Abstract 477: Comparing type 1 interferon activation in tumor cells following external beam radiotherapy versus targeted radionuclide therapy
Authors: Jagodinsky Justin C. , Arthur Ian S. , Castillo Juliana S. , Chakravarty Ishan , Zangl Luke M. , Brown Ryan J. , Patel Ravi B. , Jin Wonjon J. , Carlson Peter M. , Hernandez Reinier , Grudzinski Joseph J. , Marsh Ian R. , Weichert Jamey P. , Bednarz Bryan P. , Morris Zachary S. 
Published: September 2020

Abstract
Background: Radiation (RT) activates a type 1 interferon (IFN-1) response and, in preclinical studies, this is critical to the effect of RT in priming a response to immune checkpoint blockade. Recent studies report the impact of external beam radiotherapy (EBRT) dose and fractionation on IFN-1 response, however little is known about the time course of this effect. Clinical interest in utilizing systemically administered targeted radionuclide therapy agents (TRT) is growing. It is unclear how IFN-1 activation induced by continuous delivery of RT during exponential decay of a TRT source will compare to that induced following instantaneous EBRT. Here we report the time course of IFN-1 response following RT in vitro and in vivo.
Methods: For in vitro studies, we utilized murine models of melanoma (B16, B16 STING knockout, B78), and head and neck cancer (MOC2). EBRT was prescribed to 12 Gy, 20 Gy, or 3 fractions of 8 Gy. For in vivo studies, syngeneic C57BL/6 mice were engrafted with either B78 or MOC2 cells on the flank and RT was delivered when mean tumor size was ~ 150 mm3. EBRT was prescribed to 2 Gy or 12 Gy. For TRT, we used 90Y conjugated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells of nearly any type, including B78 and MOC2. Tumor-specific dosimetry for 90Y-NM600 was determined using sequential 86Y-NM600PET/CT imaging (3h, 24h, 72h) and a Monte Carlo based dose calculation platform. TRT was prescribed to a dose of 2 Gy or 12 Gy. Following delivery of RT in vitro or in vivo, cells or tumors were harvested at 24h, 7d, and 14 d post RT and RNA was isolated. Gene expression of Ifn-β and IFN response elements (Oas2, Oas3, and Mx1) was quantified by qPCR and normalized to untreated controls.
Results: In agreement with prior studies, we observed significant IFN-1 activation 24 hours following 8 Gy x 3 in our B16, B78, and MOC2 tumor cell lines. At 12 Gy and 20 Gy, we continued to detect significant IFN-1 activation. Peak activation was 7d following EBRT for all doses, raising the potential for a lead-time bias when comparing fractionated to single dose treatments. For all doses, select IFN response genes remained upregulated at 14d. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course of IFN-1 activation peaking 7d after RT and persisting to 14d. Using a STING knockout variant of B16 melanoma, we confirmed that activation of IFN-1 response by RT was STING dependent at all time points evaluated.
Conclusions: We report the time course for activation of a STING-dependent IFN-1 response following RT in multiple murine tumor models. We observe maximal activation 7d following both EBRT and TRT. We show the potential of TRT to generate EBRT comparable IFN-1 activation. Further studies evaluating the time course and magnitude of IFN-1 response following TRT may be critical in metastatic settings to integrating this modality with immunotherapy.
Citation Format: Justin C. Jagodinsky, Ian S. Arthur, Juliana S. Castillo, Ishan Chakravarty, Luke M. Zangl, Ryan J. Brown, Ravi B. Patel, Wonjon J. Jin, Peter M. Carlson, Reinier Hernandez, Joseph J. Grudzinski, Ian R. Marsh, Jamey P. Weichert, Bryan P. Bednarz, Zachary S. Morris. Comparing type 1 interferon activation in tumor cells following external beam radiotherapy versus targeted radionuclide therapy [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 477.