Pre-clinical evaluation of OxyChip for long-term EPR oximetry.

Tissue oxygenation is a critical parameter in various pathophysiological situations including cardiovascular disease and cancer. Hypoxia can significantly influence the prognosis of solid malignancies and the efficacy of their treatment by radiation or chemotherapy. Electron paramagnetic resonance (EPR) oximetry is a reliable method for repeatedly assessing and monitoring oxygen levels in tissues. Lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) has been developed as a probe for biological EPR oximetry, especially for clinical use. However, clinical applicability of LiNc-BuO crystals is hampered by potential limitations associated with biocompatibility, biodegradation, or migration of individual bare crystals in tissue. To overcome these limitations, we have embedded LiNc-BuO crystals in polydimethylsiloxane (PDMS), an oxygen-permeable biocompatible polymer and developed an implantable/retrievable form of chip, called OxyChip. The chip was optimized for maximum spin density (40% w/w of LiNc-BuO in PDMS) and fabricated in a form suitable for implantation using an 18-G syringe needle. In vitro evaluation of the OxyChip showed that it is robust and highly oxygen sensitive. The dependence of its EPR linewidth to oxygen was linear and highly reproducible. In vivo efficacy of the OxyChip was evaluated by implanting it in rat femoris muscle and following its response to tissue oxygenation for up to 12 months. The results revealed preservation of the integrity (size and shape) and calibration (oxygen sensitivity) of the OxyChip throughout the implantation period. Further, no inflammatory or adverse reaction around the implantation area was observed thereby establishing its biocompatibility and safety. Overall, the results demonstrated that the newly-fabricated high-sensitive OxyChip is capable of providing long-term measurements of oxygen concentration in a reliable and repeated manner under clinical conditions.

Rapamycin Reduces Carcinogenesis and Enhances Survival in Mice when Administered after Nonlethal Total-Body Irradiation.

The rationale of this study stems from the concern of a radiation-induced accident or terrorist-mediated nuclear attack resulting in large populations of people exposed to nonlethal radiation doses or after a course of definitive radiation therapy which could substantially increase the risk for cancer induction after exposure. Currently, there are no safe and effective interventions to reduce this increased cancer risk to humans. We have tested the hypothesis that the mTOR inhibitor, rapamycin, administered in the diet of mice would reduce or delay radiation-induced cancer when given after radiation exposure. A total-body irradiation (TBI) of 3 Gy was administered to female C3H/Hen mice. Immediately after TBI, along with untreated control groups, animals were placed on chow containing different concentrations of encapsulated rapamycin (14, 40, 140 mg/kg chow). Animals remained on the respective control or rapamycin diets and were followed for their entire lifespan (total of 795 mice). The endpoint for the study was tumor formation (not to exceed 1 cm) or until the animal reached a humane endpoint at which time the animal was euthanized and evaluated for the presence of tumors (pathology evaluated on all animals). Kaplan-Meier survival curves revealed that all three concentrations of rapamycin afforded a significant survival advantage by delaying the time at which tumors appeared and reduction of the incidence of certain tumor types such as hepatocellular carcinomas. The survival advantage was dependent on the rapamycin concentration used. Further, there was a survival advantage when delaying the rapamycin chow by 1 month after TBI. Rapamycin is FDA-approved for human use and could be considered for use in individuals exposed to nonlethal TBI from a nuclear accident or attack or after significant therapeutic doses for cancer treatment.

The effect of modulation of gut microbiome profile on radiation-induced carcinogenesis and survival.

Non-lethal doses of ionizing radiation (IR) delivered to humans because of terrorist events, nuclear accidents or radiotherapy can result in carcinogenesis. Means of protecting against carcinogenesis are lacking. We questioned the role of the gut microbiome in IR-induced carcinogenesis. The gut microbiome was modulated by administering broad spectrum antibiotics (Ab) in the drinking water. Mice were given Ab 3 weeks before and 3 weeks after 3 Gy total body irradiation (TBI) or for 6 weeks one month after TBI. Three weeks of Ab treatment resulted in a 98% reduction in total 16S rRNA counts for 4 out of 6 of the phylum groups detected. However, 3 more weeks of Ab treatment (6 weeks total) saw an expansion in the phylum groups Proteobacteria and Actinobacteria. The Ab treatment altered the bacteria diversity in the gut, and shortened the lifespan when Ab were administered before and after TBI. Mortality studies indicated that the adverse Ab lifespan effects were due to a decrease in the time in which solid tumors started to appear and not to any changes in hematopoietic or benign tumors. In contrast, when Ab were administered one month after TBI, lifespan was unchanged compared to the control TBI group. Use of broad-spectrum antibiotics to simulate the germ-free condition did not afford an advantage on carcinogenesis or lifespan.

