臺灣紫芝多醣體合併CTLA4阻斷劑之抗腫瘤效果

Abstract

靈芝是一種著名的藥用真菌，幾個世紀以來一直在亞洲用於治療各種疾病，而台灣紫芝 (Ganoderma formosanum) 是一種在台灣特有的靈芝品種。我們使用液態深層菌絲體培養來生產G. formosanum胞外多醣，並且經過膠體過濾法進行純化，得到三個主要分劃 (PS-F1、PS-F2、PS-F3)。本實驗室先前研究表明，G. formosanum的PS-F2具有抗腫瘤功能和免疫調節活性。在本研究中，我們研究了PS-F2結合免疫檢查點阻斷劑 (immune-checkpoint blockers; ICBs) anti-CTLA4抗體是否在CT26結直腸腫瘤小鼠中具有協同抗腫瘤作用。本研究共使用三種不同劑量anti-CTLA4抗體進行合併治療。結果表明，單獨口服PS-F2即可抑制腫瘤的生長，單獨給予anti-CTLA4抗體亦呈現劑量效應之抗腫瘤作用，並且PS-F2與anti-CTLA4抗體的合併治療可以更進一步抑制腫瘤生長。PS-F2與anti-CTLA4抗體單獨療法及合併療法皆能抑制帶腫瘤小鼠的脾臟腫大，且不會對動物之健康狀況造成顯著負面影響。在脾臟中，單獨與合併治療組其自然殺手T細胞比例有上升趨勢，而多核型骨髓衍生抑制細胞比例有下降趨勢。在腫瘤浸潤淋巴結中，合併治療組其調節型T細胞有上升趨勢。在腫瘤微環境中，單獨與合併治療組其第一型輔助T細胞及胞殺性T細胞比例有上升趨勢，而腫瘤相關巨噬細胞總數有下降趨勢。在腫瘤組織mRNA表現上，單獨給予PS-F2可顯著提升促發炎基因IL-1β、iNOS、TNF-a、IFN-β，而抑發炎基因TGF-β有顯著下降，Arginase-1則有下降趨勢。單獨給予anti-CTLA4抗體時，促發炎基因IL-1β、iNOS、TNF-a與抑發炎基因arginase-1、TGF-β對比PBS組皆顯著下降。另外合併治療對於上述基因表現之影響，則介於PS-F2與CTLA4單獨治療之間。整體來說，我們的數據證明PS-F2可通過活化抗腫瘤免疫反應發揮其抗腫瘤功能，並且當結合anti-CTLA4免疫療法時這些效果可以進一步增強，進而對於腫瘤生長產生加乘性之抑制效果，顯示PS-F2具有單獨用於輔助免疫療法，或與ICB聯合用於治療結直腸癌之潛力。

Ganoderma is a renowned medicinal fungus and has been used for treating various diseases for centuries in Asia, and Ganoderma formosanum is a native Ganoderma species isolated in Taiwan. We have used the submerged mycelial culture to produce G. formosanum polysaccharides, and three polysaccharide fractions (PS-F1, PS-F2 and PS-F3) were purified by gel filtration chromatography. Our previous study showed that PS-F2 of G. formosanum has antitumor and immunomodulating activities. In this study, we investigated whether the combination of PS-F2 and the immune checkpoint blocker (ICB), anti-CTLA4 monoclonal antibody (mAb), had a synergistic antitumor effect in CT26 colorectal tumor-bearing mice. The results showed that oral administration of PS-F2 alone could suppress the growth of established tumor, and combined treatment with an anti-CTLA4 mAb could further inhibit tumor growth. PS-F2 monotherapy and combined therapy also suppressed splenomegaly in tumor-bearing mice without causing adverse effects on animals’ health. In the spleen, the combination therapy resulted in a significant increase in the proportion of IFN-γ+ natural killer T (NKT) cells and a decrease in polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). In the tumor microenvironment, the combination therapy increased the proportion of type 1 T helper (Th1) cells and decreased the accumulation of tumor-associated macrophage (TAM). In the tumor tissue, PS-F2 administration significantly increased the expression of proinflammatory genes IL-1β, iNOS, TNF-α, and IFN-β, while downregulating the expression of anti-inflammatory genes TGF-β. In contrast, anti-CTLA4 treatment suppression the expression of both proinflammatory and anti-inflammatory genes. The combined treatment affected the expression of pro-/anti-inflammatory genes in the tumor tissue similar to anti-CTLA4 monotreatment. Overall, our data demonstrate that PS-F2 exerts its antitumor function by activating antitumor immune responses, and these effects can be further enhanced while combining the anti-CTLA4 immunotherapy, indicating that PS-F2 has the potential to be used in the adjuvant immunotherapy alone or in the combination with ICB for the colorectal cancer treatment.