To (a) evaluate whether the lysine-rich protein (LRP) magnetic resonance (MR) imaging reporter gene can be engineered into G47Δ, a herpes simplex-derived oncolytic virus that is currently being ...tested in clinical trials, without disrupting its therapeutic effectiveness and (b) establish the ability of chemical exchange saturation transfer (CEST) MR imaging to demonstrate G47Δ-LRP.
The institutional subcommittee for research animal care approved all in vivo procedures. Oncolytic herpes simplex virus G47Δ, which carried the LRP gene, was constructed and tested for its capacity to replicate in cancer cells and express LRP in vitro. The LRP gene was detected through CEST imaging of lysates derived from cells infected with G47Δ-LRP or the control G47Δ-empty virus. G47Δ-LRP was then tested for its therapeutic effectiveness and detection with CEST MR imaging in vivo. Images of rat gliomas were acquired before and 8-10 hours after injection of G47Δ-LRP (n = 7) or G47Δ-empty virus (n = 6). Group comparisons were analyzed with a paired t test.
No significant differences were observed in viral replication or therapeutic effectiveness between G47Δ-LRP and G47Δ-empty virus. An increase in CEST image contrast was observed in cell lysates (mean ± standard deviation, 0.52% ± 0.06; P = .01) and in tumors (1.1% ± 0.3, P = .02) after infection with G47Δ-LRP but not G47Δ-empty viruses. No histopathologic differences were observed between tumors infected with G47Δ-LRP and G47Δ-empty virus.
This study has demonstrated the ability of CEST MR imaging to show G47Δ-LRP at acute stages of viral infection. The introduction of the LRP transgene had no effect on the viral replication or therapeutic effectiveness. This can aid in development of the LRP gene as a reporter for the real-time detection of viral spread. Online supplemental material is available for this article.
Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MYC-driven MBs, commonly found in the group 3 MB, are aggressive and metastatic with the worst prognosis. Modeling MYC-driven ...MB is the foundation of therapeutic development. Here, we applied a synthetic mRNA-driven strategy to generate neuronal precursors from human-induced pluripotent stem cells (iPSCs). These neuronal precursors were transformed by the
oncogene combined with
loss of function to establish an MYC-driven MB model recapitulating the histologic and transcriptomic hallmarks of group 3 MB. We further show that the marine compound Frondoside A (FA) effectively inhibits this MYC-driven MB model without affecting isogenic neuronal precursors with undetectable MYC expression. Consistent results from a panel of MB models support that MYC levels are positively correlated with FA's antitumor potency. Next, we show that FA suppresses MYC expression and its downstream gene targets in MB cells, suggesting a potential mechanism underlying FA's inhibitory effects on MYC-driven cancers. In orthotopic xenografts of MYC-driven MB, intratumoral FA administration potently induces cytotoxicity in tumor xenografts, significantly extends the survival of tumor-bearing animals, and enhances the recruitment of microglia/macrophages and cytotoxic T lymphocytes to tumors. Moreover, we show that MYC levels also predict FA potency in glioblastoma and non-small cell lung cancer cells. Taken together, this study provides an efficient human iPSC-based strategy for personalizable cancer modeling, widely applicable to mechanistic studies (e.g., genetic predisposition to cancer) and drug discovery. Our preclinical results justify the clinical translation of FA in treating MYC-driven MB and other human cancers.
INTRODUCTION: The blood-brain barrier (BBB) is compromised in multiple central nervous system (CNS) disorders associated with neuroinflammation, including multiple sclerosis (MS). Currently available ...magnetic resonance imaging (MRI) methods, however, are only able to measure BBB leakage in the lower molecular size range with the use of small molecular tracers, i.e., gadolinium (Gd) agents (<1 kDa)1,2 and water (18 Da).3,4 The goal of this study is to adopt a dextran-based chemical exchange saturation transfer (CEST) MRI approach for assessing BBB leakage in the larger size range and studying the size characteristics of BBB dysfunction.
