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Results for “Bioluminescence Imaging”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Tracing the dynamic expression of the Nfκb2 gene during inflammatory processes by in vivo bioluminescence imaging in transgenic mice

Highlights: • Construct a mouse transgenic model line which could trace the p100/p50 expression by luciferase in vivo. • Nfκb2 could express in all mouse tissues and the luciferase expression could represent Nfκb2 expression in most tissues. • Lps induced inflammation could increase luciferase expression in certain stage. • In the IBD model, the fluorescence expression in mouse abdomen was coincide with the intestine after DSSfeed. Nfκb2(p52/p100) plays essential roles in many chronic inflammatory diseases. Tracing the dynamic expression of Nfκb2 during different biological processes in vivo can provide valuable clues to understand the biological functions of this gene and develop anti-inflammatory drugs. In this study, B6-Tg(Nfκb2-luc){sup Mlit} transgenic mouse line, a mouse model in which the expression of firefly luciferase gene is under the control of a 14.6-kb mouse Nfκb2 promoter, was generated to monitor the expression of p52/p100 in vivo. Bioluminescence imaging was used for tracking the luciferase signal in living mice in a variety of inflammatory processes, including LPS-induced sepsis and inflammatory bowel disease (IBD). The data of in vivo bioluminescence imaging in this mouse model showed that luciferase activity coincided with the endogenous p52/p100 expression. Moreover, dexamethasone or aspirin, two routine anti-inflammatory drugs, could decrease the high-level expression of luciferase induced by LPS. Overall, our results suggest that the B6-Tg(Nfκb2-luc){sup Mlit} mice represent a valuable reporter mouse model not only to monitor the expression of p52/p100 in physiological or pathological processes but also to evaluate the effects of various anti-inflammatory drug treatments in vivo.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Susceptibility of the wild-derived inbred CAST/Ei mouse to infection by orthopoxviruses analyzed by live bioluminescence imaging

Classical inbred mice are extensively used for virus research. However, we recently found that some wild-derived inbred mouse strains are more susceptible than classical strains to monkeypox virus. Experiments described here indicated that the 50% lethal dose of vaccinia virus (VACV) and cowpox virus (CPXV) were two logs lower in wild-derived inbred CAST/Ei mice than classical inbred BALB/c mice, whereas there was little difference in the susceptibility of the mouse strains to herpes simplex virus. Live bioluminescence imaging was used to follow spread of pathogenic and attenuated VACV strains and CPXV virus from nasal passages to organs in the chest and abdomen of CAST/Ei mice. Luminescence increased first in the head and then simultaneously in the chest and abdomen in a dose-dependent manner. The spreading kinetics was more rapid with VACV than CPXV although the peak photon flux was similar. These data suggest advantages of CAST/Ei mice for orthopoxvirus studies. - Highlights: • Wild-derived inbred CAST/Ei mice are susceptible to vaccinia virus and cowpox virus. • Morbidity and mortality from orthopoxviruses are greater in CAST/Ei than BALB/c mice. • Morbidity and mortality from herpes simplex virus type 1 are similar in both mice. • Imaging shows virus spread from nose to lungs, abdominal organs and brain. • Vaccinia virus spreads more rapidly than cowpox virus.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Preclinical evaluation of destruxin B as a novel Wnt signaling target suppressing proliferation and metastasis of colorectal cancer using non-invasive bioluminescence imaging

