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  • br didn t affect the expression pattern of


    didn't affect the o-Phenanthroline pattern of mature DCs surface markers.
    3.4. Effects of combined treatment with repeated low doses cisplatin and CSC loaded DCs on tumor growth and survival rate of SEC bearing mice We examined the therapeutic efficacy of CSC based DC vaccine to prevent tumor growth and prolong the survival of SEC bearing mice. Groups treated with cisplatin (chemo), unloaded DC (DC), CSC-loaded DC (Vacc) and a combination of cisplatin and either unloaded
    Fig. 3. The gating strategy of CD44/CD24 to determine the optimal phenotype for CSCs primed with different concentrations of chemotherapy. Expression patterns of CD24 and CD44 in EC cell line was analyzed by flow cytometry. Anti-CD44 antibody labeled with FITC and anti-CD24 antibody labeled with PE were applied to the analysis.
    On day 30, only one out of six mice vaccinated with either unloaded DC alone or in combination with cisplatin was tumor free. In contrast, two out of six mice vaccinated with either CSC-loaded DC alone or in combination with cisplatin were tumor free. (Fig. 6b) shows comparing tumor volumes of studied groups from day 14 to day 30 post-tumor induction.
    (Fig. 6c) illustrates that the induction of tumor in the untreated control group markedly decreased the percent survival rate by ↓ 66.66%. Whereas the survival rate was improved in all treated groups; chemo (83.33%), DC (83.33%), DC + chemo (83.33%), Vacc (100%), and Vacc + chemo (100%) groups. CSC loaded DC vaccinated groups showed the highest survival rate among the treated groups. These re-sults demonstrated that vaccination with CSC loaded DCs significantly augmented the therapeutic efficacy of cisplatin as evidenced by the significant reduction of tumor volume as well as improved overall 
    survival rate as compared with other treated groups in SEC tumor bearing mice.
    3.5. Beneficial immune responses after vaccination with DCs loaded with CSC enriched lysate
    3.6. Upregulation of p53 relative gene expression in co-treated group with repeated low doses cisplatin and CSC loaded DCs
    We examined the expression of p53 gene at the mRNA level by real-time PCR. As shown in Fig. (7 b), no statistically significant difference in p53 gene expression levels were found between treatment of SEC
    Fig. 4. (a) Enrichment of tumor cells bearing CSC markers identified as CD44+/CD24- by flow cytometry based on their chemotherapeutic drug resistance. Percentages of CD44+/CD24− cell population in parental EC and drug-treated cell cultures were assessed by flow cytometry. Chemotherapeutic drugs were applied with the indicated concentrations for 72 h after primary EC cell cultures. (b) Comparison between the drug-treated cell cultures with the highest expression levels of CD44+/CD24- cells. The percentage of CD44+/CD24- cells is indicated in the dot plots. Data are presented as mean ± SD. a: significant increase versus control group; b: significant decrease versus CIS 50 group. Triplicate experiments were performed. * P < 0.01. CIS, cisplatin; DOX, doxorubicin; PACLI, paclitaxel; CD, cluster of differentiation. Concentrations are expressed as μg/mL.
    A significant increase in p53 gene expression levels was also ob-served in CSC-loaded DC treated group (P < 0.05) when compared to unloaded DC treated group. Interestingly, p53 gene expression level was significantly up-regulated in co-treated vacc + chemo group (P < 0.001) when compared to other treated groups (Fig. 7b).
    4. Discussion
    Cancer stem cells are a subset of tumorigenic cells that were found to be rare representing a small proportion of the tumor mass. It was
    Fig. 5. Mature bone-marrow derived dendritic cells (BMDCs) generated from naïve mice either loaded or unloaded with enriched CSCs lysate harvested on day 9 of culture in presence of GM-CSF and IL-4 and maturation factor TNF-α. Mature loaded and unloaded DCs expressed typical mature DCs surface markers CD11c and co-stimulatory molecules CD83 and CD86. (a) Flow cytometric analysis showed that the expression pattern of mature CSC-loaded DCs exhibited almost identical phenotype as unloaded DCs group. Representative histogram plots for mature DCs surface markers stained with fluorochrome-conjugated monoclonal antibodies including anti-CD11c, CD83, and CD86 as indicated. The percentages of positive cells are indicated in the histograms. Triplicate experiments were performed. Data are presented as mean ± SD. (b) Immature loaded DCs on day 6 exhibited typical morphological changes and appeared as loosely adherent cell with branched and extended morphology (arrow) (original magnification ×20), (c) Mature unloaded DCs on day 9 (original magnification ×20), and (d) Mature CSC-loaded DCs on day 9 (original magnification ×40) by digital inverted microscope.