纳秒脉冲电场局部消融肝细胞癌诱导的肿瘤免疫原性改变的意义

We recently found nanosecond pulsed electric field (nsPEF) can trigger engulfment of hepatocellular carcinoma cells by co-culturing macrophages in vitro; and the incidence of pulmonary metastases reduced significantly while the primary tumor in subcutaneous xenoplanted model had been ablated by nsPEF with fractionated low dose remedy. It is crucial to the clinical translation of this modality that the possibility of synergistic tumor cell specific cytotoxic T cell response stimulated by this modality alone or combined with other current therapies. This proposal is designed to illustrate the relation of phosphatidylserine externalization induced by nsPEF with dispelling of other sealed molecules possessing immunogenicity in living tumor cells, and responses of macrophages and antigen presenting cells to the immuno-recognizing signals revealed by nsPEF treatment in vivo and in vitro. Researches focused on three subjects: 1. the relations of membrane phosphatidylserine translocation with other immune recognition or ectopic immuno-molecules (uric acid, ATP, Calreticulin, High mobility Group Box1, CD154 and MHC I molecules) in tumor cells treated by nsPEF. 2. M1/2 state shifts of monocytes in co-culture with treated tumor cells; responses of naive dendritic cells to stimulated cancer cells. 3. The phenotype changes of local tumor associated macrophage (TAM), antigen presenting dendritic cells and CD8+ T cells infiltration to treated tumor tissue and their environment after tumor ablated with nsPEF; as well as the serum concentration changes concerning angiogenesis and chemotaxis. To provide theoretical basis for the clinical translation of pulsed power related technology by clarify the efficiency and internal mechanism of tumor specific T cell cytotoxicity induced by nsPEF treatments with cytology, immunology and tumor biology methods.

我们发现纳秒脉电场作用于肝癌细胞后，导致共培养的单核细胞对其吞噬发生率显著提高；用于消融局部肿瘤能抑制肺转移发生。这种新的物理消融方法诱导的肿瘤细胞免疫原性改变，能否引起持续稳定的免疫识别和抗原递呈，诱导肿瘤细胞特异性免疫反应发生，对其临床转化至关重要。本项目研究纳秒脉冲电场引起肝癌细胞膜磷脂酰丝氨酸外翻与引起的其它肿瘤细胞免疫原性相关的分子如胞内尿酸、ATP、Calreticulin、HMGB1及MHC I类等空间位置变化的相关性；在诱导肝癌细胞被单核细胞吞噬的同时，对巨噬细胞M1/M2分布状态变化的影响，以及未成熟DC细胞对经电场诱导的肿瘤细胞的反应；动物实验中，治疗导致的肿瘤及微环境中巨噬细胞和CD8+ T 细胞表型变化，及其对血管新生和趋化等相关因子水平的影响。阐明基于这种新疗法诱导肿瘤细胞特异性T细胞毒性的可行性，为优化该技术的临床转化研究提供理论依据。

我们提出“等效力场”模型解释纳秒脉冲电场（nsPEF）致细胞膜损伤是其非热效应。作用于肿瘤细胞后，等效力的作用导致细胞膜内外带电荷分子异位，引起胞内ATP、Calreticulin和HMGB1等DAMP分子异位，改变肿瘤细胞免疫原性。在体外实验中，nsPEF诱导肝癌细胞产生脂质囊泡结构，能诱导巨噬细胞向抗肿瘤的M1状态偏移。在实验动物体内，nsPEF消融肝癌后，通过HGF/c-MET通路上调PDGF调节的正常肝细胞再生能力增强。这种差异杀伤可通过低剂量黄芩苷对正常肝细胞和肿瘤细胞线粒体损伤作用差异来解释。我们发现肝癌缺氧微环境中巨噬细胞招募调节性T细胞，是诱导抗PD-L1治疗抵抗的关键机制。我们同时发现nsPEF诱导PD-L1在肿瘤细胞表面的上调，并加快其释放PD-L1。nsPEF消融后，虽然浸润肿瘤的CD8+T细胞的密度增加能抑制肿瘤生长，但这种作用会被nsPEF上调的PD-L1分子升高中和，导致活化CD8+T的耗竭和失能。联合nsPEF消融和抗PD-L1治疗显着提高了肿瘤内浸润的CD8+T的数量，并改善其活化和杀伤功能，提高疗效，是一种极具转化潜力的新技术。