In continuation to our studies toward the identification of direct anti-cancer targets, here we showed that destruxin B (DB) from Metarhizium anisopliae suppressed the proliferation and induced cell cycle arrest in human colorectal cancer (CRC) HT29, SW480 and HCT116 cells. Additionally, DB induced apoptosis in HT29 cells by decreased expression level of anti-apoptotic proteins Bcl-2 and Bcl-xL while increased pro-apoptotic Bax. On the other hand, DB attenuated Wnt-signaling by downregulation of β-catenin, Tcf4 and β-catenin/Tcf4 transcriptional activity, concomitantly with decreased expression of β-catenin target genes cyclin D1, c-myc and survivin. Furthermore, DB affected the migratory and invasive ability of HT29 cells through suppressed MMPs-2 and -9 enzymatic activities. We also found that DB targeted the MAPK and/or PI3K/Akt pathway by reduced expression of Akt, IKK-α, JNK, NF-κB, c-Jun and c-Fos while increased that of IκBα. Finally, we demonstrated that DB inhibited tumorigenesis in HT29 xenograft mice using non-invasive bioluminescence technique. Consistently, tumor samples from DB-treated mice demonstrated suppressed expression of β-catenin, cyclin D1, survivin, and endothelial marker CD31 while increased caspase-3 expression. Collectively, our data supports DB as an inhibitor of Wnt/β-catenin/Tcf signaling pathway that may be beneficial in the CRC management. Highlights: ► Destruxin B (DB) inhibited colorectal cancer cells growth and induced apoptosis. ► MAPK and/or PI3K/Akt cascade cooperates in DB induced apoptosis. ► DB affected the migratory and invasive ability of HT29 cells through MMP-9. ► DB attenuated Wnt-signaling components β-catenin, Tcf4. ► DB attenuated cyclin D1, c-myc, survivin and tumorigenesis in HT29 xenograft mice.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

SU-E-J-274: Responses of Medulloblastoma Cells to Radiation Dosimetric Parameters in Intensity-Modulated Radiation Therapy

Purpose: To evaluate radiation responses of the medulloblastoma cell line Daoy in intensity-modulated radiation therapy (IMRT), quantitative variations to variable radiation dosimetic parameters were tracked by bioluminescent images (BLIs). Methods: The luciferase and green fluorescent protein positive Daoy cells were cultured on dishes. The medulloblastoma cells irradiated to different dose rate, interval of fractionated doses, field margin and misalignment, and dose uniformity in IMRT were monitored using bioluminescent images. The cultured cells were placed into a dedicated acrylic phantom to deliver intensity-modulated fluences and calculate accurate predicted dose distribution. The radiation with dose rate from 0.5 Gy/min to 15 Gy/min was irradiated by adjusting monitor unit per minute and source-to-surface distances. The intervals of fractionated dose delivery were changed considering the repair time of double strand breaks (DSB) revealed by straining of gamma-H2AX.The effect of non-uniform doses on the cells were visualized by registering dose distributions and BLIs. The viability according to dosimetric parameters was correlated with bioluminescent intensities for cross-check of radiation responses. Results: The DSB and cell responses due to the first fractionated dose delivery significantly affected final tumor control rather than other parameters. The missing tumor volumes due to the smaller field margin than the tumor periphery or field misalignment caused relapse of cell responses on BLIs. The dose rate and gradient had effect on initial responses but could not bring out the distinguishable killing effect on cancer cells. Conclusion: Visualized and quantified bioluminescent images were useful to correlate the dose distributions with spatial radiation effects on cells. This would derive the effective combination of dose delivery parameters and fractionation. Radiation responses in particular IMRT configuration could be reflected to image based-dose re-optimization.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Quantitative visualization of synchronized insulin secretion from 3D-cultured cells

Quantitative visualization of synchronized insulin secretion was performed in an isolated rat pancreatic islet and a spheroid of rat pancreatic beta cell line using a method of video-rate bioluminescence imaging. Video-rate images of insulin secretion from 3D-cultured cells were obtained by expressing the fusion protein of insulin and Gaussia luciferase (Insulin-GLase). A subclonal rat INS-1E cell line stably expressing Insulin-GLase, named iGL, was established and a cluster of iGL cells showed oscillatory insulin secretion that was completely synchronized in response to high glucose. Furthermore, we demonstrated the effect of an antidiabetic drug, glibenclamide, on synchronized insulin secretion from 2D- and 3D-cultured iGL cells. The amount of secreted Insulin-GLase from iGL cells was also determined by a luminometer. Thus, our bioluminescence imaging method could generally be used for investigating protein secretion from living 3D-cultured cells. In addition, iGL cell line would be valuable for evaluating antidiabetic drugs. - Highlights: • An imaging method for protein secretion from 3D-cultured cells was established. • The fused protein of insulin to GLase, Insulin-GLase, was used as a reporter. • Synchronous insulin secretion was visualized in rat islets and spheroidal beta cells. • A rat beta cell line stably expressing Insulin-GLase, named iGL, was established. • Effect of an antidiabetic drug on insulin secretion was visualized in iGL cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2011

