Background Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation. mTOR signaling drives several processes implicated in RIPF, including inflammatory cytokine production, fibroblast proliferation, and epithelial senescence. We sought to determine if mTOR inhibition with rapamycin would mitigate RIPF. Methods/Materials C57BL/6NCr mice received a diet formulated with rapamycin (14 mg/kg food) or control diet two days before and continuing for 16 weeks after exposure to 5 daily fractions of 6 Gy thoracic irradiation (IR). Fibrosis was assessed with Masson-Trichrome staining and hydroxyproline assay. Cytokine expression was evaluated by quantitative real time PCR. Senescence was assessed by staining for beta-galactosidase activity. Results Administration of rapamycin extended the median survival of irradiated mice compared to control diet from 116 days to 156 days (log rank p=0.006). Treatment with rapamycin reduced hydroxyproline content compared to control diet (IR+vehicle: 45.9±11.8, IR+rapamycin: 21.4±6.0, p=0.001) and reduced visible fibrotic foci. Rapamycin treatment attenuated IL-1β and TGF-β induction in irradiated lung compared to control diet. Type II pneumocyte senescence after IR was reduced with rapamycin treatment at 16 weeks (three-fold reduction at 16 weeks, p<0.001). Conclusion Rapamycin protected against RIPF in a murine model. Rapamycin treatment reduced inflammatory cytokine expression, extra cellular matrix production, and senescence in type II pneumocytes.

Purpose/Objectives Fibrosis is a late toxicity of thoracic irradiation that can result in substantial morbidity. Plasminogen activator inhibitor-1 (PAI-1) is a critical mediator of cellular senescence and fibrin stabilization. We sought to determine if the delivery of recombinant truncated PAI-1 protein (rPAI-123) would protect from the development of radiation-induced lung injury. Methods and Materials C57Bl/6 mice received intraperitoneal injections of rPAI-123 (5.4 μg/kg/day) or vehicle for 18 weeks beginning two days prior to radiation exposure (5 daily fractions of 6 Gy). Cohorts of mice were followed for survival (n=8 per treatment) and tissue collection (n=3 per treatment and time point). Fibrosis in lung was assessed with Masson-Trichrome staining and measurement of hydroxyproline content. Senescence was assessed with staining for beta-galactosidase activity in lung and primary pneumocytes. Results Hydroxyproline content in irradiated lung was significantly reduced in mice that received rPAI-123 compared to mice that received vehicle (IR+vehicle: 84.97, IR+rPAI-123: 56.2 μg/lung, p=0.001). C57Bl/6 mice exposed to IR+vehicle had dense foci of subpleural fibrosis at 19 weeks, whereas the lungs of mice exposed to IR+rPAI-123 were largely devoid of fibrotic foci. Cellular senescence was significantly decreased by rPAI-123 treatment in primary pneumocyte cultures and in lung at multiple time points after IR. Conclusions These studies identify that rPAI-123 is capable of preventing radiation-induced fibrosis in murine lungs. These anti-fibrotic effects are associated with increased fibrin metabolism, enhanced matrix metalloproteinase-3 (MMP-3) expression and reduced senescence in type II pneumocytes. rPAI-123 is a novel therapeutic option for radiation-induced fibrosis.

Fatigue is a disabling symptom in patients with multiple sclerosis and Parkinson’s Disease, and is also common in patients with traumatic brain injury, cancer, and inflammatory disorders. Little is known about the neurobiology of fatigue, in part due to the lack of an approach to induce fatigue in laboratory animals. Fatigue is a common response to systemic challenge by pathogens, a response in part mediated through action of the pro-inflammatory cytokine interleukin-1 beta (IL-1β). We investigated the behavioral responses of mice to IL-1β. Female C57Bl/6J mice of 3 ages were administered IL-1β at various doses i.p. Interleukin-1β reduced locomotor activity, and sensitivity increased with age. Further experiments were conducted with middle-aged females. Centrally administered IL-1β dose-dependently reduced locomotor activity. Using doses of IL-1β that caused suppression of locomotor activity, we measured minimal signs of sickness, such as hyperthermia, pain or anhedonia (as measured with abdominal temperature probes, pre-treatment with the analgesic buprenorphine and through sucrose preference, respectively), all of which are responses commonly reported with higher doses. We found that middle-aged orexin-/- mice showed equivalent effects of IL-1β on locomotor activity as seen in wild-type controls, suggesting that orexins are not necessary for IL-1β -induced reductions in wheel-running. Given that the availability and success of therapeutic treatments for fatigue is currently limited, we examined the effectiveness of two potential clinical treatments, modafinil and methylphenidate. We found that these treatments were variably successful in restoring locomotor activity after IL-1β administration. This provides one step toward development of a satisfactory animal model of the multidimensional experience of fatigue, a model that could allow us to determine possible pathways through which inflammation induces fatigue, and could lead to novel treatments for reversal of fatigue.

