Molecular Imaging and Biology最新文献

Purpose: Multiple myeloma (MM) affects over 35,000 patients each year in the US. There remains a need for versatile Positron Emission Tomography (PET) tracers for the detection, accurate staging, and monitoring of treatment response of MM that have optimal specificity and translational attributes. CD38 is uniformly overexpressed in MM and thus represents an ideal target to develop CD38-targeted small molecule PET radiopharmaceuticals to address these challenges.

Procedures: Using phage display peptide libraries and pioneering algorithms, we identified novel CD38 specific peptides. Imaging bioconjugates were synthesized using solid phase peptide chemistry, and systematically analyzed in vitro and in vivo in relevant MM systems.

Results: The CD38-targeted bioconjugates were radiolabeled with copper-64 (⁶⁴Cu) with100% radiochemical purity and an average specific activity of 3.3 - 6.6 MBq/nmol. The analog NODAGA-PEG4-SL022-GGS (SL022: Thr-His-Tyr-Pro-Ile-Val-Ile) had a K_d of 7.55 ± 0.291 nM and was chosen as the lead candidate. ⁶⁴Cu-NODAGA-PEG4-SL022-GGS demonstrated high binding affinity to CD38 expressing human myeloma MM.1S-CBR-GFP-WT cells, which was blocked by the non-radiolabeled version of the peptide analog and anti-CD38 clinical antibodies, daratumumab and isatuximab, by 58%, 73%, and 78%, respectively. The CD38 positive MM.1S-CBR-GFP-WT cells had > 68% enhanced cellular binding when compared to MM.1S-CBR-GFP-KO cells devoid of CD38. Furthermore, our new CD38-targeted radiopharmaceutical allowed visualization of tumors located in marrow rich bones, remaining there for up to 4 h. Clearance from non-target organs occurred within 60 min. Quantitative PET data from a murine disseminated tumor model showed significantly higher accumulation in the bones of tumor-bearing animals compared to tumor-naïve animals (SUV_max 2.06 ± 0.4 versus 1.24 ± 0.4, P = 0.02). Independently, tumor uptake of the target compound was significantly higher (P = 0.003) compared to the scrambled peptide, ⁶⁴Cu-NODAGA-PEG4-SL041-GGS (SL041: Thr-Tyr-His-Ile-Pro-Ile-Val). The subcutaneous MM model demonstrated significantly higher accumulation in tumors compared to muscle at 1 and 4 h after tracer administration (SUV_max 0.8 ± 0.2 and 0.14 ± 0.04, P = 0.04 at 1 h; SUV_max 0.89 ± 0.01 and 0.09 ± 0.01, P = 0.0002 at 4 h).

Conclusions: The novel CD38-targeted, radiolabeled bioconjugates were specific and allowed visualization of MM, providing a starting point for the clinical translation of such tracers for the detection of MM.

Purpose: In this study, we explored the role of apoptosis as a potential biomarker for cardiac failure using functional micro-CT and fluorescence molecular tomography (FMT) imaging techniques in Ercc1 mutant mice. Ercc1 is involved in multiple DNA repair pathways, and its mutations contribute to accelerated aging phenotypes in both humans and mice, due to the accumulation of DNA lesions that impair vital DNA functions. We previously found that systemic mutations and cardiomyocyte-restricted deletion of Ercc1 in mice results in left ventricular (LV) dysfunction at older age.

Procedures and results: Here we report that combined functional micro-CT and FMT imaging allowed us to detect apoptosis in systemic Ercc1 mutant mice prior to the development of overt LV dysfunction, suggesting its potential as an early indicator and contributing factor of cardiac impairment. The detection of apoptosis in vivo was feasible as early as 12 weeks of age, even when global LV function appeared normal, underscoring the potential of apoptosis as an early predictor of LV dysfunction, which subsequently manifested at 24 weeks.

