探討預先照射放射線之癌細胞與二甲雙胍對癌腫瘤生長之抑制

Abstract

研究背景與目的：癌症治療當前的困境是在癌轉移以及原位治療後癌復發的情形上無法有強而有效之抑制策略，此二現象同時亦為臨床上造成病人因癌症死亡之兩大宗理由。放射治療除了以放射線之高能量破壞癌細胞殺死癌腫瘤外，其特殊的致死機制亦會造成癌細胞免疫原性死亡，這些免疫原性死亡之癌細胞碎片被免疫系統辨識後可以被當作原位癌症疫苗，進而誘發體內的抗癌免疫反應。只是放射治療引發之抗癌免疫反應往往不夠有效，所以科學家們正致力於找出有效提升放射治療預後效果的可能方法。二甲雙胍為臨床上使用超過半世紀之糖尿病用藥，近年來研究亦相繼證明其與放射治療之協同療效、抗癌機制以及免疫調節能力，因此本研究選擇此藥物，模擬探討其是否能提升原位放射治療後對抗癌腫瘤再生成之抑制效應以及放射治療引發之旁觀者效應。 實驗材料與方法：本研究分成細胞實驗以及動物實驗，癌細胞株皆使用B16F10小鼠黑色素瘤細胞，小鼠品系為C57BL/6。細胞實驗給予二甲雙胍藥物以及將經放射線照射之癌細胞其原先培養環境裡的培養液拿去置換未照射之癌細胞的培養液，透過細胞存活率的分析，分別觀察二甲雙胍的癌毒殺效力以及放射線引發之旁觀者效應。另外，透過克隆檢驗、H&E染色以及TUNEL細胞凋亡檢定，觀察不同時間點下B16F10細胞株在不同劑量照射下之存活情形，以及接受高劑量放射線照射後之形態變化和細胞凋亡之情況。動物實驗分成兩個部分，分別討論預先給予放射線照射之癌細胞作為刺激與二甲雙胍兩者對於B16F10黑色素瘤細胞株腫瘤生成之抑制情形以及B16F10黑色素瘤之放射線引發之旁觀者效應。第一部分各組於第0天時於右側皮下接種1x105顆活的癌細胞，有預先照射放射線之癌細胞刺激的組別還會於第-14、-12、-10天分別於左背側皮下注入3x105顆甫接受75 Gy照射的癌細胞，模擬原位腫瘤接受放射治療時的原位癌症疫苗生成；第二部分除了在第0天種植活細胞時同時混合植入1x105顆甫照射完75 Gy放射線之癌細胞外，其餘條件皆與第一部分相同。兩部分動物實驗都分成四組，包括控制組、二甲雙胍組、預先注射經放射線照射之癌細胞刺激組以及預先注射經放射線照射之癌細胞刺激搭配二甲雙胍組，其中有服用二甲雙胍的組別每日給予的藥物劑量為200 mg/kg。此外，動物樣本部分，於第16天時犧牲小鼠取下腫瘤，做切片上的TUNEL檢定和H&E、Ki67、CD3+染色，並且對細胞凋亡相關標誌進行西方墨點法分析。 實驗結果：細胞存活率分析上，二甲雙胍在B16F10黑色素瘤上確實能有抑制腫瘤生長之毒殺效力；然而，培養液置換試驗中，於本研究的參數條件下並無法觀察到此細胞株的放射線引發之旁觀者效應。藉由克隆檢驗、H&E染色觀察以及TUNEL檢定分析，足以推論在照射75 Gy放射線後，B16F10黑色素瘤確實會完全死亡，同時有細胞凋亡的情形。動物實驗結果顯示，對抗遠端再生成腫瘤，使用預先注射經放射線照射之癌細胞刺激搭配二甲雙胍有較佳之腫瘤抑制效果；此外，在B16F10黑色素瘤上，放射治療的癌症旁觀者效應的確存在，並且搭配預先注射經放射線照射之癌細胞刺激還能有效提升該旁觀者效應。然而，本研究雖有觀察到程度不等的腫瘤抑制效果，但各治療對於小鼠整體存活率之提升皆相當有限。樣本分析上，TUNEL檢定在有預先注射經放射線照射之癌細胞刺激的組別中皆呈現上升的趨勢，然而caspase-3及c-PARP此二細胞凋亡之標記表現在治療組中呈現減少的趨勢，同時Ki67免疫組織化學染色亦顯示本研究之實驗設計無法在腫瘤內部減少此腫瘤增生標誌之表現；免疫螢光染色的部分則可以發現CD3+ T淋巴球於腫瘤區域浸潤之分布和腫瘤生長的抑制情形呈現正相關。 結論：本研究於動物實驗發現放射線引發之旁觀者效應於B16F10黑色素瘤中確實存在，且透過預先注射經放射線照射之癌細胞作為刺激可有效提升此效應；然而，二甲雙胍在協同給予的動物組別中，僅有有限之能力去提升放射線照射之癌細胞刺激對於腫瘤生長之抑制。本研究觀察到巨觀上腫瘤生長抑制之成效，並且在放射線引發之旁觀者效應方面，為未來之前驅研究建立了動物理論基礎。然而，在許多微觀的相關機制分析及驗證上，仍尚待分析及釐清。

