摘要:靜脈栓塞 (Venous thromboembolism, VTE) 為癌症患者的第二大死因(第一為癌症的惡化),而胰臟癌為最易發生VTE 的惡性腫瘤。癌症病人的高凝血活性狀態起因仍未明,惟腫瘤可能透過多重機制活化凝血系統導致高凝血活性狀態而引發VTE (所謂的Trousseau's syndrome),失控的凝血系統可能造成DIC (disseminated intravascular coagulation) 或致命。目前臨床使用最普遍的抗凝劑為低分子量肝素(LMWH),惟只能緩解症狀如下肢腫痛而無法有效控制後續的惡化,因此徹底瞭解癌症如何導致凝血異常而造成VTE 對研發藥物以改善癌症病人的生活品質及延長存活率極為重要。針對癌症誘發VTE的機制大多在探討凝血”外在途徑” (extrinsic or TF pathway),即高活性的組織因子(TF)主導第七因子(FVII)直接活化第十因子接續活化凝血酶產生纖維蛋白(fibrin),但臨床研究顯示TF 和VTE 風險無一定相關性,且癌症相關靜脈栓塞(癌症/VTE)中也多有TF 低或無表現(TF-)的現象,其誘發VTE 機制待釐清,因此急需探討其他機制。本計畫提出要深入探討凝血”內在途徑”及位於”內在途徑”的樞紐位置”FIX/FVIII”複合體在胰臟癌相關的VTE 的角色。立論的基礎源自幾項研究(如下圖及文獻附於”計畫背景”, p.4)包括人類胰臟癌組織染色發現腫瘤富含凝血因子FVII,FVIII, FIX, FX 等,且無抑制或溶血因子如TFPI、plasmin 等,顯示凝血調控異常;臨床流行病學調查癌症病人血中高含量的FVIII 是VTE 的獨立風險因子;文獻證實”內在途徑”參與止血機制的纖維蛋白(fibrin)生成和參與病變的血栓(thrombus,富含其它成份,如圖)生成的角色不同,譬如”內在途徑”的FXII 與止血無關但其活化後造成的血栓在癌症/VTE 扮演重要角色,因此FXII/FXI/FIX/FVIII 主導的”內在途徑”在癌症/VTE 扮演的角色是否與止血機制中作為TF 主導的”外在途徑”後的增幅作用(amplification)的角色值得探討;臨床研究證實缺乏FXI (FXII 的受質,且活化FIX)的病人其VTE 發生率較低且臨床試驗顯示抑制FXI 可降低術後靜脈栓塞,顯示FXII/FXI/FIX/FVIII 主導的”內在途徑”扮演重要角色,惟FXI 在(TF-)腫瘤造成的VTE 的角色未明;癌症患者血中偏高的負電價游離DNA 可活化”內在途徑”且和VTE 的發生具正相關,然而”內在途徑”在胰臟癌/VTE 的角色尚未被探討;腫瘤利用分泌白血球生長激素(G-CSF)促進嗜中性胞外網(neutrophil extracellular traps, NETs)的釋放,其負電荷表面會活化FXII 造成VTE,胰臟癌亦會促進VTEs的生成,然而”內在途徑”參與NETs 造成的胰臟癌/VTE 在臨床的相關性尚未被證實;組織發炎時凝血因子VWF 被証實可直接結合胞外DNA (cfDNA)及NETs 並吸引嗜中性白血球依附於內皮細胞並穿過血管,且低分子量肝素無法有效抑制此現象。已知VWF 在血中與FVIII 結合,且FIX 依附於內皮細胞,因此本計畫提出VWF/FVIII/FIX 可結合於NETs 並主導血栓生成且參與NETs 造成胰臟癌/VTE的假說具新穎性,值得探討。本計畫綜合以上論點提出假設FIX/FVIII 參與”內在途徑”及NETs 造成胰臟癌/VTE,為驗證上述假說設定三年的目標如下(詳細說明請見下頁”計畫背景”, p.1 , 及研究方法,p.13)(1)利用擬人化血友病小鼠證明並量化"內在途徑"造成胰臟癌相關靜脈栓塞的角色[第1,2 年](2)利用擬人化血友病小鼠證明"內在途徑"參與 NETs 誘發胰臟癌相關靜脈栓塞[第2,3 年](3)利用胰臟癌病人周邊血驗證NETs 所活化的內在途徑與胰臟癌相關靜脈栓塞的臨床關聯性[第1,2,3年]。本計畫的特點是將利用擬人化小鼠以活體及病人檢體探討及驗證提出的目標,本計畫最大優勢在已利用CRISPR/Cas9 剔除FVIII 及 FIX 基因而建立免疫缺陷(NSG)血友病小鼠,可避免小鼠內生性FVIII 或FIX 影響內在途徑和胰臟癌VTE 的探討,並已成功利用脾臟注射法移植周邊血液單核球入NSG 小鼠,初步偵測人類血球分佈率達60%。本計畫將利用上述成果完成設定的目標,本計畫成果將提供未來研發針對”內在途徑”或NETs 的治療方式。
Abstract: Venous thromboembolism (VTE) is the second leading cause of cancer-associated mortality in cancerpatients (the first is cancer itself). Among all types of cancers VTE is mostly found in patients withpancreatic cancer. The origin for hypercoagulant state in cancer patients remains poorly understood. Cancersmay exert its effect on coagulation through multiple mechanisms to bring about a hypercoagulant state, andthus to induces VTE (also called Trousseau’s syndrome). Uncontrolled VTE may lead to DIC (disseminatedintravascular coagulation) which is life-threatening. Currently used medication for treating cancer with VTEis the anticoagulant “low molecular weight heparin (LMWH)”. Although readily accessible, LMWH is foundto relieve edema and pain in the lower extremity at the expense of high incidence of bleeding complicationsand therefore, understanding how cancer induces overt thrombus generation leading to VTE can facilitatediscovery of more effective drugs than LMWH for treating cancer/VTE. Literatures have revealed anoverexpression of tissue factor (TF) in many types of cancers. TF in complex with FVII activates theextrinsic (or TF) pathway leading to FX activation and thrombus formation (figure below). Clinical studies,however, have not found a good correlation between TF level and the risk for VTE. Moreover, there arecancers that do not express TF (TF-) but induce VTE. These observations highlight the need to uncovermechanisms other than the “extrinsic pathway” by which cancer induces hypercoagulant state and VTE, inhope to develop novel drugs to treat VTE.This project aims to investigate the role of the coagulation “intrinsic pathway”, and theFIX/FVIII complex, functioning at the center stage of the intrinsic pathway, in the development ofhypercoagulant state and VTE in pancreatic cancer. The rationale behind this proposition stems from thefollowings (see figure below and references listed in the “background”, p.4): Immunohistochemicalstaining of human pancreatic cancer tissue sections identified coagulation factors FXI, FIX, FVIII, FX andfibrin but not natural inhibitors, such as TFPI or plasmin on tumors, indicating that fibrin coated on tumorswas unregulated; Epidemiological study revealed that high FVIII levels is an independent risk factor forsymptomatic VTE in cancer patients; Thrombus formation in cancer associated VTE through the “intrinsicpathway” may not copy what has been known to be the “amplification” step as in normal hemostasis thatleads to fibrin formation to halt bleeding. For instance, FXII of the intrinsic pathway is dispensable in normalhemostasis but its activation is critical in the formation of cancer-associated VTE. Thus, the role ofFXII/FXI/FIX/FVIII-directed “intrinsic pathway” in cancer-associated VTE needs to be explored; Epidemiology showed that FXI (substrate of FXII)-deficient patients had a low VTE incidence and a clinicaltrial showed targeting FXI was effective in reducing postoperative VTE; There is a positive correlationbetween higher plasma cell-free DNA (cfDNA) level and VTE in cancer patients. Morevoer, cfDNA canactivate “intrinsic pathway”. Thus, a role for cfDNA in the activation of the intrinsic pathway in pancreaticcancer/VTE is anticipated; Cancer cells secret granulocyte colony stimulating factor (G-CSF) to promoteneutrophils to undergo NETosis to form extracellular DNA traps (NETs) which provide a negatively-chargedsurface for factor XII/FXI activation to induce VTE. Pancreatic cancer also promotes NETs formation, butclinical association and the participation of “intrinsic pathway” in NET-induced VTE have not beendetermined in pancreatic cancer; A recent publication indicates that during inflammation coagulation factorVWF may bind directly to naked DNA and NETs to recruit neutrophils to endothelial cells for extravasationsinto tissues. This binding of VWF to naked DNA was not inhibited by LMWH. VWF is the carrier protein forFVIII. Moreover, a large proportion of FIX has been known to constantly bind to endothelial cells. Wehypothesize that FIX/FVIII in complex with VWF bound to NETs may be critical for thrombus formationleading to VTE that is not inhibited by LMWH. Thus, investigation of this possibility in association withVTE in pancreatic cancer is novel and deserves further study.Based on above literatures we propose that FVIII/FIX complex participates in the intrinsic pathway aswell as NETs formation to induce VTE in pancreatic cancer (see next page of “Background”, p1. and rationaledesign on p13. for detail and for cited references). To prove this hypothesis, we propose to investigate thefollowing 3 aims.(1) Using humanized hemophilia mice to prove and quantify the role of “intrinsic pathway” in pancreaticcancer-associated VTE [the 1st, 2nd year].(2) Using humanized hemophilia mice to uncover the role of “intrinsic pathway” in NETs-induced pancreaticcancer-associated VTE [the 2nd, 3rd year].(3) Using pancreatic patients’ samples to validate the correlation of NETs-activated “intrinsic pathway” andpancreatic cancer-associated VTE [the 1st-3rd year].The uniqueness of this grant proposal is that humanized mouse models will be used to validate ourhypotheses in vivo. Using CRISPR/Cas9 technology, the team has generated immunodeficient NSG (Nod/ScidIL2Rg)-Hemophilia mice with knockout of FVIII or FIX genes to eliminate possible ambiguity due toendogenous mouse FVIII and FIX. The team has also succeeded in repopulating the NSG mice with humanperipheral blood mononuclear cells and the proportion human blood cells have reached 60% of engrafted mice.With these established technologies and preliminary data (see “Background”), the project will deem to besuccessful and will provide clues for development of new drugs for treating cancer associated VTE.