Metabolomics reveals that tumor xenografts induce liver dysfunction.

Metabolomics, based on ultraperformance liquid chromatography coupled with electrospray ionization quadrupole mass spectrometry, was used to explore metabolic signatures of tumor growth in mice. Urine samples were collected from control mice and mice injected with squamous cell carcinoma (SCCVII) tumor cells. When tumors reached ∼2 cm, all mice were killed and blood and liver samples collected. The urine metabolites hexanoylglycine, nicotinamide 1-oxide, and 11ß,20α-dihydroxy-3-oxopregn-4-en-21-oic acid were elevated in tumor-bearing mice, as was asymmetric dimethylarginine, a biomarker for oxidative stress. Interestingly, SCCVII tumor growth resulted in hepatomegaly, reduced albumin/globulin ratios, and elevated serum triglycerides, suggesting liver dysfunction. Alterations in liver metabolites between SCCVII-tumor-bearing and control mice confirmed the presence of liver injury. Hepatic mRNA analysis indicated that inflammatory cytokines, tumor necrosis factor α, and transforming growth factor ß were enhanced in SCCVII-tumor-bearing mice, and the expression of cytochromes P450 was decreased in tumor-bearing mice. Further, genes involved in fatty acid oxidation were decreased, suggesting impaired fatty acid oxidation in SCCVII-tumor-bearing mice. Additionally, activated phospholipid metabolism and a disrupted tricarboxylic acid cycle were observed in SCCVII-tumor-bearing mice. These data suggest that tumor growth imposes a global inflammatory response that results in liver dysfunction and underscore the use of metabolomics to temporally examine these changes and potentially use metabolite changes to monitor tumor treatment response.

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.

Multiomic-Based Molecular Landscape of FaDu Xenograft Tumors in Mice after a Combinatorial Treatment with Radiation and an HSP90 Inhibitor Identifies Adaptation-Induced Targets of Resistance and Therapeutic Intervention.

Treatments involving radiation and chemotherapy alone or in combination have improved patient survival and quality of life. However, cancers frequently evade these therapies due to adaptation and tumor evolution. Given the complexity of predicting response based solely on the initial genetic profile of a patient, a predetermined treatment course may miss critical adaptation that can cause resistance or induce new targets for drug and immunotherapy. To address the timescale for these evasive mechanisms, using a mouse xenograft tumor model, we investigated the rapidity of gene expression (mRNA), molecular pathway, and phosphoproteome changes after radiation, an HSP90 inhibitor, or combination. Animals received radiation, drug, or combination treatment for 1 or 2 weeks and were then euthanized along with a time-matched untreated group for comparison. Changes in gene expression occur as early as 1 week after treatment initiation. Apoptosis and cell death pathways were activated in irradiated tumor samples. For the HSP90 inhibitor and combination treatment at weeks 1 and 2 compared with Control Day 1, gene-expression changes induced inhibition of pathways including invasion of cells, vasculogenesis, and viral infection among others. The combination group included both drug-alone and radiation-alone changes. Our data demonstrate the rapidity of gene expression and functional pathway changes in the evolving tumor as it responds to treatment. Discovering these phenotypic adaptations may help elucidate the challenges in using sustained treatment regimens and could also define evolving targets for therapeutic efficacy.

Functional RNAi screening identifies G2/M and kinetochore components as modulators of TNFα/NF-κB pro-survival signaling in head and neck squamous cell carcinoma.

Immune and radiation resistance of cancer cells to cytotoxicity mediated by Tumor Necrosis Factor-α (TNFα) is promoted by the transcription factor NF-κB in several cancers, including head and neck squamous cell carcinoma (HNSCC). Genomic alterations that converge on the TNFα/NF-κB signal axis were found in ~40% of HNSCCs by The Cancer Genome Atlas (TCGA). However, identification of therapeutic targets that contribute to aberrant TNFα/NF-κB activation and resistance has been challenging. Here, we conducted a functional RNAi screen to identify regulators of TNFα-induced NF-κB activation and cell viability, using parallel NF-κB ß-lactamase reporter and cell viability assays in a HNSCC cell line which harbors expression and genomic alterations typically found in HPV-negative HNSCC. Besides multiple components of canonical TNFα/NF-κB signaling, we identified components of the WNT, NOTCH, and TGFß pathways that we previously showed contribute to non-canonical activation of NF-κB. Unexpectedly, we also observed that multiple G2/M cell cycle kinases (AURKA, PLK1, WEE1, TTK), and structural kinetochore/microtubule components (NDC80, NUF2), modulate TNFα-induced NF-κB activation and cell viability. Several of these targets inhibit TNF-induced nuclear translocation of RELA, consistent with prior reports linking NF-B activation to G2/M kinases or microtubule assembly. Further investigation of an understudied mitotic kinase, TTK/MPS1, show that it's inhibition or depletion attenuates TNFα-induced RELA nuclear translocation, promoting cell death, DNA damage, polyploidy, and mitotic catastrophe, leading to radiosensitization. Together, our RNAi screening identifies a critical linkage between the G2/M cell cycle checkpoint/kinetochore components and NF-κB activity, and as targets that can sensitize HNSCC cells to TNFα or radiation.