Background and Purpose: Cancer therapy has become more reliable and efficient in the clinic. However, there is still a need for improving overall survival and dealing with metastasis. Radiotherapy is used to treat approximately half of cancer patients. It kills cancer cells and turns the debris into internal vaccines to patients. Metformin, a commonly used drug in patients with type two diabetes, was reported with anti-cancer and immunomodulation abilities. This study aims to investigate systemic cancer treatment regarding tumor recurrence and radiation-induced bystander effects (RIBE) by using metformin and pre-irradiated cancer cells. Materials and Methods: B16F10 melanoma was used for both in-vitro and in-vivo study. MTT assay and clonogenic assay were conducted for viability of cells treated with metformin and irradiation, respectively. Transfer medium assay (TMA) was designed to test RIBE in-vitro. Morphology and apoptosis of B16F10 melanoma after 75 Gy irradiation were examined by H&E staining and TUNEL assay. C57BL/6 mice were employed as an tumor model in-vivo. Pre-irradiated (75 Gy) stimulation was utilized to imitate in-situ vaccines formation after radiotherapy, and metformin was given in 200 mg/kg/day. In part A, mice were divided into control (Ctrl), pre-irradiated (pre-IR), metformin (Met) and pre-irradiated combined with metformin (pre-IR + Met) groups. Pre-irradiated cancer cells (3 x 105) were subcutaneously injected on left flanks of mice on Day -14, Day -12 and Day -10. Cells (1 x 105) were subcutaneously injected on right flanks of mice on Day 0. To characterize RIBE, modified setting with pre-irradiated and living B16F10 cancer cells (1:1) were inoculated together on right flank on Day 0 in part B. Four groups included mixed control (Mixed Ctrl), mixed pre-irradiated (Mixed Pre-IR), mixed metformin (Mixed Met) and mixed pre-irradiated combined with metformin (Mixed Pre-IR + Met). Tumor volume and survival fraction were examined for the efficacy study. Mice were sacrificed after cardiac perfusion on Day 16, then tumors were embedded for section staining and lysed for protein quantification. H&E staining, TUNEL assay, Ki67 immunohistochemistry staining and CD3+ immunofluorescent staining were analyzed using tumor sections. Apoptosis related markers were analyzed via Western blot. Results: The toxicity of metformin and irradiation towards B16F10 were examined by MTT assay and clonogenic assay. However, there is no significance between groups treated by TMA in-vitro. Cells were fully destroyed via apoptosis after high-dose irradiation by H&E staining and TUNEL assay. Pre-IR + Met has the best effects on inhibiting B16F10 tumor formation in-vivo in part A, while RIBE is likely to appear on B16F10 melanoma due to the slow tumor formation in part B. Collectively, pre-irradiation could probably enhance RIBE systemically, but with limited effects on overall survival. Moreover, surviving cancer cells could have a higher treatment tolerance owing to remarkable decrease of caspase-3 and c-PARP. Although mouse models showed notable tumor inhibition, data suggested that progressive abilities of tumor could not be reduced by these treatments. Conclusion: Taken together, pre-irradiation in distant sites can effectively enhance RIBE to limit tumor growth. This might be due to the systemic immunomodulation after pre-irradiation inoculation. However, further study is needed to elucidate the mechanisms. This study contributes to the knowledge of RIBE regarding B16F10 melanoma, thus provides novel insights into pre-clinical and clinical radiotherapy.