Yang Xiu-li, Chu Yi-wei, Xu Lin, Li Bao-hua, Xiong Si-dong
Department of Immunology, Shanghai Medical College, Fudan University, Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai 200032, China.
Zhonghua Yi Xue Za Zhi. 2006 Jan 17;86(3):176-81.
To explore the inhibitory effects of IP10 on the experimental tumor metastases of a mammary carcinoma cell line 4T1 in vivo.
4T1 cells were transfected with pcDNA3-IP10 plasmid and the positive clones (IP10-4T1) were screened in the presence of G418. The parental 4T1 cells and 4T1 cells transfected with pcDNA3 (pcDNA3-4T1) were used as controls. The expression of IP10 mRNA was examined with RT-PCR. The chemotactic activity of the cell-cultured supernatant for the activated lymphocytes was assessed with chemotaxis assay. All BALB/c mice were divided into 3 equal groups: IP10-4T1, parental 4T1 and pcDNA3-4T1 (6 mice in each group). Seven days after mice were inoculated with 2 x 10(6) 4T1, pcDNA3-4T1 or IP10-4T1 cells, 1 x 10(5) 4T1 cells were injected into mice in tail vein. On day 14 tumors were sectioned. On day 28 mice were sacrificed and the lung weight, metastatic forci on the lung surface, disseminated metastases in the lungs and the number of clonogenic metastases of 4T1 cells were observed. The splenocytes were isolated from tumor-bearing mice, and the cytotoxicity of the splenocytes was evaluated by CFSE/7-AAD method.
The transcription of IP10 mRNA increased in IP10-4T1 cells compared to parental 4T1 and pcDNA3-4T1 cells (P = 0.002). Moreover, accumulated lymphocytes migrated to the supernatants of IP10-4T1 cells, which can be abrogated by anti-CXCR3 (P < 0.05). Compared to controls, inoculation of IP10-4T1 cells resulted in the decreased disseminated metastases as indicated by dramatically reduced lung metastatic forci on the surface of the lungs; the lung weight was lighter (IP10-4T1, 0.27 +/- 0.02 g v.s. 4T1, 0.48 +/- 0.08 g and pcDNA3-4T1, 0.43 +/- 0.16 g, P = 0.021); the number of clonogenic metastatic 4T1 cells enumerated in ten fields decreased greatly (IP10-4T1, 2.6 +/- 1.7 v.s. 4T1, 34 +/- 6.3 and pcDNA3-4T1, 33 +/- 2.3, P < 0.05); histological assay showed that metastasis was not found in the lungs of the 4/6 of mice in IP10-4T1 group, and was seen in all the mice of parental 4T1 and pcDNA3-4T1 groups. The splenocytes from the mice inoculated with IP10-4T1 cells exhibited stronger cytotoxic activity against 4T1 target cells at different ratios of effector versus target cells (3:1, 9:1, 27:1) than controls (P < 0.05).
IP10 expressed locally could inhibit disseminated metastasis of circulating 4T1 tumor cells by enhancing anti-tumor cellular immune responses.
探讨IP10对小鼠乳腺癌细胞系4T1实验性肿瘤转移的抑制作用。
用pcDNA3 - IP10质粒转染4T1细胞，在G418存在下筛选阳性克隆（IP10 - 4T1）。以亲代4T1细胞和转染pcDNA3的4T1细胞（pcDNA3 - 4T1）作为对照。用RT - PCR检测IP10 mRNA的表达。用趋化试验评估细胞培养上清液对活化淋巴细胞的趋化活性。将所有BALB/c小鼠分为3组，每组6只：IP10 - 4T1组、亲代4T1组和pcDNA3 - 4T1组。给小鼠接种2×10⁶个4T1、pcDNA3 - 4T1或IP10 - 4T1细胞7天后，经尾静脉给小鼠注射1×10⁵个4T1细胞。第14天取肿瘤组织切片。第28天处死小鼠，观察肺重量、肺表面转移灶、肺内播散性转移灶及4T1细胞克隆形成性转移灶的数量。从荷瘤小鼠中分离脾细胞，用CFSE/7 - AAD法评估脾细胞的细胞毒性。
与亲代4T1细胞和pcDNA3 - 4T1细胞相比，IP10 - 4T1细胞中IP10 mRNA的转录增加（P = 0.002）。此外，积累的淋巴细胞迁移至IP10 - 4T1细胞的上清液，抗CXCR3可消除这种迁移（P < 0.05）。与对照组相比，接种IP10 - 4T1细胞导致肺表面转移灶减少，肺重量减轻（IP10 - 4T1组为0.27±0.02 g，4T1组为0.48±0.08 g，pcDNA3 - 4T1组为0.43±0.16 g，P = 0.021）；十个视野中计数的4T1细胞克隆形成性转移灶数量大幅减少（IP10 - 4T1组为2.6±1.7，4T1组为34±6.3，pcDNA3 - 4T1组为33±2.3，P < 0.05）；组织学检测显示，IP10 - 4T1组6只小鼠中有4只肺内未发现转移，而亲代4T1组和pcDNA3 - 4T1组所有小鼠肺内均有转移。在不同效应细胞与靶细胞比例（3:1、9:1、27:1）下，接种IP10 - 4T1细胞小鼠的脾细胞对4T1靶细胞的细胞毒性活性比对照组更强（P < 0.05）。
局部表达的IP10可通过增强抗肿瘤细胞免疫反应抑制循环中4T1肿瘤细胞的播散性转移。
