Study On Seperation And Identification And Immunotherapy Of Breast Cancer Stem Cell

Objective: Tumor stem cells, which reside in tumor tissue with self renewal and differentiated potentiality, are the initiator cells of tumor's formation and maintain tumor growth,induce recurrence and metastatic. Trough separation and identifacation on breast cancer stem cell, we could recognise those characteristic clearer and find new method to therapy tumor and elevate cure rate. In theory of tumor stem cell, tumor forms of tumor stem cell's abnormal generation and differentiation. Conventional therapy only eliminate the non-tumorigenic tumour cell in multiplication period to make the tumor diminute even disappear. However, when stop treating, the tumor stem cell with chemical sproof could generate to tumor again and disseminate like dandelion. IF we remove the tumor stem cell, the tumor will extinct forever and radical cure the tumor. Recently, immunotherapy will become another major method of cancer treatment. The effective cells using in immunotherapy include lymphakine active killer cells (LAK), tumor-infiltrating lympjocytes(TIL), cytokine induced killer cells(CIK), and specific cytotoxicity T lymphocytes(CTL). The researches have showed that dendritic cell(DC) can greatly process tumor antigen, DC loaded by tumor antigen can activate host immune, promote the activity and specificity of CTL to kill tumor cells. DC, as the professional antigen processing cells, play a center role in immune reaction. The im-matured DCs will be activated to matured DCs after captured antigen, which express MHC classⅠand classⅡmolecule,co-stimulatory molecule B7-1(CD80) and B7-2 (CD86),adhesion molecule CD54 (ICAM-1) and CD50 (ICAM-3) in the cell surface. After presenting the antigen peptide to CD4+ and CD8+ T cells, DCs induce the T cells become to specific cytotoxicity T cells,which can excrete cytokines and produce Th1 type immune response to educe the anti-tumor effect. We adopt flow cytometry determing the mean fluorecence density of cell highly exprssing CD55 in breast cancer MCF-7 cell line and sorting CD55hig cells, to analyze whether the CD55hig cells possess characteristics of cancer stem cells, detect the existence and the ratio in the primarily breast cancer and find a new method for isolate breast cancer stem cells in breast cancer tumor tissue. DCs and specific CTLs were induced from single-nucleus cells of axillary lymph nodes and analyzed by killing test in vitro. The aim is hoping to find a new method to treat breast cancer stem cells.Method: Part one: Biological characteristics of CD55hig side population in human breast cancer line MCF-7.In this rsearch, we cultivated the human breast cancer MCF-7 cells and addined nucleic dye Hoechst33342 and verapami, detected the ratio of SP and MP subpopulation cell by flow cytometry and separated them. Then SP and MP cells were labeled with anti-CD55 monoclonal antibody and detected the mean fluorescent intensity. We used anti-CD55 monoclonal antibody labling unsorted MCF-7 cells, detected the proportion of CD55hig side population cells and sorted them to detect their biological characteristics such as cell adherence efficiency, cloning efficiency and cell cycle, to analyze whether the CD55hig cells possess characteristics of cancer stem cells.Part two: Killing activity of chemotherapeutics and immunity on CD55hig side population in human breast cancer line MCF-7.Human breast cancer line MCF-7 cells were killed by docetaxel, epirubicin and fluorouracil in different blood drug level (10%, 25%, 50%, 75% and 100%). Then choosed the maximal killing blood drug level chemotherapeutics to kill CD55hig cells and CD55low cells, detected the lethal effect by Acid Phosphatase Assay; We drew-off human peripheral blood , abrupted and gathered the mononuclearcell with lympholeukocyte separating medium,cultured them with IFN-γ(1500 U/ml),anti-CD3(100 ng/ml),IL-1α(100 U/ml) and IL-2(1000 U/ml) and detected the phaenotype molecule of CIK cells such as: CD3, CD4, CD8 and CD56. After 14 days, we collected the CIK cells and kill the CD55hig cells and CD55low cells with effector cell vs target cell 40:1, the kill ratio were detected by with non-radioaction cytotoxic analytical reagent kite; 1 or 2 lymphonodes strictly with asepsis were taked in operation. Then it was separated