- 學位論文
應用過繼性自然殺手細胞結合超音波熱治療強化全身性抗癌免疫反應
Adoptive Transfer of Natural Killer Cell in Combination with Ultrasound Hyperthermia Enhances Systemic Antitumor Immunity for Cancer Treatment
摘要
研究背景與目的 根據世界衛生組織的資料統計，大腸直腸癌是全世界發生率第三高之癌症，而死亡率則是排名第二，僅次於肺癌。若在癌症初期就介入治療，病患的五年存活率高達90%，但若造成了癌症的遠端轉移，五年存活率則大幅降到僅剩14%，因此控制癌症之轉移為大腸直腸癌病患首要需解決之問題。 根據先前研究顯示，超音波熱治療可刺激腫瘤大量釋放熱休克蛋白70（heat shock protein 70, HSP70），釋放到細胞外之HSP70可活化免疫系統。因此本研究建立雙側腫瘤動物模式下，利用脈衝式超音波熱治療（pulsed wave ultrasound hyperthermia, pUSHT）作為原位癌症疫苗，並輔以過繼性自然殺手細胞治療（adoptive NK cell therapy, ACT-NK），強化pUSHT誘發之免疫反應，期許在控制原位腫瘤的同時達到抑制遠端腫瘤生長，建構一套具系統性治療效果之合併治療手段。 實驗材料與方法 本研究主要分成三部分，依序為細胞實驗、動物實驗及腫瘤分析。細胞實驗使用CT26小鼠大腸直腸癌細胞株，利用細胞存活實驗探討43°C熱治療15、30、45及60分鐘對細胞株之生長抑制能力；細胞株於43°C 60分鐘治療參數下，第2、4、8及24小時之HSP70表現量；最後探討體外NK細胞對CT26細胞株之胞殺能力在有無熱療下之差異，其中本實驗用之NK細胞皆取自小鼠脾臟，並在體外進行11天之培養擴增。 動物實驗使用國家動物中心提供周齡為8-10週之BALB/c母鼠，在治療開始前7日於小鼠大腿右側及左側皮下種植CT26細胞以建立雙側腫瘤動物模型。pUSHT參數如下：探頭發射頻率1MHz、強度：1.44W/cm2、工作週期：50%、總施打時間為15分鐘；而ACT-NK以間隔六日施打一次、共二次。動物實驗之治療成效以腫瘤生長曲線及存活率呈現，並記錄小鼠體重變化作為治療對小鼠生存壓力影響之依據。 腫瘤樣本分為治療開始後第8日進行腫瘤淋巴細胞浸潤分析及治療結束後第6日進行蘇木精-伊紅染色（hematoxylin and eosin stain, H E stain）。每組各犧牲3隻小鼠，取下其超音波治療側及非超音波治療側之腫瘤進行分析。 實驗結果 由細胞實驗結果可知，CT26細胞在43°C熱水槽加熱下30分鐘，其存活率（48.6%）即能與Control組產生統計上之顯著差異（p < 0.001），而加熱45分鐘之存活率（38.5%）及60分鐘（17.7%）之存活率亦與Control組產生統計上之顯著差異（p < 0.0001）。後續選用43°C、60分鐘之加熱條件，探討CT26細胞在加熱後2、4、8、24小時之HSP70表現量，在8小時後與Control組達顯著性統計差異（p < 0.0001），且在24小時分析HSP70之表現量為Control組之19.95倍，較8小時之表現量17.55倍高，但兩者間並無顯著差異，因此本研究之動物實驗參考HSP70表現量之實驗，以pUSHT後24小時再進行ACT-NK。 動物實驗方面，由雙側腫瘤模型可得合併治療組無論是在超音波治療側或是非超音波治療側腫瘤，皆有最佳之腫瘤生長抑制結果，與Control組達到統計上之顯著差異（超音波治療側p < 0.0001；非超音波治療側p < 0.05）在治療開始後第15日，合併治療組超音波治療側腫瘤相較於Control組減少68.4%之生長，非超音波治療側腫瘤則減少22%之生長。 在治療結束後第6日進行腫瘤H E染色分析，由超音波治療側腫瘤可見pUSHT組及合併治療組之切片有物理性破壞之痕跡，而非超音波治療側腫瘤則較無明顯差異；腫瘤淋巴細胞浸潤分析結果雖然合併治療組無論在超音波治療側或是非超音波治療側腫瘤之淋巴細胞浸潤皆為四組最高，但四組並無達到統計上顯著差異。進一步分析腫瘤浸潤之CD4+T細胞、CD8+T細胞及NK細胞，合併治療組在超音波治療側浸潤之CD8+ T細胞及IFNγ+CD8+ T細胞皆高於其他三組，且達到統計上顯著差異；而非超音波治療側在四組間IFNγ+CD8+ T細胞、IFNγ+CD4+ T細胞及IFNγ+ NK細胞浸潤之總和雖無顯著差異，合併治療組卻呈現四組最高之趨勢，可見合併治療在非超音波治療側雖無明顯之作用，但還是有活化免疫細胞的趨勢存在。 結論 本研究於細胞實驗中證實熱治療確實有增加NK細胞對CT26細胞胞殺能力之效果，在動物模型中，pUSHT結合ACT-NK無論是在超音波治療側腫瘤及非超音波治療側腫瘤皆展現出最佳之抑制腫瘤生長的能力，且小鼠之存活率相較於其他三組也顯著提升，在治療期間亦無對小鼠造成過度之負擔。腫瘤樣本分析部分，由H E可觀察到超音波熱治療對腫瘤的確能產生除熱效應外之機械效應；淋巴細胞浸潤分析方面，合併治療組在超音波治療側有顯著之CD8+T細胞、IFNγ+CD8+T細胞浸潤效果，可見合併治療的確可以活化CD8+ T細胞並增加其浸潤；非超音波治療側之T細胞及NK細胞雖在合併治療組有相對活化之趨勢，但組間無統計上差異，可見合併治療在針對非超音波直接治療的腫瘤，還是無法突破其免疫抑制環境，使更多免疫細胞可浸潤到腫瘤中。
並列摘要
Background According to the World Health Organization （WHO; 2018）, colorectal cancer is the third most common cancer in the world, and is the second leading cause of death. If treatment is performed at the early stage of cancer, the patient's five-year survival rate is as high as 90%, but if the colorectal cancer metastasizes, the five-year survival rate drops to only 14%. Therefore, controlling cancer metastasis is the first priority for colorectal cancer patients. Based on previous studies, hyperthermia can stimulate tumor to release a large amount of heat shock protein 70 （HSP70）, and thus activates the immune system. Therefore, our study established a bilateral tumor animal model, using pulsed wave ultrasound hyperthermia （pUSHT） as an in situ cancer vaccine, and combines with adoptive NK cell therapy （ACT-NK） to strengthen the immune response induced by pUSHT. Our aim is to enhance systemic antitumor immunity, which can not only control the growth of directly treated tumors but also distant tumors. Materials and Methods This research included in-vitro, in-vivo studies and analysis of tumor samples. In-vitro studies, MTT assay was used to determine the viability of CT26 mouse colorectal cancer cells for the treatment of 43°C hyperthermia（duration: 15, 30, 45 