Apoptosis Of Dendritic Cell Subsets In MCF-7Tumor-bearing Mice

Chapter1Establishment of MCF-7xenograft model in nude mice, induction and differentiation of dendritic cells and detection of its surface markerBackground and Objection:Breast cancer is the most common magnicnant in women. No matter in developed countries or developing countries, the incidence rate is increasing year by year. As operation, radiotherapy, endocrine therapy and targeted therapy are used reasonably, the mortality of breast cancer in developed countries has been declined. The clinical responds has come into a relative plateau, however, advanced and metastatic disease remains incurable and has become the main cause of threatened the life of female, especially in developing countries. On the other hand, as a promising treatment pattern, antitumor immunotherapy is received more and more attention along with the progress of medicine. Dendritic cells(DCs) is the most powerful antigen presenting cell in organism. It can uptake, processing and presentation of antigen to the T cell, and result in the T cell sensitization, activation and amplification. DCs play an important role in antitumor immunotherapy because of its potential abilities to overcome tumor tolerance and to induce antitumor immune capacity. Two main populations of DCs are recognized in mouse and human tissues:myeloid dendritic cell (mDC) and plasmacytoid dendritic cell(pDCs). The former are characterized by the high expression of CD11c and low expression of CD123. The latter express CD123at high level and CD11c at low level. There are various antitumor effect of immune responds in defferent DCs subsets. We purpose to establish MCF-7tumor-bearing mice, study the differentiation and maturation of bone marrow-derived DCs and the data of DCs subsets, in order to give the foundation information to design the new immune-therapeutic method and new vaccines in breast cancer.Methods and materials5wk-old female BALB/c mice (ten mice/group) were injected subcutaneously (s.c.) with o.2ml(1×107/ml) MCF-7cells into the flank, while mice of the control group received o.2ml serum-free medium by s.c injection. Mice were sacrificed when tumor size reached5mm in diameter. All of the experiments were conducted in randomized fashion. The isolated cells from bone marrow were plated in nonadhesive Petri dishes at a density of1×107cells/1.5ml in medium (RPMI,10%FCS). The medium was replenished every3d. After3days in culture, all medium and nonadherent cells were discarded, fresh medium supplemented with murine recombinant GM-CSF and murine recombinant IL-4was added. For DCs maturation, the cells were treated overnight with100ng/ml LPS on day7. Nonadherent DCs were harvested on day9, and culture supernatants were collected, and used for following studies. DCs were identified by flow cytometry. DC subsets were verified by analyzing anti-CD11c PE or anti-CD123PE expression using flow cytometry, respectively. Immunohistochemistry was used to detect ER, PR, Cerb-B2and Ki-67of the tumor biopsy.Comparisons of samples to establish statistical significance were determined by the two tailed Students’t-test. Results were considered to be statistically significant when the p-value was<0.05. Results:MCF-7cells were observed attached and in good shape under inverted microscope. The tumor size of tumor-bearing mice reached5mm in diameter after16days. There were no significant difference in the weight between both two groups before and after injected MCF-7cell.We can observe the single nonadherent DCs with obvious burrs protuberance on the cellular surface when the bone marrow-derived PBMC has been cultured for9days.High levels expression of CD86,CD83and MHC-Ⅱ were similarly observed in two groups, and the expression of CD83in tumor-bearing mice was significantly decreased than that in control mice.(74.61±1,26%, vs77.30±1.41%, P<0.05). Ther was a more significant descent on the expression of CD34(1.30±0.17%, vs1.71±0.18%, P<0.05). The cells of CD11c+CD123-in tumor-bearing mice was lower than that of control mice(52.01±1.43%, vs56.36±1.76%, P<0.05)。The cells of CD11c-CD123+in tumor-bearing mice was higher than that of control mice(32.19±1.71%, vs28.95±1.39%, P<0.05)。