Yannelli J R, Hyatt C, Johnson S, Hwu P, Rosenberg S A
Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
J Immunol Methods. 1993 May 5;161(1):77-90. doi: 10.1016/0022-1759(93)90199-h.
Tumor cell lines were generated from cancer patients, 17 with metastatic melanoma and one with colon adenocarcinoma. The lines were characterized as tumor cells by the presence of tumor associated antigens demonstrated by indirect immunofluorescence and analysing using a fluorescence activated cell sorter (FACS). The tumor cell lines were transduced using retroviruses encoding neomycin phosphotransferase and either human tumor necrosis factor alpha (TNF-alpha) or interleukin-2 (IL-2). Following transduction, cells were selected and grown in the neomycin analogue G418. Fibroblasts overgrew tumor cells in 6/18 cases following selection in G418 and 1/18 lines did not grow at all after selection. In the remaining 11 lines the expression of tumor associated antigens, growth, and susceptibility to lysis by LAK cells was similar between the selected transduced tumor cell lines and the nontransduced controls. Of the lines tested, all were positive for the presence of the cytokine gene by Southern blot or PCR analysis. In addition, no replication competent retrovirus was detected in the cell lines following transduction using an extended mink S+L- focus assay. The amount of specific cytokine produced per 10(5) transduced tumor cells in 24 h ranged from 0.2 ng to 5.8 ng of TNF-alpha for the TNF transduced lines and from 0.1 ng to 3.6 ng of IL-2 for the IL-2 transduced tumor cell lines. One transduced tumor cell line examined maintained consistent levels of cytokine production when studied at 15 different time intervals over a period of 198 days. Additionally, 40,000 rads of gamma irradiation did not stop cytokine production from two transduced tumor cell lines when studied over 6 days. This study demonstrates the feasibility of growing human tumor cell lines from surgical biopsies and genetically modifying those lines to produce a cytokine of choice for possible use as a tumor cell vaccine.
肿瘤细胞系源自癌症患者，其中17例为转移性黑色素瘤患者，1例为结肠腺癌患者。通过间接免疫荧光检测并使用荧光激活细胞分选仪（FACS）分析，证实这些细胞系存在肿瘤相关抗原，从而将其鉴定为肿瘤细胞。使用编码新霉素磷酸转移酶以及人肿瘤坏死因子α（TNF-α）或白细胞介素-2（IL-2）的逆转录病毒转导肿瘤细胞系。转导后，细胞在新霉素类似物G418中进行筛选和培养。在G418筛选后，6/18的病例中，成纤维细胞过度生长超过了肿瘤细胞，1/18的细胞系在筛选后根本无法生长。在其余11个细胞系中，所选转导肿瘤细胞系与未转导的对照相比，肿瘤相关抗原的表达、生长情况以及对LAK细胞裂解的敏感性相似。在检测的细胞系中，通过Southern印迹或PCR分析，所有细胞系的细胞因子基因均呈阳性。此外，使用扩展的水貂S + L-焦点测定法，在转导后的细胞系中未检测到具有复制能力的逆转录病毒。对于TNF转导的细胞系，每10(5)个转导肿瘤细胞在24小时内产生的特异性细胞因子量为0.2 ng至5.8 ng的TNF-α，对于IL-2转导的肿瘤细胞系，为0.1 ng至3.6 ng的IL-2。在198天内的15个不同时间间隔进行研究时，所检测的一个转导肿瘤细胞系维持了细胞因子产生的一致水平。此外，在6天的研究中，40,000拉德的γ射线照射并未阻止两个转导肿瘤细胞系产生细胞因子。这项研究证明了从手术活检中培养人肿瘤细胞系并对这些细胞系进行基因改造以产生一种可供选择的细胞因子，用于可能作为肿瘤细胞疫苗的可行性。
