Turner Katherine A, Kalafatis Michael
Department of Chemistry, Cleveland State University, Cleveland, OH.
Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, Cleveland, OH.
J Appl Lab Med. 2017 Sep 1;2(2):176-185. doi: 10.1373/jalm.2017.023408.
Personalized cancer treatments can be applied to the clinical use of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL). rhTRAIL holds great promise because of its selectivity for cancer cells. However, rhTRAIL clinical trials were conducted without the screening of patients' tumors for rhTRAIL-binding death receptor (DR)4 and DR5, and the unselected treatment resulted in a lack of clinical benefit. Here we propose an in vitro test to analyze tumor cells isolated from patients for the membrane expression of DRs to determine patient suitability for rhTRAIL treatment.
Using a panel of malignant melanoma cell lines, the correlation between DR membrane expression and rhTRAIL sensitivity was evaluated. The membrane expression of DR4 and DR5 was examined through staining with anti-DR4 and -DR5 antibodies followed by fluorescence-activated cell sorting. rhTRAIL sensitivity was determined through Annexin-V and propidium iodide staining and Western blotting after rhTRAIL treatment.
Here we show a direct correlation between the membrane expression of DRs and rhTRAIL sensitivity. rhTRAIL-sensitive melanoma lines, on average, had nearly 4-fold more DR4 and >2-fold more DR5 than rhTRAIL-resistant lines. For a cancer cell to display rhTRAIL sensitivity, the optimum expression of DRs is essential. To overcome the apoptotic threshold, cancer cells must express DRs >2-fold higher compared with their benign counterpart.
These data show the potential of this flow cytometry-based assay for the analysis of isolated tumor cells for DR membrane expression. By first determining a patient's susceptibility to rhTRAIL-based treatments, they can be more appropriately placed in rhTRAIL clinical trials and improve rhTRAIL as an anticancer therapeutic.
个性化癌症治疗可应用于重组人肿瘤坏死因子相关凋亡诱导配体（rhTRAIL）的临床应用。rhTRAIL因其对癌细胞的选择性而极具前景。然而，rhTRAIL临床试验在未对患者肿瘤进行rhTRAIL结合死亡受体（DR）4和DR5筛查的情况下进行，这种未经过筛选的治疗导致临床疗效不佳。在此，我们提出一种体外试验，以分析从患者分离出的肿瘤细胞中DR的膜表达情况，从而确定患者是否适合接受rhTRAIL治疗。
使用一组恶性黑色素瘤细胞系，评估DR膜表达与rhTRAIL敏感性之间的相关性。通过用抗DR4和抗DR5抗体染色，随后进行荧光激活细胞分选来检测DR4和DR5的膜表达。在rhTRAIL处理后，通过膜联蛋白V和碘化丙啶染色以及蛋白质免疫印迹法测定rhTRAIL敏感性。
我们在此表明DR的膜表达与rhTRAIL敏感性之间存在直接相关性。平均而言，对rhTRAIL敏感的黑色素瘤细胞系的DR4表达量比rhTRAIL耐药细胞系多近4倍，DR5表达量多2倍以上。癌细胞要表现出对rhTRAIL的敏感性，DR的最佳表达至关重要。为了克服凋亡阈值，癌细胞必须比其良性对应物表达高2倍以上的DR。
这些数据表明这种基于流式细胞术的检测方法在分析分离的肿瘤细胞DR膜表达方面具有潜力。通过首先确定患者对基于rhTRAIL治疗的易感性，可以更恰当地将他们纳入rhTRAIL临床试验，并改善rhTRAIL作为抗癌治疗药物的效果。
