| BackgroundThe bladder cancer is one of most common urologic cancers in the United States and the rest of the world, and radical cystectomy with pelvic lymphadenectomy is standard to treat this kind of patient. However, more than half of patients will develop local or metastatic recurrence. The MB49bladder cancer cells vaccine induced a specific antitumor immunity and was efficiently effective against metastatic bladder cancer in our earlier studies. However, we also found that a part of the mouse bladder tumor regrew after experiencing regression for a period of time. Because the cancer stem cells (CSCs), or the cancer-initiating cells, could not be eliminated. Recent findings supported the notion that the relapses of solid tumors may be attributed to the inability of traditional chemotherapies and radiotherapies to eradicate CSCs. Our former bladder cancer vaccine was not the CSCs vaccine, which was unable to induce specific immunities responsible for CSCs. Therefore, on the basis of our protein-anchor technology, we developed a technology to display streptavidin-mouse granulocyte macrophage-colony stimulating factor (SA-mGM-CSF) on the surface of biotinylated MB49bladder cancer stem cells (MCSCs), and evaluated the antitumor effects in the treatment of MCSCs metastatic mouse model.Our study was divided into four parts:Chapter1Isolation of MB49bladder cancer stem cells1. ObjectiveTo isolate MB49bladder cancer stem cells (MCSCs) from MB49bladder cancer cells.2. Methods(1) Optimal SFM for MCSCs.(2) Limited dilution method.(3) Passage culture.3. Results(1) The combination of the optimal SFM was RPMI1640+EGF (20ng/ml)+bFGF (20ng/ml)+LIF (20ng/ml)+B27(20μl/ml)+BSA (4μg/ml).(2) The limited dilution method showed that only a2-3percentages of MB49cells generated CSC spheres in SFM.(3) The passage1single MB49cell formed a CSC sphere within30days in optimal SFM. The MCSCs were passaged after15days to form new tumor spheres, and most MCSCs generated secondary spheres.4. ConclusionsMCSCs were isolated successfully with a modified method using a combination of limited dilution and SFM methods.Chapter2Characterizations of MB49bladder cancer stem cells 1. ObjectiveTo compare the characterizations of MB49bladder cancer stem cells (MCSCs).2. Methods(1) Expression of MCSCs markers, containing Fluorescence activated cell sorting (FACS), Quantitative polymerase chain reaction (qPCR), Western Blotting (WB).(2) Differentiation(3) Functional comparison, containing Cell proliferation assay, Soft agar assay, Migration abilities in vitro, Resistance to chemotherapy abilities, Tumorigenic abilities in vivo.(4) Statistical analysis:SPSS19.0software was used for the statistical evaluations. All of the data was expressed as the mean±standard deviation and analyzed using one-way ANOVA. P<0.05was considered statistically significant.3. Results(1) As demonstrated by the FACS analysis, the fraction of CD133+CD44+cells was19.83±0.68%in MCSCs and3.57±0.38%in MB49cells, which was elevated in MCSCs relative to MB49cells (t=36.173, P=0.000).(2) The relative levels of CD133, OCT4and NANOG were higher in MCSCs using the qPCR experiment, being5times as high as observed in MB49cells. However, the level of CD44was higher in MB49cells (CD133t=18.941, P=0.000-, CD44t=-16.854, P=0.000; OCT4t=15.226, P=0.000; NANOGt=12.445, P=0.000).(3) OCT4, NANOG and ABCG2were both expressed in MCSCs and in MB49cells by the WB assay. They were sparsely distributed in MB49cells, but they were abundantly expressed in MCSCs (OCT4t=2.894, P=0.044; NANOG t=14.148, P=0.000; ABCG2t=21.866, P=0.000).(4) When MCSCs were reseeded with a medium containing10%FBS, they became flat after being differentiated and attached to the culture dish. (5) MCSCs increased the proliferation as compared with MB49cells in the SFM on day4,5,6after using CCK-8in the cell proliferation assay (F=582.368, P=0.000).(6) The soft agar assay revealed that MCSCs formed bigger and more numerous colonies than MB49cells did (t=33.732, P=0.000).(7) Under the same incubation conditions, the number of invaded MCSCs was more than that of MB49cells (t=11.654, P=0.000).(8) Compared to MB49cells, MCSCs showed higher cell viabilities after being treated with different concentrations of mitomycin, cisplatin, paclitaxel, and doxorubicin (F=1753.923, P=0.000; F=1870.563, P=0.000; F=26460.000, P=0.000; F=371.314, P=0.000). MCSCs demonstrated lower susceptibility to all these traditional anticancer agents.