Purpose/Objective(s): Despite advances in surgery, radiation, and chemotherapy, glioblastoma multiforme (GBM) has a poor very prognosis. Gene expression analysis is used to group GBMs into four subtypes: classical, proneural, mesenchymal, and neural, and potentially provide information about patient prognosis and therapeutic response. New biomarkers are emerging and used with the above subtyping to derive more information about patient prognosis and guide clinical treatment. The tumor microenvironment is involved with GBM progression and collagen is a large component of the tumor microenvironment. We have shown that fibrillar collagen is a survival marker for GBM, and performed this study to assess the interaction of collagen expression and organization with the four molecular GBM subtypes. Materials/Methods: The Cancer Genome Atlas (TCGA) was used to analyze fibrillar collagen gene expression in GBM patients in the classical, proneural, and mesenchymal subtypes. GSC sphere cultures were established from patient GBM samples by marker-neutral culture in stem cell media. Orthopedic xenografts were initiated in NOD-SCID mice by implanting 2x10 5 GSCs into the right striatum. Subsequent tumor formation (10-14 weeks) was verified by MRI and animals were sacrificed when moribund. Histology and immunohistochemistry (IHC) was performed on paraffin-embedded sections. GBM subtype was determined based on IHC. Classical GBM subtype is defined by high expressions of EGFR; the proneural GBM subtype is defined by high expression of PDGFRA, P53, or OLIG2; the mesenchymal GBM subtype is defined by high expression of two or more of vimentin, PTEN, YKL40, and/or CD44. IHC was also performed on a GBM tissue microarray (TMA) with clinical data for 111 patients in order to determine patient subtype. Picrosirius red staining was performed on brain sections and the TMA to detect collagen. Custom software was used to extract collagen fiber data and to quantify collagen fiber angle, length, straightness, and width from the picrosirius images. Results: TCGA analysis found that fibrillar collagen genes were most associated with the mesenchymal subtype. More organized collagen was found in the mesenchymal GBM xenografts with less organized collagen being found in the proneural and classical subtypes. Mice with mesenchymal xenografts and more organized collagen had a longer survival. Patients with more organized collagen were found in the mesenchymal group as well and also demonstrated increased survival. Conclusion: More organized collagen is associated with the mesenchymal GBM subtype and may be a biomarker for GBM patients. Patients with mesenchymal GBM are more resistant to radiation therapy. Thus, collagen might be a useful marker to determine radiotherapeutic strategies for patients in the future.

