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PURPOSE/OBJECTIVE(S) The role of cellular senescence in radiation induced pulmonary fibrosis (RIPF) is unknown. Furthermore, clearance of senescent cells (SCs) could be an effective therapeutic strategy; however, the identification and targeted removal of only senescent cells by senolytic drugs is difficult. We isolated a pure population of radiation induced senescent and non-senescent cells using tdTOMp16+ mice and examined induction of fibrosis in target cells. MATERIALS/METHODS A stromal cell line was established from explanted tdTOMp16+ mouse bone marrow. The tdTOMp16+ stromal cell line was irradiated to each of several doses by Cesium Gamma Cell dose rate 300 cGy/min including 1.0 Gy, 2.5 Gy, 5 Gy, and 7.5 Gy under growth conditions of either 50% or 100% confluence. The number and percent of senescent cells, by p16 promoter linked to the red fluorochrome biomarker was quantitated. Irradiated red, irradiated non-red, and non-irradiated cells were sorted and cultured as single cells in 96-well plates, then monitored for cell division over four weeks. The expression of senescence markers, p16 and p21, was evaluated by qPCR, and SA-β-gal by fluorogenic substrate. To examine the effect of senescent cells on a target fibrosis-prone C57BL/6 line, sorted irradiated red, irradiated non-red, and non-irradiated cells were placed on the top level of transwells, and after 2 weeks induction of biomarkers of fibrosis including Ctgf, TGF-β, collagen 1a, and collagen 3 was measured in target cells in the lower level by qPCR. RESULTS After 5 Gy irradiation at 50% cell density, ∼9% of tdTOMp16+ cells became senescent (stably red in color) after 10 days, thus, defining the optimal conditions. Then, in bulk cultures, red cells were sorted by fluorescence automated cell sorter (FACS) to a pure population of red tdTOMp16+ cells. When cultured as single cells, none of 960 irradiated red cells divided after 2 weeks, 137 of these cells remained intact, and assume large and flat morphology. In contrast, 50 of 800 non-red irradiated cells divided, and 104 of 560 non-irradiated control cells divided. There was significant detection of senescent cell markers including: SA-β-gal, p16, and p21 in irradiated sorted red cells compared to irradiated non-red cells (P = 0.0096, P < 0.0001 and P = 0.0001 respectively based on scoring of 1000 cells in triplicate experiments for SA-β-gal and analyses from three independent experiments). Induction of biomarkers of fibrosis Ctgf, TGF-β, collagen 1a, and collagen 3 in C57BL/6 stromal target cells was significant by irradiated red, but not irradiated non-red or non-irradiated cells (P = 0.037, P = 0.14, P = 0.015, and P = 0.0259). CONCLUSION The data indicate that sorted irradiated red senescent tdTOMp16 stromal cells can be isolated as a pure non-dividing population and induce fibrotic markers in target cells in non-contact transwell cultures. This unique system can now be used to define the mechanism of senescent cell-induced radiation fibrosis, and the effects of senolytic drugs.

