| Breast carcinoma is one of the most serious malignancies among females. The current treatment include surgery, radiotherapy, chemotherapy, endocrine therapy and biological treatment, but these method are not satisfactory. To eradicate breast cancer, eradicate cancer cells from the origin should be most effective way. The theory of cancer stem cell (CSC) gives hope for the complete curing breast cancer. CSC is a group of cells which have self-renewing and non-directional differentiation potential, it is the root of tumor continued growth; it is also the "starting cell" and "motivation cell" for tumorigenesis, tumor proliferation, recurrence, etc. CSC is not only a thoroughly therapy way for breast cancer, but also has closely relation with the prognosis of breast cancer. This study first explores the values of the breast cancer CSC new markers ALDH1 and CD55 expression in breast cancer prognosis, then use the serum-free suspension mammosphere culture and chemotherapy way to enrich breast cancer CSC from 4T1 cell lines in vitro and in vivo respectively. The cell populations of CSC and mouse models of CSC were getted. At last we use Parthenolide (PTL) to targeting CSC in vitro and in vivo; it will provide the foundation for the treatment of breast cancer with PTL.This research is divided into three parts.Part 1 Prognostic significance of ALDH1 and CD55 expression in breast cancerObjective: To evaluate the relationship of new breast cancer CSC marker CD55 or ALDH1 expression in breast cancer with the diseases characteristics such as histological type, grading, etc. Identify the prognosis value of ALDH1 and CD55 for breast cancer.Methods: ALDH1 and CD55 expression in tumor tissue of 65 breast cancer patients which followed up 5 to 8 years was detected with immunohistochemical staining. ALDH1 expressed in the cytoplasm, ALDH1 positive cells showed brown-yellow, negative cells were blue-purple. Select three horizons of a representative slice, all horizons which have ALDH1 positive cells were sentenced as ALDH1 positive specimens. CD55 expressed in the cytoplasm and membrane, strong positive expression cells were brown, weakly positive expression cells were weak yellow. CD55 high expression samples were strong positive cells≥5%. According to the above criteria, determine ALDH1 expression and CD55 high expression samples, analysis the relationship of the clinical disease characteristics with CD55 high expression and ALDH1 expression. Calculated 5-year survival of patients, analysis the relevance of 5-year survival rate with CD55 high expression, ALDH1 expression and clinical disease characteristics, then use Cox proportional analysis model for single-factor analysis of breast cancer patients prognosis, on the basis of single-factor analysis use the multi-factor non-conditional Logistic regression analysis make further filter for the independent risk factors of breast cancer prognosis.Results:1 In normal breast tissue, expression of ALDH1 was negative or very weak positive, the expression rate of ALDH1 in breast cancer tissue specimens was 24.6% (16/65); expression of CD55 is low in normal breast tissue, in breast cancer tissue, high expression of CD55 specimens accounted for 33.8% (22/65).2 Expression of ALDH1 and high expression of CD55 are closely related to recurrence of the tumor(P<0.01), but they are not related with other clinical disease characteristics, such as postmenopausal status, tumor histological type, histological grade, tumor size, lymph node metastasis status, estrogen receptor and progesterone receptor expression, her2 expression, accept other forms of treatment (P>0.05).3 5-year survival rate of patients which expression of ALDH1, high expression of CD55 was significantly decreased when compared with patients which ALDH1 negative, CD55 low expression (P<0.001).4 Expression of ALDH1, high expression of CD55, tumor diameter, histological grade, her2 negative expression, patients who did not receive treatment, they are related with 5-year survival rate of breast cancer patients, they are independent factors of breast cancer prognosis.Conclusion: Expression of ALDH1 and high expression of CD55 are both closely related to recurrence of the tumor and are not related with other clinical disease characteristics of breast cancer patients. They are independent