Abstract 961: Scalable isolation of bacterial extracellular vesicles for macrophage-activating immunotherapy

Abstract The efficacy of immune checkpoint inhibitors (ICIs) is hampered by tumor-associated immunosuppressive myeloid cells. Secreted nano-sized (∼25-100 nm) bacterial extracellular vesicles (bEVs) modulate myeloid cells in the context of infection and colonization, and are being developed for cancer immunotherapy. However, lack of standardized bEV purification strategies limit reproducibility across laboratories and hamper translational development. Herein, we aimed to develop a scalable isolation workflow for bEV therapeutics. We grew the human commensal strain E. coli Nissle to early stationary phase to generate bEV-rich conditioned cell culture medium; removed bacteria by centrifugation and filtration; concentrated conditioned medium by tangential flow filtration (TFF; 100 kDa MWCO); and purified bEVs by semi-automated size-exclusion chromatography (SEC). During TFF concentration, we observed conditioned media becoming turbid, suggesting prescipitation of macromolecules, including potentially nucleic acids. Indeed, when we assessed DNA content at sequential purification steps via gel electrophoresis, and found that commercial bacterial culture media contains significant amounts of DNA which co-isolate with bEVs. We thus expanded our workflow to include clearance of baseline culture medium nucleic acid content by 30 kDa ultrafiltration of the culture medium prior to use. In addition, we treated TFF-concentrated conditioned media with the promiscuous DNAse/RNAse Benzonase (25 units/mL) which reduced nucleic acid content of bEVs by 66%, suggesting that Benzonase further increased bEV purity by depleting nuclease-sensitive non-vesicular nucleic acids. After SEC, we identified bEV-containing fractions by DNA/RNA/protein quantification, transmission electron microscopy and nanoparticle tracking analysis. Next, we assessed the immunologic activity of bEVs ex vivo by adding to cultured mouse splenocytes and bone marrow cells, and observed broad effects on myeloid cells and lymphocytes. Our optimized workflow provides a broadly applicable approach for isolating bEVs from species requiring complicated medium. This standardized method will enhance reproducibility and minimize non-specific effects of contaminants, facilitating translational development of bEV immunotherapy. Future experiments will test the role of bEVs from different microbial species in augmenting ICI efficacy in mouse models of cancer. Citation Format: Irem Karaman, Asmita Pathak, Oriana Teran Pumar, Brandon Emanuel Leon, Jonathan Mitchell, Carlos David Oliva, Selma Maacha, Wael El-Rifai, Defne Bayik, Dionysios C. Watson. Scalable isolation of bacterial extracellular vesicles for macrophage-activating immunotherapy [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 961.