Project 2 - Joshua Hood, PhD

肝细胞癌（HCC）是癌症相关死亡的第三大原因。它本质上是 由于经常同时发生基础肝硬化， 常规化疗毒性。相当多的患者仍然没有反应， “无法治疗”，到尖端免疫检查点抑制剂治疗，需要开发新的 或增强疗法。考虑到它们作为国际纳米技术会议的参与者在去除外来纳米材料方面的作用， 单核吞噬细胞系统，加上它们在介导HCC中的作用，枯否细胞（KCs）是理想的 靶向小细胞外囊泡（sEV）免疫系统。小EV富含外泌体 纳米囊泡我们以前的研究表明，黑色素瘤和肺癌sEV可以直接诱导 促肿瘤M2样Mφ表型或用蜂毒肽修饰以诱导抗肿瘤M1样Mφ。 理论上，HCC sEV也可以用蜂毒素修饰以诱导抗肿瘤M1 KC Mφ。 蜂毒肽蜂毒肽是激活M1免疫的强有力佐剂。FDA的 批准使用蜂毒注射液进行蜂蜇免疫治疗。在本提案中，我们假设 （一）HCC sEV可以转化为稳定的蜂毒肽佐剂纳米载体，和（ii.）蜂毒肽修饰 HCC sEV在体内与KCs相关并诱导KCs朝向抗肿瘤M1样表型。 在目标1中，HCC sEV将被转化为稳定的蜂毒素纳米载体。一种荧光红， 将开发生物发光的3D HepG 2-Red-Fluc球状体sEV源模型。我们将比较2D 与3D HepG 2-Red-Fluc来源的天然和蜂毒肽化sEV相比，它们影响原发性KC的能力 体外极化并使用qRT-pcr确定M1/M2极化miRNA含量的差异。 在目的2中，使用生物发光的Hepa 1 -6-Fluc-Neo细胞， 将使用免疫活性的C57 L/6小鼠。天然vs.蜂毒肽化Hepa 1 -6-Fluc-Neo sEV将 比较以确定它们是否影响KC M1/M2极化。我们还将评估预- 用蜂毒肽化的Hepa 1 -6-Fluc-Neo sEV治疗抑制随后的原位HCC生长。研究 将通过利用所有U of L肝脏生物学和毒理学COBRE核心来完成。 这些研究的结果将进一步加深我们对自然肝癌之间关系的理解， sEV和KC肿瘤支持功能，以及拮抗这种致病性的治疗手段。 还将评估使用蜂毒肽修饰的KC sEV的关系。翻译，sEV群体 可以从HCC患者的血液中收获，修饰成个性化的免疫耐受剂， 纳米药物使用蜂毒肽，或其他代理和重新管理。拟议的研究也可作为 对其他Mφ驱动的肝脏进行病理和治疗sEV研究的平台 包括肝炎、酒精性和非酒精性-SH和肝纤维化的疾病。

Hepatocellular carcinoma (HCC) is the 3rd cause of cancer-related mortality. It is inherently difficult to treat given the often-times co-occurrence of underlying cirrhosis which exacerbates conventional chemotherapeutic toxicity. A sizeable number of patients remain non-responsive or “untreatable,” to cutting edge immune checkpoint inhibitor therapy necessitating the development of new or augmentative therapies. Given their role in removing foreign nanomaterials as participants of the mononuclear phagocyte system, coupled to their role in mediating HCC, Kupffer cells (KCs) are an ideal immunotherapeutic target for small extracellular vesicles (sEVs). Small EVs are enriched in exosome nanovesicles. Our previous studies demonstrate that melanoma and lung cancer sEVs can directly induce a pro-tumor M2-like Mφ phenotype or be modified with melittin peptide to induce anti-tumor M1-like Mφs. Theoretically, HCC sEVs might also be modified with melittin to induce anti-tumor M1 KC Mφs. The bee venom peptide melittin is a powerful adjuvant for activating M1 immunity. The FDA approves the use of bee venom injections for bee sting immunotherapy. In this proposal we hypothesize that (i.) HCC sEVs can be converted into stable melittin adjuvant nanocarriers, and (ii.) melittin-modified HCC sEVs associate with and induce KCs toward an anti-tumor M1-like phenotype in vivo. In aim 1, HCC sEVs will be converted into stable melittin nanocarriers. A fluorescent red, bioluminescent, 3D HepG2-Red-Fluc spheroid sEV source model will be developed. We will compare 2D versus 3D HepG2-Red-Fluc sourced natural and melittinized sEVs for their ability to influence primary KC polarization in vitro and determine differences in M1/M2 polarizing miRNA content using qRT-pcr. In aim 2, an orthotopic syngeneic model using bioluminescent Hepa1-6-Fluc-Neo cells in immunocompetent C57L/6 mice will be used. Natural vs. melittinized Hepa1-6-Fluc-Neo sEVs will be compared to determine whether they influence KC M1/M2 polarization. We will also assess whether pre- treatment with melittinized Hepa1-6-Fluc-Neo sEVs inhibits subsequent orthotopic HCC growth. Studies will be accomplished via utilization of all U of L Hepatobiology & Toxicology COBRE cores. The results of these studies will further our understanding of the relationship between natural HCC sEVs and KC tumor supportive functions, and a therapeutic means to antagonize this pathogenic relationship using melittin-modified KC sEVs will also be evaluated. Translationally, sEV populations might be harvested from the blood of HCC patients, modified into personalized immunotolerant nanomedicines using melittin, or other agents and re-administered. The proposed studies also serve as a platform to pursue pathologic and therapeutic sEV investigations concerning other Mφ driven liver diseases including hepatitis, alcoholic and non-alcoholic-SH, and liver fibrosis.