single-nucleus cells(SNC) in albuginea rete. The SNC from lymphonodes were cultured in 10% FCS RPMI1640. After adherencing 2 hours, the attached cells were cultured with rhGM-CSF(1000U/ml), rhIL-4(200U/ml) and TNF-α(200U/ml) to be induced into DCs. The unattached cells were cultured with rhIL-2(200U/ml) induce into tumor draining lymph node cells(TDLNCs). CD55hig cells seperated and collected from MCF-7 cells were made for the breast cancer freeze-thawing antigen. DCs were stimulated by the cancer freeze-thawing antigen in order to load the tumor antigen, then, were co-cultured with TDLNCs to derivation tumor antigen specific CTLs. DCs suspension were harvested at the 1st day and the 7th day in vitro culturing and co-culturing with PE-CD1a,PE-CD83,FITC-CD86 MoAb, the cytophenotype was detected with flow cytometry(FCM). The cytotoxicity of the CTLs to CD55hig cells and CD55low cells were determined with non-radioaction cytotoxic analytical reagent kite, to analysis the function of differentiated DCs and specificity of the CTLs.Part three: The detection of stem-like cells in primary breast cancer tissue and its clinical significance.Chosen 45 Patients with breast cancer which came from the 1st surgery department in my hospital, from February in 2008 to January in 2009. Small amounts breast cancer tissue were taked in operation, Then it was separated single-nucleus cells (SNC) in albuginea rete. We joined in fluid of both immunofluorescence anti-CD55 and anti-CK19 to label the cells and detected the ratio of the high expression of CD55(CD55hig) by flow cytometry. Combined the clinical data of patients , we analyzed the experimental data. Results:1. After dyed by Hoechst33342,the percentage of SP cells contained in MCF-7 was 4.34±0.59%. The percentage of SP significantly degrated when blocked by verapamil(0.1±0.07%).2. The mean fluorescent intensity of SP and MP cells labeled with anti-CD55 monoclonal antibody was 100.85±4.57 and 50.51±4.75, respectively. Unsorted MCF-7 cells were labeled with anti-CD55 monoclonal antibody. The proportion of CD55hig cells was 2.12±0.39%.3. The adherence efficiency of CD55low cells at 6h, 12h, 18h and 24h were 40.87±1.55%, 55.40±2.46%, 85.10±3.36% and 96.3±0.96%, the adherence efficiency of CD55hig cells at 6h, 12h, 18h and 24h were 29.53±1.20%, 46.40±2.99%, 77.2±1.07% and 95.5±1.1%. The adherence efficiency of CD55low cells were higher than CD55hig cells at 6h, 12h and 18h. There was statistical difference between the two groups, (P<0.05). The adherence efficiency of CD55hig cells and CD55low cells at 24h didn't appear marked difference( P>0.05); the CD55hig cells were mostly spherica and CD55low cells were spindle after planted 12h.4. The Cloning efficiency of CD55hig cells at one week was 20.04±1.07%, which was lower than CD55low cells. There was statistical difference between the two groups(P<0.05).5. The percentage of G0/G1 in CD55hig cells was 85.4±3.37%, which was higher than CD55low cells(58.6±2.55%) and unsorted MCF-7 cells (70.73±4.21%), there was statistical difference between them, P<0.05. The percentage of S in CD55hig cells was 13.93±3.45%,which was lower than CD55low cells(40.36±2.56%) and unsorted MCF-7 cells (24.93±2.86%), there was statistical difference between them, P<0.05;6. The killing effect of chemotherapeutics on MCF-7 cells in different blood drug level were not same. In docetaxel group, the maximal blood drug level(10% blood drug level) killing MCF-7 cells was 57.3±4.75%. In epirubicin and fluorouracil group, the maximal blood drug level(100% blood drug level) killing MCF-7 cells was 40.2±2.46% and 26.6±2.44%. 7. The killing effect on CD55hig cells of docetaxel(10% blood drug level), epirubicin(100% blood drug level) and fluorouracil(100% blood drug level) were 28.5±0.04%, 18.4±0.02% and 12.4±0.01%, which were lower than on MCF-7 cells Obviorsly, there was statistical difference between them, P<0.05. The killing effect on CD55low cells of docetaxel(10% blood drug level), epirubicin(100% blood drug level) and fluorouracil(100% blood drug level) were 58.8±0.06%, 45.4±2.28% and 34.3±0.01%. There were no significant difference, P>0.05.8. The killing ratio on MCF-7 cells, CD55hig cells and CD55low cells of CIK cells were 39.15±3.30%, 42.72±4.36% and 46.41±4.67%. The killing ratio of CD55low cells group compared to MCF-7 cells group(P=0.000) and