# Project 2 - Joshua Hood, PhD

> **NIH NIH P20** · UNIVERSITY OF LOUISVILLE · 2021 · $218,400

## Abstract

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.

## Key facts

- **NIH application ID:** 10026256
- **Project number:** 2P20GM113226-06
- **Recipient organization:** UNIVERSITY OF LOUISVILLE
- **Principal Investigator:** Joshua L. Hood
- **Activity code:** P20 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $218,400
- **Award type:** 2
- **Project period:** 2016-06-10 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10026256

## Citation

> US National Institutes of Health, RePORTER application 10026256, Project 2 - Joshua Hood, PhD (2P20GM113226-06). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10026256. Licensed CC0.

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