Imiquimod-Loaded Phospholipid-Free Unilamellar Vesicles Activate the Tumor Immune Microenvironment to Treat Liver Cancer and Liver Metastases

Liver cancers are often diagnosed at advanced stages and the fourth most common cause of cancer death globally. Addition to primary tumours, liver metastases are also common from other cancer sites like colorectal cancers, where 66% of patients develop this condition. Immunotherapies for these diseases have been hindered by the immunosuppressive microenvironment. Our lab has developed phospholipid-free unilamellar vesicles (PFSUV) to localize a toll-like receptor 7 agonist Imiquimod (IMQ), to the hepatocytes. PFSUV is composed of 83 mol% cholesterol and 17 mol% Tween80 and IMQ was loaded inside the aqueous core of these 75-nm particles. When administered intravenously (i.v.), PFSUV-IMQ was found to sustain IFN-α levels in the liver over 24 hours, with significantly lower plasma IFN-α levels compared to free IMQ. In a mouse model of liver metastasis from a colorectal cancer origin generated with CT26 cells, mice treated with PFSUV-IMQ in combination with Oxaliplatin showed a significant reduction in tumour size, with increases in CD8+ T cell and a higher percentage of apoptotic cells in the tumour compared to controls. In a mouse model of primary liver cancer using HCA-1 cells, the same treatment combination also showed a significant decrease in tumour burden, increased apoptotic cell percentage in the tumour, and significantly reduced lung metastasis from the liver compared to controls. Flow cytometry analysis showed tumours treated with PFSUV-IMQ had increased infiltration of CD86+/MHC-II+ dendritic cells and IFN-y+ CD8+ T cells. RNA-seq showed an enrichment of innate immune activation genes after a single dose of PFSUV-IMQ. Overall, these data suggest that the activation of the tumour immune microenvironment through this targeted delivery of IMQ resulted in a decrease in tumour burden across both liver tumour models. Comparative gene expression profiling analysis of RNA-seq data for murine HCC tumors with or without treatment of IMQ-NP.

肝癌通常在晚期才被确诊，是全球第四大常见癌症致死病因。除原发肿瘤外，肝转移瘤也常见于结直肠癌等其他癌种来源，此类患者中66%会发生该病症。上述疾病的免疫治疗因免疫抑制微环境的存在而受到阻碍。本课题组开发了无磷脂单层囊泡（phospholipid-free unilamellar vesicles, PFSUV），用于将Toll样受体7激动剂（toll-like receptor 7 agonist）咪喹莫特（Imiquimod, IMQ）靶向递送至肝细胞。PFSUV由83 mol%胆固醇与17 mol%吐温80（Tween80）组成，IMQ被装载于此类75 nm颗粒的水相核心中。经静脉注射（intravenously, i.v.）给药后，PFSUV-IMQ可在肝脏中维持干扰素-α（IFN-α）水平超过24小时，且其血浆IFN-α水平显著低于游离IMQ。在采用CT26细胞构建的结直肠癌源性肝转移小鼠模型中，与对照组相比，PFSUV-IMQ联合奥沙利铂（Oxaliplatin）治疗的小鼠肿瘤体积显著缩小，肿瘤内CD8阳性T细胞浸润增加，凋亡细胞占比更高。在采用HCA-1细胞构建的原发性肝癌小鼠模型中，该联合治疗方案同样可显著降低肿瘤负荷，提高肿瘤内凋亡细胞占比，并较对照组显著减少肝脏来源的肺转移。流式细胞术分析显示，PFSUV-IMQ治疗后的肿瘤组织中，CD86+/MHC-II+树突状细胞与干扰素γ阳性CD8阳性T细胞的浸润水平均有所升高。RNA测序（RNA-seq）结果表明，单次给予PFSUV-IMQ后，先天免疫激活相关基因的表达出现富集。综上，上述数据表明，通过该靶向递送IMQ的方式激活肿瘤免疫微环境，可在两种肝癌模型中均实现肿瘤负荷的降低。本研究还对经或未经IMQ纳米颗粒治疗的小鼠肝细胞癌肿瘤的RNA-seq数据开展了比较基因表达谱分析。