肿瘤细胞治疗研究
英文原题：The potential of mesenchymal stem cell coexpressing cytosine deaminase and secretory IL18-FC chimeric cytokine in suppressing glioblastoma recurrence.
多形性胶质母细胞瘤（GBM）患者的复发率高达90%，5年生存率仅约5%。
多形性胶质母细胞瘤（GBM）患者复发率高达90%，5年生存率仅约5%。胞嘧啶脱氨酶（CDA）/5-氟胞嘧啶（5-FC）基因治疗是一种有前景的胶质瘤治疗方法，因为5-FC能够穿过血脑屏障（BBB），而5-氟尿嘧啶（5-FU）则不能。此外，5-FU有助于逆转冷肿瘤的免疫状态。本研究开发了共表达酵母CDA和分泌型IL18-FC超级因子的间充质干细胞（MSCs），通过同时发挥细胞毒性作用和增强免疫反应来防止肿瘤复发进展。IL18与Igk和IgG2a FC结构域融合，以增强其分泌和血清半衰期。研究证实了CDA酶的表达和活性，以及分泌型IL18和IL18-FC超级因子的表达、分泌和活性，这些均由慢病毒转导的MSCs表达。在transwell肿瘤趋向性实验中，观察到基因修饰后的MSCs在转导后仍保留其选择性肿瘤趋向能力。表达CDA的MSCs在5-FC（200 g/ml）存在下，通过间接transwell共培养系统中的旁观者效应诱导胶质瘤细胞周期阻滞和凋亡。当其在细胞群体中仅占12.5%时，即可降低直接共培养系统的细胞活力。通过将致死剂量的GL261细胞与表达CDA、或CDA和sIL18、或CDA和sIL18-FC的MSCs按1:1比例混合后脑内注射至C57BL/6小鼠，评估了工程化MSCs抑制肿瘤进展的有效性。PET扫描显示，接受5-FC治疗的MSC-CDA-sIL18-FC组未见明显肿瘤肿块。病理分析显示，该组肿瘤进展在细胞接种后第20天前受到抑制。细胞因子评估显示，与对照组相比，接受生理盐水（NS）处理的MSC-CDA-sIL18和MSC-CDA-sIL18-FC组血清中干扰素-γ（IFN-）和白细胞介素-4（IL-4）均升高。接受5-FC治疗的MSC-CDA-sIL18-FC组与对照组相比，血清IL-6水平降低，生存率显著提高。因此，共表达酵母CDA和分泌型IL18-FC、具有肿瘤趋向能力的MSCs，可作为GBM标准治疗的补充方法，有效抑制肿瘤进展并预防复发。
Glioblastoma multiforme (GBM) patients have a high recurrence rate of 90%, and the 5-year survival rate is only about 5%. Cytosine deaminase (CDA)/5-fluorocytosine (5-FC) gene therapy is a promising glioma treatment as 5-FC can cross the blood-brain barrier (BBB), while 5-fluorouracil (5-FU) cannot. Furthermore, 5-FU can assist reversing the immunological status of cold solid tumors. This study developed mesenchymal stem cells (MSCs) co-expressing yeast CDA and the secretory IL18-FC superkine to prevent recurrent tumor progression by simultaneously exerting cytotoxic effects and enhancing immune responses. IL18 was fused with Igk and IgG2a FC domains to enhance its secretion and serum half-life. The study confirmed the expression and activity of the CDA enzyme, as well as the expression, secretion, and activity of secretory IL18 and IL18-FC superkine, which were expressed by lentiviruses transduced-MSCs. In the transwell tumor-tropism assay, it was observed that the genetically modified MSCs retained their selective tumor-tropism ability following transduction. CDA-expressing MSCs, in the presence of 5-FC (200 g/ml), induced cell cycle arrest and apoptosis in glioma cells through bystander effects in an indirect transwell co-culture system. They reduced the viability of the direct co-culture system when they constituted only 12.5 % of the cell population. The effectiveness of engineered MSCs in suppressing tumor progression was assessed by intracerebral administration of a lethal dose of GL261 cells combined in a ratio of 1:1 with MSCs expressing CDA, or CDA and sIL18, or CDA and sIL18-FC, into C57BL/6 mice. PET scan showed no conspicuous tumor mass in the MSC-CDA-sIL18-FC group that received 5-FC treatment. The pathological analysis showed that tumor progression suppressed in this group until 20th day after cell inoculation. Cytokine assessment showed that both interferon-gamma (IFN- ) and interleukin-4 (IL-4) increased in the serum of MSC-CDA-sIL18 and MSC-CDA-sIL18-FC, treated with normal saline (NS) compared to those of the control group. The MSC-CDA-sIL18-FC group that received 5-FC treatment showed reduced serum levels of IL-6 and a considerably improved survival rate compared to the control group. Therefore, MSCs co-expressing yeast CDA and secretory IL18-FC, with tumor tropism capability, may serve as a supplementary approach to standard GBM treatment to effectively inhibit tumor progression and prevent recurrence.
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