Molecular Imaging and Biology最新文献

Purpose: Tumor hypoxia contributes to aggressive phenotypes and diminished therapeutic responses to radiation therapy (RT) with hypoxic tissue being 3-fold less radiosensitive than normoxic tissue. A major challenge in implementing hypoxic radiosensitizers is the lack of a high-resolution imaging modality that directly quantifies tissue-oxygen. The electron paramagnetic resonance oxygen-imager (EPROI) was used to quantify tumor oxygenation in two murine tumor models: E0771 syngeneic transplant breast cancers and primary p53/MCA soft tissue sarcomas, with the latter autochthonous model better recapitulating the tumor microenvironment in human malignancies. We hypothesized that tumor hypoxia differs between these models. We also aimed to quantify the absolute change in tumor hypoxia induced by the mitochondrial inhibitor papaverine (PPV) and its effect on RT response.

Procedures: Tumor oxygenation was characterized in E0771 and primary p53/MCA sarcomas via EPROI, with the former model also being quantified indirectly via diffuse reflectance spectroscopy (DRS). After confirming PPV's effect on hypoxic fraction (via EPROI), we compared the effect of 0 versus 2 mg/kg PPV prior to 20 Gy on tumor growth delay and survival.

Results: Hypoxic sarcomas were more radioresistant than normoxic sarcomas (p=0.0057, 2-way ANOVA), and high baseline hypoxic fraction was a significant (p=0.0063, Cox Regression Model) hazard in survivability regardless of treatment. Pre-treatment with PPV before RT did not radiosensitize tumors in the sarcoma or E0771 model. In the sarcoma model, EPROI successfully identified baseline hypoxic tumors. DRS quantification of total hemoglobin, saturated hemoglobin, changes in mitochondrial potential and glucose uptake showed no significant difference in E0771 tumors pre- and post-PPV.

Conclusion: EPROI provides 3D high-resolution pO₂ quantification; EPR is better suited than DRS to characterize tumor hypoxia. PPV did not radiosensitize E0771 tumors nor p53/MCA sarcomas, which may be related to the complex pattern of vasculature in each tumor. Additionally, understanding model-dependent tumor hypoxia will provide a much-needed foundation for future therapeutic studies with hypoxic radiosensitizers.

Purpose: Bioprinting is an additive manufacturing technology analogous to 3D printing. Instead of plastic or resin, cell-laden hydrogels are used to produce a construct of the intended biological structure. Over time, cells transform this construct into a functioning tissue or organ. The process of printing followed by tissue maturation is referred to as 4D bioprinting. The fourth dimension is temporal. Failure to provide living cells with sufficient amounts of oxygen at any point along the developmental timeline may jeopardize the bioprinting goals. Even transient hypoxia may alter cells' differentiation and proliferation or trigger apoptosis. Electron paramagnetic resonance (EPR) imaging modality is proposed to permit 4D monitoring of oxygen within bioprinted structures.

Procedures: Lithium octa-n-butoxy-phthalocyanine (LiNc-BuO) probes have been introduced into gelatin methacrylate (GelMA) bioink. GelMA is a cross-linkable hydrogel, and LiNc-BuO is an oxygen-sensitive compound that permits longitudinal oximetric measurements. The effects of the oxygen probe on printability have been evaluated. A digital light processing (DLP) bioprinter was built in the laboratory. Bioprinting protocols have been developed that consider the optical properties of the GelMA/LiNc-BuO composites. Acellular and cell-laden constructs have been printed and imaged. The post-printing effect of residual photoinitiator on oxygen depletion has been investigated.

Results: Models have been successfully printed using a lab-built bioprinter. Rapid scan EPR images reflective of the expected oxygen concentration levels have been acquired. An unreported problem of oxygen depletion in bioprinted constructs by the residual photoinitiator has been documented. EPR imaging is proposed as a control method for its removal. The oxygen consumption rates by HEK293T cells within a bioprinted cylinder have been imaged and quantified.

Conclusions: The feasibility of the cointegration of 4D EPR imaging and 4D bioprinting has been demonstrated. The proof-of-concept experiments, which were conducted using oxygen probes loaded into GelMA, lay the foundation for a broad range of applications, such as bioprinting with many types of bioinks loaded with diverse varieties of molecular spin probes.

Purpose: Our study aimed to accelerate the acquisition of four-dimensional (4D) spectral-spatial electron paramagnetic resonance (EPR) imaging for mouse tumor models. This advancement in EPR imaging should reduce the acquisition time of spectroscopic mapping while reducing quality degradation for mouse tumor models.

Procedures: EPR spectra under magnetic field gradients, called spectral projections, were partially measured. Additional spectral projections were later computationally synthesized from the measured spectral projections. Four-dimensional spectral-spatial images were reconstructed from the post-processed spectral projections using the algebraic reconstruction technique (ART) and assessed in terms of their image qualities. We applied this approach to a sample solution and a mouse Hs766T xenograft model of human-derived pancreatic ductal adenocarcinoma cells to demonstrate the feasibility of our concept. The nitroxyl radical imaging agent ²H,¹⁵N-DCP was exogenously infused into the mouse xenograft model.