METHODS: All animal experiments will be approved by the Animal Care and Use Committee of Johns Hopkins University. EAE MS mouse model: C57Bl/6 mice (F/6-10w), were injected s.c. with myelin peptide (MOG35-55, 200 μL, 0.5 mg/mL) emulsified in incomplete Freund's adjuvant supplemented with M. tuberculosis H37Ra (5 mg/mL) and i.p. with 300 ng of pertussis toxin on days 0 and 2. Mice were observed daily for signs of paralysis using a 0-5 rating system. Fluorescent imaging. EAE mice (n=3) were injected with the combination of fixable Dex40-TRITC and Dex3-FITC (i.v.) at the dose of 80 mg/kg, and sacrificed at 30 min after injection (without perfusion) to collect brains. Fluorescence microscopy was then performed on tissue sections. MRI: all in vivo MRI was acquired using a Biospec 11.7 T horizontal MRI scanner (Bruker, Ettlingen, Germany). According to our previously reported protocol,5 CEST MRI was performed before and after the i.v. injection of 200 µL dex40 saline solution (750 mg/kg b.w), using parameters: B1=1.8 µT, Tsat=3 s, Δω=-3 to +3 ppm with a step size of 0.2 ppm. MTRasym=(S-Δω–S+Δω)/S0 was computed after the B0 correction using the WASSR method. ΔMTRasym (1 ppm) at each time point was calculated by MTRasym (t)- MTRasym (pre).
RESULTS: 1. The size-dependent BBB disruption in MS can be detected by fluorescent dextran-tracers of different sizes: Immunofluorescent results show dextrans of smaller sizes (e.g., 3 kDa) penetrated the brain parenchyma deeper than larger sizes (e.g., 40 kDa). Our study proves the feasibility to use dextrans as a group of tracers with different sizes for probing the size effect of BBB dysfunction. 2. Dex-enhanced CEST MRI: As shown in Figure 1, mice with high clinical disability scores have BBB impairment in the mouse brain, confirmed with Gd-enhanced MRI (Figure 1B). Dex-enhanced MRI results (Figure 1C) showed substantial contrast enhancement in the corresponding brain regions. Interestingly, while the size of Dex (40 kDa) is larger than the size of Gd-DOTA (559 Da), the area showing enhanced Dex-CEST signal is slightly larger than that of Gd-enhancement, suggesting that, besides size, other particle properties such as shape and surface properties of a given agent/particle may also contribute to the permeation across BBB.
CONCLUSIONS: We have established a dextran-based imaging protocol for assessing the biodistribution of dextrans in the brains of EAE mice. We will continue studying the size effect of dextrans and determining the optimal dextran size for accurately mentoring the disease progression.
The measurement of extracellular pH (pHe) has potential utility for cancer diagnoses and for assessing the therapeutic effects of pH-dependent therapies. A single magnetic resonance imaging (MRI) ...contrast agent that is detected through paramagnetic chemical exchange saturation transfer (PARACEST) was designed to measure tumor pHe throughout the range of physiologic pH and with magnetic resonance saturation powers that are not harmful to a mouse model of cancer. The chemical characterization and modeling of the contrast agent Yb3+-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, 10-o-aminoanilide (Yb-DO3A-oAA) suggested that the aryl amine of the agent forms an intramolecular hydrogen bond with a proximal carboxylate ligand, which was essential for generating a practical chemical exchange saturation transfer (CEST) effect from an amine. A ratio of CEST effects from the aryl amine and amide was linearly correlated with pH throughout the physiologic pH range. The pH calibration was used to produce a parametric pH map of a subcutaneous flank tumor on a mouse model of MCF-7 mammary carcinoma. Although refinements in the in vivo CEST MRI methodology may improve the accuracy of pHe measurements, this study demonstrated that the PARACEST contrast agent can be used to generate parametric pH maps of in vivo tumors with saturation power levels that are not harmful to a mouse model of cancer.
Citicoline (CDPC) is a natural supplement with well-documented neuroprotective effects in the treatment of neurodegenerative diseases. In the present study, we sought to exploit citicoline as a ...theranostic agent with its inherent chemical exchange saturation transfer (CEST) MRI signal, which can be directly used as an MRI guidance in the citicoline drug delivery. Our in vitro CEST MRI results showed citicoline has two inherent CEST signals at +1 and +2 ppm, attributed to exchangeable hydroxyl and amine protons, respectively. To facilitate the targeted drug delivery of citicoline to ischemic regions, we prepared liposomes encapsulating citicoline (CDPC-lipo) and characterized the particle properties and CEST MRI properties. The in vivo CEST MRI detection of liposomal citicoline was then examined in a rat brain model of unilateral transient ischemia induced by a two-hour middle cerebral artery occlusion. The results showed that the delivery of CPDC-lipo to the brain ischemic areas could be monitored and quantified by CEST MRI. When administered intra-arterially, CDPC-lipo clearly demonstrated a detectable CEST MRI contrast at 2 ppm. CEST MRI revealed that liposomes preferentially accumulated in the areas of ischemia with a disrupted blood-brain-barrier. We furthermore used CEST MRI to detect the improvement in drug delivery using CDPC-lipo targeted against vascular cell adhesion molecule (VCAM)-1 in the same animal model. The MRI findings were validated using fluorescence microscopy. Hence, liposomal citicoline represents a prototype theranostic system, where the therapeutic agent can be detected directly by CEST MRI in a label-free fashion.
