| Human immune system has an important role in identifying abnormal mutant cells or tumor cells, can monitor and kill tumor cells by cellular immune mechanisms. Dendritic cells (DCs) is the most powerful professional antigen presenting cells (APC). They are able to uptake and processing antigen, with a strong ability to activate CD8+cytotoxic T cells and CD4+helper T cells. They control the process of immune response in vivo and play a central role in the immune response. Now, DCs is considered to be an ideal tool for the development of anti-tumor vaccine therapy. A large number of clinical trials have evaluated the DC tumor vaccine's safety and efficacy for cancer patients. DCs can be induced effect and memory T cells but can also produce tolerance-type T cells, its causes and mechanisms are still unclear.The monocytes is the important immune cells to kill tumor cells, is the precursor cells of DCs and macrophages. They have the roles of phagocytosis, presenting antigens to initiate the immune response, anti-tumor, regulation immune. Human peripheral blood monocytes are mainly divided into CD14+CD16-and CD14+CD16+two subsets according to the expression of CD16. The CD16+monocytes can be subdivided into CD14++CD16+and CD14+CD16+monocytes. Many studies found that the different monocyte subsets have the different function in cytokine secretion, antigen presentation, migration, differentiation of DC in vitro, and so on. In particular, the CD16+monocyte-derived DC (16+mDC) stimulated naive T cells to produce higher levels of IL-4and induced T cell polarization to Th2direction. CD16-monocyte-derived DC (16-mDC) stimulated T cells to produce a high level of IL-12and inducd a Thl immune response. These results suggest that the different phenotypes of monocytes may be have the different role of tumor immunity, but right now these researches have not yet been reported.The CD14+CD16+monocytes accounted for2.2%of the mononuclear cells, accounting for about10%of CD14+monocytes in healthy human peripheral blood. The CD14+CD16+monocytes have a significant increase in a variety of inflammatory diseases such as sepsis, asthma, arterial atherosclerosis and other diseases. Studies have found that CD14+CD16+monocytes increased to40%or more in the advanced gastric cancer patients, but the mechanism of CD14+CD16+monocytes increase and the correlation on tumor risk are still unclear.Tumours can mimic key features of lymph nodes and create a tolerant microenvironment, allowing tumours to be better able to escape from immunological attack. Chemokines are a superfamily of low molecular weight cytokines that selectively attract and activate different cell types. Many pathophysiological conditions require the participation of chemokines, including malignant tumours. Chemokines play two contradictory roles in tumour immunity activity:they may enhance innate or specific host anti-tumour immunity, while they may also favour tumour growth and metastasis. Monocyte chemoattractant protein-1(MCP-1) is one of the key chemokines produced by immune cells, and is over-expressed in breast tumour cells and some other tumour cells. MCP-1can shift the balance between host anti-tumour immunity and tumour tolerance by increasing the presence of harmful tumour-associated macrophages (TAM) and by inhibiting anti-tumour T cell activities. However, it remains unclear whether these CD14+CD16+monocytes are associated with varying and different levels of risk of cancer. It also remains unclear whether the level of MCP-1in co-cultured tumour cells interferes with monocyte heterogeneity.In this study, we hypothesize that the monocytes heterogeneity may play an important role in the immunopathologeneis of breast cancer, and MCP-1maybe regulate the CD14+CD16+monocytes in breast cancer. We firstly examined the level of CD14+CD16+and CD14+CD16-monocytes in breast cancer patients and healthy human, followed by in vitro experiments to study the role of breast cancer cell line MCF-7cell supernatant on monocyte phenotype, and finally discuss a role of MCP-1of MCF-7tumor supernatant in regulating monocyte heterogeneity. The results of this study provide valuable data and new research ideas for the mechanism of tumor immune tolerance