Conclusions: This study highlights the utility of combined functional micro-CT and FMT imaging in assessing cardiac function and detecting apoptosis, providing valuable insights into the potential of apoptosis as an early biomarker for cardiac failure.

The incidence of melanoma is continuously increasing over time. Melanoma is the most aggressive skin cancer, significantly reducing quality of life and survival rates of patients at advanced stages. Therefore, early diagnosis remains the key to change the prognosis of patients with melanoma. In this context, advanced technologies are under evaluation to increase the accuracy of the diagnostic, to better characterize the lesions and visualize their possible invasiveness in the epidermis. Among the innovative methods, because melanin is paramagnetic, clinical low frequency electron paramagnetic resonance (EPR) that characterizes the melanin content in the lesion has the potential to be an adjunct diagnostic method of melanoma. In this review, we first summarize the challenges faced by dermatologists and oncologists in melanoma diagnostic and management. We also provide a historical perspective on melanin detection with a focus on EPR spectroscopy/imaging of melanomas. We describe key elements that allow EPR to move from in vitro studies to in vivo and finally to patients for melanoma studies. Finally, we provide a critical view on challenges to meet to make EPR operational in the clinic to characterize pigmented lesions.

Purpose: Electron Paramagnetic Resonance Imaging (EPRI) can image the partial pressure of oxygen (pO₂) within in vivo tumor models. We sought to develop Oxygen Enhanced (OE) EPRI that measures tumor pO₂ with breathing gases of 21% O₂ (pO₂^21%) and 100% O₂ (pO₂^100%), and the differences in pO₂ between breathing gases (ΔpO₂). We applied OE EPRI to study the early change in tumor pathophysiology in response to radiotherapy in two tumor models of pancreatic cancer.

Procedures: We developed a protocol that intraperitoneally administered OX071, a trityl radical contrast agent, and then acquired anatomical MR images to localize the tumor. Subsequently, we acquired two pO₂^21% and two pO₂^100% maps using the T1 relaxation time of OX071 measured with EPRI and a R₁-pO₂ calibration of OX071. We studied 4T1 flank tumor model to evaluate the repeatability of OE EPRI. We then applied OE EPRI to study COLO 357 and Su.86.86 flank tumor models treated with 10 Gy radiotherapy.

Results: The repeatability of mean pO₂ for individual tumors was ± 2.6 Torr between successive scans when breathing 21% O₂ or 100% O₂, representing a precision of 9.6%. Tumor pO₂^21% and pO₂^100% decreased after radiotherapy for both models, although the decreases were not significant or only moderately significant, and the effect sizes were modest. For comparison, ΔpO₂ showed a large, highly significant decrease after radiotherapy, and the effect size was large. MANOVA and analyses of the HF10 hypoxia fraction provided similar results.

Conclusions: EPRI can evaluate tumor pO₂ with outstanding precision relative to other imaging modalities. The change in ΔpO₂ before vs. after treatment was the best parameter for measuring the early change in tumor pathophysiology in response to radiotherapy. Our studies have established ΔpO₂ from OE EPRI as a new parameter, and have established that OE EPRI is a valuable new methodology for molecular imaging.

Purpose: Patients with hyper- vs. hypo-inflammatory subphenotypes of acute respiratory distress syndrome (ARDS) exhibit different clinical outcomes. Inflammation increases the production of reactive oxygen species (ROS) and increased ROS contributes to the severity of illness. Our long-term goal is to develop electron paramagnetic resonance (EPR) imaging of lungs in vivo to precisely measure superoxide production in ARDS in real time. As a first step, this requires the development of in vivo EPR methods for quantifying superoxide generation in the lung during injury, and testing if such superoxide measurements can differentiate between susceptible and protected mouse strains.

