Department of Orthopaedic Surgery, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands.
Br J Cancer. 2013 Oct 15;109(8):2142-54. doi: 10.1038/bjc.2013.578. Epub 2013 Sep 24.
Osteosarcoma (OS) is the most common bone tumour in children and adolescents. Despite aggressive therapy regimens, treatment outcomes are unsatisfactory. Targeted delivery of drugs can provide higher effective doses at the site of the tumour, ultimately improving the efficacy of existing therapy. Identification of suitable receptors for drug targeting is an essential step in the design of targeted therapy for OS.
We conducted a comparative analysis of the surface proteome of human OS cells and osteoblasts using cell surface biotinylation combined with nano-liquid chromatography - tandem mass spectrometry-based proteomics to identify surface proteins specifically upregulated on OS cells. This approach generated an extensive data set from which we selected a candidate to study for its suitability as receptor for targeted treatment delivery to OS. First, surface expression of the ephrin type-A receptor 2 (EPHA2) receptor was confirmed using FACS analysis. Ephrin type-A receptor 2 expression in human tumour tissue was tested using immunohistochemistry. Receptor targeting and internalisation studies were conducted to assess intracellular uptake of targeted modalities via EPHA2. Finally, tissue micro arrays containing cores of human OS tissue were stained using immunohistochemistry and EPHA2 staining was correlated to clinical outcome measures.
Using mass spectrometry, a total of 2841 proteins were identified of which 156 were surface proteins significantly upregulated on OS cells compared with human primary osteoblasts. Ephrin type-A receptor 2 was highly upregulated and the most abundant surface protein on OS cells. In addition, EPHA2 was expressed in a vast majority of human OS samples. Ephrin type-A receptor 2 effectively mediates internalisation of targeted adenoviral vectors into OS cells. Patients with EPHA2-positive tumours showed a trend toward inferior overall survival.
The results presented here suggest that the EPHA2 receptor can be considered an attractive candidate receptor for targeted delivery of therapeutics to OS.
骨肉瘤（OS）是儿童和青少年中最常见的骨肿瘤。尽管采用了积极的治疗方案，但治疗效果仍不理想。药物的靶向递送可以在肿瘤部位提供更高的有效剂量，最终提高现有治疗的疗效。鉴定合适的药物靶向受体是骨肉瘤靶向治疗设计的重要步骤。
我们使用细胞表面生物素化结合纳升液相色谱-串联质谱蛋白质组学对人骨肉瘤细胞和成骨细胞的表面蛋白质组进行了比较分析，以鉴定在骨肉瘤细胞上特异性上调的表面蛋白。这种方法从大量数据中选择了一个候选物，用于研究其作为受体用于靶向治疗骨肉瘤的适用性。首先，使用流式细胞术分析确认 Ephrin 型-A 受体 2（EPHA2）受体的表面表达。使用免疫组织化学检测人肿瘤组织中 Ephrin 型-A 受体 2 的表达。进行受体靶向和内化研究，以评估通过 EPHA2 对靶向模式的细胞内摄取。最后，使用免疫组织化学染色对包含人骨肉瘤组织芯的组织微阵列进行染色，并将 EPHA2 染色与临床结果测量相关联。
使用质谱法共鉴定出 2841 种蛋白质，其中 156 种是与人类原代成骨细胞相比在骨肉瘤细胞上显著上调的表面蛋白。Ephrin 型-A 受体 2 高度上调，是骨肉瘤细胞上最丰富的表面蛋白。此外，Ephrin 型-A 受体 2 在绝大多数人骨肉瘤样本中表达。Ephrin 型-A 受体 2 有效地介导靶向腺病毒载体内化进入骨肉瘤细胞。EPHA2 阳性肿瘤患者的总生存期有下降趋势。
这里提出的结果表明，Ephrin 型-A 受体 2 可被视为骨肉瘤靶向治疗的有吸引力的候选受体。
