Abstract 3329: Ionizing radiation modifies the molecular composition of exosomes derived from breast cancer cells
Authors: Diamond Julie M. , Chapman Jessica R. , Ueberheide Beatrix M. , Demaria Sandra 
Published: August 2015

Abstract
Metastatic breast cancer fosters an immunosuppressive tumor microenvironment (TME). Local ionizing radiotherapy (RT) has the potential to convert the tumor into an in situ vaccine by facilitating efficient uptake of tumor neoantigens by dendritic cells and enhancing antigen presentation to promote robust effector T cell responses. Exosomes are microvesicles (30-100nm) released from living cells that shuttle and transfer selected intracellular components, including cytokines, growth factors, mRNA, and miRNA. Tumor-derived exosomes (TEX) allow for a sophisticated means of communication with a variety of cells, including immune cells, within the TME. We hypothesized that TEX released from irradiated tumors may play a role in altering the susceptibility of tumor cells to immune-mediated rejection.
To determine if RT changes TEX composition, we treated mouse mammary carcinoma cells TSA in vitro with sham RT, 1 dose of 20Gy, or 3 fractions of 8Gy given in three consecutive days (8Gyx3). Cells were transferred to exosome-depleted media following RT and cell-free supernatant was collected 48hr later. TEX were isolated using differential ultracentrifugation and purified with a sucrose gradient. Electron microscopy was used to confirm the expected size and morphology. TEX were lysed for identification of proteins using label-free quantitation mass spectrometry (LFQ-MS) followed by MS/MS analyses. To characterize miRNA signatures of TEX and their parent cells, RNA was isolated for nanoString miRNA expression assay, which includes a panel of 578 mouse miRNAs. Normalized results were analyzed with MultiExperiment Viewer.
LFQ-MS revealed that TEX from 20Gy- and 8Gyx3-irradiated TSA shared 10 unique proteins not present in TEX from non-irradiated TSA. In addition, 17 unique proteins were present only in TEX from 8Gyx3-treated TSA. Among them were proteins involved in T cell development, MHC class I peptide processing, and proinflammatory lipid signaling. Radiation induced downregulation of 73% of miRNAs expressed in untreated TSA cells. Many of the changes in miRNA expression seen in irradiated cells were also seen in the TEX derived from these cells. Interestingly, unique expression patterns emerged with different miRNA subsets expressed in TEX depending on RT regimen used. Interesting candidates are being analyzed for functional relevance.
This data suggests that irradiation of tumor cells results in changes in protein and miRNA composition of TEX, with differences dependent on the RT regimen used. We have previously shown that 8Gyx3 RT synergized with anti-CTLA-4 antibody and induced an abscopal effect in mice, while 20Gy did not (Dewan et al, Clin Cancer Res, 2009). We are testing whether TEX contain biomarkers that define the ability of RT regimens to synergize with immunotherapy. Exosome-mediated communication networks may provide a new therapeutic target for treatment of metastatic cancer.
Citation Format: Julie M. Diamond, Jessica R. Chapman, Beatrix M. Ueberheide, Sandra Demaria. Ionizing radiation modifies the molecular composition of exosomes derived from breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2015-3329