(9) MCSCs caused a more remarkable tumor volume than MB49cells did despite the same number of injections (F=288.012, P=0.000).(10) The morphology of H&E stained xenograft tumor sections from MCSCs resembled tumor tissue from MB49cells.4. ConclusionsMCSCs contained characteristics resembling CSCs such as in vitro self-renewal, a differentiation potential, chemotherapy resistance and in vivo tumorigenic capacity.Chapter3Preparation and biological activity of GM-CSF modified MB49bladder cancer stem cells vaccine1. ObjectiveTo prepare GM-CSF modified MB49bladder cancer stem cells (MCSCs) vaccine and study their biological activity.2. Methods(1) Vaccines preparation. (2) Evaluation of SA-mGM-CSF on the surface of MCSCs.(3) Bioactive assay of SA-mGM-CSF immobilized on the surface of MCSCs.(4) Statistical analysis:SPSS19.0software was used for the statistical evaluations. All of the data was expressed as the mean±standard deviation and analyzed using one-way ANOVA. P<0.05was considered statistically significant.3. Results(1) As demonstrated by the FCM analysis, the percentage of MCSCs anchored with SA-mGM-CSF was89.5±1.5%.(2) The membrane-bound mGM-CSF stimulated the proliferation of BMCs in a dosage dependent manner using CCK-8assay (F=1546.522, P=0.000)4. ConclusionsSA-mGM-CSF could be efficiently anchored on the surface of MCSCs and retained its bioactivity.Chapter4Animal experiment1. ObjectiveTo evaluate the GM-CSF surface-modified MB49MCSCs vaccines in the treatment of metastatic bladder cancer.2. Methods(1) Subcutaneous and lung metastatic mouse model of MCSCs.(2) Protective immune response experiment.(3) Immunotherapy experiment.(4) Tumor-specific lymphocyte cytotoxicity assay.(5) FCM of dendritic cells (DCs).(6) FCM of T cell subsets.(7) Memory immune response experiment. (8) Specific immune response experiment.(9) Statistical analysis:SPSS19.0software was used for the statistical evaluations. Statistical analyses of survival were performed using Kaplan-Meier analyses. All of the data was expressed as the mean±standard deviation and analyzed using one-way ANOVA. Tumor volume between groups were compared using Repeated Measures. P <0.05was considered statistically significant.3. Results(1) Protective immune response with MCSCs vaccines.The pulmonary model mice in the treated group had a significantly longer survival compared to other four control groups (x2=39.656,-P=0.000). The subcutaneous model mice in the treated group reached a maximum mean volume, and had a significantly smaller tumor volumes trend compared to other four control groups (F=311.723, P=0.000).(2) Immunotherapy with MCSCs vaccines.The pulmonary model mice in the treated group had a significantly longer survival compared to other four control groups (x2=42.591, P=0.000). The subcutaneous model mice in the treated group reached a maximum mean volume, and had a significantly smaller tumor volumes trend compared to other four control groups(F=81.825, P=0.000).(3) The percentage of CTL in the treated group was higher significantly compared to other four control groups (F=6505.314, P=0.000).(4) As demonstrated by the FCM analysis, the number of the mature DCs (CD11c+CD80+) was more significantly in the treated group than other four control groups (F=333.398, P=0.000; F=277.693, P=0.000).(5) As demonstrated by the FCM analysis, the proportion of CD4+and CD8+T cells in the treated group was more obviously than other four control groups (CD4+T: F=582.514, P=0.000; CD8+T:F=154.142, P=0.000; CD4+T:F=248.893, P=0.000; CD8+T:F=70.438, P=0.000).(6) Memory immune response with MCSCs vaccines. The survival time of mice in the experimental group was significantly longer than the control group after a second challenge of MCSCs (x2=26.800, P=0.000).(7) Specific immune response with MCSCs vaccines.The volume of tumor at the MCSCs injecting side in right leg was smaller obviously than that at the RM-1cells injecting side in left leg (F=6009.684, P=0.000).4. Conclusions(1) SA-mGM-CSF vaccine could establish a stronger tumor-specific immunity.(2) SA-mGM-CSF vaccine could inhibit the tumor.(3). SA-mGM-CSF vaccine could establishT-specific immunity.(4). SA-mGM-CSF vaccine could increase the mature DCs population.(5) SA-mGM-CSF vaccine could increase the mature CD4+and CD8+T lymphocytes population.(6) SA-mGM-CSF vaccine could produce long memory immunity to MCSCs.(7) SA-mGM-CSF vaccine could establish a same tumor specific immunity to protect mice against MCSCs. |
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