and efficacy of autologous DCs vaccine therapy to improve. Part I The level of monocyte subsets in breast cancer and their clinical significanceObjective:To determine the level of CD14+CD16+and CD14+CD16" monocytes subsets in the peripheral blood of healthy controls or breast cancer and in tumor tissues. To analysis the correlation between elevated CD14+CD16+monocytes in breast cancer and patients'clinicopathological data. To explore the role of CD14+CD16+monocytes in the incidence and development of breast cancer.Methods:1. The detection of CD14+CD16+and CD14+CD16-monocytes subsets in peripheral blood of breast cancer patients and healthy controls. Peripheral blood samples were obtained from patients with breast cancer and healthy adult controls. The level of CD14+CD16+and CD14+CD16-monocytes was determined by flow cytometry.2. The detection of CD14+CD16+and CD14+CD16-monocytes in local tumor tissues from breast cancer. We prepared the single cell suspension of tumour tissues and then detected the CD14+CD16+and CD14+CD16-monocytes by flow cytometry.3. The correlation between CD14+CD16+monocytes and patients' clinicopathological data. ROC curves were used to evaluate the performance of CD14+CD16+monocytes in diagnosing breast cancer. The sensitivity and specificity of CD14+CD16+monocytes were analysed using variable cut-off values. Intergroup comparisons of the clinical and pathological variables were analysed using a two-tailed λ2test for discrete variables.Results:1. The level of CD14+CD16+and CD14+CD16-monocytes subsets in peripheral blood of breast cancer patients and healthy controls. The results show that CD14+CD16+monocytes exist in the peripheral blood of both healthy donors and patients with breast cancer. Furthermore, the frequency of CD14+CD16+monocytes in patients with breast cancer were increased significantly by comparison with healthy controls (16.96±7.7%, n=96versus10.84±3.6%, n=54)(P<0·0001). The levels of CD14+CD16-monocytes were not significantly different among cancer and healthy subjects.2. The level of CD14+CD16+and CD14+CD16-monocytes subsets in tumor tissue. There existed a considerable proportion of CD14+CD16+monocytes in tumour tissues.3. The correlation between CD14+CD16+monocytes and patients' clinicopathological data. The ROC curve analysis used breast cancers as the end-point for detection compared with healthy donors. The data showed that the area under the ROC curve (AUC) was0·805(P=0·0001) for the CD14+CD16+monocytes assay. Specifically, when the cut-point (13·31%) was used as the cut-off value, a sensitivity of68.7%and specificity of79.6%were achieved for the overall samples. We used the cut-off value of13.31%in the ROC curve as the cut-point. A significant relationship was found between the CD14+CD16+monocyte<13.31%group and the CD14+CD16+monocyte>13.31%group in respect of tumour size and pathological staging (P<0-05). However, menstruation, lymph node metastasis, oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor2(HER2) showed no such significant association with the level of CD14+CD16+monocytes. In early-stage breast cancer patients, especially those with stage I and/or small tumour size (T1-T2), had high levels of CD14+CD16+monocytes. The mean level of CD14+CD16+monocytes was increased significantly in stage I patients (19.39±9.4%) compared with stages II-IV patients.Conclusions:1. The pro inflammatory CD14+CD16+monocytes significantly increased in the peripheral blood of breast cancer and they can migrate to the local tumor tissue to play an important role in rumor immune.2. CD14+CD16+monocytes were significantly negatively correlated with tumour size and TNM staging in breast cancer patients. Early-stage (pstage I) patients have higher rates of CD14+CD16+monocytes. Overall, these results suggest that CD14+CD16+monocytes play a critical role in tumorigenesis.3. In the future, the increased level of CD14+CD16+monocytes in the peripheral blood may be a useful indicator in early diagnosis of breast cancer with a certain degree of sensitivity and specificity.4. CD14+CD16+monocytes could be induced and expanded in breast cancer patients by the tumor microenvironment. Part II Mechanisms of CD14+CD16+monocytes increased in breast cancer