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.

Combined Inhibition of IAPs and WEE1 Enhances TNFα- and Radiation-Induced Cell Death in Head and Neck Squamous Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) remains a prevalent diagnosis with current treatment options that include radiotherapy and immune-mediated therapies, in which tumor necrosis factor-α (TNFα) is a key mediator of cytotoxicity. However, HNSCC and other cancers often display TNFα resistance due to activation of the canonical IKK-NFκB/RELA pathway, which is activated by, and induces expression of, cellular inhibitors of apoptosis proteins (cIAPs). Our previous studies have demonstrated that the IAP inhibitor birinapant sensitized HNSCC to TNFα-dependent cell death in vitro and radiotherapy in vivo. Furthermore, we recently demonstrated that the inhibition of the G2/M checkpoint kinase WEE1 also sensitized HNSCC cells to TNFα-dependent cell death, due to the inhibition of the pro-survival IKK-NFκB/RELA complex. Given these observations, we hypothesized that dual-antagonist therapy targeting both IAP and WEE1 proteins may have the potential to synergistically sensitize HNSCC to TNFα-dependent cell death. Using the IAP inhibitor birinapant and the WEE1 inhibitor AZD1775, we show that combination treatment reduced cell viability, proliferation and survival when compared with individual treatment. Furthermore, combination treatment enhanced the sensitivity of HNSCC cells to TNFα-induced cytotoxicity via the induction of apoptosis and DNA damage. Additionally, birinapant and AZD1775 combination treatment decreased cell proliferation and survival in combination with radiotherapy, a critical source of TNFα. These results support further investigation of IAP and WEE1 inhibitor combinations in preclinical and clinical studies in HNSCC.

Inhibition of USP14 promotes TNFα-induced cell death in head and neck squamous cell carcinoma (HNSCC).

TNFα is a key mediator of immune, chemotherapy and radiotherapy-induced cytotoxicity, but several cancers, including head and neck squamous cell carcinomas (HNSCC), display resistance to TNFα due to activation of the canonical NFκB pro-survival pathway. However, direct targeting of this pathway is associated with significant toxicity; thus, it is vital to identify novel mechanism(s) contributing to NFκB activation and TNFα resistance in cancer cells. Here, we demonstrate that the expression of proteasome-associated deubiquitinase USP14 is significantly increased in HNSCC and correlates with worse progression free survival in Human Papillomavirus (HPV)- HNSCC. Inhibition or depletion of USP14 inhibited the proliferation and survival of HNSCC cells. Further, USP14 inhibition reduced both basal and TNFα-inducible NFκB activity, NFκB-dependent gene expression and the nuclear translocation of the NFκB subunit RELA. Mechanistically, USP14 bound to both RELA and IκBα and reduced IκBα K48-ubiquitination leading to the degradation of IκBα, a critical inhibitor of the canonical NFκB pathway. Furthermore, we demonstrated that b-AP15, an inhibitor of USP14 and UCHL5, sensitized HNSCC cells to TNFα-mediated cell death, as well as radiation-induced cell death in vitro. Finally, b-AP15 delayed tumor growth and enhanced survival, both as a monotherapy and in combination with radiation, in HNSCC tumor xenograft models in vivo, which could be significantly attenuated by TNFα depletion. These data offer new insights into the activation of NFκB signaling in HNSCC and demonstrate that small molecule inhibitors targeting the ubiquitin pathway warrant further investigation as a novel therapeutic avenue to sensitize these cancers to TNFα- and radiation-induced cytotoxicity.

The antioxidant tempol transforms gut microbiome to resist obesity in female C3H mice fed a high fat diet.