Background: Fibrosis can develop as a late side effect of radiation exposure in a variety of tissues, including lung and skin. Late radiation injury and fibrosis are characterized by parenchymal cell depletion, inflammation, senescence, fibroblast proliferation, and excessive deposition of collagen. Plasminogen activator inhibitor 1 (PAI-1) is a critical mediator of cellular senescence and fibrin stabilization, and its expression is increased in experimental fibrosis models. We sought to determine if inhibition of PAI-1 signaling with a recombinant truncated protein (rPAI-123) would protect from the development of radiation-induced lung injury. Methods: C57Bl/6 mice received intraperitoneal injections of rPAI-123 (5.4 μg/kg/day) or vehicle (PBS) for 18 weeks beginning two days prior to radiation exposure. Cohorts of mice treated with rPAI-123 or vehicle were exposed to thoracic irradiation in 5 daily fractions of 6 Gy (RT), and followed for survival (n = 8 per group) and tissue collection (n = 3 per each time point). Histologic changes in irradiated lungs were evaluated by Masson-Trichrome staining at 19 weeks after RT. Senescence was assessed with staining for beta-galactosidase activity in lung tissue and primary pneumocytes. To define the roles of rPAI-123 in the fibroproliferative response, cell proliferation (MTT assay) and collagen deposition (Hydroxyproline assay) were examined in a mouse fibroblast cell line in vitro. Results: Administration of rPAI-123 increased C57Bl/6 mice survival from 37.5% to 62.5% at 19 weeks after radiation exposure. At 19 weeks after irradiation, hydroxyproline content was markedly decreased in mice received rPAI-123 compared to mice received vehicle (RT+rPAI-123: 56.2±10.79, RT+vehicle: 84.97±2.98, μg/lung respectively, p = 0.001 between RT+vehicle and RT+rPAI-123). C57Bl/6 mice exposed to RT+vehicle had dense foci of subplueral fibrosis at 19 weeks, whereas the lungs of mice exposed to RT+rPAI-123 were largely devoid of fibrotic foci. Cellular senescence in response to radiation was significantly decreased by rPAI-123 treatment in primary type2 pneumocyte culture (2-fold reduction at 5 days after RT, p = 0.036), and in lung tissues (&gt;2-fold reduction at 4, 8, and 16 weeks after RT, p&lt;0.001 at each time point). Treatment of NIH-3T3 fibroblasts with rPAI-123 resulted in decreased collagen production, but had no effect on proliferation.Conclusions: These studies identify that rPAI-123 has a novel protection mechanism against radiation-induced fibrosis in murine lungs due to its ability to reduce senescence in type2 pneumocytes, and the potential to be an effective therapy option for radiation induced fibrosis. Citation Format: Eunjoo Chung, Ayla White, Bradley T. Scroggins, Grace B. McKay-Corkum, Mary Jo Mulligan-Kehoe, Deborah E. Citrin. A truncated Plasminogen Activator Inhibitor-1 protein protects from pulmonary fibrosis mediated by irradiation in a murine model. [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 1803. doi:10.1158/1538-7445.AM2015-1803

These results support SP1 as a target for radiation sensitization and confirm MTA as a radiation sensitizer in human tumor models.

No abstract

e22004 Background: RMS is the most common pediatric cancer of the soft tissues. Patients who present with metastatic disease or experience recurrence have a poor prognosis; survivors often suffer from long-term systemic effects resulting from cytotoxic chemotherapy. Thus, novel therapies are needed. NAMPT inhibitors are a class of drugs targeting a key metabolic enzyme in the production of NAD+, a coenzyme critical to energy generation in some cancer cells. In this study, we evaluated the mechanism and functional outcomes of treatment with the clinical NAMPT inhibitor, OT-82, in preclinical models of RMS. Methods: Live cell analysis via IncuCyte was used to determine the temporal effects of OT-82 on cell growth in a diverse panel of ten fusion positive and negative RMS cell lines. Analysis of the proposed mechanism of action was performed using NAD/NADH detection assays and rescue experiments with nicotinamide mononucleotide (NMN), the product of NAMPT. Markers of cellular apoptosis and necrosis were quantified with flow cytometric assays. Specific metabolic effects of OT-82 were determined with ATP quantification and real-time extracellular flux analysis of oxidative phosphorylation and glycolysis. In vivo studies were performed in orthotopic RMS models. Tumor dimensions were measured with calipers, and toxicity was assessed by observation and body weight measurement. Results: Treatment of RMS cell lines with OT-82 dosed in the low nanomolar range resulted in time- and dose-dependent decreases in NAD+ levels and proliferation in all cell lines tested. Addition of NMN rescued cell growth, confirming the on-target activity and functional effect of OT-82. Flow cytometric assays revealed cell-line dependent differences in cell fates, with a subset of cell lines staining positive for markers of necrosis, and the other subset staining negative for markers of necrosis and apoptosis. Functional investigation verified that necrotic cell lines did not regrow after withdrawal of OT-82 in culture (durable responders), whereas non-necrotic cell lines recovered growth (transient responders). Additionally, ATP levels in durable responders decreased with OT-82 treatment but remained stable in transient responders. Extracellular flux analysis revealed that both durable and transient responders experienced inhibition of glycolysis, but that oxidative phosphorylation was only reduced in the durable responders. In vivo studies using OT-82 on a clinically-relevant schedule demonstrated that all RMS xenografts underwent complete tumor regression, with durable responder models experiencing a longer tumor-free period following discontinuation of treatment. Conclusions: In vitro and in vivo efficacy of OT-82 suggest that targeting NAD+ metabolism through NAMPT inhibition may be a promising approach for the treatment of RMS.