結合治療用超音波與CXCL10質體轉染於癌腫瘤之治療

Abstract

研究背景：手術、化學治療和放射治療，是目前癌腫瘤的主要療法。而腫瘤的復發的情況，一直是目前癌症治療的主要議題。希望藉由全身性癌症治療的免疫療法，能夠降低腫瘤復發的機率。 研究目標：利用超音波的聲孔作用及熱效應，破壞腫瘤微環境並將CXCL10質體轉染進入腫瘤細胞。腫瘤釋放CXCL10趨化因子後，進而吸引並活化自然殺手細胞去毒殺腫瘤細胞，提高治療效果。 材料與方法：在in-vitro實驗中，先測試自然殺手細胞對不同濃度CXCL10的趨化能力是否有所差異。使用不同比例的自然殺手細胞與腫瘤細胞量，評估自然殺手細胞對腫瘤細胞的細胞毒殺性。在in-vitro和in-vivo中，測試給予CXCL10質體後施打超音波，是否能提高的CXCL10的濃度。將實驗分為四組，分別為：控制組(Control)、CXCL10質體組(Plasmid)、超音波熱治療組(Ultrasound hyperthermia, pUH)、質體加超音波組(Plasmid +pUH)。在in-vivo實驗中，當腫瘤大小為50 mm3，開始第一次治療，一週治療兩次，總共治療兩週。每隔兩天記錄腫瘤體積及小鼠體重。此外，會利用非侵入性活體分子影像觀察腫瘤的冷光表現的強度。 實驗結果：研究結果顯示，自然殺手細胞會隨著CXCL10的濃度上升，其趨化性愈強，被吸引的自然殺手細胞數目也愈多。當自然殺手細胞數目愈多，其對腫瘤細胞的毒殺效果也愈顯著。在in-vitro和in-vivo中，給予CXCL10質體後施打超音波(Plasmid +pUH)，其CXCL10的濃度和其他三組都具有顯著性差異。趨化測試和免疫組織切片染色也發現，Plasmid +pUH組自然殺手細胞浸潤程度有顯著差異。觀察腫瘤大小變化發現，Plasmid +pUH組和Control組在第17天開始出現顯著性差異，而在第23天和Plasmid組出現顯著性差異，和pUH組並無明顯差異。利用IVIS系統觀察第13天及第20天的冷光表現值和腫瘤大小相類似。 結論：利用超音波熱治療可傷害腫瘤，破壞腫瘤的微環境，增強CXCL10質體轉染進入腫瘤細胞，進而可提高腫瘤細胞釋放的CXCL10濃度，吸引自然殺手細胞，提高自然殺手細胞的浸潤程度，以達到較好的腫瘤治療效果。

Background: Surgery, chemotherapy and radiotherapy are the major modalities for cancer tumor treatment, but the development of tumor recurrence is still the major issue for cancer treatment. Immunotherapy is a systemic cancer treatment and it may be useful for reducing relapse of the treated tumors. Purpose: To use ultrasound sonication/hyperthermia to disrupt the microenvironment of tumor and to transfect CXCL10 plasmid into cancer cells, and then the release of CXCL10 from cancer cells to attract and activate natural killer(NK) cells to kill the remaining cancer cells and enhance the immune system to improve the treatment efficacy. Materials and Methods: In in-vitro study, different concentration of CXCL10 was used to evaluate the chemotaxis of NK cells and different ratio of NK cells to cancer cells was used to assess the cytotoxicity of NK cells. The transfect of CXCL10 plasmid into cancer cells by ultrasound sonication was investigated both in-vitro and in-vivo studies. The studies included four groups: control, plasmid only, ultrasound hyperthermia (pUH), and plasmid +pUH. For the in-vivo study, the first treatment was conducted when the tumor grew up to 50 mm3, and treatment was conducted twice a week for two weeks. Body weight and tumor volume were measured every two days. The tumors were also observed by In-vivo Image System (IVIS) during the treatment. Results: The results showed that the chemotaxis of NK cells increased with the concentration of CXCL10, and the cytotoxicity of NK cells to cancer cells also increased with the NK/cancer cell ratio. Both in-vitro and in-vivo results displayed that the concentration of CXCL10 increased after plasmid injection plus ultrasound hyperthermia (plasmid +pUH) and there was a significant difference compared with the other groups. The chemotaxis assay and immunohistochemical staining indicated that the increased tumor infiltration of NK cells in the plasmid +pUH group and it had a significant difference. For the change of tumor volumes, the plasmid +pUH group was significantly smaller than the control group since day 17 and significantly smaller than the plasmid group since day 23. However, there was no significant difference between the pUH group and the plasmid +pUH group. Similar results were observed in the expression of bioluminescence (IVIS image). Conclusion: Ultrasound hyperthermia might damage tumor to disrupt its microenvironment and enhance the CXCL10 plasmid transfection to cancer cells to release CXCL10 to attract NK cells for a better cancer tumor treatment.