and 60 minutes）; HSP70 expression levels at 2, 4, 8 and 24 hours after 43°C 60 minutes hyperthermia was determined by western blot. Lastly, the difference of NK cell cytotoxicity between heated and unheated-CT26 was investigated. NK cells used in this experiment were all taken from the spleen of mice, which was expanded for 11 days before experimentation. For in-vivo studies, CT26 cells were subcutaneously inoculated on the right and left back of BALB/c mice 7 days before the start of treatment. The parameters of pUSHT were set at 1MHz of frequency, 1.44W/cm2 of intensity, 50% of duty cycle and15 minutes of treatment duration; ACT-NK was delivered two times（on second day and seventh day after therapy initiation）. During the treatment, tumor volume and survival rate were recorded to evaluate the treatment efficacy, and body weight of mice was also recorded to test toxicity of treatment. For tumor sample analysis, three mice from each group were sacrificed 6 days after final treatment and the treated and untreated tumors were collected for hematoxylin and eosin stain （H E stain）. On the other hand, three mice from each group were sacrificed 8 days after therapy initiation and the treated and untreated tumors were collected to test the number of infiltrating lymphocytes in tumor. Results According to the in-vitro results, the survival rate of CT26 cells was significantly lower than control group when heated in water bath for 30, 45 and 60 minutes, by 48.6%, 38.5% and 17.7% respectively. The expression of HSP70 of CT26 cells was increased 4 hours after 43°C hyperthermia and reached the highest record （19.95 fold of control） at 24 hour; as a result, performed ACT-NK at 24 hour after pUSHT might be the best combined strategy. In-vivo results, the combined group showed the best outcome on tumor growth inhibition both treated and untreated sides, which were 68.4% and 22% decrease compared to the control group. Tumor sample H E analysis was performed 6 days after the end of treatment. It showed signs of physical damage in pUSHT directly treated tumors; the lymphocyte infiltration was performed 8 days after treatment initiation. The infiltrating lymphocytes of the combined group at the ultrasound-treated and non-ultrasound-treated tumors were the highest among four groups but not statistically significant. Further analysis of infiltration at the ultrasound-treated side, CD8+ T cells showed significant difference between the combine group and the other three groups. Moreover, the sum of infiltrated IFNγ+CD4+ T cell, IFNγ+CD8+ T cell and IFNγ+ NK cell showed the highest tendency on the combined group. Conclusion In-vitro result showed that hyperthermia increased the cytotoxicity of NK cells on CT26 cells, and corresponding result was found in in-vivo study. pUSHT combined with ACT-NK did inhibit tumor growth in both ultrasound treated and untreated tumors compared to control group; the survival rate of the combined group was also significantly improved, and did not cause excessive burden to mice. H E showed that pUSHT can indeed produce non-thermal effects on the directly treated tumor. Furthermore, in terms of the percentage of CD8+ T cells, there was a significant difference between the control group and the other three groups at the ultrasound-treated side; however, non-ultrasound-treated side didn’t present a significant difference but the highest IFNγ+ lymphocytes infiltrating tendency. As a result, further methods must be taken to break through the tumor's immunosuppressive environment, allowing more immune cells to infiltrate into the non-ultrasound-treated tumor.