Purpose/Objective(s) We delivered intraoral (gavage) of LR-IL-22 (Zhang, et al., In Vivo, 34: 139-150, 2020) prior to whole abdominal irradiation (WAI) of C57BL/6J mice and quantitated survival, intestinal barrier integrity, and levels of irradiation-induced inflammatory cytokines. We measured the effect on fractionated WAI of LR-IL-22 in transgenic C57BL/6J-MUC1 (human-MUC1) female mice with widespread intra-abdominal cisplatin-resistant ovarian cancer. Materials/Methods C57BL/6J mice were irradiated to 19.75 Gy WAI, then evaluated for the primary endpoint of improved 30-day survival. Fluorescent beads (F13083 FluoSpheres™ Polystyrene Microspheres, 1.0 μm, red fluorescent (580/605), for tracer studies) were administered intraorally three hours prior to sacrifice at days 2 or 5 after WAI, and blood was assayed for beads. Luminex assay of plasma and intestinal cells were also assayed at days 0, 1, 2, 3, and 5 after WAI for radiation-induced inflammatory cytokines. Murine ovarian cancer 2F8cis cells were injected intraperitoneally into female transgenic C57BL/6J-MUC1 mice (1 × 106 cells per mouse). Seventy-two hours later, mice were irradiated to 16 Gy WAI and followed for tumor growth. In a separate experiment, female mice harboring 2F8cis tumors were irradiated to 6 Gy WAI daily for 4 successive days. Overall survival was compared between groups: irradiation only, LR-IL-22 gavage three hours prior to each fraction, and/or weekly anti-PD-L1 antibody treatment. Results Mice receiving LR-IL-22 (109 bacteria in 100 µL of saline) 24-hours and 3-hours prior to a single fraction of 19.75 Gy WAI, and 24-hours following irradiation, showed improved survival and barrier integrity compared to those receiving control LR or IL-22 protein (P = 0.0167 or 0.0276, n = 15). LR-IL-22 gavage maintained intestinal barrier integrity and decreased levels of intestinal pro-inflammatory cytokines, including KC (CXCL1) (P = 0.002), and IFN- γ (P = 0.0024), as well as profibrotic plasma Eotaxin (CCL11) (P = 0.0088). In MUC1 transgenic female mice with tumors, nonirradiated mice had over 100 disseminated tumor nodules throughout the abdomen at day 10, while control WAI mice or those treated with intraoral LR-IL-22 had less than 10 nodules at day 10 (P = 0.0471); furthermore, LR-IL-22 treated mice showed reduced intestinal edema. C57BL/6J-MUC1 female mice with intra-abdominal 2F8cis tumor showed significantly improved 30-day survival by adding LR-IL-22 and anti-PD-L1 to 6 Gy × 4 WAI (P = 0.0004). Conclusion Intraoral LR-IL-22 prior to WAI protects the intestine from radiation-induced toxicity and facilitates fractionated therapeutic WAI to improve survival in cis-platinum resistant ovarian cancer. Author Disclosure D.F. Hamade: None. M. Epperly: None. R. Fisher: None. W. Hou: None. J.S. Greenberger: None.

Senescence is an irreversible cell cycle arrest induced by various triggers, including aging and irradiation. Fanconi Anemia (FA) is a genetic disorder associated with defects in 22 proteins in the scaffold DNA double strand break repair. FA mice develop anemia and marrow failure and have been considered as a potential model for accelerated aging. Fancd2-/- murine bone marrow (BM) stromal cell lines are radiosensitive in clonogenic survival curves compared to wild type (Berhane et al, Radiat Res, 2014). We tested the Fanca-/- mouse model for irradiation induced senescence. 129Sv Fanca+/+ and Fanca-/- BM stromal cell lines were irradiated to 5Gy, then assayed for senescence at various timepoints. Cells were stained with a chromogenic beta-galactosidase assay. Cells were also stained by immunofluorescence (IF) to determine percent of cells positive for p16 and p21. For total body irradiation (TBI) studies, 129Sv Fanca+/+ and Fanca-/- mice were given 7Gy. Organs were harvested at Day 7, then assayed for senescence. Statistical analysis was carried out using student's t-test. Baseline levels of beta-gal positive Fanca-/- BM stromal cells were not significantly different from Fanca+/+ (respectively, 0.88% ± 0.22% vs 0.74% ± 0.23%). Fanca-/- and Fanca+/+ IF also showed no significant differences in baseline p16 levels (4.98%±2.5 vs 14.37%± 4.06%) and p21 levels (14.57% ± 7.61% vs. 11.7% ± 3.48%). In contrast, Fanca-/- cells showed significantly higher beta-gal staining compared to Fanca+/+ at days 1, 3, and 5 after irradiation (p=0.0042, p Citation Format: Aranee P. Sivananthan, Xichen Zhang, Wen Hou, Donna Shields, Renee Fisher, Michael Epperly, Joel S. Greenberger. Increased irradiation-induced senescence in Fanconi anemia (FA) mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 842.