2014-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647758摘要：血管新生抑制劑是目前治療晚期肝細胞癌最常使用的標靶治療藥物。我們最近的研究顯示，使用免疫調控劑增加肝細胞癌腫瘤微環境的免疫作用，可以有效提高標靶藥物sorafenib﹝是第一個被核准用於治療晚期肝細胞癌病患的標靶治療藥物﹞的療效。我們因此假設，合併血管新生抑制劑與免疫調控治療應可改善晚期肝細胞癌病患的療效。本計畫中我們將進一步探討血管新生抑制劑-免疫調控治療的協同抗癌作用的作用機轉，並且發展可以即時偵測肝細胞癌腫瘤微環境中兩種藥物可能交互作用的超音波影像技術。本計畫的特定目標包括﹝1﹞探討血管新生抑制劑-免疫調控治療對於肝細胞癌腫瘤微環境中T淋巴球的活化、移行、組織浸潤等影響以澄清兩種藥物協同抗癌的作用機轉；﹝2﹞發展可以即時偵測腫瘤為環境中血管新生以及免疫細胞分布的微氣泡顯影超音波探測技術。將比較由超音波所偵測得的藥物治療後腫瘤血管新生變化以及免疫細胞分布的改變與藥物療效之間的關聯，從此找出與藥物療效最有關聯的免疫相關生物指標，並探討相關檢測與指標在臨床應用的可行性。本計畫將使用免疫健全的「正位」﹝orthotopic﹞小鼠肝癌實驗模式。將使用人工表現gp33病毒蛋白之Hepa1-6 肝癌細胞株，植入P14CD8﹝具有可辨識gp33蛋白之T細胞接受體﹞基因轉殖鼠的肝臟內以產生肝內腫瘤評估藥物療效。藥物治療對於具有gp33抗原特異性之T淋巴球的活化、移行、組織浸潤等功能之影響將以具有腫瘤特異性抗原之小鼠正位肝癌實驗模式來偵測。在微氣泡顯影超音波部分，將利用可辨識不同T淋巴球的抗體鍵結於微氣泡上，並以超音波偵測腫瘤在藥物治療後﹝1﹞血液灌流狀態的改變與療效之關聯性﹝2﹞可辨識不同T淋巴球的微氣泡所偵測的超音波訊號改變與腫瘤微環境中淋巴球﹝tumor-infiltrating lymphocytes﹞分布變化的關聯性。並將針對腫瘤組織進行免疫化學染色以及流式細胞儀分析以發展免疫相關之生物指標。本計畫的研究結果將有助於發展即時偵測血管新生抑制劑-免疫調控治療的療效及作用機轉的臨床前肝細胞癌動物實驗模式，生物指標研究結果將有助於相關藥物臨床試驗的發展。<br> Abstract: Anti-angiogenic therapy is the mainstream molecular targeted therapy for patients with advanced hepatocellular carcinoma (HCC). We hypothesized that the anti-tumor efficacy of molecular targeted therapy for HCC can be improved by increasing the anti-tumor immune effects in the tumor micro-environment. This hypothesis was supported by the synergistic anti-tumor activity between sorafenib, the first approved molecular targeted therapy for patients with advanced HCC, and the immune modulatory agents in our pre-clinical studies.In this study we will explore the mechanisms of anti-angiogenic/immune-modulatory synergy and develop a new sonography technology for real-time analysis of the interaction between anti-angiogenic agents and immune modulators in HCC tumor micro-environment. Our specific aims include (1) to explore the synergistic effect of the anti-angiogenic/immune-modulatory combination therapy on activation, differentiation, and acquisition of migration/tumor-infiltrating and tumor-killing capabilities of tumor-specific T cells in HCC microenvironment; (2) to develop targeting microbubles for simultaneous sonographic analysis of tumor angiogenesis and immune effector cells in the HCC micro-environment. Correlation of the sonographic findings with treatment efficacy of different combination regimens (anti-angiogenic agents and immune modulators) to identify pertinent immune effector cells and immune-related biomarkers that can help predict treatment efficacy in the clinic.In this project an orthotopic immune-competent liver cancer model (implantation of Hepa1-6 gp33 cells into the liver capsule of P14CD8 transgenic mice (carrying rearranged TCR specific for gp33)) will be used to evaluate the efficacy and safety of drug treatment. Antigen-specific synergy of different drug combination regarding trafficking, priming, and activation of cytotoxic T cell will be evaluated by using a murine orthotopic, tumor antigen-specific liver cancer model. Biotinylated lipid-formed microbubbles conjugated with antibodies targeting different lymphocyte subpopulations will be prepared. Sonographic evaluation will be done to (1) correlate changes in tumor blood flow and tumor volume after drug treatment; (2) compare signals detected by microbubbles targeting different lymphocyte sub-populations and changes of tumor-infiltrating lymphocytes after drug treatment. Immunohistochemical and flow cytometry studies on tumor tissues will be done to explore potential biomarkers.Results from this project will help develop real-time measurement of anti-angiogenic and immune-modulatory effects of molecular targeted therapy in pre-clinical HCC models. The immune-related effector cells and biomarkers found in this project will help predict treatment efficacy in the clinic.肝細胞癌血管新生抑制劑標靶免疫治療微氣泡顯影超音波hepatocellular carcinomaanti-angiogenic agentsmolecular targeted immunotherapymicrobubble contrast enhanced ultrasound