應用免疫刺激劑與自然殺手細胞來強化超音波治療所引發的抗癌免疫反應

Abstract

研究背景：時至今日，對於腫瘤共同特徵的研究，普遍已將「調控抗癌免疫機制」視為設計新一代癌症治療策略時的關鍵考量因素，希望藉由重新導正患者的免疫平衡，走向增強自身抗癌免疫力，進而達到提升腫瘤控制率，降低復發和轉移機率，延長存活期，提升整體治療效益之目的。而近年來，有越來越多的研究證據顯示超音波熱治療於「增強全身性或局部性抗癌免疫力」方面獨具潛力，主要透過揭露腫瘤抗原、活化發炎細胞、重新調整免疫系統平衡等方式，使得腫瘤細胞更容易被免疫系統清除。 研究目的：本研究利用脈衝式超音波熱治療（pUSHT）的熱效應與機械力作為披露腫瘤特異性抗原的主要工具，加上OK-432免疫刺激劑，刺激多種免疫細胞活化與擴增，誘發多種抗癌細胞因子分泌，再輔以過繼性自然殺手細胞轉移療法（ACT-NK），給予大量健康且胞殺能力增強的自然殺手細胞（NK），取代本身疲累且虛弱的NK細胞，可望達到大幅提升抗癌免疫力之效。 材料方法：為了能探討抗癌免疫力的引發成效，本研究使用兩種不同的腫瘤模式：雙側遠端腫瘤模式、再刺激腫瘤模式。在雙側遠端腫瘤模式部分，小鼠雙側背部被皮下植入CT26-luc-GFP大腸直腸癌細胞，右側作為治療側，左側作為遠端非治療側。治療側腫瘤被給予為期10天的治療，包含4次超音波熱治療（每3天1次）、4次OK-432皮下注射（每次熱治療前3小時）、2次NK細胞腫瘤內注射；遠端側腫瘤模擬影像上難見的小腫塊，作為評估抗癌免疫反應程度的依據。在再刺激腫瘤模式方面，小鼠右側背部皮下植入腫瘤細胞並待長成50~100 cm3實質腫瘤後，進行為期5天的合併治療，治療結束後隔天手術切除腫瘤及周圍皮下組織（含表皮），並待縫合傷口恢復並確定無原位腫瘤復發後，第10天於對側（左側）背部皮下重新植入腫瘤細胞，作為再刺激測試，觀察經OK+pUSHT+NK治療後，是否能建立足夠有效且長期的抗癌免疫力，抑制腫瘤復發機率。所有的實驗資料皆以獨立T檢定或ANOVA（搭配Fisher's LSD test多重比較）進行統計分析，P value設為0.05；存活率部分則使用Kaplan-Meier法進行統計檢定。 研究結果：根據實驗結果，在雙側遠端腫瘤模式部分，無論是在治療側或遠端側，OK+pUSHT+NK組的腫瘤生長和活性皆受到顯著性抑制，且該抑制效果有延長之趨勢。H&E切片染色可看出，OK+pUSHT+NK合併治療使得腫瘤組織內壞死情形更為嚴重、廣泛且提早；IF染色結果則顯示，經OK+pUSHT+NK合併治療後，治療側和遠端側腫瘤內NK細胞浸潤量皆大幅提升。而存活率於OK+pUSHT和OK+pUSHT+NK兩組也都明顯增加。此外，根據腫瘤再刺激測試結果，和Control組相比，OK+pUSHT和OK+pUSHT+NK組的再刺激腫瘤皆無法形成實質腫塊，顯示本研究治療策略確實能引發有效、長期且具記憶性的免疫反應。 結論：本研究證實了超音波熱治療的確具有作為癌症免疫治療輔助工具的高度潛力，能透過抗原揭露和破壞免疫抑制微環境，重新調整免疫系統平衡，活化抗癌相關免疫反應，再結合多種免疫療法或刺激劑進一步增強此抗癌效益，實為未來足具發展潛力的抗癌合併治療策略。

Background: The capability of tumors for modulating anti-tumor immune response and orchestrating an immune permissive microenvironment was reported as one of the cancer hallmarks. Therefore, unlike the past concept of cancer therapy which simply aims at killing tumor cells and ignores the tolerance of the host itself to the immune-mediated attack, the emerging strategy for cancer treatment includes not only extirpating all localized tumor cells, but also arousing a systemic, long-lasting, and tumor-specific immune response synchronously, against tumor growth, recurrence and metastasis to achieve the ultimate objective: prolongation of survival times. In addition, ultrasound hyperthermia has been used against various types of cancer and recent studies demonstrated its potential of activating a systemic anti-tumor immune response. Purpose: To evaluate whether an inducible anti-tumor immunity could be enhanced systemically through combining immunotherapies, in this study we exposed immunogenic moieties from tumor cells by sonicating tumors with pulsed ultrasound hyperthermia (pUSHT) and strengthened the anti-cancer immunity in an all-round way by injecting tumors with OK-432 immunostimulant and adoptive natural killer cell transfer therapy (ACT-NK). Methods and Materials: A bilateral distant tumor model and a rechallenging tumor model were applied in the studies. For the bilateral distant tumor model, male BALB/c mice were inoculated CT26-luc-GFP tumors on both flanks. The treated tumor (right side) underwent a 10-day treatment including four times of pUSHT (once per 3 days), four times of subcutaneous OK-432 injection (3 hours before each pUSHT) and two times of local ACT-NK injection. The distant untreated tumor (left side) was regarded as a non-visualized small tumor used to assess the degree of anti-tumor immune response. For the rechallenging tumor model, a rechallenge tumor was implanted contralaterally on day 10 after the right-sided tumor had experienced a 5-day treatment (days 0~4) and then a surgical removal on day 5. This model was designed to evaluate whether or not the establishment of a long-term active tumor-specific immune memory is strong enough to prevent recurrence. All data was analyzed statistically by independent T-test or ANOVA with Fisher's LSD test as a multiple comparison tool alternatively, and survival time was evaluated by the Kaplan-Meier method. Results: The results of bilateral distant tumor model showed that the tumor growth rate and growth activity of both treated and distant tumors could be significantly inhibited with OK+pUSHT+NK combined therapy (p value＜0.05) and the systemic anti-tumor effect seemed to be prolonged as well. H&E staining results implicated that the necrosis area appeared earlier after the combination treatment. IF staining results also showed a remarkable increase of NK cell infiltration in the central zone of tumors due to the OK+pUSHT+NK therapy. Survival rates significantly increased in both OK+pUSHT and OK+pUSHT+NK groups. In the rechallenging test, all the reimplanted tumors failed to form solid tumors as compared with the control group due to an effective long-lasting anti-tumor immune response which was induced by the combinational treatment. Conclusion: Combining ultrasound hyperthermia and multiple immunotherapies could lead immune system toward an anti-tumor regulation to overcome an immunosuppressive milieu of tumors. This combined approach has a potential to play an important role in the future cancer treatment of solid malignancy to improve the overall therapeutic benefit.