| 摘要: | 背景:
克服免疫抑制性的腫瘤微環境對於實現癌症免疫治療策略的潛力至關重要。最近的研究表明，環狀二鳥苷酸（c-di-GMP）是一種分子佐劑，可通過抗原呈現細胞中(APC)干擾素基因（STING）路徑的刺激物誘導產生I型干擾素（IFN），導致增強腫瘤免疫原性。然而，帶負電的c-di-GMP的功效可能受到一些固有缺點的限制，包括膜滲透性差，被快速清除和低效率的細胞質傳遞。因此，我們以奈米技術為基礎嘗試發展替代的“原位疫苗接種”方法，以啟動癌症治療的抗腫瘤免疫反應。
目的:
本實驗的目的為合成帶正電的中孔洞二氧化矽奈米粒子(MSN)，將c-di-GMP帶入4T1乳癌接種小鼠中，接著引發腫瘤免疫原性，以及成功抑制腫瘤生長。
材料與方法:
透過水解縮合形成MSN，接著在表面修飾上PEGylated和帶正電的分子(N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride) TA，並且接上螢光標記物RITC，來合成RMSN-PEG/TA。利用穿透式電子顯微鏡(TEM)、動態光散射儀(DLS)和氮吸脫附等溫線確定奈米材料的特徵。藉由正負電吸引作用將帶負電的c-di-GMP裝載至帶正電的RMSN-PEG/TA中形成c-di-GMP @ RMSN-PEG / TA。利用qPCR和ELISA測定由c-di-GMP @ RMSN-PEG / TA誘導Raw 267.4細胞產生的細胞因子。西方墨點法檢測了STING蛋白的表現程度。4T1乳癌細胞接種BALB/c小鼠在第8、11、14天腫瘤內注射c-di-GMP @ RMSN-PEG / TA。腫瘤移植後的抗腫瘤效用包括腫瘤大小，小鼠體重與病理分析被追蹤。另外，透過流式細胞儀了解細胞吞噬奈米材料和免疫細胞滲透浸潤的比例。
結果:
我們合成了RMSN-PEG/TA奈米顆粒，並且帶有均勻的孔洞，平均大小為 25 nm，c-di-GMP在c-di-GMP @ RMSN-PEG / TA上的裝載重量百分比為約3wt％。用c-di-GMP @ RMSN-PEG / TA治療的RAW264.7細胞明顯增加IL-6，IL-1β和IFN-β的產生，以及磷酸化STING（Ser365）蛋白的表現。動物實驗結果顯示明顯抑制腫瘤生長，伴隨著腫瘤部位的活化的CD11c +樹突細胞和CD8 + T細胞的滲透浸潤。
討論:
我們透過STING路徑確認原位疫苗接種確實可以為乳腺癌提供一種有吸引力的治療方法，而且也凸顯出改善癌症免疫療法的臨床潛力。
Background:
Overcoming the immunosuppressive tumor microenvironment is critical to realizing the potential of cancer immunotherapy strategies. Recent evidences are emerging to show that the cyclic di-guanylate (c-di-GMP), a molecular adjuvant, could induce the production of type I interferons (IFNs) via the stimulator of interferon genes (STING) pathway in antigen presenting cells (APC), leading to enhance the tumor immunogenicity. However, the efficacy of negatively charged c-di-GMP may be limited by some inherent shortcomings, including the poorly membrane permeable、rapid clearance and the inefficiency of cytosolic delivery. Hence, we attempt to develop an alternative “in situ vaccination” approach based on nanotechnology to initiate an antitumor immune response for cancer therapy.
Aim:
The aim of this study to synthesize cationic mesoporous silica nanoparticles (MSNs), which are employed to deliver c-di-GMP into 4T1 breast tumor-bearing mice, followed by triggering the tumor immunogenicity, as well as the inhibition of tumor growth successfully.
Material and Methods:
PEGylated RITC fluorescent MSN with a positively charged molecule (N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride, TA) by co-condensation was synthesized to form RMSN-PEG/TA. The characteristics of nanoparticles were determined by transmission electron microscopy (TEM), dynamic light scattering (DLS) and nitrogen adsorption-desorption isotherm. The anionic c-di-GMP was loaded into cationic RMSN-PEG/TA via electrostatic interactions (c-di-GMP@RMSN-PEG/TA). The production of cytokines induced by c-di-GMP@ RMSN-PEG/TA was analyzed by real-time PCR and ELISA assay in Raw 264.7 cells, respectively. Western blot investigated the expression level of STING protein. The mouse 4T1 breast tumor-bearing Balb/c mice received intratumoral injection of c-di-GMP@ RMSN-PEG/TA on day 8, day 11, and day 14. Antitumor effects, including tumor size, body weight and pathologic analysis had been tracked after tumor implantation. Also, the proportion of cellular uptake and infiltration of activated immune cells were detected by flow cytometry.
Results:
We synthesized well-ordered RMSN-PEG/TA nanoparticles with an average diameter of 25 nm. The loading weight percent of c-di-GMP on c-di-GMP@ RMSN-PEG/TA was around 3 wt%. RAW264.7 cells treated with c-di-GMP@ RMSN-PEG/TA obvious increased the production of IL-6, IL-1β, and IFN-β, as well as the phospho-STING (Ser365) protein expression. In vivo results revealed dramatically tumor growth inhibition accompanied by the infiltration of activated CD11c+ dendritic cells and CD8+ T cells at the tumor site.
Conclusion:
We validate this “in situ vaccination” by STING pathway activation provides an attractive therapeutics for breast cancer, highlighting its potential to improve clinical outcomes of cancer immunotherapy. |
