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.