DEVELOPMENT OF A DUAL MODALITY TOMOGRAPHIC IMAGING SYSTEM FOR BIOLUMINESCENCE AND PET

The goal of this proposal was to develop a new hybrid imaging modality capable to simultaneously image optical bioluminescence signals, as well as radionuclide emissions from the annihilation of positrons originating from molecular imaging probes in preclinical mouse models. This new technology enables the simultaneous in-vivo measurements of both emissions that could be produced from a single or a combination of two different biomarkers. It also facilitates establishing the physical limitations of bioluminescence imaging, its tomographic and spectral image reconstruction potential and the quantification of bioluminescence signals.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2017

Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions

We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion. - Highlights: • The fused protein of proglucagon to Gaussia luciferase was used as a reporter. • The fusion protein was highly expressed using a preferred human-codon optimized gene. • Glucagon secretion stimulated by depolarization was determined by luminescence. • Glucagon secretion in α cells was visualized by bioluminescence imaging. • Glucagon secretion sites were localized in the intercellular contact regions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Bioluminescent system for dynamic imaging of cell and animal behavior

Highlights: Black-Right-Pointing-Pointer We combined a yellow variant of GFP and firefly luciferase to make ffLuc-cp156. Black-Right-Pointing-Pointer ffLuc-cp156 showed improved photon yield in cultured cells and transgenic mice. Black-Right-Pointing-Pointer ffLuc-cp156 enabled video-rate bioluminescence imaging of freely-moving animals. Black-Right-Pointing-Pointer ffLuc-cp156 mice enabled tracking real-time drug delivery in conscious animals. -- Abstract: The current utility of bioluminescence imaging is constrained by a low photon yield that limits temporal sensitivity. Here, we describe an imaging method that uses a chemiluminescent/fluorescent protein, ffLuc-cp156, which consists of a yellow variant of Aequorea GFP and firefly luciferase. We report an improvement in photon yield by over three orders of magnitude over current bioluminescent systems. We imaged cellular movement at high resolution including neuronal growth cones and microglial cell protrusions. Transgenic ffLuc-cp156 mice enabled video-rate bioluminescence imaging of freely moving animals, which may provide a reliable assay for drug distribution in behaving animals for pre-clinical studies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Caged luciferins for bioluminescent activity-based sensing

Bioluminescence imaging is a powerful modality for in vivo imaging owing to its low background and high signal-to-noise ratio. Because bioluminescent emission occurs only upon the catalytic reaction between the luciferase enzyme and its luciferin substrate, caging luciferins with analyte-reactive triggers offers a general approach for activity-based sensing of specific biochemical processes in living systems across cell, tissue, and animal models. In this review, we summarize recent efforts in the development of synthetic caged luciferins for tracking enzyme, small molecule, and metal ion activity and their contributions to physiological and pathological processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Real-time Tumor Oxygenation Changes After Single High-dose Radiation Therapy in Orthotopic and Subcutaneous Lung Cancer in Mice: Clinical Implication for Stereotactic Ablative Radiation Therapy Schedule Optimization

Purpose: To investigate the serial changes of tumor hypoxia in response to single high-dose irradiation by various clinical and preclinical methods to propose an optimal fractionation schedule for stereotactic ablative radiation therapy. Methods and Materials: Syngeneic Lewis lung carcinomas were grown either orthotopically or subcutaneously in C57BL/6 mice and irradiated with a single dose of 15 Gy to mimic stereotactic ablative radiation therapy used in the clinic. Serial [{sup 18}F]-misonidazole (F-MISO) positron emission tomography (PET) imaging, pimonidazole fluorescence-activated cell sorting analyses, hypoxia-responsive element-driven bioluminescence, and Hoechst 33342 perfusion were performed before irradiation (day −1), at 6 hours (day 0), and 2 (day 2) and 6 (day 6) days after irradiation for both subcutaneous and orthotopic lung tumors. For F-MISO, the tumor/brain ratio was analyzed. Results: Hypoxic signals were too low to quantitate for orthotopic tumors using F-MISO PET or hypoxia-responsive element-driven bioluminescence imaging. In subcutaneous tumors, the maximum tumor/brain ratio was 2.87 ± 0.483 at day −1, 1.67 ± 0.116 at day 0, 2.92 ± 0.334 at day 2, and 2.13 ± 0.385 at day 6, indicating that tumor hypoxia was decreased immediately after irradiation and had returned to the pretreatment levels at day 2, followed by a slight decrease by day 6 after radiation. Pimonidazole analysis also revealed similar patterns. Using Hoechst 33342 vascular perfusion dye, CD31, and cleaved caspase 3 co-immunostaining, we found a rapid and transient vascular collapse, which might have resulted in poor intratumor perfusion of F-MISO PET tracer or pimonidazole delivered at day 0, leading to decreased hypoxic signals at day 0 by PET or pimonidazole analyses. Conclusions: We found tumor hypoxia levels decreased immediately after delivery of a single dose of 15 Gy and had returned to the pretreatment levels 2 days after irradiation and had decreased slightly by day 6. Our results indicate that single high-dose irradiation can produce a rapid, but reversible, vascular collapse in tumors.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2011

