發展AlphaLISA分析平台定量血清岩藻醣化上皮鈣粘蛋白作為肺腺癌轉移之生物標誌

Abstract

肺癌是全世界癌症致死的首要原因。其中非小細胞肺癌因個體差異性極大，不論在預後或個人對治療的反應都有顯著差異。因此，若能判別病人是否屬於轉移的高風險族群或能評估其手術後復發率，將對療程有更好的掌控。近年來已有許多研究及越來越多的證據表明，醣基化參與在許多生物反應過程中，當中包含癌症病程轉化和癌細胞轉移等重要機轉。本研究的特定目標是建立一個有效的平台，同時篩選和鑑定肺癌轉移標誌物的血清醣蛋白。首先以刀豆素A親和性層析法純化出肺腺癌患者的血清中的N-醣基化修飾蛋白。得到的醣蛋白經由二維螢光差異電泳法分離再結合奈流液相層析質譜儀分析，最後從數據庫檢索鑑定出蛋白身分。我們鑑定到一些表現量有差異的癌症相關醣蛋白，包括α-1-抗胰蛋白酶，補體C3c，結合球蛋白，上皮鈣粘蛋白等。經由西方墨點法和橙黃網孢盤菌 (Aleuria aurantia)凝集素染色法驗證後，發現包括上皮鈣粘蛋白在內的這些醣蛋白，其核心岩藻醣化的現象隨著肺癌病程進展而增加。我們接著檢測154例肺腺癌病人血清的上皮鈣粘蛋白的岩藻醣化指數（fucosylation index, FI），岩藻醣化指數較低的患者（FI≤ 1.5），其存活率高於岩藻醣化指數較高的患者（FI>1.5）。同時我們建立了一個AlphaLISA檢測平台，量測上皮鈣粘蛋白的岩藻醣化指數。目前的研究結果顯示，血清中上皮鈣粘蛋白的岩藻醣化指數可能是一個有潛力的轉移性肺腺癌的預後指標。除此之外，AlphaLISA檢測平台的發展也已經可以達到許多其他疾病診斷上的高通量篩選需求。

Lung cancer is the leading cause of cancer-associated deaths worldwide. Non-small cell lung cancer is a heterogeneous condition with significant variability in prognosis and in individual response to treatment. Thus, the identification of patients with a high risk of metastasis or relapse after surgery would allow better management. There is increasing evidence that glycosylation plays a significant role in biological processes including oncogenic transformation and metastasis. The specific aim of our study is to set up a platform to screen and identify serum glycoproteins as metastasis biomarkers of lung cancer. Concanavalin A affinity chromatography was used to enrich N-linked glycoproteins from pooled serum of lung adenocarcinoma patients. The captured glycoproteins were separated with 2-D DIGE combined with nano-LC-MS/MS and identified by database searching. Some differentially expressed cancer-related glycoproteins, such as α-1-antitrypsin, complement C3c, haptoglobin, and E-cadherin, were identified. These glycoproteins were evaluated by Western blotting and Aleuria aurantia lectin staining and several, including E-cadherin, showed increased core-fucosylation during lung cancer progression. We then measured the fucosylation index (FI) of E-cadherin in 154 lung adenocarcinoma patients. The survival rate of patient with lower fucosylation index (FI≤1.5) of e-cadherin is higher than that with higher fucosylation index (FI>1.5). In addition, a homogeneous proximity-based AlphaLISA assay to measure the FI of E-cadherin was established. The present study indicates that the FI of E-cadherin could be a potential prognostic marker of metastatic lung adenocarcinoma. Furthermore, the AlphaLISA assay is ready to meet the high throughput screening requirements for the diagnosis of many other diseases.