prognostic factors of breast cancer patients.Part 2 CSC enrich of mouse breast cancer cell line 4T1 and the animal model which enriched with CSC was establishedObjective: Use serum-free suspension mammosphere culture and chemotherapy way to enrich breast cancer CSC from 4T1 cell lines in vitro and in vivo respectively, from that cell populations and mouse model which enriched with breast cancer CSC were getted. This foundation can use for anti-breast cancer CSC therapy.Methods:1 Mammosphere culture: 4T1 cells were inoculated in serum-free medium(SFM) with cell density of 2×104/ml, change medium with centrifuge after 3 days, when the cell cultured to mammosphere with 100 to 200 cells, digestion with diluted trypsin-EDTA and passage, about 6 to 7 days passage one generation.2 The establish of mouse model which enriched with breast cancer CSC: 0.2ml 4T1 cell suspension (1.5×106 cells) inoculated in the right shoulder of mouse subcutaneously. Observe the formation of tumor , when the tumor grows about 0.1cm, give the mouse 0.2mg/ml 5-FU 0.2ml intraperitoneally once a day. Seven days later, the administration way was changed to once a week. Four weeks later the mouse were sacrificed. Sterile removal mouse tumor, the tumor was grinded with organize grinders and make cell suspension, adjust the living cell concentration to 7.5×106/ml with PBS. Repeat the above procedure make the second generation mouse model, four generations of mouse model were making at last.3 CSC detection: CD44+CD24-/low cell content was detected with flow cytometry, non-fluorescent dye staining side population(SP) cell ratio were counted after cells stained with HOECHST33342. MDR1, BCRP, ALDH1 mRNA expression in enriched CSC cells were detected with fluorescence quantitative PCR. CD55 and ALDH1 protein expression in tumor tissue were detected with immunohistochemistry. The tumorigenicity of the enriched CSC was detected with tumorigenicity test in mouse.Results:1 4T1 cells in SFM can form continuous passage mammosphere, In 10th generation mammosphere, the content of CD44+CD24-/low cell was 68.9±3.78%, SP cell ratio was 56.1±5.18%, MDR1, BCRP, and ALDH1 mRNA expression was compared with the control cells 4T1 cultured in SSM raised for 1.63±0.21 fold, 2.14±0.21 fold, 3.41±0.53 fold respectively. Tumorigenic test showed that 2×103 mammosphere cells can form tumor in mice, whereas the control 4T1 cells cultured in SSM requires at least 2×105 cells to form tumor.2 Mouse model enriched with breast cancer CSC has established with 5-Fu. In the third generation of mouse tumor, CD44+CD24-/low cell ratio was 69.0±1.6%, SP cell ratio was 61.3±2.6%, MDR1, BCRP, and ALDH1 mRNA expression was raised by 4.35±0.21 fold 6.14±0.47 fold, 3.78±0.32 fold, compared with the control of the tumor tissue(P <0.01). ALDH1 expression was positive, CD55 strong positive cell ratio was 17.3±1.9%, they express much stronger than the control tumor tissue. Tumorigenicity test showed that 2×103 mouse model cells can form tumor, whereas the control tumor requires at least 2×105 cells to form tumor.Conclusion: The CSC induced for 4T1 cell line with serum-free suspension mammosphere culture in vitro, the mouse model which enriched CSC with chemotherapy drugs was established. Through a number of target detection and tumorigenicity test we confirmed that the way we used in this study was successful for the enrich of breast cancer CSC. This study provides basis for further research target killing CSC.Part 3 Research for Parthenolide activity of mouse breast cancer CSCObjective: To investigate the effects of Parthenolide(PTL) to breast cancer CSC in vivo and in vitro, provide basis for the treatment of breast cancer with PTL on clinical.Method:1 Effect of PTL to kill CSC from 4T1 cell lines: the 10th generation of mammosphere cells inoculated in 25ml flasks with the cell concentration 5×104/mL in SFM culture medium, 25 bottles cells were randomly divided into control group, 5-FU group, PTL group, 5-FU+PTL group, PTL+NAC(N-acetyl cysteine)Group,altogether 5 groups. Control group adds normal saline 20μl, 5-FU group adds 0.1mg/ml 5-FU20μl, PTL group adds 1mg/ml PTL 20μl, 5-FU+PTL group adds 0.2mg/ml 5-FU 10μl and 2mg/ml PTL 10μl, PTL+NAC group adds 2mg/ml PTL 10μl and 20mg/ml NAC 10μl. After culture 48 hours, centrifuged and changed to SFM for continued culture. After 7 days, the cells were observed for the mammosphere forming and then cells were collected for further testing.2 Experiment of PTL kill CSC in mouse model: 25 female Balb/c mice were randomly divided into control group, 5-FU group, PTL group, 5-FU+PTL group, PTL+NAC group. Sterile removal mouse tumor of the third generation mouse model(Part 2, 2.1), the tumor was grinded with organize grinders and make cell suspension. Absorb 0.2ml cell suspension (containing 1.5×106 cells) were inoculated subcutaneously. 