patientsObjective:The rugulation of monocyte subsets by human breast cancer cell supernatant or the cytokines in the supernatant in vitro. To explore the mechanism of CD14+CD16+monocytes elevated in breast cancer patients.Methods:1. Isolation of monocytes. Human peripheral blood was drawn from healthy adult volunteers under written informed consent. PBMC were prepared by Ficoll gradient centrifugation. Sorting of CD14+monocytes was performed by magnetic cell sorting (MACS) according to the manufacturer's instructions. The purity of the cell population was>96%, as determined by flow cytometry.2. Human breast cancer MCF-7cell conditioned medium (MCF-CM) preparation. The human mammary gland adenocarcinoma cell line MCF-7was grown in an logarithmic phase. The cells were seeded at2×106cells/75cm2and cultivated until60-70%confluence was reached. The medium was replaced and the supernatants were harvested after48h of further incubation.3. Study on the regulation of monocyte subsets by tumor supernatant MCF-CM in vitro. Freshly purified CD14+monocytes which served as primary monocytes were cultured in RPMI1640media either in the absence or in the presence of different concentration MCF-CM. Cells were cultured at37℃in a5%C02atmosphere for24hours and were then harvested. The changes of CD14+CD16+and CD14+CD16-monocytes were analyzed by flow cytometry.4. Study on the regulation of monocyte subsets by human recombinant cytokines in vitro. Freshly purified CD14+monocytes were cultured in RPMI1640media either in the absence or in the presence of human recombinant TNF-a, IL-2, osteopontin protein (OPN), MCP-1mAb. Cells were cultured at37℃in a5%C02atmosphere for24hours and were then harvested. The changes of CD14+CD16+and CD14+CD16-monocytes were analyzed by flow cytometry.5. Detection the level of MCP-1. Culture supematants of monocytes, MCF-CM and in the co-culture supernatant of monocytes and MCF-CM were assayed for MCP-1using a Quantikine enzyme-llinked immunosorbent assay (ELISA) kit according to the manufacturer's instructions.6. Study on the effect of anti-MCP-1mAb on rugulation of monocyte subsets. Freshly purified CD14+monocytes were treated with25%MCF-CM either in the absence or in the presence of anti-MCP-1mAb for24hours. Then these cells were harvested. The changes of CD14+CD16+and CD14+CD16-monocytes were analyzed by flow cytometry.Results:1. Regulation of monocyte subsets by tumor supernatant MCF-CM in vitro. The flow cytometry analyses results showed that, compared with the group that was treated with normal medium, the proportion of CD14+CD16+monocytes was increased by almost three times in the presence of25%MCF-CM and by almost five times in50%and75%MCF-CM. While the proportion of CD14+CD16-monocytes was significantly decreased (P<0.05).2. Regulation of monocyte subsets by human recombinant cytokines in vitro. Freshly purified CD14+monocytes were treated with human recombinant TNF-a, IL-2, osteopontin protein (OPN), MCP-1mAb for24hours. The frequency of CD14+CD16+monocytes was increased significantly when stimulated by rh-MCP-1(P<0.05), not by other cytokines TNF-a, IL-2and OPN (P>0.05). Moreover, the effect of MCP-1on the frequency of CD14+CD16+monocytes was dose-dependent.3. The level of MCP-1in culture supematants. MCP-1in monocytes treated with25%MCF-CM for24h was raised compared with monocytes and MCF-CM (P<0.05).4. The effect of anti-MCP-1mAb on rugulation of monocyte subsets. The anti- MCP-1mAb can partly inhibit the increased CD14+CD16+monocyte frequency by MCF-CM (56.43%versus43.09%, P<0.05).Conclusion:1. MCF-CM can significantly increase the CD14+CD16+monocyte and with the percentage of MCF-CM increasing, the level of CD14+CD16+monocyte was more elevated in vitro culture system.2. Human recombinant MCP-1can significantly increase CD14+CD16+monocytes. Moreover, the effect of MCP-1on the frequency of CD14+CD16+monocytes was dose-dependent.3. MCF-CM can stimulate monocytes to secret more MCP-1, which can increase CD14+CD16+monocytes, whereas anti-MCP-1can inhibit the increase of CD14+CD16+monocytes by MCF-CM. So we think MCP-1may be one of the reasons why CD14+CD16+monocytes increased in patients with breast cancer.
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