An adaptive regularization parameter choice strategy for multispectral bioluminescence tomography

Purpose: Bioluminescence tomography (BLT) provides an effective tool for monitoring physiological and pathological activities in vivo. However, the measured data in bioluminescence imaging are corrupted by noise. Therefore, regularization methods are commonly used to find a regularized solution. Nevertheless, for the quality of the reconstructed bioluminescent source obtained by regularization methods, the choice of the regularization parameters is crucial. To date, the selection of regularization parameters remains challenging. With regards to the above problems, the authors proposed a BLT reconstruction algorithm with an adaptive parameter choice rule. Methods: The proposed reconstruction algorithm uses a diffusion equation for modeling the bioluminescent photon transport. The diffusion equation is solved with a finite element method. Computed tomography (CT) images provide anatomical information regarding the geometry of the small animal and its internal organs. To reduce the ill-posedness of BLT, spectral information and the optimal permissible source region are employed. Then, the relationship between the unknown source distribution and multiview and multispectral boundary measurements is established based on the finite element method and the optimal permissible source region. Since the measured data are noisy, the BLT reconstruction is formulated as l{sub 2} data fidelity and a general regularization term. When choosing the regularization parameters for BLT, an efficient model function approach is proposed, which does not require knowledge of the noise level. This approach only requests the computation of the residual and regularized solution norm. With this knowledge, we construct the model function to approximate the objective function, and the regularization parameter is updated iteratively. Results: First, the micro-CT based mouse phantom was used for simulation verification. Simulation experiments were used to illustrate why multispectral data were used rather than monochromatic data. Furthermore, the study conducted using an adaptive regularization parameter demonstrated our ability to accurately localize the bioluminescent source. With the adaptively estimated regularization parameter, the reconstructed center position of the source was (20.37, 31.05, 12.95) mm, and the distance to the real source was 0.63 mm. The results of the dual-source experiments further showed that our algorithm could localize the bioluminescent sources accurately. The authors then presented experimental evidence that the proposed algorithm exhibited its calculated efficiency over the heuristic method. The effectiveness of the new algorithm was also confirmed by comparing it with the L-curve method. Furthermore, various initial speculations regarding the regularization parameter were used to illustrate the convergence of our algorithm. Finally, in vivo mouse experiment further illustrates the effectiveness of the proposed algorithm. Conclusions: Utilizing numerical, physical phantom and in vivo examples, we demonstrated that the bioluminescent sources could be reconstructed accurately with automatic regularization parameters. The proposed algorithm exhibited superior performance than both the heuristic regularization parameter choice method and L-curve method based on the computational speed and localization error.

