臺灣紫芝多醣體對腫瘤相關巨噬細胞的影響

Abstract

臺灣紫芝 (Ganoderma formosanum) 是於臺灣發現的靈芝屬特有種。在本實驗室前人的研究中，臺灣紫芝藉由深層發酵培養所產生的胞外多醣體、再經膠體過濾分劃所得之PS-F2於小鼠體內實驗中具抗腫瘤的效果。在本研究之中，我們探討了PS-F2的抗腫瘤效果是否為透過調節具免疫抑制性的腫瘤相關巨噬細胞 (tumor associated macrophages, TAMs) 來達成。將分離自小鼠C26腫瘤組織的TAM (tumor TAMs, tTAMs) 與naïve小鼠之脾臟巨噬細胞做比較，可得知iNOS、IL-6、IL-1β、TNF-α、arginase-1、IL-10、VEGF-A、CCL-3、CXCL-4、MMP-9、MMP-12、osteopontin (OPN) 等基因於tTAMs之中表現上升。在PS-F2的刺激下，可發現其M1相關基因如iNOS與IL-6等有進一步的表現上升，而arginase-1則下降。tTAMs能抑制CD4+ T cell之增生，此抑制效果能被PS-F2所逆轉。另一方面，本研究亦透過將發炎性單核球 (inflammatory monocytes, IM) 與腫瘤細胞共培養，於體外產生腫瘤相關巨噬細胞 (cultured TAM, cTAMs)。結果顯示，在共培養3天以後與單獨培養組相比，可發現IM已分化為cTAMs，且CD80、CD206等表面分子於cTAMs之表現上升；此外，iNOS、arginase-1、VEGF-A、CXCL-4、OPN等基因表現也較單獨培養組提升。另方面，cTAMs對於抑制T 細胞增生的能力也大於tTAMs。總的來說，PS-F2能促使tTAMs極化為M1，並能減緩tTAMs抑制CD4+ T細胞增生的作用；此外也初步建立了cTAMs之體外分化系統，能作為體外研究TAMs之平台。

Ganoderma formosanum is a native species of Ganoderma first discovered in Taiwan. Our previous studies showed that PS-F2, a polysaccharide fraction purified from the submerged fermentation broth of G. formosanum, exhibits antitumor activities in mice. In this study, we investigated whether PS-F2 may exert the antitumor function by modulating the immunosuppressive phenotypes of tumor-associated macrophages (TAMs). Compared with splenic macrophages, TAMs purified from C26 colon carcinoma (tumor TAMs, tTAMs) expressed elevated levels of iNOS, IL-6, IL-1β, TNF-α, arginase-1, IL-10, VEGF-A, CCL-3, CXCL-4, MMP-9, MMP-12, and osteopontin (OPN). Upon PS-F2 stimulation, the expression of iNOS and IL-6 were further upregulated, and the expression of arginase-1 was downregulated. tTAMs inhibited the proliferation of CD4+ T cells, which could be reversed by PS-F2 treatment. In this study, we also generated TAMs in vitro (cultured TAMs, cTAMs) by coculturing inflammatory monocytes with tumor cells. After coculturing with tumor cells for 3 days, inflammatory monocytes differentiated into macrophages with elevated surface expression of CD80 and CD206 compared with monocultured macrophages. In addition, cTAMs also expressed higher levels of iNOS, arginase-1, VEGF-A, CXCL-4 and OPN than monocultured macrophages. Moreover, compared with TAMs purified from C26 tumors, cTAMs showed stronger suppression on T cell proliferation. In conclusion, our results showed that PS-F2 stimulation promotes M1 polarization of TAMs and alleviates the immunosuppression exerted by TAMs, and the cTAMs differentiation system may serve as a platform for identifying modulatory agents targeting TAMs.