Isolation And Identification Of Breast Cancer Stem-like Cells And Dendritic Cells Vaccination Targeting Bs

[Purpose]According to the annual report of Chinese cancer statistics 2014, breast cancer is one of the most common malignant tumor in women. Breast cancer has the highest incidence, and at the fifth position of the cause of death in woman cancer patients in China. There remains a high rate of recurrences despite effective treatment of some patients with breast cancer. Current treatment options are often palliative.Breast stem cells (BSC) which reside in tumor tissue with self-renewal and differentiated potentiality, are the initiator cells of tumor formation, chemotherapy and radiotherapy resistance, tumor growth, and angiogenesis, which induces recurrence and metastatic. Breast cancer-initiating cells have been recently identified in breast carcinoma as CD44+/CD24-/low cells, which exclusively retain tumorigenic activity and display stem cell-like properties. Trough isolation and identification of breast cancer stem-like cells, we could recognize those characteristics clearer and find new therapy method of tumor and elevate cure rate.Immunotherapies harness the body’s own immune system to target cancer and could overcome the limitations of conventional treatments. An immunotherapeutic strategy currently explores the use of dendritic cell (DC)-based vaccination to initiate T-cell-mediated antitumor immunity. However, whether using human BSC antigens may improve the antitumor effect of DC vaccination against BSC remains unclear. In this study, we isolated and evaluated BSCs derived from breast cancer patients, and explored the suitability of BSCs as sources of antigens for DC vaccination against human BSCs, with the aim of achieving BSC targeting and enhanced antitumor immunity.In this study, we have collected tumor tissues obtained by clinical breast cancer patients after surgery, isolated and cultured breast cancer stem-like cells using a mammosphere cell culture model. The mammospheric cells were assessed stem cell phenotype and tumorigenicity in vitro. The in vitro cytotoxic experiments analyzed the immunogenicity induced by purified BSCs as special antigens to prime DCs, which activated cytotoxic T lymphocytes (CTL) and induced interferon y (IFN-y) production against BSCs. In vivo experiments of bearing-BSC animals, we further studied the survival time of BSCs-bearing NOD/SCID mice to observe the anti-tumor ability of DC vaccination. The aim is to find a new method and supply theoretic foundation to treat breast cancer stem cells.[Method]Tumor specimens were derived from consenting patients according to the Internal Review and the Medical Ethics Committee of Yanbian University. Sixteen breast lesions, from the histological diagnostic assessment and sampled by pathologists, were received in the laboratory in 2 hours and immediately disaggregated mechanically. The enzymatic digestion of tissue fragments was incubated in a solution of collagenase/hyaluronidase. After filtration, single cells were plated in serum-free DMEM-F12 (1:1), supplemented with bFGF, EGF, insulin and B27. Cells grown in these conditions were known as non-adherent spherical clusters of cells.FACS was used to assess the expression of a panel of stem cells markers in mammospheres in serum-free conditions or adherent cells cultured in serum conditions. The antibodies used were anti-CD44-PE, anti-CD24-FITC, and anti-CD 133-PE. In vivo tumorigenesis was analyzed by injecting mammospheric cells into NOD/SCID mice.Peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were prepared by Ficoll/Paquedensity gradient centrifugation. PBMCs were seeded and induced for immature DC generation. Mature DCs were pulsed with BSC lysates and stimulated T lymphocytes for three times and obtained CTLs. The cytotoxicity against BSCs was tested using the lactate dehydrogenase (LDH) release assay by CytbTox 96 Non-Radioactive Cytotoxicity kits. The release of EFN-y was measured by commercial enzyme-linked immunospot assay (ELISPOT) kit.In animal model, BSCs-bearing NOD/SCED mice received 1 x 105/g DC-stimulated cytotoxic T cells vaccinations into tail intravenous injection. We studied survival time to observe the anti-tumor ability of the DC vaccination targeting BSCs in vivo.[Results](1) Some cells from the tumor samples gradually formed small mammospheres in culturing for 48 hours, while others underwent apoptosis or remained undifferentiated. Those small mammospheres were able to form larger mammospheres. Patient-derived breast cancer mammospheres expressed CD44 and CD24 markers, as determined by flow cytometry. Mammospheres from 6 patients (37.5%) were demonstrated a higher proportion of CD44+/CD24-/low. In these 6 patients,4 were HER-2(-), ER (-), PR (-),1 was HER-2 (+/++), ER (-), PR(-), and 1 was HER-2(+), ER(+), PR(-). Figure 1 shows that the percentage of CD44+/CD24-/low mammosphere cells derived from patient 7 and patient 11 was 97.69% and 99.45%, respectively.(2) The percentage of CD44+/CD24- cells in mammospheres and adherent monolayer were 91.87% and 0.08%, respectivel. However, the percentage of CD44+/CD24+double positive cells was found in adherent cells (61.59%) versus mammospheric cells (0.77%). CD133 was highly expressed in mammospheres when compared to adherent cells. CD 133 expression was found 96.67% in mammospheric cells and 43.78% in adherent cells, respectively in the cells from patient 9. To study whether cancer stem cells (CSCs) express certain tumor associated antigens (TAAs), we measured the expression levels of several GBM-associated tumor antigens in both mammospheric cells and adherent cells using immunofluorescence. We observed that adherent cells, but not mammospheres, express ER, PR, and HER-2.