61 RADIATION PROTECTION AND DOSIMETRY↗
DOE OSTI2019

Yttrium-90-Labeled Anti-Glypican 3 Radioimmunotherapy Halts Tumor Growth in an Orthotopic Xenograft Model of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the second most lethal malignancy globally and is increasing in incidence in the United States. Unfortunately, there are few effective systemic treatment options, particularly for disseminated disease. Glypican-3 (GPC3) is a proteoglycan cell surface receptor overexpressed in most HCCs and provides a unique target for molecular therapies. We have previously demonstrated that PET imaging using a 89 Zr-conjugated monoclonal anti-GPC3 antibody ( α GPC3) can bind to minute tumors and allow imaging with high sensitivity and specificity in an orthotopic xenograft mouse model of HCC and that serum alpha-fetoprotein (AFP) levels are highly correlated with tumor size in this model. In the present study, we conjugated 90 Y, a high-energy beta-particle-emitting radionuclide, to our α GPC3 antibody to develop a novel antibody-directed radiotherapeutic approach for HCC. Luciferase-expressing HepG2 human hepatoblastoma cells were orthotopically implanted in the livers of athymic nude mice, and tumor establishment was verified at 6 weeks after implantation by bioluminescent imaging and serum AFP concentration. Tumor burden by bioluminescence and serum AFP concentration was highly correlated in our model. Yttrium-90 was conjugated to α GPC3 using the chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and injected via the tail vein into the experimental mice at a dose of 200 μ Ci/mouse or 300 μ Ci/mouse. Control mice received DOTA- α GPC3 without radionuclide. At 30 days after a single dose of the radioimmunotherapy agent, mean serum AFP levels in control animals increased dramatically, while animals treated with 200 μ Ci only experienced a minor increase, indicating cessation of tumor growth, and animals treated with 300 μ Ci experienced a reduction in serum AFP concentration, indicating tumor shrinkage. Mean tumor-bearing liver weight in control animals was also significantly greater than that in animals that received either dose of 90 Y- α GPC3. These results were achieved without significant toxicity as measured by body condition scoring and body weight. The results of this preclinical pilot demonstrate that GPC3 can be used as a target for radioimmunotherapy in an orthotopic mouse model of HCC and may be a target of clinical significance, particularly for disseminated HCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

TH-C-17A-02: New Radioluminescence Strategies Based On CRET (Cerenkov Radiation Energy Transfer) for Imaging and Therapy

Purpose: Cerenkov photons are produced when charged particles, emitted from radionuclides, travel through a media with a speed greater than that of the light in the media. Cerenkov radiation is mostly in the UV/Blue region and, thus, readily absorbed by biological tissue. Cerenkov Radiation Energy Transfer (CRET) is a wavelength-shifting phenomenon from blue Cerenkov light to more penetrating red wavelengths. We demonstrate the feasibility of in-depth imaging of CRET light originating from radionuclides realized by down conversion of gold nanoclusters (AuNCs, a novel particle composed of few atoms of gold coated with serum proteins) in vivo. Methods: Bovine Serum Albumin, Human Serum Albumin and Transferrin conjugated gold nanoclusters were synthesized, characterized and examined for CRET. Three different clinically used radiotracers: 18F-FDG, 90Y and 99mTc were used. Optical spectrum (440–750 nm) was recorded by sensitive bioluminescence imaging system at physiological temperature. Dose dependence (activity range from 0.5 up to 800uCi) and concentration dependence (0.01 to 1uM) studies were carried out. The compound was also imaged in a xenograft mouse model. Results: Only β+ and β--emitting radionuclides (18F-FDG, 90Y) are capable of CRET; no signal was found in 99mTc (γ-emitter). The emission peak of CRET by AuNCs was found to be ∼700 nm and was ∼3 fold times of background. In vitro studies showed a linear dependency between luminescence intensity and dose and concentration. CRET by gold nanoclusters was observed in xenografted mice injected with 100uCi of 18F-FDG. Conclusion: The unique optical, transport and chemical properties of AuNCs (gold nanoclusters) make them ideal candidates for in-vivo imaging applications. Development of new molecular imaging probes will allow us to achieve substantially improved spatiotemporal resolution, sensitivity and specificity for tumor imaging and detection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Systematic study of target localization for bioluminescence tomography guided radiation therapy

