Abstract 2263: Characteristics of the interferon-stimulatory DNA cargo of exosomes produced by irradiated breast cancer cells
Authors: Spada Sheila , Zumbo Paul , Betel Doron , Zhang Tuo , Rudqvist Nils-Petter , Demaria Sandra 
Published: September 2020

Abstract
Radiation therapy (RT) used at immunogenic doses (8GyX3) leads to cytosolic accumulation of DNA that binds to the DNA sensor cGAS and activates the cGAMP synthesis, resulting in STING activation and downstream interferon type I (IFN-I) production by cancer cells [1-3]. RT-induced IFN-I is critical for effective anti-tumor immune responses in combination with immune-checkpoint blockade therapy [1]. We have recently demonstrated that tumor-derived exosomes (TEX) secreted by irradiated (8GyX3) TSA murine carcinoma cells (RT-TEX) contain more DNA compared to TEX produced by untreated cells (UT-TEX). Interestingly, only the DNA carried by RT-TEX is capable of STING-dependent activation of recipient dendritic cells (DCs) resulting in CD40, CD80 and CD86 upregulation and IFN-I production. Mice vaccination with RT-TEX, but not UT-TEX, elicits tumor-specific CD8+ T-cell responses that protect mice from tumor development [4]. Here, we tested the hypothesis that not only quantitative but also qualitative differences between the DNA cargo of RT-TEX and UT-TEX may explain its differential ability to activate DCs. To this end, internal DNA purified from TEX and from the cytosolic fraction of the parent TSA cells was characterized for length using Agilent Bioanalyzer. Furthermore, DNA TEX was analyzed by whole-genome sequencing (WGS) and by whole-genome bisulfite sequencing for methylation status. In addition, the percentage of global methylation of DNA of the TSA cells was quantified by 5-methyl cytosine DNA Elisa kit. All experiments were performed in biological triplicates. DNA size analysis revealed an enrichment of DNA fragments with size between 60 and 250 bp in RT-TEX compared to UT-TEX, and in the cytosolic fraction of irradiated compared to untreated TSA cells. The WGS showed that the entire genome was represented in DNA contained within TEX, regardless of the treatment of the parent cells. More than 99% of TEX DNA was of nuclear origin, but mitochondrial DNA was increased in RT-TEX (6.2 fold change, p=0.006). Interestingly, the RT treatment increases the DNA abundance of several regions (median p=0.006). Interestingly, we found reduced levels of methylation in exosomal and total DNA purified from irradiated compared to untreated TSA cells. Our data support the hypothesis that there are potentially important qualitative differences in the DNA cargo of TEX derived from irradiated compared to untreated cancer cells that reflect molecular changes occurring in parent cells. For instance, the enrichment in DNA fragments with size between 60-250 bp in RT-TEX is especially intriguing in light of the recent report that cGAS is optimally activated by this DNA length range [5]. The impact of these qualitative differences in the cargo DNA on activation of the IFN-I pathway in innate immune cells that uptake TEX is under investigation. Identification of DNA signature associated with TEX ability to activate the cGAS/STING pathway could provide a blood-based biomarker for the immunogenic tumor response to radiotherapy.
Citation Format: Sheila Spada, Paul Zumbo, Doron Betel, Tuo Zhang, Nils-Petter Rudqvist, Sandra Demaria. Characteristics of the interferon-stimulatory DNA cargo of exosomes produced by irradiated breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2263.