The blood-brain barrier (BBB) prevents effective delivery of most therapeutic agents to the brain. Intra-arterial (IA) infusion of hyperosmotic mannitol has been widely used to open the BBB and ...improve parenchymal targeting, but the extent of BBB disruption has varied widely with therapeutic outcomes often being unpredictable. In this work, we show that real-time MRI can enable fine-tuning of the infusion rate to adjust and predict effective and local brain perfusion in mice, and thereby can be allowed for achieving the targeted and localized BBB opening (BBBO). Both the reproducibility and safety are validated by MRI and histology. The reliable and reproducible BBBO we developed in mice will allow cost-effective studies on the biology of the BBB and drug delivery to the brain. In addition, the IA route for BBBO also permits subsequent IA delivery of a specific drug during the same procedure and obtains high targeting efficiency of the therapeutic agent in the targeted tissue, which has great potential for future clinical translation in neuro-oncology, regenerative medicine and other neurological applications.
Responsive magnetic resonance imaging (MRI) contrast agents can change MR image contrast in response to a molecular biomarker. Quantitative detection of the biomarker requires an accounting of the ...other effects that may alter MR image contrast, such as a change in the agent’s concentration, magnetic field variations, and hardware sensitivity profiles. A second unresponsive MRI contrast agent may serve as an “internal control” to isolate the detection of the molecular biomarker. Chemical exchange saturation transfer (CEST) MRI contrast agents can be selectively detected, providing the opportunity to combine a responsive CEST agent and an unresponsive CEST agent during the same MRI scan session. When two CEST MRI contrast agents are used for molecular imaging applications, the CEST agents should be designed to maximize accurate quantification of the concentrations of the two agents. From a chemical perspective, CEST agents behave like enzymes that catalyze the conversion of an unsaturated water “substrate” into a saturated water “product”. The analysis of CEST agent kinetics parallels the Michaelis−Menten analysis of enzyme kinetics, which can be used to correlate the CEST effect with the concentration of the agent in solution. If the concentration of water “substrate” that is available to the CEST agent is unknown, which may be likely for in vivo MRI studies, then only a ratio of concentrations of the two CEST agents can be measured. In both cases, CEST agents should be designed with minimal T 1 relaxivity to improve concentration quantifications. CEST agents can also be designed to maximize sensitivity. This may be accomplished by incorporating many CEST agents within nanoparticles to create a large number of exchangeable protons per nanoparticle. Finally, CEST agents can be designed with rapid detection in mind. This may be accomplished by minimizing T 1 relaxivity of the CEST agent so that MRI acquisition methods have time to collect many MRI signals following a single selective saturation period. In this Account, we provide an example that shows the sensitive and rapid detection of two CEST agents in an in vivo MRI study of a mouse model of mammary carcinoma. The ratio of the concentrations of the two CEST agents was quantified with analysis methods that parallel Michaelis−Menten enzyme kinetic analysis. This example demonstrates current limitations of the method that require additional research, but it also shows that two CEST MRI contrast agents can be detected and quantitatively assessed during in vivo molecular imaging studies.
In experiments involving transgenic animals or animals treated with transgenic cells, it is important to have a method to monitor the expression of the relevant genes longitudinally and ...noninvasively. An MRI-based reporter gene enables monitoring of gene expression in the deep tissues of living subjects. This information can be co-registered with detailed high-resolution anatomical and functional information. We describe here the synthesis of the reporter probe, 5-methyl-5,6-dihydrothymidine (5-MDHT), which can be used for imaging of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene expression in rodents by MRI. The protocol also includes data acquisition and data processing routines customized for chemical exchange saturation transfer (CEST) contrast mechanisms. The dihydropyrimidine 5-MDHT is synthesized through a catalytic hydrogenation of the 5,6-double bond of thymidine to yield 5,6-dihydrothymidine, which is methylated on the C-5 position of the resulting saturated pyrimidine ring. The synthesis of 5-MDHT can be completed within 5 d, and the compound is stable for more than 1 year.
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