The nitroxide, Tempol, prevents obesity related changes in mice fed a high fat diet (HFD). The purpose of this study was to gain insight into the mechanisms that result in such changes by Tempol in female C3H mice. Microarray methodology, Western blotting, bile acid analyses, and gut microbiome sequencing were used to identify multiple genes, proteins, bile acids, and bacteria that are regulated by Tempol in female C3H mice on HFD. The effects of antibiotics in combination with Tempol on the gut microflora were also studied. Adipose tissue, from Tempol treated mice, was analyzed using targeted gene microarrays revealing up-regulation of fatty acid metabolism genes (Acadm and Acadl > 4-fold, and Acsm3 and Acsm5 > 10-fold). Gene microarray studies of liver tissue from mice switched from HFD to Tempol HFD showed down-regulation of fatty acid synthesis genes and up-regulation of fatty acid oxidation genes. Analyses of proteins involved in obesity revealed that the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) and fasting induced adipose factor/angiopoietin-like protein 4 (FIAF/ANGPTL4) was altered by Tempol HFD. Bile acid studies revealed increases in cholic acid (CA) and deoxycholic acid (DCA) in both the liver and serum of Tempol treated mice. Tempol HFD effect on the gut microbiome composition showed an increase in the population of Akkermansia muciniphila, a bacterial species known to be associated with a lean, anti-inflammatory phenotype. Antibiotic treatment significantly reduced the total level of bacterial numbers, however, Tempol was still effective in reducing the HFD weight gain. Even after antibiotic treatment Tempol still positively influenced several bacterial species such as as Akkermansia muciniphila and Bilophila wadsworthia. The positive effects of Tempol moderating weight gain in female mice fed a HFD involves changes to the gut microbiome, bile acids composition, and finally to changes in genes and proteins involved in fatty acid metabolism and storage.

Intracellular hypoxia of tumor tissue estimated by noninvasive electron paramagnetic resonance oximetry technique using paramagnetic probes.

Electron paramagnetic resonance (EPR) oximetry at 700 MHz operating frequency employing a surface coil resonator is used to assess tissue partial pressure of oxygen (pO(2)) using paramagnetic media whose linewidth and decay constant are related to oxygen concentration. Differences in extracellular and intracellular pO(2) in squamous cell carcinoma (SCC) tumor tissue were tested using several types of water-soluble paramagnetic media, which localize extracellularly or permeate through the cell membrane. The nitroxide carboxy-PROXYL (CxP) can only be distributed in blood plasma and extracellular fluids whereas the nitroxides carbamoyl-PROXYL (CmP) and TEMPOL (TPL) can permeate cell membranes and localize intracellularly. EPR signal decay constant and the linewidth of the intravenously administered nitroxides in SCC tumor tissues implanted in mouse thigh and the contralateral normal muscle of healthy mice breathing gases with different pO(2) were compared. The pO(2) in the blood can depend on the oxygen content in the breathing gas while tissue pO(2) was not directly influenced by pO(2) in the breathing gas. The decay constants of CmP and TPL in tumor tissue were significantly larger than in the normal muscles, and lower linewidths of CmP and TPL in tumor tissue was observed. The SCC tumor showed intracellular hypoxia even though the extracellular pO(2) is similar to normal tissue in the peripheral region.

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.

Halofuginone mediated protection against radiation-induced leg contracture.

Fibrosis of normal tissues often accompanies radiation treatment of cancer. Activation of the transforming growth factor-beta (TGF-beta) signaling pathway is thought to play a major role in radiation-induced fibrosis and has prompted the development and assessment of low molecular weight inhibitors of the pathway. Previous studies with halofuginone have shown it to inhibit TGF-beta signaling in vitro and protect mice from radiation-induced leg contraction (a model for soft tissue fibrosis). The current study confirms these findings for HaCaT cells stimulated with exogenous TGF-beta treatment. Reducing the halifuginone treatment from 7 days/week (used previously) to 5 days/week post-radiation exposure provided significant protection against radiation-induced leg contraction in mice 3 and 4 months post-radiation treatment. Halofuginone treatment was shown to attenuate TGF-beta signaling molecules taken from irradiated skin including TGF-betaRII, pSmad3, Smad7, and TSP1. The latter, TSP1, a co-activator of TGF-beta may serve as a suitable biomarker for monitoring the efficacy of halofuginone should it be evaluated in a clinical setting for protection against radiation-induced fibrosis.

Differential radiation protection of salivary glands versus tumor by Tempol with accompanying tissue assessment of Tempol by magnetic resonance imaging.