The tumor biopsy can observe breast cancer cell, and immunohistochemistry shows that ER(+++), PR(++), Cerb-B2(-) and Ki-67(+++) in tumor cells.Conclusion:The MCF-7Tumor Xenograft Model was established successfully. In vitro, bone marrow-derived PBMC can be induced high quality and high purity mature DCs.The immature status of bone marrow-derived DCs in MCF-7tumor-bearing mice resulted in decreased the ability of tumor antigen presentation and subsequent lack the ability to induce T-cell activation and proliferation. The efficiency of immune killing tumor cells declined.The number of mDC decreasing in MCF-7tumor-bearing mice showed the antitumor immune responses was diminished, which generated by presentation tumor antigen to T cell, in turn, the level of Il-12secreted and actived by mDC reduced, the effect of immune killing tumor cells lessened. While pDC has a weak immune responses against tumor, on the other hand, it has a strong ability to inhibit antitumor immune responses, so the high quantity of pDC can not enhance the antitumor immune function.Chapter2The detection of apoptosis of dendritic cell subsets in MCF-7tumor-bearing miceBackground and Objection:Programmed cell death is important for maintaining the homeostasis of DCs in vivo. Dysregulated programmed cell death in DCs may change the lifespan of DCs and lead to the imparement of immune responses. Recent research suggests that induction apoptosis of DCs is another important mechanism of tumor escape immune recognition. In vitro, the interactions between tumor cells and tumor-derived cytokines can induce apoptosis of DCs.There are two subsets T cell in human tissues:Thl cells and Th2cells. The function of Thl cells and Th2cells remains dynamic balance in normal organism. The antitumor immune responses is mainly depending on cellular immunity mediated by Thl cells. Matuer DCs not only can induce cytotoxic T lymphocyte(CTL) proliferation, also induce CTL secreting cytokines (such as IL-12).11-12has a strong capability of stimulating cytotoxic T cells, natural killer cells and lymphokine-activated killer cells, result in generating abundant of IFN-y, perforin and granzyme, the effect of lysing the target cell enhances. A Thl response is induced to antitumor. Different DCs subsets express various cellutoxic molecule. It was believed that pDC induce a Th2response, whereas mDC induce a Thl response.Using DCs as antitumor immunotherapy is a powerful approach to eradicate tumor cells by enhancing the immune responses of the organism. The vaccines of tumor antigen with mature DCs have been applied in several cancers, however, although the technique of the therapy is ripe, the clinical effect is not ideal. More reasonable and more effectively DCs vaccines exploration would be required. We purpose to study the apoptosis of DCs subsets, in order to provide more accurate and more scientific iomformation on antitumor immune therapy.Methods and materials:DCs suspensions were prepared as above. Cells were treated with CD11c-PC5and CD123-PE, respectively. And then were put at room temperature for30min. Annexin V Binding Buffer and FITC Annexin V were added after being washed with PBS, Three colour analysis was performed using flow cytometry for apoptosis of DC subsets.Comparisons of samples to establish statistical significance were determined by the two tailed Students’t-test. Results were considered to be statistically significant when the p-value was<0.05.Results:The proportion of apoptotic mDCs in tumor-bearing mice was significantly increased (23.64±2.43%, vs20.05±2.49%, P<0.05). In contrast, in tumor-bearing mice, the percentage of apoptotic pDC was lower than that in control mice(11.53±2.92%, vs14.96±2.96%, P<0.05).Conclusion The rate of apoptotic mDCs in tumor-bearing mice is higher significantly than that in control mice. It suggested that mDC in MCF-7breast cancer mice induced a weak Thl response, and the level of cytokines secreted by Thl cells including IL-12decreased. The consequence on one hand impaired the T-cell activation and proliferation, result in decreasing the capability of stimulating cytotoxic T cells, natural killer cells and lymphokine-activated killer cells to generate IFN-y, perforin and granzyme, in turn, the effect of lysing the target cell declined, on the other hand, changed the balance of Thl/Th2in the organism, induces immune tolerance and inhibited antitumor immune responses.The rate of apoptotic pDCs in tumor-bearing mice is lower significantly than that in control mice. It suggested that pDC promoted ThO cell differentiation into Th2cells. Cytokines secreted by Th2response increased, induced producing CD4+/CD25+regulatory T cells to escape antitumor immune response. The high percentage of apoptotic pDC can also induce immune tolerance by declining the ability of antigen presentation, T-cell activation and proliferation, participate and promote breast cancer growth and metastasis by angiogenesis. There was another important factor on the descent of antitumor immune ability.The rate of apoptotic mDC, which is mainly important in killing tumor cell by immune responses, in MCF-7breast cancer mice increasing can impair directly antitumor immune responses. However, decreasing of apoptotic pDC can inhibit monocyte cell differentiation into DCs, and decline the