探討地西他濱對肺癌受到γδ T細胞毒殺之增強作用

Abstract

肺癌在台灣為十大癌症死亡原因第一名，且有逐年增加的趨勢。癌細胞具有和其他正常細胞不同的蛋白質表現，可被免疫細胞識別並進行攻擊。然而，藉由不正常的表觀遺傳基因調控，癌細胞能隱藏這些細胞表面標記的表達，進而躲過免疫細胞的攻擊。DNA甲基化為細胞內重要的表觀遺傳修飾機轉，由DNA甲基化轉移酶（DNA methyltransferase, DNMT）催化，如果在錯誤的地方甲基化，或未進行甲基化，都可能造成細胞癌化。目前Decitabine (DAC)為已上市的DNMT 抑制劑（inhibitor），臨床上使用於原發性或續發性急性骨髓性白血病病患，在高劑量之下會造成DNA損傷，產生細胞毒性，但是在低劑量時，卻能產生有記憶性的持續抗腫瘤效果。本實驗室於先前之SILAC蛋白質體分析中，發現肺癌細胞株在給予DAC之後，其細胞表面改變的免疫相關蛋白，以伽瑪-德耳塔（gamma-delta, γδ）T細胞活化的路徑最為顯著。γδ T在人體內淋巴細胞中僅佔1-3%，此類細胞與一般αβ T不同，不需藉由樹突細胞等抗原呈現細胞提供敵物抗原，即可以活化並毒殺癌細胞，而存在肺部的多屬於Vδ1+型，根據先前文獻指出，和其他γδ T亞群相比，以Vδ1+治療癌症的存活率最高。因此本研究主要目的為，觀察受DAC處理過的肺癌細胞是否較易受到Vδ1+型γδ T細胞攻擊，並深入探討其分子機轉。 研究發現，藉由Annexin V 和 Propidium iodide的細胞凋亡實驗結果得知，DAC合併Vδ1+細胞治療，對肺癌的毒殺效果 比單獨使用DAC或是Vδ1+細胞治療更佳，而在給予DAC之後，可以發現肺癌細胞與Vδ1+細胞間形成功能性免疫突觸（functional immune synapses）的比例明顯增加；我們也進一步以縮時攝影的方式觀察到，DAC 治療後的癌細胞，被Vδ1+細胞毒殺的數目增加。我們根據先前SILAC蛋白質體分析的資訊，發現細胞黏附因子ICAM-1與DAC導致之Vδ1+細胞毒殺增強效應最具相關性。我們進一步剔除或過度表達肺癌細胞株上的ICAM-1，發現當ICAM-1被剔除後，Vδ1+對癌細胞的毒殺能力明顯降低，證實ICAM-1在γδ T對癌細胞的毒殺作用扮演重要角色。此外，我們也藉由螢光染色影像分析，發現在DAC處理後的癌細胞表面免疫突觸處，纖維形肌動蛋白（filamentous actin, F-actin）的訊號會明顯增加，這些觀察顯示，表觀遺傳藥物可藉由重塑癌細胞免疫突觸細胞骨架（cytoskeletal remodeling），增加癌細胞受到免疫毒殺的作用。動物實驗亦證實，表觀遺傳藥物與γδ T聯合治療可增加肺癌小鼠之存活率。綜合上述研究結果可知，肺癌細胞經由DAC處理之後，能夠促進Vδ1+的毒殺效果，而ICAM-1在其中扮演重要角色。未來希望能更深入了解DAC對於Vδ1+的活化機轉，並進行分子機制的觀察，以期能應用於臨床肺癌治療。

Lung cancer is the leading cause of cancer death in Taiwan, and its incidence is on the rise. Cancer cells may express surface molecules distinctive from those on normal cells and thus can be recognized and killed by immune cells. However, many cancer cells develop strategies to escape from immunosurveillance. For example, DNA methylation-mediated downregulation of surface molecules is one of the escape mechanisms that render tumor cells invisible to the body's immune system. DNA methylation is an epigenetic mechanism in which DNA methyltransferases (DNMTs) transfer methyl groups to DNA. Two DNA methyltransferase inhibitors (DNMTis), decitabine (DAC) and azacitidine (AZA), are used clinically in treating hematological malignancies. Previous studies showed that at high doses, DAC inhibits cell proliferation via incorporation into DNA, leading to DNA damages. At low doses, the drug can exert sustained changes in gene expression and produce a memory type of antitumor response. Through Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based quantitative surface proteomics analysis, our lab demonstrated that DAC modulates surface immune proteomes in lung cancer cell lines. Notably, the alterations are highly associated with γδ T cell activation. γδ T cells are a subset of T cells with anti-tumor effects that do not rely on major histocompatibility complexes (MHC) for antigen-recognition. They account for only 1-3% of all lymphocytes in the circulation. The majority of resident γδ T cells in the lungs are the Vδ1 subtype. We found that pretreatment of lung cancer cells with 100 nM DAC can enhance their susceptibility to Vδ1-enriched γδ T cell killing in vitro using Annexin V and propidium iodide apoptosis assays. This potentiation effect appears to result from enhanced formation of immune synapses and the cytoskeleton remodeling at the interface between cancer and γδ T cells. Furthermore, knockout of ICAM-1, an adhesion molecule at immune synapses, diminished the potentiating effects of DAC on lung cancer cells for γδ T killing. In addition, we observed a significant increase of F-actin at immune synapses, which was accompanied by global transcriptomic changes of the cytoskeleton following DAC treatment. Collectively, these data indicated that DAC could regulate patterns of cytoskeleton to stabilize the structure of immune synapses. Most importantly, combination therapy of DNMTi and adoptive γδ T transfer offered a significant survival benefit in a mouse lung cancer model. Our data suggested that DNMTis may couple with adoptive γδ T cell transfer in cancer therapeutics.