Results: The computation code of 4D spectral-spatial imaging was tested with numerically generated spectral projections. In the linewidth mapping of the sample solution, we achieved a relative standard uncertainty (standard deviation/| mean |) of 0.76 μT/45.38 μT = 0.017 on the peak-to-peak first-derivative EPR linewidth. The qualities of the linewidth maps and the effect of computational synthesis of spectral projections were examined. Finally, we obtained the three-dimensional linewidth map of ²H,¹⁵N-DCP in a Hs766T tumor-bearing leg in vivo.

Conclusion: We achieved a 46.7% reduction in the acquisition time of 4D spectral-spatial EPR imaging without significantly degrading the image quality. A combination of ART and partial acquisition in three-dimensional raster magnetic field gradient settings in orthogonal coordinates is a novel approach. Our approach to 4D spectral-spatial EPR imaging can be applied to any subject, especially for samples with less variation in one direction.

Purpose: Oxidative stress is proposed to be critical in acute lung disease, but methods to monitor radicals in lungs are lacking. Our goal is to develop low-frequency electron paramagnetic resonance (EPR) methods to monitor radicals that contribute to the disease.

Procedures: Free radicals generated in a lipopolysaccharide-induced mouse model of acute respiratory distress syndrome reacted with cyclic hydroxylamines CPH (1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride) and DCP-AM-H (4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid), which were converted into the corresponding nitroxide radicals, CP• and DCP•. The EPR signals of the nitroxide radicals in excised lungs were imaged with a 1 GHz EPR spectrometer/imager that employs rapid scan technology.

Results: The small numbers of nitroxides formed by reaction of the hydroxylamine with superoxide result in low signal-to-noise in the spectra and images. However, since the spectral properties of the nitroxides are known, we can use prior knowledge of the line shape and hyperfine splitting to fit the noisy data, yielding well-defined spectra and images. Two-dimensional spectral-spatial images are shown for lung samples containing (4.5 ± 0.5) ×10¹⁴ CP• and (9.9 ± 1.0) ×10¹⁴ DCP• nitroxide spins. These results suggest that a probe that accumulates in cells gives a stronger nitroxide signal than a probe that is more easily washed out of cells.

Conclusion: The nitroxide radicals in excised mouse lungs formed by reaction with hydroxylamine probes CPH and DCP-AM-H can be imaged at 1 GHz.

Within this special issue, many eminent investigators report on measurements of oxygen (O₂) levels in tissues. Given the complexities of spatial and temporal heterogeneities of O₂ in tissues and its many sources, this commentary draws attention to what such measurements do and do not actually assess regarding O₂ levels in tissues. Given this limitation, it also discusses how these results can be used most effectively. To provide a convenient mechanism to discuss these issues more fully, this analysis focuses on measurements using EPR oximetry, but these considerations apply to all other techniques. The nature of the delivery of O₂ to tissues and the mechanisms by which O₂ is consumed necessarily result in very different levels of O₂ within the volume of each voxel of a measurement. Better spatial resolution cannot fully resolve the problem because the variations include O₂ gradients within each cell. Improved resolution of the time-dependent variation in O₂ is also very challenging because O₂ levels within tissues can have fluctuations of O₂ levels in the range of milliseconds, while most methods require longer times to acquire the data from each voxel. Based on these issues, we argue that the values obtained inevitably are complex aggregates of averages of O₂ levels across space and time in the tissue. These complexities arise from the complex physiology of tissues and are compounded by the limitations of the technique and its ability to acquire data. However, one often can obtain very meaningful and useful results if these complexities and limitations are taken into account. We illustrate this, using results obtained with in vivo EPR oximetry, especially utilizing its capacity to make repeated measurements to follow changes in O₂ levels that occur with interventions and/or over time.

Molecular oxygen and its thermodynamic transformation drive nearly all life processes. Quantitative measurement and imaging of oxygen in living systems is of fundamental importance for the study of life processes and their aberrations-disease- many of which are affected by hypoxia, or low levels of oxygen. Cancer is among the disease processes profoundly affected by hypoxia. Electron paramagnetic resonance has been shown to provide remarkably accurate images of normal and cancerous tissue. In this review, we emphasize the reactivity of molecular oxygen particularly highlighting the metabolic processes of living systems to store free energy in the reactants. The history of hypoxic resistance of living systems to cytotoxic therapy, particularly radiation therapy is also reviewed. The measurement and imaging of molecular oxygen with pulse spin lattice relaxation (SLR) electron paramagnetic resonance (EPR) is reviewed briefly. This emphasizes the advantages of the spin lattice relaxation based measurement paradigm to reduce the sensitivity of the measurement to the presence of the oxygen sensing probe itself. The involvement of a novel small mammal external beam radiation delivery system is described. This enables an experimental paradigm based on control by radiation of the last resistant clonogen. This is much more specific for tumor cure than growth delay assays which primarily reflects control of tumor cells most sensitive to therapy.