Procedures: In WT mice, mice lacking total body extracellular superoxide dismutase (EC-SOD) (KO), or mice overexpressing lung EC-SOD (Tg), lung injury was induced with intraperitoneal (IP) lipopolysaccharide (LPS) (10 mg/kg). At 24 h after LPS treatment, mice were injected with the cyclic hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) or 4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid (DCP-AM-H) probes to detect, respectively, cellular and mitochondrial ROS - specifically superoxide. Several probe delivery strategies were tested. Lung tissue was collected up to one hour after probe administration and assayed by EPR.

Results: As measured by X-band EPR, cellular and mitochondrial superoxide increased in the lungs of LPS-treated mice compared to control. Lung cellular superoxide was increased in EC-SOD KO mice and decreased in EC-SOD Tg mice compared to WT. We also validated an intratracheal (IT) delivery method, which enhanced the lung signal for both spin probes compared to IP administration.

Conclusions: We have developed protocols for delivering EPR spin probes in vivo, allowing detection of cellular and mitochondrial superoxide in lung injury by EPR. Superoxide measurements by EPR could differentiate mice with and without lung injury, as well as mouse strains with different disease susceptibilities. We expect these protocols to capture real-time superoxide production and enable evaluation of lung EPR imaging as a potential clinical tool for subphenotyping ARDS patients based on redox status.

Purpose: Hypoxia and acidosis are recognized tumor microenvironment (TME) biomarkers of cancer progression. Alterations in cancer redox status and metabolism are also associated with elevated levels of intracellular glutathione (GSH) and interstitial inorganic phosphate (Pi). This study aims to evaluate the capability of these biomarkers to discriminate between stages and inform on a switch to malignancy.

Procedures: These studies were performed using MMTV-PyMT( +) female transgenic mice that spontaneously develop breast cancer and emulate human tumor staging. In vivo assessment of oxygen concentration (pO₂), extracellular acidity (pH_e), Pi, and GSH was performed using L-band electron paramagnetic resonance spectroscopy and multifunctional trityl and GSH-sensitive nitroxide probes.

Results: Profiling of the TME showed significant deviation of measured biomarkers upon tumor progression from pre-malignancy (pre-S4) to the malignant stage (S4). For the combined marker, HOP: (pH_e × pO₂)/Pi, a value > 186 indicated that the tumors were pre-malignant in 85% of the mammary glands analyzed, and when < 186, they were malignant 42% of the time. For GSH, a value < 3 mM indicated that the tumors were pre-malignant 74% of the time, and when > 3 mM, they were malignant 80% of the time. The only marker that markedly deviated as early as stage 1 (S1) from its value in pre-S1 was elevated Pi, followed by a decrease of pH_e and pO₂ and increase in GSH at later stages.

Conclusion: Molecular TME profiling informs on alteration of tumor redox and metabolism during tumor staging. Early elevation of interstitial Pi at S1 may reflect tumor metabolic alterations that demand elevated phosphorus supply in accordance with the high rate growth hypothesis. These metabolic changes are supported by the following decrease of pH_e due to a high tumor reliance on glycolysis and increase of intracellular GSH, a major intracellular redox buffer. The appreciable decrease in TME pO₂ was observed only at malignant S4, apparently as a consequence of tumor mass growth and corresponding decrease in perfusion efficacy and increase in oxygen consumption as the tumor cells proliferate.

Purpose: The goal of this work was to compare pO₂ measured using both continuous wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopy. The Oxychip particle spin probe enabled longitudinal monitoring of pO₂ in murine pancreatic tumor treated with gemcitabine during the course of therapy.

Procedures: Pancreatic PanO2 tumors were growing in the syngeneic mice, in the leg. Five doses of saline in control animals or gemcitabine were administered every 3 days, and pO₂ was measured after each dose at several time points. Oxygen partial pressure was determined from the linewidth of the CW EPR signal (Bruker E540L) or from the T₂ measured using the electron spin echo sequence (Jiva-25™).

Results: The oxygen sensitivity was determined from a calibration curve as 6.1 mG/mm Hg in CW EPR and 68.5 ms^-1/mm Hg in pulse EPR. A slight increase in pO₂ of up to 20 mm Hg was observed after the third dose of gemcitabine compared to the control. The maximum delta pO₂ during the therapy correlated with better survival.

