Abstract 2493: Fractionated but not single dose radiation is an optimal adjuvant for in situ tumor vaccination
Authors: Vanpouille-Box Claire Isabelle , Aryankalayil Molykutty , Pilones Karsten A. , Formenti Silvia C. , Coleman Norman , Demaria Sandra 
Published: August 2015

Abstract
Local radiotherapy (RT) has the potential to generate an in situ tumor vaccine by inducing an immunogenic cell death (ICD) of the cancer cells. In vitro, generation of the signals that define ICD is radiation dose-dependent. However, in vivo we have previously shown that radiation doses of 8 and 6 Gy given in 3 to 5 consecutive days were more effective at inducing anti-tumor T cell responses in combination with anti-CTLA-4 antibody than a single larger dose of 20 Gy (Dewan et al., Clin Cancer Res 2009).
To understand the mechanisms underlying the different outcome obtained with fractionated versus single dose RT, TSA tumors growing in syngeneic immunocompetent BALB/c mice were harvested at 4, 24 and 48 hrs post irradiation with 8 Gy X 3 or 20 Gy X 1 for analysis of gene expression using Agilent mouse mRNA Microarray Kit (V2). Subsequent data were analyzed using Ingenuity Pathway Analysis (IPA) and gene ontology classification. Expression of key immune genes in TSA cells irradiated in vitro was assessed by RT-qPCR. Tumor-infiltrating dendritic cells (TIDC) were analyzed 5 days after the last radiation exposure by flow cytometry. IFNβ production by irradiated TSA cells was measured by Procarta™ immunoassay.
Over 100 immune response genes were differentially expressed in tumors irradiated with 8Gyx3 but not 20Gyx1, with a dominant type I interferon (IFN) response at 4 and 24 hours, which was confirmed by RT-qPCR. Tumor infiltration by CD8α+ dendritic cells (DC), which are the subset of DC cross-presenting tumor cell-derived antigens, was only slightly increased by 20Gyx1 but markedly increased by 8Gyx3. Moreover, DCs present in tumors treated with 8Gyx3 showed a significant upregulation of activation markers CD40 and CD70. Importantly, the in vitro setting (devoid of an immune infiltrate) demonstrated expression of IFNβ and downstream immune genes, including chemokines CXCL9, CXCL10 and CXCL11 by TSA cells irradiated with 8Gyx3 but not 20Gyx1. Production of IFNβ by TSA cells was confirmed with an increased secretion of IFNβ in the supernatant only after 8Gyx3 irradiation.
Overall, data indicate that fractionated RT can mimic, at least in part, a viral infection and activate canonical defense pathways in neoplastic epithelial cells with induction of type-I IFN. In vivo this leads to recruitment and activation of DC cross-presenting tumor antigens. Thus, fractionated-RT acts as an optimal immune adjuvant for in situ tumor vaccination. We propose that repeated cycles of RT-induced cell death may better mimick cycles of viral infection and are currently defining the molecular mechanisms involved. Results are important for understanding the determinant of response in patients currently treated wit RT and anti-CTLA-4 in clinical trials at our institution.
Citation Format: Claire Isabelle Vanpouille-Box, Molykutty Aryankalayil, Karsten A. Pilones, Silvia C. Formenti, Norman Coleman, Sandra Demaria. Fractionated but not single dose radiation is an optimal adjuvant for in situ tumor vaccination. [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 2493. doi:10.1158/1538-7445.AM2015-2493