Main Session
Sep
29
QP 24 - Novel Delivery, Metabolism, and Immune Modulation
1141 - Mapping the Genomic Landscape of Particle Therapy: Nucleotide-Resolution DSB Patterns Following Carbon Ion vs. Photon Irradiation
Presenter(s)
Aleksei Smirnov, MD - German Cancer Research Center (DKFZ), Heidelberg, Baden-Wurt
A. Smirnov1,2, M. Allers1,2, L. Corazzi3, G. Di Muzio3, B. Ding3, L. Liu2, S. Brons1, J. Debus1, R. Lopez Perez2, P. C. Wei3, and P. E. Huber1,2; 1Department of Radiation Oncology, University Hospital Heidelberg, Heidelberg, Germany, 2Division of Molecular Radiooncology at German Cancer Research Center (DKFZ), Heidelberg, Germany, 3Brain Mosaicism and Tumorigenesis at German Cancer Research Center (DKFZ), Heidelberg, Germany
Purpose/Objective(s):
Carbon ion radiotherapy demonstrates increased relative biological effectiveness (RBE) and superior dose conformity compared to photon irradiation. While DNA DSBs are the principal determinants of radiation-induced cell fate, their genome-wide spatial distribution at nucleotide resolution remains largely inaccessible with conventional methodologies. We introduce Linear Amplification-Mediated High-Throughput Genome-Wide Translocation Sequencing (LAM-HTGTS) as a novel platform for radiobiological research. This technique enables unbiased, genome-wide detection of radiation-induced DSBs by introducing a defined Cas9-mediated “bait” break to capture translocation events across the genome. We applied LAM-HTGTS to directly compare carbon ion and photon irradiation in tumor and normal cells.Materials/Methods:
Human U2OS osteosarcoma cells and murine JM8 embryonic stem cells were irradiated with 0–10 Gy 225 kVp X-rays (LET ~0.2 keV/µm) or 0–5 Gy carbon ions (LET ~100 keV/µm) at the Heidelberg Ion Beam Therapy Center. Twenty-four hours post-irradiation, DSBs were mapped using LAM-HTGTS with three independent Cas9 bait sites per cell line, generating normalized libraries averaging 3.7 million reads. Genome-wide DSB junction profiles were correlated with functional endpoints including clonogenic survival, ?H2AX foci formation, cell cycle distribution, micronuclei formation, and apoptosis (caspase-3), assessed by flow cytometry and immunofluorescence microscopy.Results:
LAM-HTGTS was successfully established as a high-resolution radiogenomic platform, enabling quantitative and spatial discrimination of DNA damage patterns induced by different radiation qualities. Carbon ions produced a markedly steeper dose-dependent increase in total DSB junction counts compared to photons in both cell types. This molecular signature was consistent with enhanced biological effectiveness, reflected by greater clonogenic cell kill (RBE 3.5 at SF10 and 4.5 at SF50), stronger G2 phase arrest, elevated ?H2AX levels, increased micronuclei formation, and increased apoptosis. While most biological endpoints qualitatively mirrored RBE values, JM8 cells exhibited comparatively higher apoptosis sensitivity to photons.Conclusion:
LAM-HTGTS represents a transformative technology for radiobiology, enabling nucleotide-resolution mapping of radiation-induced DSB landscapes. By directly capturing primary lesions and translocation patterns, this approach may help reveal fundamental differences in damage induction and downstream signaling between carbon ions and photons.