Abstract 1844: From barrier to bullet: Genetically engineered CAF-derived exosomes for PDAC therapy

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers, with a five-year survival rate of less than 10%. This poor prognosis is due in part to the tumor's dense desmoplastic stroma, which impedes nanoparticle penetration and immune cell infiltration, rendering conventional therapies ineffective. The current therapeutic paradigm, which directly targets PDAC cells with nanoparticles or relies on immune modulation, often fails due to the "immune cold" PDAC tumor microenvironment (TME) characterized by low infiltration of cytotoxic T lymphocytes (CTLs) and abundance of cancer-associated fibroblasts (CAFs) that create a formidable stromal barrier. Therefore, there is a critical need for a paradigm shift in PDAC treatment—moving beyond traditional strategies that directly target cancer cells or modulate the immune system, and instead, focusing on harnessing the unique properties of the TME itself to combat the tumor. Therefore, we transformed the inherent limitations of the PDAC TME into therapeutic opportunities by reprogramming CAFs to act as endogenous factories of cytotoxic exosomes capable of selectively targeting and killing cancer cells. By leveraging the natural abundance and strategic location of CAFs within the TME, we employed exosome fusion protein technology to engineer CAFs to produce exosomes loaded with cytotoxic proteins, such as granzyme B (GrB), or pro-melittin, fused to one of the exosomal components e.g., CD63 on gene level, and specific targeting motifs e.g., B7-H3 single-chain variable fragment (scFv) to ensure selective uptake by PDAC cells inducing their apoptosis. These cytotoxic exosomes bypass the need for CTL infiltration, thereby overcoming the challenges posed by the "immune cold" TME. We engineered CAF-targeted lipid nanoparticles (LNPs) for delivering plasmid DNA (pDNA) encoding the fusion proteins into PDAC patient-derived CAFs. The cells could be successfully transfected in vitro and subsequently produced the desired cytotoxic exosomes. The expression of fusion proteins in the exosomes was confirmed in vitro via western blot and proteomic analysis. The GFP-expressing exosomes showed efficient internalization into PDAC cells as evidenced by immunofluorescence imaging and flow cytometry. The genetically engineered CAF-derived exosomes induced efficient apoptosis of patient-derived PDAC cells in vitro revealing their cytotoxic power. Moreover, intravenous administration of the pDNA-loaded lipid nanoparticles into pancreatic tumor mouse model significantly suppressed tumor growth. Histopathological studies and biochemical analysis showed the safety of our engineered nanoparticles. This approach leverages the unique properties of the TME to provide a new paradigm in cancer therapy that avoids the need for direct targeting of cancer cells or enhancement of immune cell infiltration. Citation Format: Leyla Escalante Gonzalez, Ahmed Elzoghby, Subin Yu, Alaa Bakr, Aya Jaros, Cuiyan Xin, Luke Lee. From barrier to bullet: Genetically engineered CAF-derived exosomes for PDAC therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1844.