Animals.
A total of 40 female mice were selected, aged 6-8 weeks, with a body mass of 18-20 g, purchased from Hunan slack Jingda Experimental animal Co., Ltd. During the experiment, all raw materials, feed and drinking water were disinfected at super pressure-high temperature and dried in an oven at 180˚C. Mice were fed in a specific pathogen‑free (sPF) barrier system, had free access to food and water and were kept at 23‑25˚C, on a 12 h light/12 h dark cycle, and humidity 55±10%. The study was approved by the Ethics Committee of Xiangyang No. 1 People's Hospital (Xiangyang, China). The experimental procedures were in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.
Tumor model establishment.
Mouse tumor models were established. Mouse GC cells (MFC; cat. no. BNCC100207) in logarithmic growth phase were purchased from BeNa Culture Collection. Saline was used for dilution to prepare the cell suspension, the concentration of which was adjusted to ~1010/l. The left abdomen skin of the mice was disinfected with iodophor, and 0.2 ml of cell suspension was inoculated in mice 13. The needle was kept as oblique as possible during the operation. After experimental inoculation, mice were immediately returned to their original place to continue feeding, if their behavioral state was normal. Tumors formed after ~1 week (the tumor nodule was approximately 8-10 mm in diameter). Then, the growth of xenograft tumors in mice was examined on the 2nd day. The behavioral changes of the mice during the experiment were observed and recorded, mainly in terms of activity, body mass, mental state, eating and drinking, with timely measures taken. Vernier caliper was used for measuring the long and short diameters of xenograft tumors, and the tumor volumes were calculated.
Experimental grouping and treatment measures.
When the xenograft tumor volume in mice reached 8-10 mm3, the 40 tumor-bearing mice were randomly divided into control group, endostar group, radiotherapy group, and endostar combined with radiotherapy group (referred to as combination group); 10 mice in each group. There was no statistically significant difference in the body mass of mice among the groups before the experiment (p>0.05). i) Control group: saline (0.2 ml/day) was injected through the vena caudalis for 14 consecutive days. ii) Endostar group: endostar (Shandong Xiansheng Maidejin biological Pharmaceutical Co., Ltd.; sFDa approval no. s20050088) of 5 mg/kg was injected through the vena caudalis, with each ~0.2 ml, for 14 consecu- tive days. iii) Radiotherapy group: saline (0.2 ml/day) was injected through the vena caudalis for 14 consecutive days. On the 7th day of the experiment, 1% chloral hydrate (Qingdao Yulong Seaweed Co., Ltd.; SFDA approval no. H37022673) was used for anesthesia in mice, who were fixed in appropriate body position. At the same time, mice were covered with tissue equivalent materials (~1 cm in thickness), and irradiated with 6 MV X-rays. The total dose of radiotherapy in the experiment was 10 Gy, and the source-skin distance was 100 cm, for 1 day of radiotherapy. iv) Combination group: endostar (5 mg/kg) was injected through the vena caudalis for 14 consecutive days, ~0.2 ml/mouse. Radiotherapy was given on the 7th day of administration, with the same specific treatment measures as those in the radiotherapy group.
At the same time, the growth of xenograft tumors and behavioral changes of mice in each group were observed. Mice were sacrificed by cervical dislocation at 24 h after the last experimental operation. Their tumor tissues were removed, and weighed with an electronic balance after washing with saline. Filter paper was used to absorb moisture. Then, these tumor tissues were numbered and recorded, and stored in a freezer at ‑80˚C.
Observation indicators.
i) The growth of xenograft tumors in each group of mice was observed. ii) The behavioral state, such as mental state, eating, drinking, activity and body weight, was The vernier caliper was used for measuring (a) the long diameter and (b) short diameter of the xenograft tumors every 3 days and the values were recorded. The formula V = ab2/2 was used for calculating the tumor volume, and the average value was also calculated for plotting the tumor growth curve of mice in each group. iii) The tumor mass was weighed with an electronic balance and the related formula was used to obtain the tumor growth inhibition rate (IR). Formula: IR = [1 - average tumor mass in experimental group (endostar group, radiotherapy group, or combination group)/average tumor mass in blank control group] x100%.
Detection of hematology and serum protein levels.
Blood was taken by eyeball removal method at 24 h after the last administration. The leucocyte count, erythrocyte count, platelet count, and expression levels of serum total protein and serum albumin in each group were detected.
Detection of TGF‑β1 and IL‑10 expression in the supernatant of tumor tissues. Enzyme-linked immunosorbent assay (ELISA) was used to determine the serum TGF-β1 and IL-10 levels. TGF-β1 and IL-10 kits were provided by Moshake Biotechnology Co., Ltd. (item nos. 69-36961 and 69-99847, respectively). The instrument was BS-1101 micro- plate reader from Beijing Linmao Technology Co., Ltd. All operations were strictly in accordance with the manufacturer's instructions.
Statistical analysis.
sPss 17.0 statistical software (Tianjin Network Technology Co., Ltd.) was used to statistically analyze the experimental data. Measurement data were expressed as mean ± sD. The analysis of variance of two‑factor factorial design was used for the comparison of the expression levels of TGF-β1 and IL-10 among multiple groups, one-way analysis of variance for the comparison of the measurement data of hematology and serum protein level among multiple groups and LSD-t test was the post hoc test. t-test was used for the comparison of body weight before and after treatment in the same group. p<0.05 was considered to indicate a statistically significant difference.