ability of antigen presentation of mDC even make mDC mediate immune tolerance, further impair antitumor immune responses in vivo.Chapter3The detection of the levels of cytokines in MCF-7murine serum and DCs-derived supernatansBackground and Objection:Cytokines is a kinds of polypeptide substances with varied biological effects. IL-6, TNF-a and11-12are the important members of the cytokines net. They not only mediate the anti-infection immunity and autoimmunity, but also contribute to differentiation, metastasis and angiogenesis of tumor cells. They play a important role in surveillance and killin g tumor cells.Now the role of TNF-a in breast cancer remains controversy. High dose of TNF-a can cause the vascular degeneration and hemorrhagic necrosis in tumor. However, other studies showed endogenousTNF-a in tumor cell can not kill tumor cell, but induce malignant mammary epithelial cell proliferation and promote angiogenesis, invasion and metastasis of tumor cells. IL-6involves in the antitumor immune responses. It can promote breast cancer stem cell growth and progression, and promote angiogenesis, and invasion of tumor cells. It plays an important role in tumor progression and estrogen regulation. As a key role of trigger cellular immunity, IL-12promotes NK-cell mediated killing of HER2-positive tumor cells. IL-12appears to contribute to humoral immunity in humans through a direct path in DC-B interaction, and an indirect path in DC-T cell interaction and induction of Th cells.Varied cytokines play important roles in antitumor therapy. We purpose to study the change of these cytokines in tumor-bearing organism, and then modify the level of cytokines in vivo in reasonable ways and combine other immune therapy to enhance antitumor immune responses.Methods and materials:For measurements of TNF-a, IL-6and IL-12secretion, murine serum and DCs-derived supernatans were collected, and assayed using commercially available ELISA kits. Samples were analyzed in duplicate according to manufacturer’s instruction.Comparisons of samples to establish statistical significance were determined by the two tailed Students’t-test. Results were considered to be statistically significant when the p-value was<0.05.Results:No major difference in the levels of TNF-a in serum was observed between control mice and tumor-bearing mice(314.81±103.36pg/ml, vs371.87±115.30pg/ml, P>0.05). however, TNF-a concentration in DCs-derived supernatans of tumor-bearing mice was higher than that of control mice (1.88±0.1Ong/ml, vs1.72±0.07ng/ml, P<0.05). The IL-6level was significantly increased in serum of tumor-bearing mice(170.72±54.51pg/ml, vs122.35±20.65pg/ml, P<0.05). A similar increase in the IL-6level in DCs-derived supernatans was also observed in tumor-bearing mice(57.18±5.06pg/ml, vs44.53±4.86pg/ml, P<0.05).On the other hand, IL-12level was significantly decreased either in serum or in DCs-derived supernatans in tumor-bearing mice(119.80±33.53pg/ml, vs186.22±51.91pg/ml, P<0.05)(89.76±8.89pg/ml, vs157.98±48.31pg/ml, P<0.05).Conclusion:There was not significantly differences in TNF-α concentration in serum between tumor-bearing mice and control mice. It probably correlate with a variety production ways. TNF-α can product by the way of autocine, paracrine and endocrine. So a variety of cells and other cytokines involved in producting the TNF-α in MCF-7breast cancer. That is to say the role of TNF-α in breast cancer is more complex and need more comprehensive study. The level of TNF-α in DCs-derived supernatans of tumor-bearing mice was higher than that of control mice. DCs maybe participate to produce TNF-α. Further investigations in the role of TNF-α in antitumor immune in breast cancer should be needed, and the role maybe correlate with the dose of TNF-α and the type of tumor.The level of IL-6showed a significant increase in serum in tumor bearing mice when compared with control mice. IL-6plays an important role in participating the breast cancer cell growth and promoting breast cancer cells infiltrating and metastasis as described above previous mechanism. IL-6concentration in DCs-derived supernatans of tumor-bearing mice was higher than that of control mice. Our data revealed that that IL-6may also display its suppressive ability in antitumor immune by decreasing the level of IL-6secreted from DCs.IL-12concentration in serum of tumor-bearing mice was lower significantly than that of control mice. The ability of killing tumor cell by T cells and NK cells decreased because of low-level IL-12. In our study, the level of IL-12in DCs-derived supernatans had s significant reduction in tumor-bearing mice when compared with control mice. The result demonstrate that decreasing of IL-121evel may suppress exerting potent antitumor activity