Dessureault S, Graham F, Gallinger S
Department of Surgery, Mount Sinai Hospital, University of Toronto, Ontario, Canada.
Ann Surg Oncol. 1996 May;3(3):317-24. doi: 10.1007/BF02306289.
Transfection of the costimulatory molecule B7-1 into some murine tumors can increase antitumor immunity and eradicate tumor growth. The purpose of this work was to construct an adenovirus-B7 (Ad-B7) expression vector and study B7-1 gene transfer into human cancer cells.
The human B7-1 cDNA was ligated into an expression cassette containing the human cytomegalovirus immediate early gene promoter and then inserted into the E1 region of the Ad5 genome by homologous recombination. The resulting Ad-B7 vector was used to infect established cancer cell lines and freshly resected cancers. Resected tumors were disaggregated into single cell suspensions by mechanical mincing and enzymatic digestion. Surface expression of B7-1 after infection was verified by flow cytometry.
Expression kinetics in three cell lines showed that infected cells began to express B7-1 within 24 h. The proportion of B7-1+ cells continued to increase during the next 48 h, after which expression remained relatively constant during the next 5 days (up to 98% B7-1+ cells). Fresh tumor cells from various cancers displayed similar kinetics, but with greater variability in the proportion of cells expressing B7-1 (13% to 95% B7-1+ cells). Cancers which were successfully infected included 3 colorectal adenocarcinomas, 2 leiomyosarcomas, 2 lung squamous cell carcinomas, and 1 renal cell carcinoma.
The Ad-B7 vector is a rapid and efficient means of gene transfer which does not require host cell proliferation. The ultimate objective is to engineer autologous tumors to express B7-1 and vaccinate cancer patients in an adjuvant or palliative setting.
将共刺激分子B7-1转染至某些小鼠肿瘤中可增强抗肿瘤免疫力并消除肿瘤生长。本研究旨在构建腺病毒-B7（Ad-B7）表达载体，并研究B7-1基因向人癌细胞的转移。
将人B7-1 cDNA连接至包含人巨细胞病毒立即早期基因启动子的表达盒中，然后通过同源重组插入Ad5基因组的E1区域。所得的Ad-B7载体用于感染已建立的癌细胞系和新鲜切除的癌症组织。通过机械切碎和酶消化将切除的肿瘤组织解离成单细胞悬液。通过流式细胞术验证感染后B7-1的表面表达。
在三种细胞系中的表达动力学显示，感染的细胞在24小时内开始表达B7-1。在接下来的48小时内，B7-1+细胞的比例持续增加，之后在接下来的5天内表达保持相对恒定（高达98%的B7-1+细胞）。来自各种癌症的新鲜肿瘤细胞表现出相似的动力学，但表达B7-1的细胞比例变化更大（13%至95%的B7-1+细胞）。成功感染的癌症包括3例结直肠腺癌、2例平滑肌肉瘤、2例肺鳞状细胞癌和1例肾细胞癌。
Ad-B7载体是一种快速有效的基因转移手段，不需要宿主细胞增殖。最终目标是改造自体肿瘤以表达B7-1，并在辅助或姑息治疗环境中为癌症患者接种疫苗。
