Project 2 - Joshua Hood, PhD

肝细胞癌是癌症相关死亡的第三大原因。它与生俱来地 很难治疗，因为潜在的肝硬变经常多次并存，这会加剧 常规化疗毒性。相当数量的患者仍然没有反应或 “无法治愈”的尖端免疫检查点抑制剂疗法需要开发新的 或强化疗法。考虑到它们作为 单核吞噬细胞系统，再加上它们在介导肝细胞癌中的作用，库普弗细胞(KCs)是理想的 针对细胞外小泡(SEV)的免疫治疗靶点。小型电动汽车富含外切体 纳米车。我们之前的研究表明，黑色素瘤和肺癌SEV可以直接诱导 亲瘤M2样Mφ表型或用蜂毒素修饰诱导抗肿瘤M1样MφS。 从理论上讲，肝癌SEV也可以用蜂毒素修饰来诱导抗肿瘤细胞M1、KC、M、φS。 蜂毒肽蜂毒素是一种激活M1免疫的强佐剂。美国食品和药物管理局 批准使用蜂毒注射进行蜜蜂叮咬免疫治疗。在这个提案中，我们假设 这(i.)肝癌SEV可以转化为稳定的蜂毒素佐剂纳米载体，以及(Ii)蜂毒素修饰 肝细胞癌SEV在体内结合并诱导KCs向抗肿瘤M1样表型转化。 在目标1中，肝癌SEV将被转化为稳定的蜂毒素纳米载体。荧光红， 将开发生物发光的三维HepG2-Red-Fluc球体SEV源模型。我们将比较2D 对比3D HepG2-Red-Fluc来源的天然和熔融SEV影响原发KC的能力 体外极化，并用qRT-PCR测定M1/M2极化miRNA含量的差异。 在目标2中，使用生物发光的Hepa1-6-Fluc-Neo细胞建立了原位同基因模型。 将使用免疫活性C57L/6小鼠。天然与蜂蜜酸化的Hepa1-6-Fluc-Neo SEV将是 比较它们是否影响KCM1/M2极化。我们还将评估是否会预先 用蜂毒素化的Hepa1-6-Fluc-Neo SEV治疗可抑制随后的原位肝细胞癌生长。研究 将通过利用L肝脏生物学和毒理学核心的所有U来实现。 这些研究结果将进一步加深我们对自然肝细胞癌之间关系的理解 SEVS和KC的肿瘤支持功能以及拮抗该致病的治疗手段 还将评估使用蜂毒素修饰的KC SEV的关系。翻译过来就是SEV人群 可能是从肝癌患者的血液中提取的，改装成个性化的免疫耐受药 使用蜂毒素或其他试剂的纳米药物，并重新给药。建议的研究亦可作为 对其他Mφ驱动的肝脏进行病理和治疗性SEV研究的平台 疾病包括肝炎、酒精性和非酒精性-SH和肝纤维化。

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.