Purpose: Molecular oxygen, besides a photosensitizer and light of appropriate wavelength, is one of the three factors necessary for photodynamic therapy (PDT). In tumor tissue, PDT leads to the killing of tumor cells, destruction of endothelial cells and vasculature collapse, and the induction of strong immune responses. All these effects may influence the oxygenation levels, but it is the vasculature changes that have the main impact on pO₂. The purpose of our study was to monitor changes in tumor oxygenation after PDT and explore its significance for predicting long-term treatment response.

Procedures: Electron paramagnetic resonance (EPR) spectroscopy enables direct, quantitative, and sequential measurements of partial pressure of oxygen (pO₂) in the same animal. The levels of chlorophyll derived photosensitizers in tumor tissue were determined by transdermal emission measurements.

Results: The noninvasive monitoring of pO₂ in the tumor tissue after PDT showed that the higher ΔpO₂ (pO₂ after PDT minus pO₂ before PDT), the greater the inhibition of tumor growth. ΔpO₂ also correlated with higher levels of the photosensitizers in the tumor and with the occurrence of a severe edema/erythema after PDT.

Conclusion: Monitoring of PDT-induced changes in tumor oxygenation is a valuable prognostic factor and could be also used to identify potentially resistant tumors, which is important in predicting long-term treatment response.

Purpose: This study aimed to develop a biocompatible oximetric electron paramagnetic resonance (EPR) spin probe with reduced self-relaxation, and sensitivity to oxygen for a higher signal-to-noise ratio and longer relaxation times at high oxygen concentration, compared to the reference spin probe OX071.

Procedures: SOX71 was synthesized by succinylation of the twelve alcohol groups of OX071 spin probe and characterized by EPR at X-Band (9.5 GHz) and at low field (720 MHz). The biocompatibility of SOX71 was tested in vitro and in vivo in mice. A pharmacokinetic study was performed to determine the best time frame for EPR imaging. Finally, a proof-of-concept EPR oxygen imaging was performed on a mouse model of a fibrosarcoma tumor.

Results: SOX71 was synthesized in one step from OX071. SOX71 exhibits a narrow line EPR spectrum with a peak-to-peak linewidth of 66 mG, similar to OX071. SOX71 does not bind to albumin nor show cell toxicity for the concentrations tested up to 5 mM. No toxicity was observed after systemic delivery via intraperitoneal injection in mice at twice the dose required for EPR imaging. After the injection, the probe is readily absorbed into the bloodstream, with a peak blood concentration half an hour, post-injection. Then, the probe is quickly cleared by the kidney with a half-life of ~ 45 min. SOX71 shows long relaxation times under anoxic condition (T_1e = 9.5 µs and T_2e = 5.1 µs; [SOX71] = 1 mM in PBS at 37 °C, pO₂ = 0 mmHg, 720 MHz). Both the relaxation rates R_1e and R_2e show a decreased sensitivity to pO_2, leading to twice longer relaxation times under room air conditions (pO₂ = 159 mmHg) compared to OX071. This is ideal for oxygen imaging in samples with a wide range of pO₂. Both the relaxation rates R_1e and R_2e show a decreased sensitivity to self-relaxation compared to OX071, with a negligible effect of the probe concentration on R_1e. SOX71 was successfully applied to image oxygen in a tumor.

Conclusion: SOX71, a succinylated derivative of OX071 was synthesized, characterized, and applied for in vivo EPR tumor oxygen imaging. SOX71 is highly biocompatible, and shows decreased sensitivity to oxygen and self-relaxation. This first report suggests that SOX71 is superior to OX071 for absolute oxygen mapping under a broad range of pO₂ values.

Purpose: Low frequency EPR can noninvasively detect endogenous free radical melanin in melanocytic skin lesions and could potentially discriminate between benign atypical nevi and malignant melanoma lesions. We recently succeeded in demonstrating the ability of clinical EPR to noninvasively detect the endogenous melanin free radical in skin lesions of patients. However, the signal-to-noise ratio (SNR) was extremely low warranting further research to boost the sensitivity of detection. In the present study, we assessed the performance of a clinical EPR system with the capability to perform multi-harmonic (MH) analysis for the detection of melanin.

Procedures: The sensitivity of MH-EPR was compared with a classical continuous wave (CW)-EPR (1st harmonic) detection in vitro in melanin phantoms, in vivo in melanoma models with cells implanted in the skin, in lymph nodes and having colonized the lungs, and finally on phantoms placed at the surface of human skin.

Results: In vitro, we observed an increase in SNR by a factor of 10 in flat melanin phantoms when using MH analysis compared to CW combined with an increase in modulation amplitude. In B16 melanomas having grown in the skin of hairless mice, we observed a boost in sensitivity in vivo similar to that observed in vitro with the capability to detect melanoma cells at an earlier stage of development. MH-EPR was also able to detect non-invasively the melanin signal coming from melanoma cells present in lymph nodes as well as in lungs. We also observed a boost of sensitivity using phantoms of melanin placed at the surface of human skin.

Conclusions: Overall, our results are paving the way for new clinical trials that will use MH clinical EPR for the characterization of pigmented skin lesions.