The Anti-tumor Effect Of PA-MSHA In Breast Cancer Mouse Model And Its Impact On Peripheral Blood Treg Cell

Objective:To establish the high spontaneous metastasis breast cancer mouse model. To observe the effects on local and lung metastatic tumors of PA-MSHA and its impact on Treg cell in peripheral blood in breast cancer mouse model.Material and methods:We selected randomly 32 from 40 syngeneic BALB/c mice to establish the breast cancer model. Using the method of cell suspension injection,4T1 cells were injected into subcutaneous of right upper inguinal region of 32 BALB/c mice. The 7th day, the aimed model was established. Then we divided randomly them into four groups:A, B, C, D and given therapy as follows:Group A, named as untreated group,0.3ml normal sodium(NS) subcutaneous injection around tumors; Group B 0.1ml (20mg/kg) CTX intraperitoneal injection; Group C 0.3ml PA-MHSA subcutaneous injection around tumors; Group D 0.1ml (20mg/kg) CTX intraperitoneal injection and 0.3ml PA-MSHA subcutaneous injection around tumors. Every therapic way was given once per two days and five times in all. At the same time, the rest of eight healthy BALB/c mice were not given treatment, named as the normal control Group E. All the mice were raised under the same situation. We observed the growth of tumors every day until the 17th day. We selected randomly four mice from every group and acquired orbital venous blood by enucleateing eyeballs. We used the flow cytometry to detect the percentage of Treg cell in peripheral blood CD4+ lympholeukocyte as soon as the blood examples were acquired. Harvested tumors, lungs and spleens after they died, weighted tumors and calculated spleen index. We observed the metastatic situation in lungs with immuno-histochemistry method. We monitored the survival time of the remains tumor-bearing groups until they died.All data was analyzed by SPSS 13.0 for windows. Results:(1)After injected 4T1 cells suspension,32 BALB/c mice achieved tumors on the 7th day. The tumor formation rate is 100%.The range of tumor volume is from 21.10mm3 to 22.63mm3.We randomly divided into four groups, the average of tumor volumes in four groups are not significantly statistical different (P=0.999).(2) After different treatment methods, the average of tumor volumes in Group A, B, C, D are as follows:495.60mm3,293.87mm3,346.68 mm3,216.71mm. Group C is smaller than Group A (P<0.001) and bigger than Group D (P<0.05). It showed no statistical difference between Group C and Group B (P=0.160). After different treatment methods, the minimum of tumor weight is 0.15g, the maximum is 0.93g. Average of tumor weights in tumor-bearing mice were in order:Group A 0.78g,Group B 0.47g,Group C 0.51g,Group E 0.26g. Group C is lower than Group A (P<0.01) and higher than Group D(P<0.01), but is not statistically different with Group B (P=0.508).(3) After different treatment methods, the lung metastasis rate were: Group A 4/4, Group B 1/4, Group C 2/4, Group D 0/4, respectively. Group E was lower than Group A (P=0.014). It showed no statistical difference among others (P>0.05).(4) After different treatment methods, the median survival time in Group A, B, C, D were as follows:21.0 days,36.5 days,29.5 days,46.5 days. Group C is higher than Group A (P<0.05) and lower than Group D (P<0.01), but it considered to be no statistical difference with Group B (P=0.056).(5)The average of spleen index in every group are as follows:Group A 9.62 mg/g, Group B 14.67 mg/g, Group C 16.89 mg/g, Group D 27.57 mg/g, Group E 4.44 mg/g. Group C is higher than Group A (P=0.000) and lower than Group D (P=0.000), but is not statistically different with Group B(P=0.352).(6) The percentage of Treg cell in CD4+ lympholeukocyte in every group was as follows:Group A 5.83%, Group B 4.95%, Group C 4.92%, Group D 4.73%, Group E 4.20%. Group E is lower than others (all P<0.05). Group C is lower than Group A (P=0.000) and higher than Group E (P<0.05), and compare with Group B and Group D, the differences are considered to be not statistically significant (P=0.847, 0.304).The percentage is negatively related to the survival time in Pearson correlation analysis (r=-0.618, P=0.011).Conclusions:1. PA-MSHA could depress the growth of tumour, prolong the survival time in breast cancer mouse by subcutaneous injection around tumors. Especially, combined with the low-dose CTX, the efficacy is better.2. The percentage of Treg cell in peripheral blood CD4+ lymphocyte increases in 4T1 established high spontaneous metastasis breast cancer mouse model, and considered as a negative factor in the prognosis of breast cancer mice. PA-MSHA can depress increase of the percentage.