Parney Ian F, Farr-Jones Maxine A, Koshal Anu, Chang Lung-Ji, Petruk Kenneth C
Division of Neurosurgery, Department of Surgery, University of Alberta, Edmonton, Alberta, Canada.
Neurosurgery. 2002 May;50(5):1094-102. doi: 10.1097/00006123-200205000-00027.
Immunogene therapy is a novel cancer treatment strategy based on vaccination with irradiated autologous tumor cells transduced with immunostimulatory genes. To characterize such cells before clinical applications, we studied a human glioma cell line (D54 MG) and early passage human glioma (Ed147.BT, Ed149.BT) and melanoma (Ed141.MEL) cultures after immunostimulatory gene transfer.
Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-12 (IL-12), and B7-2 genes were retrovirally transferred to tumor cells. Gene expression before and after irradiation (200 Gy) was assessed by enzyme-linked immunosorbent assay (GM-CSF, IL-12) and flow cytometry (B7-2). Viability and clonogenicity were determined via trypan blue staining before and after irradiation. Growth rates were determined by serial cell counts.
GM-CSF expression was high in GM-CSF-transduced (10.36-162.10 ng/10(6) cells/d preirradiation and 10.22-122.02 ng/10(6) cells/d postirradiation) but lower in B7-2/GM-CSF-transduced cultures (1.41-2.90 ng/10(6) cells/d preirradiation, 1.96-5.02 ng/10(6) cells/d postirradiation). IL-12 expression also was lower (1.30-2.10 ng/10(6) cells/d preirradiation, 0.47-1.70 ng/10(6) cells/d postirradiation). B7-2 expression was high (one- to two-logarithm increase in fluorescence) and unaffected by radiation. Postirradiation viability was initially high (94.20 +/- 8.46%, Day 1) but decreased rapidly (28.13 +/- 4.64%, Day 10). No cultures demonstrated evidence of clonogenicity (i.e., cell division) after 200-Gy irradiation. Growth rates were similar in wild-type and gene-transduced Ed141.MEL, Ed147.BT, and Ed149.BT. However, D54MG-IL-12 growth was slower than that of wild-type D54MG.
GM-CSF, IL-12, and B7-2 genes can be transferred to human glioma and melanoma cell cultures efficiently by use of our retroviral vectors. Irradiation (200 Gy) does not significantly alter therapeutic gene expression. Irradiated cells remain viable for several days but cannot undergo further cell division. Early passage culture growth rates are not altered by therapeutic gene expression but are decreased by IL-12 in an immortalized cell line (D54MG). These results suggest that it is feasible to create vaccines with irradiated, autologous, genetically modified brain tumor cells.
免疫基因治疗是一种基于用转导了免疫刺激基因的经辐照自体肿瘤细胞进行疫苗接种的新型癌症治疗策略。为了在临床应用前对这类细胞进行表征，我们研究了免疫刺激基因转移后的人胶质瘤细胞系（D54 MG）以及早期传代的人胶质瘤（Ed147.BT、Ed149.BT）和黑色素瘤（Ed141.MEL）培养物。
将粒细胞 - 巨噬细胞集落刺激因子（GM - CSF）、白细胞介素 - 12（IL - 12）和B7 - 2基因通过逆转录病毒转移至肿瘤细胞。通过酶联免疫吸附测定法（GM - CSF、IL - 12）和流式细胞术（B7 - 2）评估辐照（200 Gy）前后的基因表达。通过台盼蓝染色测定辐照前后的活力和克隆形成能力。通过连续细胞计数确定生长速率。
GM - CSF转导的细胞中GM - CSF表达较高（辐照前10.36 - 162.10 ng/10⁶细胞/天，辐照后10.22 - 122.02 ng/10⁶细胞/天），但在B7 - 2/GM - CSF转导的培养物中较低（辐照前1.41 - 2.90 ng/10⁶细胞/天，辐照后1.96 - 5.02 ng/10⁶细胞/天）。IL - 12表达也较低（辐照前1.30 - 2.10 ng/10⁶细胞/天，辐照后0.47 - 1.70 ng/10⁶细胞/天）。B7 - 2表达较高（荧光增加一到两个对数）且不受辐射影响。辐照后的活力最初较高（第1天为94.20±8.46%），但迅速下降（第10天为28.13±4.64%）。200 - Gy辐照后，没有培养物显示出克隆形成能力（即细胞分裂）的证据。野生型和基因转导的Ed141.MEL、Ed147.BT和Ed149.BT中的生长速率相似。然而，D54MG - IL - 12的生长比野生型D54MG慢。
使用我们的逆转录病毒载体可将GM - CSF、IL - 12和B7 - 2基因有效地转移至人胶质瘤和黑色素瘤细胞培养物中。辐照（200 Gy）不会显著改变治疗性基因表达。辐照后的细胞在数天内仍保持活力，但不能进行进一步的细胞分裂。早期传代培养物的生长速率不受治疗性基因表达的影响，但在永生化细胞系（D54MG）中会因IL - 12而降低。这些结果表明，用经辐照的、自体的、基因修饰的脑肿瘤细胞制备疫苗是可行的。