Pharmacological Inhibition of HSP90 Radiosensitizes Head and Neck Squamous Cell Carcinoma Xenograft by Inhibition of DNA Damage Repair, Nucleotide Metabolism, and Radiation-Induced Tumor Vasculogenesis.

PURPOSE: Recent preclinical studies suggest combining the HSP90 inhibitor AT13387 (Onalespib) with radiation (IR) against colon cancer and head and neck squamous cell carcinoma (HNSCC). These studies emphasized that AT13387 downregulates HSP90 client proteins involved in oncogenic signaling and DNA repair mechanisms as major drivers of enhanced radiosensitivity. Given the large array of client proteins HSP90 directs, we hypothesized that other key proteins or signaling pathways may be inhibited by AT13387 and contribute to enhanced radiosensitivity. Metabolomic analysis of HSP90 inhibition by AT13387 was conducted to identify metabolic biomarkers of radiosensitization and whether modulations of key proteins were involved in IR-induced tumor vasculogenesis, a process involved in tumor recurrence. METHODS AND MATERIALS: HNSCC and non-small cell lung cancer cell lines were used to evaluate the AT13387 radiosensitization effect in vitro and in vivo. Flow cytometry, immunofluorescence, and immunoblot analysis were used to evaluate cell cycle changes and HSP90 client protein's role in DNA damage repair. Metabolic analysis was performed using liquid chromatography-Mass spectrometry. Immunohistochemical examination of resected tumors post-AT13387 and IR treatment were conducted to identify biomarkers of IR-induced tumor vasculogenesis. RESULTS: In agreement with recent studies, AT13387 treatment combined with IR resulted in a G2/M cell cycle arrest and inhibited DNA repair. Metabolomic profiling indicated a decrease in key metabolites in glycolysis and tricarboxylic acid cycle by AT13387, a reduction in Adenosine 5'-triphosphate levels, and rate-limiting metabolites in nucleotide metabolism, namely phosphoribosyl diphosphate and aspartate. HNSCC xenografts treated with the combination exhibited increased tumor regrowth delay, decreased tumor infiltration of CD45 and CD11b+ bone marrow-derived cells, and inhibition of HIF-1 and SDF-1 expression, thereby inhibiting IR-induced vasculogenesis. CONCLUSIONS: AT13387 treatment resulted in pharmacologic inhibition of cancer cell metabolism that was linked to DNA damage repair. AT13387 combined with IR inhibited IR-induced vasculogenesis, a process involved in tumor recurrence postradiotherapy. Combining AT13387 with IR warrants consideration of clinical trial assessment.

Dynamic EPR Oximetry of Changes in Intracerebral Oxygen Tension During Induced Thromboembolism.

Cerebral tissue oxygenation (oxygen tension, pO2) is a critical parameter that is closely linked to brain metabolism, function, and pathophysiology. In this work, we have used electron paramagnetic resonance oximetry with a deep-tissue multi-site oxygen-sensing probe, called implantable resonator, to monitor temporal changes in cerebral pO2 simultaneously at four sites in a rabbit model of ischemic stroke induced by embolic clot. The pO2 values in healthy brain were not significantly different among the four sites measured over a period of 4 weeks. During exposure to 15% O2 (hypoxia), a sudden and significant decrease in pO2 was observed in all four sites. On the other hand, brief exposure to breathing carbogen gas (95% O2 + 5% CO2) showed a significant increase in the cerebral pO2 from baseline value. During ischemic stroke, induced by embolic clot in the left brain, a significant decline in the pO2 of the left cortex (ischemic core) was observed without any change in the contralateral sites. While the pO2 in the non-infarct regions returned to baseline at 24-h post-stroke, pO2 in the infarct core was consistently lower compared to the baseline and other regions of the brain. The results demonstrated that electron paramagnetic resonance oximetry with the implantable resonator can repeatedly and simultaneously report temporal changes in cerebral pO2 at multiple sites. This oximetry approach can be used to develop interventions to rescue hypoxic/ischemic tissue by modulating cerebral pO2 during hypoxic and stroke injury.