CD55hig cells group(P=0.046), there was statistical difference between them, P<0.05, there were significant difference. Compared the killing ratio of MCF-7 cells group with CD55hig cells group, P=0.053, there were no significant difference.9. The percentage of CD3+ and CD8+ T cells in TDLNCs were 73.93±2.18 and 32.78±3.21% before stimulation with tumor antigen. and were 82.67±2.79% and 62.54±2.51% after stimulation with rhIL-2 and Ag-DCs. The percentage of CD3+ and CD8+ T cell could be increased by the Ag-DCs. There was statistical difference between them, P<0.01. The percentage of CD4+ T cells in TDLNCs was 27.3±2.58% before stimulation with tumor antigen, however the percentage were 17.49±4.21% after stimulation. The percentage of CD4+ T cells wasn't heighten after induced by comparison, there were no significant difference, P>0.05.10. The killing ratio on MCF-7 cells, CD55hig cells and CD55low cells of special CTL cells were 52.86±4.45%,22.41±2.83% and 21.67±4.15%. The killing ratio of CD55hig cells group was higher than MCF-7 cells group and CD55low cells group, there was statistical difference between them, P<0.001; Compared MCF-7 cells group with CD55low cells group, P=0.629, there were no significant difference.11. The killing ratio of docetaxel(10% blood drug level), CIK cells and special CTL cells on CD55hig cells were 28.5±0.04%, 42.72±4.36% and 52.86±4.45%; Compared special CTL cells group with docetaxel(10% blood drug level) and CIK cells group, P=0.000. Compared docetaxel(10% blood drug level) and CIK cells group, P=0.000.12. The mean of the rate of CD55hig cells in primary breast cancer tissue was 0.21%, which was lower than the rate in breast cancer MCF-7 cells (2.12±0.39%).13. The ratio of CD55hig cells were increased with increased the number of the transferring axillary lymph nodes. There were statistically significance among non-metastatic axillary lymph node group,one to three axillary lymph nodes metastasis group and more than three axillary lymph nodes metastasis group, P =0.000.14. The ratio of CD55hig cells in C-erbB2(++~+++) group was 0.277±0.034%, higher than the ratio of CD55hig cells in C-erbB2(-~+) group the difference was statistical significant, P =0.000.15. There were no relationship between the ratio of CD55hig cells and pathology category, clinical stage, ER(-) and ER(+~+++), PR(-) and PR(+~+++), Ki-67(-) and Ki-67(+~+++), P>0.05.16. In the research, there was no relationship between the rate of CD55hig cells and preoperative chemotherapy,P =0.448. Conclusions:1. Hypochromatic sp cells are detect in human breast cancer line MCF-7 cells dyed by Hoechst33342. The percentage of SP cells obviously decreased when blocked by verapamil. SP cells contain cancer stem cells.2. The mean fluorescent intensity of SP is higher than MP cells when labeled with anti-CD55 monoclonal antibody. Unsorted MCF-7 cells are labeled with anti-CD55 monoclonal antibody by flow cytometer, we find a small amount of CD55hig cells.3. CD55hig cells are more nonadherent, quiensent and clonogenetic. So CD55hig cells have biological characteristics of cancer stem cells. 4. Three chemotherapeutics have certainly killin effect on MCF-7 cells, CD55low cells and CD55hig cells. But most CD55hig cells don't divide, the killing effect of chemotherapeutics on CD55hig cells is not significant.5. Although the killing effect of CIK cells on MCF-7 cells, CD55low cells and CD55hig cells are significant, which are higher than chemotherapeutics, the special killing effect are not significant.6. DCs induced by CD55hig cells freeze-thawing antigen could make TDLNCs generate to tumor antigen special CTLs, which have significant killing effect on CD55hig cells and non significant killing effect on other category tumor cells.7. Special CTLs have special killing effect on CD55hig cells, heigher than CIK cells and chemotherapeutics.This provids new method to cure breast cancer stem cells.8. The rate of CD55hig cells in breast cancer tissue is 0.21±0.20%.9. The rate of CD55hig cells in breast cancer tissue relates to the axillary lymph nodes metastasis and C-erbB2 protain expressions. There are no relationship to pathology category, clinical stage, expression of ER, PR and Ki-67 and preoperative chemotherapy. Those suppose that the rate of CD55hig cells relates to the metastasis and advancement of breast cancer.