Chemical Exchange Saturation Transfer Magnetic Resonance Research Articles

L-DOS47 Elevates Pancreatic Cancer Tumor pH and Enhances Response to Immunotherapy.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds to CEACAM6, a cell-surface protein that is highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Using chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) to measure the tumor extracellular pH (pHe), we confirmed that L-DOS47 raises the tumor pHe from 4 h to 96 h post injection in acidic tumors (average increase of 0.13 units). Additional studies showed that combining L-DOS47 with anti-PD1 significantly increases the efficacy of the anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

Abstract B125: Neutralizing acidosis with L-DOS47 urease immunoconjugate enhances responses to anti-PD1 checkpoint blockade in a preclinical orthotopic model of pancreatic adenocarcinoma

Abstract Background: Acidosis in the tumor microenvironment is an important immunosuppressive mechanism that leads to tumor growth. We have shown that neutralization of tumor pH using sodium bicarbonate improves responses to immune checkpoint blockade (ICB) in pre-clinical models, but phase I/IIa clinical trials failed due to poor patient compliance. Thus, we aimed to test clinically translatable agents to neutralize tumor acidity and improve cancer outcomes in response to ICB. We tested L-DOS47, a targeted urease immunoconjugate designed to directly neutralize tumor acidity that has been well-tolerated in phase I/IIa trials. The nanobody component of L-DOS47 binds carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6), a cell surface protein highly expressed in gastrointestinal and lung cancers; the urease moiety cleaves endogenous urea into two NH4+ and one CO2 thereby raising local pH. Materials and Methods: Immunohistochemistry screening for CEACAM6 expression and L-DOS47 binding was performed on tumor microarrays from pancreatic ductal adenocarcinoma (PDAC) cases (n=10 plus 2 normal tumor adjacent cases). We established a preclinical in vivo PDAC model by orthotopically inoculating human CEACAM6-expressing KPC961 into immunocompetent mice. Pharmacodynamic studies were performed using chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) to measure tumor extracellular pH (pHe) at baseline (pre-L-DOS47) and at various time points post-L-DOS47 (4, 18, 24, 48, 72 and 96 hours). Mean tumor pHe and histograms of pHe distributions were compared among groups. Additional biological replicates were conducted to assess therapeutic efficacy. For these experiments, mice were randomized, after measurable tumors are established, into groups with equal tumor volume averages to initiate twice-weekly therapies with 300 µg anti-PD1 or 90 µg/kg L-DOS47 monotherapies, 300 µg anti-PD1 + 90 µg/kg L-DOS47 compared to a control group receiving no therapy. Tumor volumes were measured weekly by ultrasound imaging up to 4 weeks. Results: Immunohistochemistry screening of patient tumor microarrays confirmed high CEACAM6 expression and L-DOS47 binding to PDAC tissues. CEST-MRI results showed that L-DOS47 increased tumor pHe from 4h to 96h post-injection; however, pHe increases were only evident in tumors with baseline pHe ≤ 6.60. Efficacy studies in the PDAC model showed that the combination of anti-PD1 + L-DOS47 significantly reduced tumor growth when compared with control, L-DOS47 and anti-PD1 groups at week 3 (p<.0001; <.0001 and 0.0368, respectively) and week 4 (p<.0001; <.0001 and 0.0142, respectively). Conclusion: These studies provide strong evidence that L-DOS47, by neutralizing acidic tumor pH, significantly improves responses to anti-PD1 immunotherapy. Citation Format: Bruna V Jardim-Perassi, Pietro Irrera, Dominique Abrahams, Christopher J Whelan, Matthew S Beatty, Samantha R Byrne, Andrew A Odeja, Dario L Longo, Young Ou, Kim Gaspar, Martin Koebel, Shari A Pilon-Thomas, Christof Boehler, Gabrielle M Siegers, Robert J Gillies, Arig A Ibrahim-Hashim. Neutralizing acidosis with L-DOS47 urease immunoconjugate enhances responses to anti-PD1 checkpoint blockade in a preclinical orthotopic model of pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B125.