(3) Mice inoculated with 1×104 mammospheric cells formed tumors in 5 days, while inoculated with 1×106 adherent cells formed tumors after 7 days, suggesting that the mammospheres have a higher tumorigenic capacity than adherent cells. Lung metastasis was observed in one mouse when inoculated with 1×106 adherent cells group, while liver and lung metastases was observed in all mice by day 10 when mice were inoculated with mammospheric cells. Mammospheric cells have a characteristic of cancer stem cells. As patient-derived breast cancer mammosphere shows the characteristics of breast cancer stem cells, mammosphere (sphere) also be described as breast cancer stem-like cells (BSCs) in this article for more convenient.(4) CTL activities were stimulated by dendritic cells, which were loaded lysates of mammospheric cells or adherent cells, or saline as blank. Cytotoxicity against tumor cells increased with a growing ratio of effectontarget (E:T) cells. We also found that as below:1) The cytotoxicity of BSC-CTL against the mammopheric cells was significantly higher than that of BAC-CTL (P<0.01).2) The cytotoxicity of BSC-CTL against the mammopheric cells was also significantly higher than that of Blank-CTL (P<0.01).3) The cytotoxicity of BAC-CTL against the mammopheric cells was similar to that of Blank-CTL.4) The cytotoxicity of BAC-CTL against the adherent cells was higher than that of BSC-CTL (P<0.05).5) The cytotoxicity of BAC-CTL against the adherent cells was also higher than that of Blank-CTL (P<0.05).6) The cytotoxicity of BSC-CTL against the adherent cells was similar to that of Blank-CTL.7) The cytotoxicity of BAC-CTL against the adherent cells was signifcantly higher than against the mammopheric cells (P<0.01).8) The cytotoxicity of BSC-CTL against the mammopheric cells was higher than against the adherent cells (P<0.05).(5) BSC lysates-loaded DCs stimulated Thl response and induced significant EFN-y production. Stimulation of DCs using each of the two breast cancer cells antigens induced significant cytotoxicity. By using a ratio of E:T=10:1, the spot counts produced by BSC-CTL and BAC-CTL against mammospheric cells were 52 and 31 spots, respectively. The IFN-y production of BSC-CTL was also significantly higher than BAC-CTL as observed by spot counts (P<0.05) and area coverage (P<0.05). The IFN-y production of both BSC-CTL and BAC-CTL were significantly higher than Blank-CTL, determined by spot counts and area coverage (P<0.01 and P<0.05). Moreover, a higher ratio of effector to target (E:T) also increased the spot counts and area coverage as determined by IFN y ELISPOT.(6) BSC-bearing mice vaccinated with CTLs stimulated by DCs pulsed with saline as blank had a median survival of 31.4 days. Moreover, BSC-bearing mice vaccinated with CTLs stimulated by DCs pulsed with BAC lysate had a median survival time of 35 days. In contrast, BSC-bearing mice vaccinated with CTLs stimulated by DCs pulsed with BSC lysate had a median survival of 56.1 days. Up to 70 days after inoculation,40% of the BSC-bearing mice vaccinated with CTLs stimulated by DCs pulsed with BSC lysate were still alive. The Kaplan-Meier survival curve showed that BSC-bearing mice treated with CTL stimulated by DCs pulsed with BSC lysate have longer survival time than other groups (P<0.001).[Conclusion]We demonstrated that mammospheric cells express high levels of stem cell-associated molecules, CD44+/CD24-/low and CD133+, while adherent cells express CD44+/CD24+ and low CD133+. In vivo, the enriched mammospheric cells exhibited a stronger tumorigenic capacity than adherent cells. DC vaccination using BSC lysates elicited specific T-cell responses against BSCs. DC vaccination stimulated Thl response and induced significant IFN-y production which positively correlated with the number of activated cytotoxic T lymphocytes (CTLs). In a BSC-bearing mouse model, we also demonstrate that vaccination with CTLs stimulated by DCs pulsed with BSC lysate, but not with adherent cells lysates, was associated with prolonged survival time. Therefore, the results of this study demonstrated that CD44+/CD24"/low/CD133+ mammospheric cells have stem cells properties and DC immunization with BSCs generates superior antitumor immunity. This study may accelerate development of BSC-specific immunotherapies and cancer vaccines.[Purpose]The objective of the present study was to investigate the effects of CYP2C9*3 polymorphisms on the therapeutic response to gliclazide in type 2 diabetes patients.[Methods]A total of 746 incident type 2 diabetes patients were included in this study. After enrolment, patients went on 4-week gliclazide monotherapy. Fasting plasma glucose was measured before and after treatment. Hypoglycemia episodes and lifestyle information were collected by weekly follow up. Genotyping of rs 1057910 was carried out using the single base primer extension method. The t-test, analysis of variance and chisquare-test were used to evaluate the effects of rs 1057910 alleles on the therapeutic response to gliclazide.[Results]After the therapy, fasting plasma glucose decreased significantly from 11.2-2.7 mmol/L to 8.0-2.2 mmol/L (P< 0.001). Patients with AC/CC genotypes of rs 1057910 had a greater reduction of fasting plasma glucose (3.6 vs 3.0 mmol/L, P< 0.001; 31.4 vs 24.5%, P< 0.001) and a higher rate of treatment success (54.7 vs 37.5%,P< 0.001; 51.4 vs 32.3%, P< 0.001; 71.6 vs 48.3%, P< 0.001 for criterion 1,2 and 3,respectively).[Conclusions]The present study showed that the polymorphism at rs 1057910 significantly affected the therapeutic response of gliclazide in type 2 diabetes mellitus patients. The risk allele is associated with a greater decrease of fasting blood glucose and a higher rate of treatment success with gliclazide monotherapy.