| OBJECTIVEOur previous studies revealed that after high intensity focused ultrasound (HIFU) treatment for tumors, peripheral blood lymphocytes presented obvious anti-tumor immunity against early-stage homogeneous tumors in mice. This study was designed to investigate anti-tumor effects of HIFU-activated lymphocytes on middle- and advanced-stage homogeneous tumors, and to provide laboratory evidences regarding the adoptive immunotherapy of HIFU-activated lymphocytes in mice bearing homogeneous tumor.MATERIAL AND METHODS1. Establishment of middle- and advanced-stage tumor models with implanted H22 hepatic carcinoma in mice.A total of 180 normal C57/6J mice received H22 hepatic carcinoma implantation using the hypodermic injection of 0.05 ml H22 tumor cells suspension(6×107个/ml)in the right back of each mouse. All mice were followed up to observe tumor incidence, body weight and tumor volume on the 5th, 7th, 10th, 14th, 18th, 21st, 25th and 30th day of tumor implantation respectively. At each measurement day 20 mice were killed with standard decapitation, and pathological examination was performed to observe histological changes in the implanted tumors, and tumor local invasion and metastasis.2. Anti-tumor effects of HIFU-activated lymphocytes on middle- and advanced-stage homogeneous tumor in miceFifty normal C57/6J mice were randomly divided into following five groups:①blank group, in which normal saline was injected via the caudal vein in 10 mice on the 11th and 12th day of tumor implantation;②control group, in which lymphocytes, purified from the peripheral blood of normal mice, were injected via the caudal vein in 10 mice on the 11th and 12th day of tumor implantation;③early-stage HIFU group, in which HIFU-activated lymphocytes, purified from the peripheral blood of tumor-bearing mice after HIFU treatment, were injected via the caudal vein in 10 mice on the 3rd and 4th day of tumor implantation;④middle-stage HIFU group, in which the activated lymphocytes, purified from the peripheral blood of tumor-bearing mice after HIFU treatment, were injected via the caudal vein in 10 mice on the 11th and 12th day of tumor implantation;⑤advanced-stage HIFU group, in which the activated lymphocytes, purified from the peripheral blood of tumor-bearing mice after HIFU treatment, were injected via the caudal vein in 10 mice on the 21st and 22nd day of tumor implantation. The mice in the HIFU groups received a 0.05ml lymphocyte suspension (2×107/ml) via the caudal vein. Those in the blank group and control group received either same amount of normal saline or normal lymphocytes. The injection was once a day, for 2 days without any interval. All mice in each group were followed up to observe tumor incidence, tumor extinction rate, tumor growth curve, survival rate, and survival curve respectively.RESULTS1. Establishment of middle- and advanced-stage tumor models with implanted H22 hepatic carcinoma in mice.On the 5th day of tumor implantation, tumor mass was macroscopically observed in the local region of mouse back, and total tumor incidence was 88.9%. Five days after tumor implantation, body weight increased continuously in mice, and on the 10th , 21st and 30th day of tumor implantation, the body weights were 28.56±1.17g,31.39±2.45g and 37.58±1.58g respectively. Tumor volume measured grossly were 156.09±54.40mm3, 619.62±179.21mm3, 2288.93±525.42mm3 and 5391.48±1972.31mm3 on the 5th, 10th, 21st and 30th day of tumor implantation respectively. Pathological examination revealed that a new-growth tumor mass was observed at the local site of mouse back on the 5th day of tumor implantation. The tumor started to invade surrounding muscle tissue, blood vessels and nerves on the 10th day of tumor implantation. A few bloody ascites, which was caused by peritoneal metastasis, were observed in mice on the 21st day, and the amount of bloody ascites was obviously increased on the 30th day of tumor implantation, with inguinal lymph node metastasis.2. Anti-tumor effects of HIFU-activated lymphocytes on middle- and advanced-stage homogeneous tumor in mice①On the 5 rd and 10 rd day of tumor implantation, the incidences of tumor were observed in 100% of mice in all groups.②Tumor growth was slower in the early- and middle-stage HIFU groups, and average tumor volume was significantly lower in the early- and middle-stage HIFU groups than those in the blank, control and advanced-stage groups; While there was no statistical difference of average tumor volume among the blank, control and advanced-stage HIFU groups. Compared to the middle-stage HIFU group, average tumor volume was significantly higher in the early-stage HIFU group on the 30th of tumor implantation, but no statistical differences were detected on the 40th and 50th day.③Tumor extinction rates on the 70th day of tumor implantation were 80% of mice in the early-stage HIFU group, 10% in the middle-stage HIFU group, and 0% in the advanced-stage HIFU, blank and control groups, respectively. Compared to the blank and control groups, tumor extinction rate in the early-stage HIFU group was significantly higher, and statistical differences were observed (X2= 16.9124,P=0.0001). There was a significant difference of tumor extinction rate between the early- and middle-stage HIFU groups (X2=11.0158 , P=0.0009). However, no significant differences were observed among the middle- and advanced-stage HIFU groups, the blank and control groups in tumor extinction rate respectively (X2=1.4390, P=0.2303).④Seventy-day survival rates were 80% of mice in the early-stage HIFU group, 50% in the middle-stage HIFU group, 0% in the blank, control and advanced-stage HIFU groups respectively. Compared to the blank, control and advanced-stage HIFU groups, 70-day survival rate was significantly higher in the early-stage HIFU group (X2=16.9124, P=0.0001), and in the middle-stage HIFU group (X2=8.6305,P=0.0033); while no statistical difference was observed in 70-day survival rate between the early- and middle-stage HIFU groups (X2= 2.0269, P=0.1545). There were no significant differences of the survival rates among the advanced-stage HIFU, blank and control groups (P=1).CONCLUSIONS1. Mice bearing H22 hepatic carcinoma on the 5th– 7th day of tumor implantation can be regarded as an early-stage tumor model; while mice bearing H22 tumor can be an animal model for middle-stage tumor on the 10th– 18th day, and an advanced-stage animal tumor model on the 21st– 30th of tumor implantation, respectively.2. Peripheral blood lymphocytes of mice bearing tumor were activated after HIFU treatment for tumor. They can present obvious immunotherapeutic effect on the early-stage homogeneous tumor in mice. However, this therapeutic effect decreases in mice bearing middle-stage homogeneous tumor, and disappears in the mice bearing advanced-stage tumor.
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