycling enzymes in human laryngeal cancer

Aberrant protein O-GlcNAcylation may contribute to the development and malignant behavior of many cancers. This modification is controlled by O-linked β-N-acetylglucosamine transferase (OGT) and O-GlcNAcase (OGA). The aim of this study was to determine the expression of O-GlcNAc cycling enzymes mRNA/protein and to investigate their relationship with clinicopathological parameters in laryngeal cancer. The mRNA levels of OGT and MGEA5 genes were determined in 106 squamous cell laryngeal cancer (SCLC) cases and 73 non-cancerous adjacent laryngeal mucosa (NCLM) controls using quantitative real-time PCR. The level of OGT and OGA proteins was analyzed by Western blot. A positive expression of OGT and MGEA5 transcripts and OGT and OGA proteins was confirmed in 75.5 and 68.9 % and in 43.7 and 59.4 % samples of SCLC, respectively. Higher levels of mRNA/protein for both OGT and OGA as well as significant increases of 60 % in total protein O-GlcNAcylation levels were noted in SCLC compared with NCLM (p < 0.05). As a result, an increased level of OGT and MGEA5 mRNA was related to larger tumor size, nodal metastases, higher grade and tumor behavior according to TFG scale, as well as incidence of disease recurrence (p < 0.05). An inverse association between OGT and MGEA5 transcripts was determined with regard to prognosis (p < 0.05). In addition, the highest OGT and OGA protein levels were observed in poorly differentiated tumors (p < 0.05). No correlations with other parameters were noted, but the results showed a trend of more advanced tumors to be more frequently OGT and OGA positive. The results suggest that increased O-GlcNAcylation may have an effect on tumor aggressiveness and prognosis in laryngeal cancer.This work was supported, in part, by a grant from the National Science Council, Poland (N403 043 32/2326), by the statutory fund of the Department of Cytobiochemistry, University of Łódź, Poland (506/811

The role of tumor cells in the modification of T lymphocytes activity &amp;#8212; the expression of the early CD69&lt;sup&gt;+&lt;/sup&gt;, CD71&lt;sup&gt;+&lt;/sup&gt; and the late CD25&lt;sup&gt;+&lt;/sup&gt;, CD26&lt;sup&gt;+&lt;/sup&gt;, HLA/DR&lt;sup&gt;+&lt;/sup&gt; activation markers on T CD4&lt;sup&gt;+&lt;/sup&gt; and CD8&lt;sup&gt;+&lt;/sup&gt; cells in squamous cell laryngeal carcinoma. Part I

The role of interactions between tumor cells and autologous immunocompetent cells, the impact on the modulation of the activity of T CD4&lt;sup&gt;+&lt;/sup&gt; and CD8&lt;sup&gt;+&lt;/sup&gt; lymphocytes, as well as the influence on the regulation and determination of antitumor cellular immune response in patients with head and neck squamous cell carcinomas (HNSCC) is not completely clear. The aim of this study was to analyze early and late activation antigens expression on T cells subpopulations modified under the influence of the presence of cancer cells to investigate the regulatory mechanisms of the local cellular immune response in carcinoma of the larynx. Cytofluorymetric analysis of the early (CD69&lt;sup&gt;+&lt;/sup&gt;, CD71&lt;sup&gt;+&lt;/sup&gt;) and late activation markers (CD25&lt;sup&gt;+&lt;/sup&gt; &lt;sub&gt;high&lt;/sub&gt;, CD26&lt;sup&gt;+&lt;/sup&gt;, HLA/DR&lt;sup&gt;+&lt;/sup&gt;) expression on T CD3&lt;sup&gt;+&lt;/sup&gt;CD4&lt;sup&gt;+&lt;/sup&gt; and CD3&lt;sup&gt;+&lt;/sup&gt;CD8&lt;sup&gt;+&lt;/sup&gt; cells subpopulations in mixed cellular cultures of freshly isolated tumor cells (MLTMC) and non-cancerous normal epithelial cells (MLNCC) with immunocompetent cells was performed in 55 cases of squamous cell laryngeal carcinoma. The whole peripheral blood concentrations of IL-10 and IFN-&amp;#947; in 21 h and 72 h of experiments were also measured by ELISA. The relationships between the activation markers expression depending on the type of cells used in co-cultures, as well as the level of secreted cytokines, were investigated. Our work has revealed a statistically significant dependence of cytofluorymetric results on the presence of TMC or NCC in mixed cellular cultures. Increased expression of CD69&lt;sup&gt;+&lt;/sup&gt;, CD71&lt;sup&gt;+&lt;/sup&gt; and CD25&lt;sup&gt;+&lt;/sup&gt; &lt;sub&gt;high&lt;/sub&gt;, CD26&lt;sup&gt;+&lt;/sup&gt;, HLA/DR&lt;sup&gt;+&lt;/sup&gt; antigens on T CD3&lt;sup&gt;+&lt;/sup&gt;CD4&lt;sup&gt;+&lt;/sup&gt; and CD3&lt;sup&gt;+&lt;/sup&gt;CD8&lt;sup&gt;+&lt;/sup&gt; cells was higher in MLTMC cultures, in comparison with MLNCC. We demonstrated negative significant relationships of IFN-&amp;#947; and IL-10 secretion with regard to CD4&lt;sup&gt;+&lt;/sup&gt;CD69&lt;sup&gt;+&lt;/sup&gt;, CD8&lt;sup&gt;+&lt;/sup&gt;CD69&lt;sup&gt;+&lt;/sup&gt;, CD4&lt;sup&gt;+&lt;/sup&gt;CD71&lt;sup&gt;+&lt;/sup&gt;, CD8&lt;sup&gt;+&lt;/sup&gt;CD71&lt;sup&gt;+&lt;/sup&gt; antigens expression in 21 h of experiments without mitogenic stimulation. Furthermore, this study revealed negative significant relationships of IFN-g secretion with regard to CD4&lt;sup&gt;+&lt;/sup&gt;HLA/DR&lt;sup&gt;+&lt;/sup&gt; and CD8&lt;sup&gt;+&lt;/sup&gt;HLA/DR&lt;sup&gt;+&lt;/sup&gt; as well as between IL-10 concentration and CD4&lt;sup&gt;+&lt;/sup&gt;HLA/DR&lt;sup&gt;+&lt;/sup&gt; in trials without PHA stimulation. Our findings have confirmed a key role for tumor cells in determining the function of T cells involved in the immunological processes and impact of neoplastic cells on modulating the activity of T CD4&lt;sup&gt;+&lt;/sup&gt; and CD8&lt;sup&gt;+&lt;/sup&gt; lymphocytes in laryngeal carcinoma. (&lt;i&gt;Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 4, pp. 579&amp;#8211;592&lt;/i&gt;