| Colorectal cancer (CRC) is one of the most commonly diagnosed and lethal cancers worldwide. Its incidence and mortality increase significantly in China. The traditional treatment of surgery combining with radiotherapy and chemotherapy can not completely prevent tumor metastasis and recurrence. Immunotherapy which induces tumor-specific killing effect by improving the body’s immunity is expected to be a new treatment.Cancer stem cell theory presents a new paradigm for the development of cancer immune treatments. CSCs may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease. These stem cells have been shown to be relatively resistant to conventional chemotherapeutic regimens and radiation. Increase risk of relapse, metastasis and progression of cancers after such therapies. The targeting cancer stem cell therapy may be a promising approach treatment for tumor. Thus, the CSC hypothesis promises the development of more effective treatments, aimed not at reducing tumor bulk, but rather at targeting the beating "heart" of the tumor, CSCs. Studies have shown that cancer stem cell vaccination confers significant antitumor immunity based dendritic cells. These dendritic cell vaccine based CSCs antigen maybe an effective approach to target cancer stem cells therapy. Dendritic cells as the most important antigen-presenting cells are in center of the immune system. Many studies have confirmed that immunotherapy with one kind of immune cells may be insufficient to induce effective anti-tumor immunity. Synergizing several kinds of immune cells to induce effective cellular immunity and humoral immunity may be new ideas for the treatment of tumors.iNKT is a bridge connection inherent immunity and adaptive immunity as DCs play key roles in modulating different immune responses, such as, inflammation, tumors, and autoimmune diseases. Invariant natural killer T (iNKT) cells are a subset of innate-like lymphocytes that recognize CD1d lipid antigens, such as:α-galactosylcer-amide (a-Galcer), a nonmammalian glycosphingolipid (GSL). iNKT cells produce large amounts of interferon IFN-y and interleukin IL-4 which lead to downstream activation of DCs, NK cells, B cells, and conventional T cells.Here we combine cancer stem cell as antigen loading dendritic cell vaccine and α-Galcer aimed to boost anti-tumor immunity, especially to induce target cancer stem cells antitumor immunity in a murine colon cancer model. The immunological targeting of CSCs may provide a novel approach for the development of more effective cancer immuno therapies.PartⅠ:Sorting and identification of cancer stem cellObjective:Cancers are composed of heterogeneous cancer cell clones that differ with respect to proliferation, differentiation, and ability to initiate daughter cancers. Cancer stem cells (CSCs) are dedined as a unique subpopulation in tumors that have the ability to initiate tumor grwth and sustain tumor self-renewal. Abundand evidence suggests that high aldehyde dehydrogenase (ALDH) is a hallmark of CSCs can be measured by the ALDEFLUOR assay. In this study we have sorted and identified CSCs using stem cell marker ALDH from murine colorectal cancer cell line (MC38) and murine carcinoma. Preparation for further study in process, CSCs as resourse of antigen loading dendritic cells vaccine confers immunity in murine mode.Method:The ALDEFLUOR kit was used to isolate the subpopulation with high ALDH enzymatic activity. Cells obtained from MC38 cell line, fresh dissociated murine colorectal cancer in the homologous murine. Cells were incubated with ALDEFLUOR reagent, ALDH+ and ALDH- cells were sorted and analyzed by flow cytometry. Sorted cells including ALDH+ and ALDH" were inoculated in nonadhernt conditions with serum-free media extra EGF and β-FGF. Tumor spheroid colony formation in serum-free media was observed in vitro. We performed transplantation of sorted cells in homologous mice to test the tumorigenicity in vivo. Tumor size was monitored every 3days.Results:The ALDEFLUOR kit was used to isolate the subpopulation tumor cells. ALDH+ cells were sorted and analyzed by flow cytometry. We identified ALDH+ CSC-enriched subpopulations in MC38 cell line and fresh dissociated murine colorectal cancer in the homologous murine. MC38 cell line had a stable subpopulation of ALDH+ cells (approximately 5-7%), with the rest (approximately 94%) being ALDH". There existence approximately 5% of ALDH+ cells in established murine tumors was confirmed by analyzing freshly harvested tumor cells, the rest approximately 95% were ALDH-. The sorted cells were re-stained with ALDEFLUOR reagent using the same staining protocol confirmed the purity of originally sorted cells was more than 98%. Sorted ALDH+ cells showed spheroid colony formation in serum-free media with extra EGF and β-FGF in vitro in the same number cells and same condition with ALDH-, while ALDH- failed. The tumor igenicuty of sorted ALDH+ was evaluated in the C57BL/6 hosts. Only ALDH+ cells generated tumors while equal numbers or even much greater numbers of ALDH- cells