PURPOSE: The nitroxide free radical, Tempol, was evaluated for potential differential radiation protection of salivary glands and tumor using fractionated radiation. Mechanistic information was explored by monitoring the presence and bioreduction of Tempol in both tissues noninvasively by magnetic resonance imaging (MRI). EXPERIMENTAL DESIGN: Female C3H mice were immobilized using custom-made Lucite jigs for localized irradiation (five daily fractions) either to the oral cavity or tumor-bearing leg. Tempol (275 mg/kg) was administered (i.p.) 10 min before each radiation fraction. Salivary gland damage was assessed 8 weeks after radiation by measuring pilocarpine-mediated saliva output. Tumor growth was assessed by standard radiation regrowth methods. Dynamic T1-weighted magnetic resonance scans were acquired before and after Tempol injection using a 4.7T animal MRI instrument. RESULTS: Tempol treatment was found to protect salivary glands significantly against radiation damage (approximately 60% improvement); whereas no tumor protection was observed. Intracellular reduction of Tempol to the nonradioprotective hydroxylamine as assessed by MRI was 2-fold faster in tumor compared with salivary glands or muscle. CONCLUSIONS: Tempol provided salivary gland radioprotection and did not protect tumor, consistent with the hypothesis that differential radioprotection by Tempol resides in faster reduction to the nonradioprotective hydroxylamine in tumor compared with normal tissues. The unique paramagnetic properties of Tempol afforded noninvasive MRI monitoring of dynamic changes of Tempol levels in tissue to support the finding. These data support further development and consideration of Tempol for human clinical trials as a selective protector against radiation-induced salivary gland damage.

Assessment of tissue redox status using metabolic responsive contrast agents and magnetic resonance imaging.

Regulation of tissue redox status is important to maintain normal physiological conditions in the living body. Disruption of redox homoeostasis may lead to oxidative stress and can induce many pathological conditions such as cancer, neurological disorders and ageing. Therefore, imaging of tissue redox status could have clinical applications. Redox imaging employing magnetic resonance imaging (MRI) with nitroxides as cell-permeable redox-sensitive contrast agents has been used for non-invasive monitoring of tissue redox status in animal models. The redox imaging applications of nitroxide electron paramagnetic resonance imaging (EPRI) and MRI are reviewed here, with a focus on application of tumour redox status monitoring. While particular emphasis has been placed on differences in the redox status in tumours compared to selected normal tissues, the technique possesses the potential to have broad applications to the study of other disease states, inflammatory processes and other circumstances where oxidative stress is implicated.

Cancer Incidence in C3H Mice Protected from Lethal Total-Body Radiation after Amifostine.

Amifostine is a potent antioxidant that protects against ionizing radiation effects. In this study, we evaluated the effect of Amifostine administered before total-body irradiation (TBI), at a drug dose that protects against TBI lethality, for potential protection against radiation-induced late effects such as a shortened lifespan and cancer. Three groups of mice were studied: 0 Gy control; 10.8 Gy TBI with Amifostine pretreatment; and 5.4 Gy TBI alone. Animals were monitored for their entire lifespan. The median survival times for mice receiving 0, 5.4 or 10.8 Gy TBI were 706, 460 and 491 days, respectively. Median survival of both irradiated groups was significantly shorter compared to nonirradiated mice ( P < 0.0001). Cancer incidence (hematopoietic and solid tumors) was similar between the irradiated groups and was significantly greater than for the 0 Gy controls. The ratio of hematopoietic-to-solid tumors differed among the groups, with the 5.4 Gy group having a higher incidence of hematopoietic neoplasms compared to the 10.8 Gy/Amifostine group (1.8-fold). Solid tumor incidence was greater in the 10.8 Gy/Amifostine group (1.6-fold). There are few mouse lifespan studies for agents that protect against radiation-induced lethality. Mice treated with 10.8 Gy/Amifostine yielded a lower incidence of hematopoietic neoplasms and higher incidence of solid neoplasms. In conclusion, mice protected from lethal TBI have a shortened lifespan, due in large part to cancer induction after exposure compared to nonexposed controls. Amifostine treatment did protect against radiation-induced hematopoietic tumors, while protection against solid neoplasms was significant but incomplete.

The influence of tumor oxygenation on hypoxia imaging in murine squamous cell carcinoma using [64Cu]Cu-ATSM or [18F]Fluoromisonidazole positron emission tomography.

