B. I. Bell1,2, S. Pandey1, B. Malachowska1, M. M. Schumacher1,2, D. Young1,3, V. Kumar1, C. Velten1, M. Moustafa4,5, S. Sidoli3, P. Guida6, A. Abdollahi4,5, and C. Guha1,2; 1Department of Radiation Oncology, Montefiore Medical Center, Bronx, NY, 2Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, 3Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY, 4Divisions of Molecular & Translational Radiation Oncology, Heidelberg Faculty of Medicine (MFHD) and Heidelberg University Hospital (UKHD), Heidelberg Ion-Beam Therapy Center (HIT), Heidelberg, Germany, 5German Cancer Consortium (DKTK) Core-Center, German Cancer Research Center (DKFZ),Heidelberg Institute of Radiation Oncology (HIRO), National Center for Radiation Research in Oncology (NCRO), German Cancer Research Center (DKFZ), Heidelberg, Germany, 6Department of Biology, Brookhaven National Laboratory, Upton, NY
Purpose/Objective(s): Protons and carbon ions each confer dosimetric advantages over photons, yet of these modalities, only carbon ion radiotherapy (CIRT) delivers densely ionizing, high linear energy transfer (LET) radiation which results in more complex DNA damage and an increased relative biological effectiveness (RBE) compared to low LET photons. However, our classical understanding of RBE does not account for potential microenvironmental effects. We therefore performed a multi-institutional, multi-omics analysis to understand how CIRT shifts the contexture of the tumor immune microenvironment in pancreatic cancer.Materials/
Methods: Murine pancreatic cancer cell lines derived from KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-CreERTM;Rosa26YFP/YFP (KPCY) mice which exhibit low T-cell infiltration (6419c5) vs. high T-cell infiltration (2838c3) were studied. These tumor cell lines were implanted subcutaneously into wild-type (WT) C57BL/6J or immunodeficient NSG mice and irradiated 7 days later. CIRT was performed at an LETd of ~100 keV/µm in the mid-spread-out Bragg peak (SOBP) while low LET photon treatments were performed using orthovoltage X-rays or 137Cs ?-rays. Clonogenic assays were performed to determine the RBE, and proteomics was performed by mass spectrometry on cell culture supernatant 24 hours post-radiation. 6419c5 flank tumors were dissociated 7 days post-10 Gy of CIRT or X-rays and single cell RNA-sequencing (scRNA-seq) was performed on sorted live cells. Results: Clonogenic assays in both cell lines revealed a CIRT RBE of 1.76 compared to photons. Despite these cell lines exhibiting similar clonogenic survival after CIRT in vitro, the response to CIRT in vivo was notably different between them in tumor growth experiments. There was a similar tumor growth delay with 10 Gy CIRT in both WT and NSG mice with 6419c5 tumors. However, 2838c3 responded to 10 Gy CIRT with a significantly longer tumor growth delay in WT vs. NSG mice (P < 0.05), suggesting that tumor infiltrating T lymphocytes contribute to tumor control after CIRT. Overlap analysis of common differentially expressed proteins related to CIRT from both cell lines identified a unique signature of CIRT with 25 upregulated and 5 downregulated proteins. scRNA-seq identified that CIRT upregulates genes and pathways associated with the response to type I interferons and with antigen presentation across populations including tumor cells, dendritic cells, and T/NK cells 7 days post-irradiation when compared to an equivalent physical dose of X-rays. Conclusion: CIRT not only exhibited an increased RBE, but it also generated a unique tumor immune microenvironment characterized by transcriptional evidence of a type I interferon response and enhanced antigen presentation when compared to low LET radiation. Tumor growth experiments further suggest that effective immune activation could contribute to the augmented in vivo RBE of CIRT.
