Abstract B022: Systematic analysis of tumor and immune EV subset changes with radiation therapy
Authors: George Jubin , Chidester Stephanie , Pleet Michelle L. , Camphausen Kevin , Escorcia Freddy , Jones Jennifer C. 
Published: January 2025

Abstract
Background: Although radiation is known to modulate immune responses, it remains challenging to identify effective combinations of radiation and immune-based therapies. Because tumor, immune, and other cells release extracellular vesicles (EVs) continuously and because those vesicles are composed of lipid membrane-bound complexes of proteins and nucleic acids, we have developed a suite of tools to enable systematic subset characterization of tumor and immune EV subsets. Broadly speaking, these tools provide a new, informative approach for interrogating tumor and immune biomarkers that may be used not only for predictive and prognostic purposes, but also for adaptive treatment strategies. In this study, we hypothesized that EV subsets could be used to monitor tumor and immune responses to radiation. The objective of this study was to evaluate radiation-induced changes in tumor-cell derived EVs from cell culture and from patient serum EV subsets. Methods: For these studies, we have developed and refined a set of rigorous tools and protocols for the enumeration, labeling, and sorting of EVs individually or as subsets. We applied these methods to EVs isolated at a series of timepoints from a panel of tumor cell lines irradiated at different doses. In a parallel fashion, we also applied these methods to clinical serum samples obtained from patients before, at the completion of, and one month following radiation therapy. For all samples, EV repertoires were evaluated by multiplex EV repertoire assays, and selected EV subsets were further enriched by immunoaffinity for cargo analysis. Results: EV surface marker repertoires demonstrated both donor-specific and treatment response-specific signatures, for both tumor-associated and immune-associated EVs. For patients with bladder and colon cancer, characteristic EV tumor and immune marker signatures were noted between patients, including CD326 and CD69, and various donor-specific changes in tetraspanins (CD63, -81, and -9), CD42a, and CD62P were observed at the completion of radiation treatment. Certain EV subsets could be consistently isolated with affinity chromatography from donor serum samples but were not strongly represented in multiplex assay based on co-expression of tetraspanins. Conclusions: Our results establish feasibility for future EV subset studies, particularly in clinical trials investigating tumor and immune biomarkers for patients receiving combined radiation and immunotherapy treatments. Specifically, we demonstrate a robust method for evaluation of individual donor responses, including kinetic changes in tumor-associated markers (CD326) and immune-associated markers (CD69). Furthermore, each patient’s serum EVs demonstrated distinctive treatment-response patterns, with inter-donor differences appearing to relate to tumor-specific attributes and/or radiation parameters, such as marrow volume irradiated. Ongoing studies are further exploring EV-borne RNA signatures using a total RNAseq pipeline that is customized for EV RNA.
Citation Format: Jubin George, Stephanie Chidester, Michelle L. Pleet, Kevin Camphausen, Freddy Escorcia, Jennifer C. Jones. Systematic analysis of tumor and immune EV subset changes with radiation therapy. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B022.