[64Cu]Cu(II)-ATSM (64Cu-ATSM) and [18F]-Fluoromisonidazole (18F-FMiso) tumor binding as assessed by positron emisson topography (PET) was used to determine the responsiveness of each probe to modulation in tumor oxygenation levels in the SCCVII tumor model. Animals bearing the SCCVII tumor were injected with 64Cu-ATSM or 18F-FMiso followed by dynamic small animal PET imaging. Animals were imaged with both agents using different inspired oxygen mixtures (air, 10% oxygen, carbogen) which modulated tumor hypoxia as independently assessed by the hypoxia marker pimonidazole. The extent of hypoxia in the SCCVII tumor as monitored by the pimonidazole hypoxia marker was found to be in the following order: 10% oxygen>air>carbogen. Tumor uptake of 64Cu-ATSM could not be changed if the tumor was oxygenated using carbogen inhalation 90 min post-injection suggesting irreversible cellular uptake of the 64Cu-ATSM complex. A small but significant paradoxical increase in 64Cu-ATSM tumor uptake was observed for animals breathing air or carbogen compared to 10% oxygen. There was a positive trend toward 18F-FMiso tumor uptake as a function of changing hypoxia levels in agreement with the pimonidazole data. 64Cu-ATSM tumor uptake was unable to predictably detect changes in varying amounts of hypoxia when oxygenation levels in SCCVII tumors were modulated. 18F-FMiso tumor uptake was more responsive to changing levels of hypoxia. While the mechanism of nitroimidazole binding to hypoxic cells has been extensively studied, the avid binding of Cu-ATSM to tumors may involve other mechanisms independent of hypoxia that warrant further study.

High-resolution mapping of tumor redox status by magnetic resonance imaging using nitroxides as redox-sensitive contrast agents.

PURPOSE: There is considerable research directed toward the identification and development of functional contrast agents for medical imaging that superimpose tissue biochemical/molecular information with anatomical structures. Nitroxide radicals were identified as in vivo radioprotectors. Being paramagnetic, they can provide image contrast in magnetic resonance imaging (MRI) and electron paramagnetic resonance imaging (EPRI). The present study sought to determine the efficacy of nitroxide radioprotectors as functional image contrast agents. EXPERIMENTAL DESIGN: Nitroxide radioprotectors, which act as contrast agents, were tested by EPRI and MRI to provide tissue redox status information noninvasively. RESULTS: Phantom studies showed that the nitroxide, 3-carbamoyl-PROXYL (3CP), undergoes time-dependent reduction to the corresponding diamagnetic hydroxylamine only in the presence of reducing agents. The reduction rates of 3CP obtained by EPRI and MRI were in agreement suggesting the feasibility of using MRI to monitor nitroxide levels in tissues. The levels of 3CP were examined by EPRI and MRI for differences in reduction between muscle and tumor (squamous cell carcinoma) implanted in the hind leg of C3H mice simultaneously. In vivo experiments showed a T1-dependent image intensity enhancement afforded by 3CP which decreased in a time-dependent manner. Reduction of 3CP was found to be the dominant mechanism of contrast loss. The tumor regions exhibited a faster decay rate of the nitroxide compared to muscle (0.097 min(-1) versus 0.067 min(-1), respectively). CONCLUSIONS: This study shows that MRI can be successfully used to co-register tissue redox status along with anatomic images, thus providing potentially valuable biochemical information from the region of interest.

Radiation-induced changes in gene-expression profiles for the SCC VII tumor cells grown in vitro and in vivo.

SCCVII tumor cells that grow in vitro or in vivo as a solid tumor were used to compare and contrast geneexpression profiles with or without exposure to two doses of ionizing radiation. Exponentially growing SCCVII cell cultures and tumors (1 cm diameter) were treated with 0, 2, or 10 Gy, and RNA was collected 1, 3, 6, 12, and 24 h after treatment. Growth under in vitro conditions increased the expression of genes associated with the unfolded protein response (UPR) including ATF4, Ero-1 like, and cystathionase. Growth in vivo indicated that the HIF-1a genes were not upregulated, whereas genes such as hemoglobin alpha and crystallin alpha B were significantly upregulated. Ninety genes of 16K were found to be significantly modulated under either growth condition by radiation treatment. Gene expression was not dose dependent. Sixty percent of these genes exhibited similar modulation under both in vitro and in vivo conditions; however, 29% of the genes were modulated by radiation under in vivo conditions only. Gene-expression profiles for the same tumor cells can differ, dependent on growth conditions, underscoring the influence that the tumor microenvironment exerts on gene expression for both growth of solid tumors and their response to radiation treatment.