Purpose: To overcome the limitation of CT/cone-beam CT (CBCT) in guiding radiation for soft tissue targets, the authors developed a spectrally resolved bioluminescence tomography (BLT) system for the small animal radiation research platform. The authors systematically assessed the performance of the BLT system in terms of target localization and the ability to resolve two neighboring sources in simulations, tissue-mimicking phantom, and in vivo environments. Methods: Multispectral measurements acquired in a single projection were used for the BLT reconstruction. The incomplete variables truncated conjugate gradient algorithm with an iterative permissible region shrinking strategy was employed as the optimization scheme to reconstruct source distributions. Simulation studies were conducted for single spherical sources with sizes from 0.5 to 3 mm radius at depth of 3–12 mm. The same configuration was also applied for the double source simulation with source separations varying from 3 to 9 mm. Experiments were performed in a standalone BLT/CBCT system. Two self-illuminated sources with 3 and 4.7 mm separations placed inside a tissue-mimicking phantom were chosen as the test cases. Live mice implanted with single-source at 6 and 9 mm depth, two sources at 3 and 5 mm separation at depth of 5 mm, or three sources in the abdomen were also used to illustrate the localization capability of the BLT system for multiple targets in vivo. Results: For simulation study, approximate 1 mm accuracy can be achieved at localizing center of mass (CoM) for single-source and grouped CoM for double source cases. For the case of 1.5 mm radius source, a common tumor size used in preclinical study, their simulation shows that for all the source separations considered, except for the 3 mm separation at 9 and 12 mm depth, the two neighboring sources can be resolved at depths from 3 to 12 mm. Phantom experiments illustrated that 2D bioluminescence imaging failed to distinguish two sources, but BLT can provide 3D source localization with approximately 1 mm accuracy. The in vivo results are encouraging that 1 and 1.7 mm accuracy can be attained for the single-source case at 6 and 9 mm depth, respectively. For the 2 sources in vivo study, both sources can be distinguished at 3 and 5 mm separations, and approximately 1 mm localization accuracy can also be achieved. Conclusions: This study demonstrated that their multispectral BLT/CBCT system could be potentially applied to localize and resolve multiple sources at wide range of source sizes, depths, and separations. The average accuracy of localizing CoM for single-source and grouped CoM for double sources is approximately 1 mm except deep-seated target. The information provided in this study can be instructive to devise treatment margins for BLT-guided irradiation. These results also suggest that the 3D BLT system could guide radiation for the situation with multiple targets, such as metastatic tumor models.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Brominated Luciferins Are Versatile Bioluminescent Probes

Here, we report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed by using a common synthetic route. All analogues produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed by computation and photophysical measurements. The brightest brominated luciferin was further evaluated in cell and animal models. At low doses, the analogue outperformed the native substrate in cells. The remaining luciferins, although weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Investigating the FLASH Effect in a Rat Brain Organotypic Model With a Novel High-Energy Electron Beam

Ultrahigh dose rate (FLASH) radiation therapy is reported to reduce normal tissue toxicity while maintaining tumor control; however, mechanism(s) remain obscure. To study FLASH mechanisms in brain tissue, we developed a novel experimental platform featuring a specialized high-energy electron linear accelerator, High Intensity Gamma Ray Source (HIGS), paired with an organotypic ex vivo brain metastasis model. We varied interpulse spacing to modulate the mean dose rate (MDR) of our unique 35 MeV electron beam, while maintaining extremely high instantaneous dose rate (IDR). We characterized dosimetry and targeting accuracy of the FLASH beam with film dosimetry. We combined this FLASH beam with an organotypic rat brain slice/breast carcinoma coculture model of brain metastasis to assess effects on normal and neoplastic tissues. Live-cell and bioluminescence imaging demonstrated cancer cell growth effects, whereas normal tissue responses and immune activation were assessed using live-cell imaging, cytokine profiles, and confocal microscopy. Here, we performed comparison experiments with 20 MeV electrons from a Varian clinical linear accelerator (VCLA) using conventional dose rates. The highest IDR of the FLASH beam to date was 20.7 ± 0.6 MGy/s, with maximum MDR of 20.7 MGy/s delivered in 1 pulse of 1 µs duration. Beam targeting was accurate to <1 mm and reproducible. HIGS-FLASH and VCLA dose rates equivalently decreased cancer cell growth. HIGS-FLASH irradiation significantly increased tumor necrosis factor α and fractalkine levels and confocal microscopy revealed distinct changes in microglial morphology slices suggesting microglia activation. Our novel experimental platform produces extremely high dose rates and rapid normal/neoplastic tissue readouts for mechanistic research into the effects of FLASH radiation in the brain. HIGS-FLASH irradiation induces comparable cancer cell growth inhibition but differential effects on cytokines and microglial morphology, suggesting that acute innate immune responses may be involved in FLASH normal tissue effects in the brain.