failed to establish tumor. Even 200 ALDH+ established tumor while 200000 ALDH" failed to establish tumor at the same host abdomen opposite site. Only ALDH+ initiated daughter tumor in the immunocompetent murine host indicate that ALDH can serve as a reliable marker of enrichment of MC38 CSCs.Summary:ALDH+ cells were sorted and analyzed by flow cytometry by ALDEFLUOR kit. ALDH+ cells were identified in MC38 colorectal cancer model. Only ALDH+ generated daughter tumor in the immunocompetent murine host and formed spheroid colony in serum-free media with extra EGF and p-FGF in vitro. These date demonstrate that CSCs can be sorted by ALDH as a reliable marker of murine colorectal cancer. It has allowed us to invetigate CSC-induced targeted CSCs anti-tumor immunity in the immunocompetent murine host in the subsequent experiments.PartⅡ:Combination cancer stem cell vaccine and a-GC enhances immunity in vivo.Objective:According cancer stem cell definition, cancer stem cells are a subpopulation of tumor cells that resist conventional therapy such as chemotherapy and radiotherapy, have abiliy high tumorigenicity in vivo, and are responsible for tumor relapse and metastasis simultaneously. Thus, the cancer theory promises the development of more effective and target treatment to cancer stem cell -"the heart of tumor". Dendritic cells as the special antigen presenting cells can effectively capture and present antigen to effector cell and induce immune response of T cell special for killing the antigen. The use of dendritic cells in cancer immunotherapy is based on their potent abilities to present antigens, so they can act as’natural adjuvants’to enhance immunogenicity of tumor antigens and stimulate specific cytotoxic T-cells. The DC can then be pulsed with tumor antigens and re-infused. In vitro, antigen-pulsed DC can stimulate allogeneic T-cell proliferation and induction of autologous specific cytotoxic T-cells; in vivo, DC loading with antigen as vaccine can apparently supress tumor growth and mobilize the immunity response to inhibit the growth of tumors or protect hosts (i.e. mice) from development of inoculated tumors. However, the rates of clinical responses are low. It has become quite clear that some key reasons for unsatisfactory clinical results are:loading tumor antigen to DCs appears in low antigen presenting efficiency and insufficient CSCs antigen to induce target CSC specific cytotoxic T-cells; tumor-induced immuno suppress ion. To overcome this dilemma, we combine α-galactosylceramide (α-GC) which can be presented by CD1d express dendritc cell to NKT cell and stimulated iNKT to mature. Based on an elevated aldehyde dehydrogenase (ALDH) activity in stem ALDH+ cells have been identified and isolated from tumors and shown to have characteristics of CSCs. Coperation CSCs antigen loading to dendritic cells as vaccine and a-GC to detect the response of immunity, such as DC, iNKT and NK cell maturation and function.Method:Based on sorting ALDH+ cells, we identified such subpopulation tumor cells characteristic of cancer stem cells. ALDH+, ALDH- and unsorted cells were frozen and thawed 3 times to make cell lysate. Bone marrow-derived DCs were cultured in interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor (GM-CSF). When cultured on 6 days DCs be identified by morphology with dendritic appearance and flow cytometry by cell surface marker such as CD80, CD86. DCs were plused with a-GC and tumor cells lysate as antigen, after 24 hours coculture in different groups. Normal animal were vaccinated 3 times everyone week. After 24 hours of the last vaccination the spleen, liver and peripheral blood were harvested to test the iNKT, NK, dendritic cells maturation and function such as CD69, CD40L, CD80, CD86 and secretion of cytokine such as IFN-y IL-12, IL-2, TNF-α,IL-4, IL5.Results:Vaccines were successfully prepared with CSCs lysate loading with DCs. Combination DC vaccine loading with cancer stem cells antigen and a-GC can increase the number of iNKT:the experimental group vs other groups except α-GC group (P<0.05). Combination DC vaccine loading with cancer stem cells antigen and a-GC can upregulate iNKT cells express CD69:the experimental group vs other groups (P<0.05). Combination DC vaccine loading with cancer stem cells antigen and a-GC can upregulate the iNKT cells express of CD40L:the experimental group vs other groups (P<0.01). Combination DC vaccine loading with cancer stem cells antigen and α-GC can promote iNKT secretion of IFN-γ, the expression level of IFN-γ more than α-GC, however there was no significant difference with a-GC. There were significant difference betweent experimental group vs other groups except a-GC group (P<0.01). Combination DC vaccine loading with cancer stem cells antigen and a-GC also can improve NK secretion of IFN-y the experimental group vs other groups (P<0.05). Combination DC vaccine loading with cancer stem cells antigen and a-GC can upregulate the DC cells express of CD80, CD86. The experimental group, the expression level of