Glycerol-weighted chemical exchange saturation transfer nanoprobes allow 19F/1H dual-modality magnetic resonance imaging-guided cancer radiotherapy

Recently, radiotherapy (RT) has entered a new realm of precision cancer therapy with the introduction of magnetic resonance (MR) imaging guided radiotherapy systems into the clinic. Nonetheless, identifying an optimized radiotherapy time window (ORTW) is still critical for the best therapeutic efficacy of RT. Here we describe pH and O2 dual-sensitive, perfluorooctylbromide (PFOB)-based and glycerol-weighted chemical exchange saturation transfer (CEST) nano-molecular imaging probes (Gly-PFOBs) with dual fluorine and hydrogen proton based CEST MR imaging properties (19F/1H-CEST). Oxygenated Gly-PFOBs ameliorate tumor hypoxia and improve O2-dependent radiotherapy. Moreover, the pH and O2 dual-sensitive properties of Gly-PFOBs could be quantitatively, spatially, and temporally monitored by 19F/1H-CEST imaging to optimize ORTW. In this study, we describe the CEST signal characteristics exhibited by the glycerol components of Gly-PFOBs. The pH and O2 dual-sensitive Gly-PFOBs with19F/1H-CEST MR dual-modality imaging properties, with superior therapeutic efficacy and biosafety, are employed for sensitive imaging-guided lung cancer RT, illustrating the potential of multi-functional imaging to noninvasively monitor and enhance RT-integrated effectiveness.

Amide proton transfer (APT) and magnetization transfer (MT) in predicting short-term therapeutic outcome in nasopharyngeal carcinoma after chemoradiotherapy: a feasibility study of three-dimensional chemical exchange saturation transfer (CEST) MRI

BackgroundThe three-dimensional chemical exchange saturation transfer (3D CEST) technique is a novel and promising magnetic resonance sequence; however, its application in nasopharyngeal carcinoma (NPC) lacks sufficient evaluation. This study aimed to assess the feasibility of the 3D CEST technique in predicting the short-term treatment outcomes for chemoradiotherapy (CRT) in NPC patients.MethodsForty NPC patients and fourteen healthy volunteers were enrolled and underwent the pre-treatment 3D CEST magnetic resonance imaging and diffusion-weighted imaging (DWI). The reliability of 3D CEST was assessed in healthy volunteers by calculating the intra- and inter-observer correlation coefficient (ICC) for amide proton transfer weighted-signal intensity (APTw-SI) and magnetization transfer ratio (MTR) values. NPC patients were divided into residual and non-residual groups based on short-term treatment outcomes after CRT. Whole-tumor regions of interest (ROIs) were manually drawn to measure APTw-SI, MTR and apparent diffusion coefficient (ADC) values. Multivariate analysis and the receiver operating characteristic curve (ROC) were used to evaluate the prediction performance of clinical characteristics, APTw-SI, MTR, ADC values, and combined models in predicting short-term treatment outcomes in NPC patients.ResultsFor the healthy volunteer group, all APTw-SI and MTR values exhibited good to excellent intra- and inter-observer agreements (0.736–0.910, 0.895–0.981, all P > 0.05). For NPC patients, MTR values showed a significant difference between the non-residual and residual groups (31.24 ± 5.21% vs. 34.74 ± 1.54%, P = 0.003) while no significant differences were observed for APTw-SI and ADC values (P > 0.05). Moreover, the diagnostic power of MTR value was superior to APTw-SI (AUC: 0.818 vs. 0.521, P = 0.017) and comparable to ADC values (AUC: 0.818 vs. 0.649, P > 0.05) in predicting short-term treatment outcomes for NPC patients. The prediction performance did not improve even when combining MTR values with APTw-SI and/or ADC values (P > 0.05).ConclusionsThe pre-treatment MTR value acquired through 3D CEST demonstrated superior predictive performance for short-term treatment outcomes compared to APTw-SI and ADC values in NPC patients after CRT.

Hierarchical K-means clustering method for accelerated Lorentzian estimation (KALE) in chemical exchange saturation transfer-magnetic resonance imaging quantification.