Kay, Tyler V. [Duke University, Durham, NC (United↗
DOE OSTI2014

Hydroxychavicol, a betel leaf component, inhibits prostate cancer through ROS-driven DNA damage and apoptosis

Dietary phytochemicals are excellent ROS-modulating agents and have been shown to effectively enhance ROS levels beyond toxic threshold in cancer cells to ensure their selective killing while leaving normal cells unscathed. Here we demonstrate that hydroxychavicol (HC), extracted and purified from Piper betel leaves, significantly inhibits growth and proliferation via ROS generation in human prostate cancer, PC-3 cells. HC perturbed cell-cycle kinetics and progression, reduced clonogenicity and mediated cytotoxicity by ROS-induced DNA damage leading to activation of several pro-apoptotic molecules. In addition, HC treatment elicited a novel autophagic response as evidenced by the appearance of acidic vesicular organelles and increased expression of autophagic markers, LC3-IIb and beclin-1. Interestingly, quenching of ROS with tiron, an antioxidant, offered significant protection against HC-induced inhibition of cell growth and down regulation of caspase-3, suggesting the crucial role of ROS in mediating cell death. The collapse of mitochondrial transmembrane potential by HC further revealed the link between ROS generation and induction of caspase-mediated apoptosis in PC-3 cells. Our data showed remarkable inhibition of prostate tumor xenografts by ∼ 72% upon daily oral administration of 150 mg/kg bw HC by quantitative tumor volume measurements and non-invasive real-time bioluminescent imaging. HC was well-tolerated at this dosing level without any observable toxicity. This is the first report to demonstrate the anti-prostate cancer efficacy of HC in vitro and in vivo, which is perhaps attributable to its selective prooxidant activity to eliminate cancer cells thus providing compelling grounds for future preclinical studies to validate its potential usefulness for prostate cancer management. - Highlights: • HC perturbs cell-cycle progression by induction of reactive oxygen species (ROS). • HC mediated cytotoxicity by ROS-induced DNA damage leading to apoptosis. • HC induced ROS-mediated autophagic response. • It inhibited prostate tumor growth by ∼ 72% without any observable toxicity. • Its anticancer efficacy is likely due to its selective prooxidant activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Intravenous administration of bone marrow-derived multipotent mesenchymal stromal cells enhances the recruitment of CD11b{sup +} myeloid cells to the lungs and facilitates B16-F10 melanoma colonization

The discovery that the regenerative properties of bone marrow multipotent mesenchymal stromal cells (BM-MSCs) could collaterally favor neoplastic progression has led to a great interest in the function of these cells in tumors. However, the effect of BM-MSCs on colonization, a rate-limiting step of the metastatic cascade, is unknown. In this study, we investigated the effect of BM-MSCs on metastatic outgrowth of B16-F10 melanoma cells. In in vitro experiments, direct co-culture assays demonstrated that BM-MSCs stimulated the proliferation of B16-F10 cells in a dose-dependent manner. For in vivo experiments, luciferase-expressing B16-F10 cells were injected through tail vein and mice were subsequently treated with four systemic injections of BM-MSCs. In vivo bioluminescent imaging during 16 days demonstrated that BM-MSCs enhanced the colonization of lungs by B16-F10 cells, which correlated with a 2-fold increase in the number of metastatic foci. Flow cytometry analysis of lungs demonstrated that although mice harboring B16-F10 metastases displayed more endothelial cells, CD4 T and CD8 T lymphocytes in the lungs in comparison to metastases-free mice, BM-MSCs did not alter the number of these cells. Interestingly, BM-MSCs inoculation resulted in a 2-fold increase in the number of CD11b{sup +} myeloid cells in the lungs of melanoma-bearing animals, a cell population previously described to organize “premetastatic niches” in experimental models. These findings indicate that BM-MSCs provide support to B16-F10 cells to overcome the constraints that limit metastatic outgrowth and that these effects might involve the interplay between BM-MSCs, CD11b{sup +} myeloid cells and tumor cells. - Highlights: • BM-MSCs enhanced B16-F10 proliferation in a dose-dependent manner in vitro. • BM-MSCs facilitated lung colonization by B16-F10 melanoma cells. • BM-MSCs administration did not alter the number of endothelial cells and T lymphocytes in the lungs. • BM-MSCs enhanced the recruitment of CD11b{sup +} myeloid cells during tumor colonization.

60 APPLIED LIFE SCIENCES↗