應用超音波結合微泡進行原位肝腫瘤的抗血管新生基因治療

Abstract

肝細胞癌是一個遍布於世界各地的疾病，特別在亞洲和非洲地區，肝癌更是好發的癌症之ㄧ；由於肝細胞癌常伴隨著血管異常增生，所以抗血管新生的策略被認為是一種很有潛力的治療方式。超音波目前則被認為是一個有效的工具，能夠將基因轉殖到特定的組織或腫瘤之中；超音波結合微泡(SonoVue®)，可藉由產生空化效應使得細胞膜之通透性短暫的改變，而促進質體DNA進入到細胞內表現。本研究的第一個部分，我們探討三種不同的位置，如原位肝腫瘤、背部肝腫瘤以及肌肉部位，比較其超音波轉殖基因效率的差異。我們分別將Luciferase、IL-12、EGFP的質體DNA與SonoVue®混合後打入BALB/c小鼠的肝腫瘤或肌肉組織，再給予照射超音波；我們發現在肌肉部位相較於肝腫瘤部位，可產生較高的表現量。本研究的第二部份是嘗試利用超音波在肌肉轉殖基因的技術，去治療三種不同的肝腫瘤動物模式：分別是背部肝腫瘤模式、原位肝腫瘤模式以及原發性多位點肝腫瘤模式。我們各別轉殖兩種不同的抗血管新生基因，名為血管內膜阻生素(endostatin, ED)以及鈣網蛋白(calreticulin, CRT)。動物實驗的結果顯示，在分別經過四次的超音波基因治療後，對於上述三種不同的肝腫瘤動物模式都可以產生顯著的療效，並且有效地延長小鼠的存活時間。接著我們從免疫組織化學染色分析的結果觀察到，有較多的T細胞以及凋亡細胞出現在治療過的腫瘤中，此凋亡現象很有可能是由於腫瘤內的血管數目減少所造成。此外，我們結合了免疫療法與抗血管新生療法，先在肝腫瘤內打入攜帶GM-CSF和IL-12的重組腺病毒；而後再使用超音波結合微泡的技術轉殖ED或CRT。結果證明了不論是對於原位肝腫瘤或是原發性多位點肝腫瘤，結合治療都比單一種治療更能有效抑制腫瘤的生長。在治療組的腫瘤中我們也發現到，有明顯的T細胞浸潤以及更顯著減少的血管分佈。除此之外，在另外一個合併治療的實驗中，我們證實了結合化療藥物小紅莓(doxorubicin, Dox)與抗血管新生療法，可以比單一種療法發揮更顯著的療效。綜合以上結果，我們的研究首次證明了將ED或CRT的DNA與混合SonoVue®打入肌肉組織中，再以超音波照射該部位，可以有效地治療原位肝腫瘤，而這樣的策略或許將有潛力能夠應用於臨床治療。

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, particularly in Asia and Africa. Due to its highly neovascularization, anti-angiogenic approach could be a potential therapeutic strategy. Ultrasound (US) is an effective tool to locally deliver genes into target tumors or organs. US, in combination with microbubbles SonoVue®, can temporarily change the permeability of cell membranes by cavitation and thus enhances the delivery of plasmid DNA into cells. In the first part of this study, we compared the efficiency of US-mediated gene delivery at different sites, i.e., the orthotopic liver tumor, the subcutaneous tumor or the muscle. We injected luciferase, IL-12 or EGFP plasmid DNA mixed with SonoVue® into BALB/c mice either intramuscularly (IM) or intratumorally (IT), followed by exposure to US. We found that intramuscular injection provided better expression results than intratumoral injection. In the second part of this research, we assessed the therapeutic effects of US-mediated IM gene therapy on three previously established liver tumor models, namely subcutaneous tumor model, implanted liver tumor model and primary multifocal HCC model. We delivered the endostatin (ED) or the calreticulin (CRT) gene, both of which are anti-angiogenic factors through IM injection. The experimental results showed that four times of IM ultrasound treatment yielded significant therapeutic effects on these three liver tumor models and also prolonged the survival time in mice. Furthermore, immunohistochemical staining of the tumors revealed increased tumor-infiltrating T lymphocytes and apoptotic cells which were probably induced by the decreased neovascularization. We also combined immunotherapy with anti-angiogenic therapy, using recombinant adenoviruses carrying GM-CSF and IL-12 genes followed by ultrasound delivery of ED or CRT gene. The combined therapy showed much better therapeutic effects on both implanted and primary multifocal HCC models than either therapy alone. In this combined therapy, a very significant increase in tumor-infiltrating T cells and a further decrease in tumor vascularization were observed. In addition, combination of chemotherapy using doxorubicin with anti-angiogenic therapy also provided a better therapeutic effect than either therapy alone. Taken together, we have proved for the first time that co-injection of microbubbles and plasmid DNA intramuscularly followed by ultrasound exposure can effectively provide a cure for liver cancers, and this strategy may have therapeutic potential for the clinical treatment.