Quantification of in vivo chemical exchange saturation transfer (CEST) magnetic resonance signals is challenging due to contamination from coexisting effects, including the direct water effect and asymmetric magnetization transfer. Fitting-based analysis allows the calculation of multiple types of signals from the line shape of Z-spectra. However, the conventional voxelwise method has several drawbacks, including its long computation time and its susceptibility to image noise and Z-spectra oscillations, and it is difficult to determine the initial fitting parameters. Herein, we propose a K-means clustering method for accelerated Lorentzian estimation (KALE) in CEST quantification. Briefly, voxels in CEST images are clustered into K groups according to their Z-spectra characteristics. A 'groupwise' fitting process is then performed with preset initial values, yielding a set of fitted spectra and fitted parameters for each group. With the updated initial values, each group is further clustered into subgroups, and groupwise fitting is performed again. This hierarchical K-means clustering and parameter updating process continues until the pixel number or intensity error meets the termination criteria. Voxelwise fitting could be further conducted to improve the quantification images (termed voxel-K) by utilizing the previous groupwise KALE results as the initial values (termed group-K). Incorporated with Lorentzian difference (LD) quantification, KALE was first optimized and evaluated on 5 healthy human brain datasets at 3 Tesla. Compared with traditional voxel-by-voxel LD quantification, the computation times of group-K and voxel-K were significantly reduced by ~85% and ~70%, respectively (P<0.001). Furthermore, the group-K images exhibited better denoising performance than traditional LD and voxel-K. KALE was further validated on six ischemic rat brains acquired at 7 Tesla, with both LD_group-K and LD_voxel-K displaying almost identical contrast maps with traditional voxelwise maps. When incorporated with the five-pool Lorentzian fitting (LF), KALE exhibited an improved contrast-to-noise ratio (CNR) for amplitude maps of each pool [P=0.003, 0.015, 0.047, and 0.047 for amide, nuclear Overhauser effect (NOE), magnetic transfer (MT) and guanidine amine, respectively] and improved fitting goodness (P=0.033). KALE quantification provides comparable or even superior contrast maps to traditional voxelwise fitting, with significantly reduced computation time. The 'smart' and hierarchical voxel-clustering and parameter updating process of KALE may facilitate more preclinical and clinical CEST applications.

Intraperitoneal Delivery of Iopamidol to Assess Extracellular pH of Orthotopic Pancreatic Tumor Model by CEST-MRI.

The extracellular pH (pHe) of solid tumors is often acidic, as a consequence of the Warburg effect, and an altered metabolic state is often associated with malignancy. It has been shown that acidosis can promote tumor progression; thus, many therapeutic strategies have been adopted against tumor metabolism; one of these involves alkalinization therapies to raise tumor pH to inhibit tumor progression, improve immune surveillance, and overcome resistance to chemotherapies. Chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) is a noninvasive technique that can measure pH in vivo using pH-sensitive contrast agents. Iopamidol, an iodinated contrast agent, clinically used for computed tomography (CT), contains amide group protons with pH-dependent exchange rates that can reveal the pHe of the tumor microenvironment. In this study, we optimized intraperitoneal (IP) delivery of iopamidol to facilitate longitudinal assessments of orthotopic pancreatic tumor pHe by CEST-MRI. Following IV-infusion and IP-bolus injections, we compared the two protocols for assessing tumor pH. Time-resolved CT imaging was used to evaluate the uptake of iopamidol in the tumor, revealing that IP-bolus delivered a high amount of contrast agent 40 min postinjection, which was similar to the amounts reached with the IV-infusion protocol. As expected, both IP and IV injection protocols produced comparable measurements of tumor pHe, showing no statistically significant difference between groups (p=0.16). In addition, we showed the ability to conduct longitudinal monitoring of tumor pHe using CEST-MRI with the IP injection protocol, revealing a statistically significant increase in tumor pHe following bicarbonate administration (p < 0.001). In conclusion, this study shows the capability to measure pHe using an IP delivery of iopamidol into orthotopic pancreatic tumors, which is important to conduct longitudinal studies.

Angiopep-2, an MRI Biomarker, Dynamically Monitors Amyloid Deposition in Early Alzheimer's Disease.

The reliable and dynamic detection of amyloid β-protein (Aβ) deposition using imaging technology is necessary for preclinical Alzheimer's disease (AD), which may significantly improve prognosis. The present study aimed to evaluate the feasibility of applying angiopep-2 (ANG), a chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) biomarker, for monitoring Aβ deposition in vivo. ANG exerted a good chemical exchange saturation transfer (CEST) effect and displayed a moderate binding affinity to Aβ1-42 in vitro. Six-month-old mice with AD injected with ANG exhibited a significantly enhanced CEST effect than controls in vivo; this effect gradually became more apparent at 8, 10, and 12 months. Spatial learning impairment caused by abundant Aβ deposition (representing mild cognitive impairment in AD patients) develops at 12 months in APPswe/PSEN1dE9 (line 85) AD mice. To conclude, the CEST of ANG could display very earlier age-related Aβ pathological progress in mice with AD, consistent with immunohistochemistry. ANG has extraordinary potential for clinical transformation as an imaging biomarker to diagnose early AD and track its progress dynamically and nonradiationally.