CD80, CD86more than ALDH+ group, howerver there was no significant difference. There were significant difference between experimental group vs other groups except ALDH+ group (CD80 P<0.05; CD86 P<0.01). Combination DC vaccine loading with cancer stem cells antigen and a-GC also can enhance DCs secretion IL-12. The expression level of IL-12 in experimental group was more than ALDH+ group in supernatant of DCs culture system, howerver there was no significant difference. There were significant difference of expression level of IL-12 between experimental group and other groups except of ALDH+ supernatant of DCs culture system, (P<0.01). Combination DC vaccine loading with cancer stem cells antigen and α-GC can transfer the differentiation of Thl. The experimental group can strengthen secretion cytokines IL-12 and IFN-γ, TNFα, IL-2(P<0.05), and suppress secretion cytokines such as IL-4, IL-5.Summary:Combination DC vaccine loading with cancer stem cells antigen and α-GC can effectively induce NKT, NK, and DCs maturation and enhance function of anti-tumor immunity. Combination DC vaccine loading with cancer stem cells antigen and a-GC can enhance Th1 type cytokines and suppress Th2 type cytokines secretion. This transformation was conductive to antitumor immunity.PartⅢ Combination cancer stem cell vaccine and a-GC confers anti-tumor immunity in colorectal cancer model.Objective:Dendritic cells vaccines were successfully prepared with CSCs lysate antigen. Combination DCs vaccine was plused antigen of CSCs and a-GC can effectively induce iNKT, NK, and DCs maturation and enhance function of immunity. To verify the antitumor effect of this new vaccine Combination DCs loading with cancer stem cells antigen and α-GC, we tested it in miurine colorectal cancer model, investigated the survival rate and monitored the the tumor size. We explored the possible mechanisms underlying ALDH+ CSC-plused DC vaccine combination with a-GC induced protective immunity and tested the CSC-induced antitumor immunity is due to direct targeting of CSCs.Method:In the therapy model, MC38 cells were inoculated to the C67BL/6 subcutaneous of abdomen. Animal were vaccinated 3 times every week 3 days later. The tumors were monitored and tested, the survival datas were recorded. In the preventive mode C67BL/6 were vaccinated 3 times every week, MC38 cells were inoculated at the last time. The tumor sizes were measured. The spleen and peripheral blood and were harvested to test the CD3,CD4 and CD8 T cells rate in the peripheral blood and MDSC in the spleen on endpoint 50 days after tumor cell inoculation. The spleen cells as effector cells, the sorting ALDH+ as target cells, assessed for cytotoxicity against ALDH+ CSCs according different rate.Results:In the therapy model. Combination the vaccine of DCs loading with cancer stem cells antigen and a-GC can reduce the tumorgenic and suppresse the tumor growth.There were significant difference with other gropus(P<0.05). The experimental group survival rate was higher than ALDH+ group without statistic difference. However, there were significant difference with other groups (P<0.01). In the preventive model, the experimental group significantly inhibited the tumor growth and delayed the tumor occurrence (P<0.01).On endpoint the tumor size was larger than ALDH+ group without statistic difference, however there were significant difference with other groups(P<0.05). Combination the vaccine of DCs loading with cancer stem cells antigen and a-GC can increase CD8+ T cell and decrease MDSC ratio than ALDH+ group without significant statistic difference, but with other groups(P<0.05). The results demonstrated combination the vaccine of DCs loading with cancer stem cells antigen and a-GC induced anti-tumor immunity direct targeting of ALDH+ CSCs primed CTLs killed CSCs efficiently (approximately 73.6±3.00%) significantly more than other groups (P<0.01).Summary:Combination DC vaccine loading with cancer stem cells antigen and a-GC can effectively induce anti-tumor immunity, significantly prolonged survival of tumor-bearing mice and inhibited growth of tumor. Incresing the CTLs of CD8+ T cells and dcreasing the MDSCs ratio, induced anti-tumor immunity direct targeting of ALDH+ CSCs.Conclusion:ALDH is a reliable cancer stem cell marker of murine colorectal cancer. ALDH+SCs were sorted and identified successfully. Vaccine of dendritic cells were plused ALDH+CSCs antigen combined a-GC can effectively induce anti-tumor immunity direct targeting of ALDH+CSCs significantly prolonged survival of tumor-bearing mice and inhibited growth of tumor. The potential mechanism of combination DC vaccine loading with cancer stem cells antigen and a-GC maybe include promoting iNKT cells, NK cells, DCs maturation and enhaning function, incresing the CTLs of CD8+ T cells and decreasing the MDSCs ratio, inducing anti-tumor immunity direct targeting of ALDH+ CSCs. The immunological targeting of CSCs may provide a novel approach for the development of effective cancer immunotherapies. |
PDF Full Text Request