24 hours after inoculation, control group mice were given normal saline 0.4ml, intra-abdominal 0.2ml and the tumor cell inoculation site 0.2ml respectively. 5-FU group mice were given 0.2mg/ml of 5-FU 0.4ml, PTL group were given 1mg/ml of PTL 0.4ml, 5-FU+PTL group were given 0.2mg/ml 5-FU and 1mg/ml PTL mixture 0.4ml, PTL+NAC group were treated with 1mg/ml PTL and 10mg/ml NAC mixture 0.4ml, the drugs given site and dose were identified with control group. Time for given drugs were once a day, sharing seven days, seven days later changed to once a week. Four weeks later, the mice were sacrificed, tumors were taken for testing.3 The residual CSC detection after PTL killed: CD44+CD24-/low cell content was detected with flow cytometry, non-fluorescent dye staining side population(SP) cells ratio were counted after cells stained with HOECHST33342, MDR1, BCRP, ALDH1 mRNA expression were detected with fluorescence quantitative PCR. CD55 and ALDH1 protein expression in tumor tissue were detected with immunohistochemistry.Results:1 PTL killed CSC from 4T1 cell lines in vitro: After 5-FU, PTL, 5-FU+PTL or PTL+NAC treatment and cultured in SFM, control group, 5-FU group, PTL+NAC group can form mammospheres clearly, PTL group formed only fewer mammospheres in SFM, there was only scattered mammospheres existence, 5-FU+PTL group live cells were too little, the cells were lysised due to apoptosis. The CD44+CD24-/low cell contents of control group, 5-FU group, PTL group, PTL+NAC group were 71.2±2.3%, 75.6±3.1%, 12.3±1.9%, 58.1±2.6%, respectively; non-fluorescent staining(SP) cells percentages were 56.7±3.4%, 62.0±2.7%, 8.1±1.1%, 51.5±2.3%, respectively, PTL group was significantly lower than the control group, 5-FU group and PTL+NAC group(P<0.01).2 Animal experiment for PTL killed CSC in mouse model: After 5-FU, PTL, 5-FU+PTL or PTL+NAC treatment, the velocity and diameter of the tumor forming in control group and 5-FU group are much larger than PTL group, 5-FU+PTL group, PTL+NAC group(P<0.01). The CD44+CD24-/low cell contents in tumor of control group, 5-FU group, PTL group, 5-FU+PTL group, PTL+NAC group were 57.3±4.1%, 68.7±3.2%, 42.5±3.7%, 39.1±2.9%, 50.1±3.1%, respectively; non-staining(SP) cell percentages were 58.1±4.1%, 61.3±2.6%, 36.9±3.5%, 39.2±1.8%, 42.7±2.5%, respectively. Both CD44+CD24-/low cell contents and SP cell percentages, PTL group, 5-FU+PTL group, PTL+NAC group were significantly lower than control group, 5-FU group (P<0.05). MDR1 gene expression in tumor tissue for 5-FU group, PTL group, 5-FU+PTL group, PTL + NAC group compared with control group was 1.11±0.11 fold, 0.92±0.12 fold, 1.03±0.15 fold, 0.94±0.08 fold, all the groups have no significant difference(P>0.05); BCRP gene expression in tumor tissue for 5-FU group, PTL group, 5-FU+PTL group, PTL + NAC group compared with control group was 1.09±0.16 fold, 0.97±0.10 fold, 1.13±0.11 fold, 0.95±0.13 fold, all the groups have no significant difference (P>0.05). ALDH1 gene expression in tumor tissue for 5-FU group, PTL group, 5-FU+PTL group, PTL + NAC group compared with control group was 1.16±0.12 fold, 0.38±0.07 fold, 0.42±0.09 fold, 0.57±0.04 fold. PTL group, 5-FU+PTL group, PTL+NAC group were significantly lower than control group and 5-FU group (P<0.05).Conclusions: PTL could target to breast cancer CSC, change the state of CSC, so CSC contents in the cultured mamosphere cells or tumor tissue significantly reduced. The roles of PTL targeting CSC is mainly due to oxidative stress. Anti-oxidant drug NAC can clearly antagonistic the effect in vitro, but in mice it is not obvious. Combination with other chemotherapy drugs PTL may enhance the effects of other chemotherapy drugs; PTL could be used as a chemotherapy sensitizer.
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