結合超音波腫瘤碎片與免疫刺激劑強化抗腫瘤免疫效應

Combination of Ultrasonicated Tumor Lysate and Immunostimulant for Boosting Antitumor Immunity

張定洋(Ting-Yang Chang)

指導教授 ： 林文澧

共同指導教授 ： 繆希椿(Shi-Chuen Miaw)

國立臺灣大學/工學院/醫學工程學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202002168

摘要

研究背景與目的:
根據中華民國衛生福利部國民健康署2019的統計，大腸直腸癌已經連續為國人10大癌症之首。目前大腸直腸癌的治療主要以手術切除配合放射線療法及化療的聯合治療方式。近年來日漸興起的免疫療法在大腸直腸癌的研究上，主要是以免疫檢查點抑制劑搭配放射線治療或是化療的聯合治療。因此目前可以看出在治療晚期的腸癌，免疫療法是一個重要的輔助角色。在本研究中將使用超音波結合免疫刺激劑，刺激免疫細胞以及提供腫瘤相關抗原，觀察對腫瘤的治療效果。超音波可震碎腫瘤細胞，提供腫瘤相關抗原，作為免疫細胞辨識的材料，達成強化抗腫瘤免疫的效果。
材料與方法:
在小鼠皮下植入CT26腫瘤細胞，待成長至50-100mm3時將小鼠分成控制組、OK-432組、Lysate組以及OK-432+Lysate組。控制組在皮下注射生理食鹽水、OK-432組注射0.25K.E./100μL的OK-432、Lysate組注射106 CT26 cell-lysate/100μL的細胞碎片懸浮液、OK-432+Lysate組注射0.25K.E.+106 CT26 cell lysate/100μL。紀錄小鼠腫瘤生長趨勢並將動物犧牲後取下腫瘤，製作切片觀察腫瘤的型態以及細胞凋亡狀況，最後分析腫瘤組織內細胞激素的變化。
實驗結果:
本研究統計結果顯示施打免疫刺激劑(Ok-432)及腫瘤碎片的結合組在腫瘤抑制效果上顯著高於控制組、腫瘤碎片組以及免疫刺激劑組。小鼠存活率的表現上結合組也優於其他組別。而其他組別之間無論是腫瘤體積或是存活率都沒有統計上的顯著差異。不過在非侵入式影像系統的表現上，結合組的腫瘤活性在統計上顯著低於免疫刺激劑組，與其他組並無顯著差異。本研究針對腫瘤組織的切片進行分析，結合組的細胞凋亡的表現也高於其他三組。細胞激素的聚合酶連鎖反應分析結果顯示IL-6 、IL-12以及iNOS的RNA表現都有顯著上升。
結論:
腫瘤經過超音波結合免疫刺激劑的治療後，腫瘤生長在外觀上確實有受到抑制。受到結合治療的影響，抗腫瘤免疫效應的細胞激素產生可判斷為第一型巨噬細胞有活化的現象。

關鍵字

超音波機械力 ； OK-432免疫刺激劑 ； 內源性危險因子 ； 抗癌免疫反應 ； 癌細胞碎片

並列摘要

Background and purpose:
According to statistics of cancer from Health Promotion Administration, Ministry of Health and Welfare in 2019, incidence of colorectal cancer has ranked top one for several years. Therapies performed routinely for colorectal cancer are tumor resection combined with chemotherapy or irradiation. Beside routine therapy, immunotherapy for colorectal cancer has been developing currently. Immunotherapy plays an vital role in the late stage of colorectal cancer. In this study, we combined ultrasonicated tumor lysate and immunostimulant to boost antitumor immunity, in order to inhibit tumor growth.
Materials and methods:
In this study, we implanted 106 CT26 tumor cells subcutaneously on the right flank of each BALB/c mouse. After the tumor volume reached 50-100mm3, we then divided the mice into control, Lysate, OK-432 and Lysate+OK-432 group. Cell lysate was prepared with a Bioruptor, sonicated 35 minutes (duty cycle: 50%, high intensity). Injectable saline, CT26 cell lysate (106 cells/100μL), OK-432 (0.25 K.E./100μL) and Lysate+OK-432 were given subcutaneously once every two days for three times. Tumor volume was assessed by Vernier caliper and IVIS as well. The tumor was resected to perform H E stain, TUNEL assay (IF) in order to observe morphology and cell apoptosis. Real time PCR was performed to analyze inflammatory mediators, in order to evaluate the boosting antitumor effect of Lysate and immunostimulant.
Results:
The results show that the tumor growth is significantly slower in the lysate+OK-432 group than the other three groups. The IVIS images also reveal that the light intensity of tumor in the Lysate+OK-432 group is milder than the other three groups. The survival rate of the lysate+OK-432 group is the highest among all groups within a month of observation. The TUNEL assay demonstrates that DNA double strands break in the lysate+OK-432 group is significantly higher than the other three groups. The outcome of real-time PCR reveals that IL-6, IL-12 and iNOS show a significant increase in the combined group. It may refer to type one macrophages activation. IL-12 secretion can activate cytotoxic T cells, helper T cells and natural killer cells, thus the adaptive immune may be activated by the treatment.
Conclusion:
The results show that lysate cancer cells plus immunostimulant can inhibit tumor growth. Antitumor immunity was boosted by the combined treatment since inflammatory cytokines increased. The mechanism of how cytokines affect immunity should be further investigated.

並列關鍵字

ultrasound mechanical force ； OK-432 immunostimulant ； danger associated signal ； antitumor immunity ； cancer cell lysate

參考文獻

[1] Ganesh, K., et al. " Immunotherapy in colorectal cancer: rationale, challenges and potential". Nature Reviews Gastroenterology Hepatology, 2019; 16(6): 361-375.

[2] Le, D. T., et al. "Programmed death-1 blockade in mismatch repair deficient colorectal cancer." Journal of Clinical Oncology 2016; 34(15): 103-103.

[3] Le, D. T., et al. "Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade." Science 2017; 357(6349): 409-413.

[4] Hochster, H. S., et al. "Efficacy and safety of atezolizumab (atezo) and bevacizumab (bev) in a phase Ib study of microsatellite instability (MSI)-high metastatic colorectal cancer (mCRC)." American Society of Clinical Oncology 2017; 35(4): 673-673.

[5] Bendell, J. C., et al. "Safety and efficacy of MPDL3280A (anti-PDL1) in combination with bevacizumab (bev) and/or FOLFOX in patients (pts) with metastatic colorectal cancer (mCRC)." American Society of Clinical Oncology 2015; 33(3): 704-704.