Combination of ceramide and immunotherapy in treatment of hepatocellular cancer

摘要 在操纵免疫系统以消除已建立的肿瘤方面取得了重大进展 包括肝细胞癌。然而，缺乏临床相关的动物模型在很大程度上阻碍了 我们对肝细胞癌诱导的免疫逃避的理解。因此，预防肿瘤诱导的免疫耐受 开发有效的抗肝癌免疫疗法仍然是一项具有挑战性的任务。我们最近创造了一种新的小鼠 免疫功能正常的小鼠模型。该模型反映了包括免疫在内的人类肝细胞癌最典型的特征 逃走。该模型中的肿瘤生长诱导了深刻的免疫耐受，其特征是 肿瘤相关巨噬细胞(TAMs)、调节性T细胞(Tregs)和髓系来源的抑制物的频率 细胞(MDSCs)、肝窦内皮细胞(LSECs)以及效应CD8+T细胞的深度耗尽。 因此，我们建立了一个独特的平台来研究肿瘤诱导免疫耐受的机制 并为最终的临床翻译开发肝细胞癌的概念验证治疗策略。 神经酰胺具有抗肿瘤活性。我们使用先进的纳米技术来制造纳米脂质体载C6- 礼仪(LipC6)。该配方的LipC6克服了神经酰胺细胞的不通透性和水沉淀， 同时增强细胞滞留，促进其作为药物的临床应用。脂肪C6与过继疗法的结合 肿瘤抗原特异性CD8+T细胞的转移加上肿瘤特异性抗原免疫不仅延缓 肿瘤生长但也逆转了已建立的肿瘤，这表明LipC6与 免疫治疗在控制肝细胞癌方面具有协同治疗作用。这项提议的总体目标是 剖析LipC6破坏肿瘤诱导的免疫耐受挽救抗肿瘤的潜在机制 免疫活性和开发临床有用的针对肝癌的LipC6综合免疫疗法。 进一步的研究表明，LipC6治疗在细胞水平上调节和LSec；并调节 活性氧(ROS)、干扰素调节因子(IRF)和脂肪酰辅酶A还原酶2(FAR2)、PD1 在肝细胞癌的分子水平上。抗PD1抗体治疗有效地阻碍了肝癌的生长。因此，我们假设 LipC6靶向和LSec通过调节ROS、IRF和FAR2来恢复抗肿瘤免疫反应 因此，LipC6联合抗PD1抗体不仅可以强烈地阻断PD1/PD-L1通路 激活抗肝癌免疫的同时也产生了前所未有的治疗效果。我们将检验我们的假设 通过目标1：剖析LipC6调节和LSec影响抗肿瘤的细胞机制 荷瘤小鼠的免疫功能。2.剖析LipC6调节巨噬细胞的分子机制 通过ROS和IRF途径，以及LSEC通过Far2途径在肝细胞癌的发生中起重要作用。3.定义治疗性 LipC6联合抗PD-1单抗治疗肝癌的疗效及机制探讨。 这项研究将为有效的LipC6-免疫治疗干预的机制提供更好的见解 在肝细胞癌对照组。这些发现将导致新的治疗方法和治疗目标，以及更好的抗癌策略。

ABSTRACT Great progress has been made in manipulating the immune system to eliminate established tumors including hepatocellular cancer (HCC). However, the lack of a clinically-relevant animal model largely impedes our understanding of HCC-induced immune evasion. Therefore, prevention of tumor-induced immunotolerance to develop effective immunotherapies against HCC is still a challenging task. We recently created a novel murine model with immunocompetent mice. This model reflects most typical features of human HCC including immune escape. Tumor growth in this model induces profound immune tolerance characterized by increase in the frequency of tumor-associated macrophages (TAMs), regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), liver sinusoidal endothelial cells (LSECs) as well as profound exhaustion of effector CD8+T cells. We have thus established a unique platform to investigate the mechanism of tumor-induced immune tolerance and to develop proof-of-concept therapeutic strategies for HCC toward eventual clinical translation. Ceramide has tumoricidal activity. We use advanced nanotechnology to make nanoliposome-loaded C6- ceremide (LipC6). The formulated LipC6 overcomes ceramide cell impermeability and aqueous precipitation, while enhancing cellular retention, promoting its clinical use as a drug. The combination of LipC6 with adoptive transfer of tumor antigen-specific CD8+ T-cell plus immunization with tumor-specific antigens not only retarded tumor growth but also regressed established tumors, suggesting that the combination of LipC6 with immunotherapies has a synergistic therapeutic effect in HCC control. The overall objective of this proposal is to dissect the underlying mechanisms by which LipC6 breaks tumor-induced immune tolerance to rescue antitumor immune activity and develop a clinically useful LipC6-integrated immunotherapy for HCC. Further studies reveal that LipC6 treatment modulates TAM and LSEC in cellular level; and regulates reactive oxygen species (ROS), interferon regulator factor (IRF), and fatty acyl-COA reductase 2(Far2), PD1 in molecular level in HCC. Anti-PD1 antibodies treatment effectively impedes HCC growth. Thus, we hypothesize that LipC6 targets TAM and LSEC to resurrect anti-tumor immune reactivity by regulating ROS, IRF, and Far2 pathways; thus LipC6 in combination with anti-PD1 antibodies to block PD1/PD-L1 pathway not only strongly activate anti-HCC immunity but also result in unprecedented therapeutic efficacy. We will test our hypothesis through aims 1: Dissect the cellular mechanisms by which LipC6 modulates TAM and LSEC to impact anti-tumor immunity in tumor-bearing mice. 2. Dissect the molecular mechanisms by which LipC6 modulates macrophages through ROS and IRF pathways, and LSEC through Far2 pathway in the setting of HCC. 3. Define the therapeutic efficacy of LipC6 in combination with anti-PD-1 Abs for HCC treatment and elucidate the underlying mechanisms. This study will provide better insights into the mechanisms of an effective LipC6-immunotherapeutic intervention in HCC control. The findings will lead to new treatments and therapeutic targets, and better anticancer strategies.