Evaluation of radiation-induced oral mucositis by optical coherence tomography.

PURPOSE: Optical coherence tomography (OCT) imaging was evaluated to determine if radiation-induced mucosal damage could be noninvasively monitored in real time and correlated with histopathologic findings. EXPERIMENTAL DESIGN: Female C3H mice, ages 7 to 9 weeks, four per group, were immobilized in a custom-made Lucite jig and received 0, 15, 22.5, and 25 Gy in a single fraction to their oral cavity. OCT images were acquired of proximal, middle, and distal aspects of the dorsum of the tongue on days 0, 1, 3, 5, and 7 post-irradiation. Animals were sacrificed on day 7 and samples taken for histologic evaluation. OCT images were visually examined and also quantified by image analysis and compared with histologic findings. RESULTS: Tongues removed 7 days post-irradiation showed no visible damage; however, upon staining with toluidine blue, ulcers at the base of the tongue became visible (100% for 25 Gy, 75% after 22.5 Gy, and 0% after 15 Gy). Visual inspection of OCT images qualitatively compared with histologic findings and quantitative image analysis of the OCT images (effective light penetration depth) revealed significant changes 7 days post-irradiation compared with unirradiated controls for the base of the tongue. CONCLUSIONS: OCT allows for direct noninvasive real-time acquisition of digitally archivable images of oral mucosa and can detect radiation-induced changes in the mucosa before visual manifestation. OCT may be a useful technique to quantify subclinical radiation-induced mucosal injury in experimental chemoradiation clinical trials.

Kinetics of tempol for prevention of xerostomia following head and neck irradiation in a mouse model.

PURPOSE: Radiotherapy is commonly used to treat the majority of patients with head and neck cancers. Salivary glands in the radiation field are dramatically affected by this procedure. The purpose of this study was to examine pharmacokinetic characteristics of the stable nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol) with respect to radioprotection of the salivary glands. EXPERIMENTAL DESIGN: To evaluate the effect of different doses and times of administration, the heads of C3H mice were exposed to a single irradiation dose of 15 Gy, with i.p. tempol injection. To analyze other routes of administration, we injected 275 mg/kg tempol by an i.m., i.v., or s.c. route, 10 minutes before irradiation. We also tested whether oral administration of tempol in a topical form (either in a mouthwash or gel) provided any salivary gland protection. RESULTS: Tempol treatment (137.5 or 275 mg/kg, i.p., 10 minutes before irradiation) significantly reduced irradiation-induced salivary hypofunction (approximately 50-60%). I.v. or s.c. administration of tempol also showed significant radioprotection, whereas i.m. administration proved to be ineffective. Topical use of tempol, either as a mouthwash or gel, also was radioprotective. CONCLUSIONS: Our results strongly suggest that tempol is a promising candidate for clinical application to protect salivary glands in patients undergoing radiotherapy for head and neck cancers.

Mass Spectrometry-Based Metabolomics Identifies Longitudinal Urinary Metabolite Profiles Predictive of Radiation-Induced Cancer.

Nonlethal exposure to ionizing radiation (IR) is a public concern due to its known carcinogenic effects. Although latency periods for IR-induced neoplasms are relatively long, the ability to detect cancer as early as possible is highly advantageous for effective therapeutic intervention. Therefore, we hypothesized that metabolites in the urine from mice exposed to total body radiation (TBI) would predict for the presence of cancer before a palpable mass was detected. In this study, we exposed mice to 0 or 5.4 Gy TBI, collected urine samples periodically over 1 year, and assayed urine metabolites by using mass spectrometry. Longitudinal data analysis within the first year post-TBI revealed that cancers, including hematopoietic, solid, and benign neoplasms, could be distinguished by unique urinary signatures as early as 3 months post-TBI. Furthermore, a distinction among different types of malignancies could be clearly delineated as early as 3 months post-TBI for hematopoietic neoplasms, 6 months for solid neoplasms, and by 1 year for benign neoplasms. Moreover, the feature profile for radiation-exposed mice 6 months post-TBI was found to be similar to nonirradiated control mice at 18 months, suggesting that TBI accelerates aging. These results demonstrate that urine feature profiles following TBI can identify cancers in mice prior to macroscopic detection, with important implications for the early diagnosis and treatment.