in breast cancer. Enhancement the level of IL-12in vivo, especially combining chemotherapy and target therapy, can powerfully improve the clinical effectiveness of the breast cancer.Chapter4The relationships between apoptosis of DC subsets and cytokines levelBackground and Objection:The interplays between DCs and cytokines network can influence the antitumor immune response in organism. On one hand, cytokines play an important regulating role in the differentiation and maturation of DCs, arious tumor-derived factors (VEGF, IL-6, IL-10, M-CSF, and GM-CSF) can inhibit DC differentiation from hematopoietic progenitor cells (HPCs) in vitro and in vivo. On the other hand, defferent mature DC subsets can secret various cytokines to enhance or decrease the ability of immune killing response or immune tolerance responses.Tumor can generate a series of immunosuppressive factors to form a immunosuppression environment. The changes of various soluble cytokines, including decreasing of inflammatory factors, increasing anti-inflammatory factors and increasing of Treg regulated T cell, can inhibit differentiation and maturation of DCs. With detrimental effects on DC quantity and function can significantly prevent the establishment of effective antitumor immune responsesThe effects of various receptors, cytokines and chemokines may affect the lifespan of DCs at various stages of immune responses. Although the precise mechanism for the maintenance of DC homeostasis is not clear, emerging evidence suggests that cytokines may be important regulators. The investigation of interaction between DCs subsets and key cytokines in MCF-7breast cancer can bring important significance in designing composite antitumor immune therapy. On one hand, we can use the more effective DCs subsets to present antigen, induce T cell to generate specific antitumor immune responses, enhance cellular immunity mediated by CTL. On the other hand, combining cytokines can further enhance the immune killing response.Methods and materials:Detecting the rate of mDC and oDC in tumor-bearing mice and control mice as described above, assayed the levels of TNF-a, IL-6and IL-12in serum and DCs-derived supernatans using commercially available ELISA kits.Comparisons of samples to establish statistical significance were determined by the two tailed Students’t-test. Pearson correlation analysis was used to assess associations between apoptosis of DC subsets and cytokines levels in DCs-derived supernatans. Results were considered to be statistically significant when the p-value was<0.05.Results:The negative correlation betweenTNF-alevel in serum and proportion of apoptotic mDC was shown. r=-0.70, P=0.00. There was the negative correlation between IL-6level in serum and proportion of apoptotic pDC. r=-0.83,P=0.00. The IL-12level in serum and proportion of apoptoticmDC also showed a negative correlation. r=-0.83, P=0.00.There was the negative correlation between TNF-a level in DCs-derived supernatans and proportion of apoptotic pDC. r=-0.91, P=0.00. The negative negative correlation betweenIL-6in in DCs-derived supernatans and proportion of apoptotic pDC was shown. r=-0.85, P=0.00. IL-12level and the percentage of apoptotic mDC in DCs-derived supernatans have also been shown negative correlation. r=-0.70, P=0.00.Conclusion:TNF-a level and the percentage of apoptotic mDC in serum showed negative correlation, and the correlation has no significance because of various cells and factors participating the secretion of TNF-a. There was the negative correlation between TNF-a level in DCs-derived supernatans and proportion of apoptotic pDC. It showed that the level of TNF-a were significantly increased as proportion of apoptotic pDC was reduction, however, because of the bidirectional action on antitumor immunity in breast cancer, more investigations on the role of TNF-a in defferent molecule isoforms of breast cancer would be need.Thl/Th2was imbalance due to reduction of apoptosis of pDC in MCF-7breast cancer and deflected to Th2status. As the main cytokines secreted by Th2cell, the level of IL-6increased significantly. IL-6can promote breast cancer stem cell growth and progression, and promote angiogenesis. It plays an important role in breast cancer progression and invasive. Moreover, IL-6can produce a marked effect with other cytokines, impair natural killer ability and promote breast cancer cell invasion and metastasis.The level of IL-12decreased significantly due to the proportion of apoptotic mDC increased in MCF-7breast cancer mice. The status can stimulate ineffectively T cell proliferation and inhibit the maturation and activation of mDC, in turn, the balance of Thl/Th2changed, and immune tolerance induced, Finally lead to tumor progression and metastasis.