Conclusions: Both techniques offer fast and reliable oximetry in vivo, allowing to follow the effects of pharmaceutic intervention.

Purpose: Progress toward developing a novel radiocontrast agent for determining pO₂ in tumors in a clinical setting is described. The imaging agent is designed for use with electron paramagnetic resonance imaging (EPRI), in which the collision of a paramagnetic probe molecule with molecular oxygen causes a spectroscopic change which can be calibrated to give the real oxygen concentration in the tumor tissue.

Procedures: The imaging agent is based on a nanoscaffold of aluminum hydroxide (boehmite) with sizes from 100 to 200 nm, paramagnetic probe molecule, and encapsulation with a gas permeable, thin (10-20 nm) polymer layer to separate the imaging agent and body environment while still allowing O₂ to interact with the paramagnetic probe. A specially designed deuterated Finland trityl (dFT) is covalently attached on the surface of the nanoparticle through 1,3-dipolar addition of the alkyne on the dFT with an azide on the surface of the nanoscaffold. This click-chemistry reaction affords 100% efficiency of the trityl attachment as followed by the complete disappearance of the azide peak in the infrared spectrum. The fully encapsulated, dFT-functionalized nanoparticle is referred to as RADI-Sense.

Results: Side-by-side in vivo imaging comparisons made in a mouse model made between RADI-Sense and free paramagnetic probe (OX-071) showed oxygen sensitivity is retained and RADI-Sense can create 3D pO₂ maps of solid tumors CONCLUSIONS: A novel encapsulated nanoparticle EPR imaging agent has been described which could be used in the future to bring EPR imaging for guidance of radiotherapy into clinical reality.

Purpose: Spatial heterogeneity in tumor hypoxia is one of the most important factors regulating tumor growth, development, aggressiveness, metastasis, and affecting treatment outcome. Most solid tumors are known to have hypoxia or low oxygen levels (pO₂ ≤10 torr). Electron paramagnetic resonance oxygen imaging (EPROI) is an emerging oxygen mapping technology. EPROI utilizes the linear relationship between the relaxation rates of the injectable OX071 trityl spin probe and the partial oxygen pressure (pO₂). However, most of the EPROI studies have been limited to mouse models of solid tumors because of the instrument-size limitations. The purpose of this work was to develop a human-sized 9-mT (250 MHz resonance frequency, 60 cm bore size) pulse EPROI instrument and evaluate its performance with rabbit VX-2 tumor oxygen imaging.

Methods: A New Zealand white rabbit with a 3.2-cm VX-2 tumor in the calf muscle was imaged using the human-sized EPROI instrument and a 2.25-in. ID volume coil. The animal received a ~8-min intravenous injection of OX071 (5.2 mL total volume at 72 mM concentration) and, after 75 min, an intratumoral injection (120 μL total at 5 mM OX071 concentration) and underwent EPROI. At the end of the experiments, MRI was performed using a preclinical 9.4-T MRI system to outline the tumor boundaries.

Results: For the first time, a human-sized pulse EPROI instrument with a 60-cm bore size/250-MHz frequency was built and evaluated using rabbit tumor oxygen imaging. For the first time, the systemic IV injection of the oxygen-sensitive trityl OX071 spin probe was used for an animal of this size. The resulting EPROI image from the IV injection showed complete tumor coverage. The image obtained after intratumoral injection showed localized coverage in the upper lobe of the tumor, demonstrating the need for improved intratumoral injection protocol.

Conclusions: This study demonstrates the performance of the world's first human-sized pulse EPROI instrument. It also demonstrates that the EPROI of larger animals can be performed using the systemic injection of a manageable amount of the spin probe. This brings EPROI one step closer to clinical applications in cancer therapies. Oxygen imaging is a platform technology, and the instrument and techniques developed here will also be useful for other clinical applications.

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.