Chemical Exchange Saturation Transfer Magnetic Resonance Imaging for Longitudinal Assessment of Intracerebral Hemorrhage and Deferoxamine Treatment at 3T in a Mouse Model.

Noninvasive imaging of molecular alterations after intracerebral hemorrhage (ICH) could provide valuable information to guide and monitor treatments. Chemical exchange saturation transfer (CEST) magnetic resonance imaging has demonstrated promises in identifying proliferation, necrosis, and changes in cellularity in brain tumors. Here, we applied CEST magnetic resonance imaging to monitor molecular changes in hematoma without and with treatment noninvasively over 2 weeks at 3T using endogenous contrast. CEST contrast related to proteins at 3.5 ppm (amide proton transfer) and proteins/lipids at -3.5 ppm (relayed nuclear overhauser effect [rNOE]) were examined over 14 days in a collagenase-induced ICH mouse model. Imaging findings were validated with immunohistochemistry based on the ICH neuropathology. We also examined iron-containing phantoms that mimicked iron concentrations in hematoma to ensure the iron will not attenuate the CEST contrast during disease progression. Based on the validity of the CEST contrast of hematoma, we further examined related molecular alterations under iron-chelation treatment with deferoxamine. We observed the temporal and spatial differences of CEST contrasts between rNOE at -3.5 ppm and amide proton transfer at 3.5 ppm, in which the core and perihematoma could be identified by rNOE on day 3 and day 14, and amide proton transfer on day 1, day 7, and day 14. Moreover, we observed a 25.7% significant reduction (P<0.05) of rNOE contrast after deferoxamine treatment to the ICH mice on day 3, which was not observable in amide proton transfer contrast. Our histology data indicated that rNOE primarily correlated with the myelin pathology, and amide proton transfer could reflect the cellularity increase at hematoma up to day 7. Significant rNOE changes correlated well with histologic findings, especially myelin lipids, and regional characteristics in hematoma indicate the uniqueness of CEST magnetic resonance imaging in monitoring molecular changes during ICH and treatment.

Quantitative imaging of apoptosis following oncolytic virotherapy by magnetic resonance fingerprinting aided by deep learning.

Oncolytic virotherapy is a promising anticancer strategy, however, non-invasive imaging methods that detect intratumoral viral spread and an individual’s response to therapy are either slow, lack specificity or require the use of radioactive or metal-based contrast agents, hindering their clinical translation. Here, we describe a fast chemical-exchange saturation transfer magnetic resonance fingerprinting (CEST-MRF) approach that selectively labels the exchangeable amide protons of endogenous proteins and the exchangeable macromolecule protons of lipids, without requiring an exogenous contrast agent. A deep neural network, previously trained with simulated MR-fingerprints, then generates, using CEST-MRF image data, quantitative maps of intratumoral pH, and protein and lipid concentrations. In a mouse model of glioblastoma multiforme, the maps enabled the detection of the early apoptotic response to oncolytic virotherapy, characterized by decreased cytosolic pH and reduced protein synthesis. Imaging of a healthy human volunteer yields molecular parameters in good agreement with literature values, demonstrating the clinical potential of artificial intelligence-based CEST-MRF, which may be further applicable to the imaging of a wide range of pathologies, including stroke, cancer, and neurological disorders.

Accelerated multi-target chemical exchange saturation transfer magnetic resonance imaging of the mouse heart

Cardiac chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) has been used to probe levels of various metabolites that provide insight into myocardial structure and function. However, imaging of the heart using CEST-MRI is prolonged by the need to repeatedly acquire multiple images for a full Z-spectrum and to perform saturation and acquisition around cardiac and respiratory cycles. Compressed sensing (CS) reconstruction of sparse data enables accelerated acquisition, but reconstruction artifacts may bias subsequently derived measures of CEST contrast. In this study, we examine the impact of CS reconstruction of increasingly under-sampled cardiac CEST-MRI data on subsequent CEST contrasts of amine-containing metabolites and amide-containing proteins. Cardiac CEST-MRI data sets were acquired in six mice using low and high RF saturation for single and dual contrast generation, respectively. CEST-weighted images were reconstructed using CS methods at 2–5× levels of under-sampling. CEST contrasts were derived from corresponding Z-spectra and the impact of accelerated imaging on accuracy was assessed via analysis of variance. CS reconstruction preserved myocardial signal to noise ratio as compared to conventional reconstruction. However, greater absolute error and distribution of derived contrasts was observed with increasing acceleration factors. The results from this study indicate that acquisition of radial cardiac CEST-MRI data can be modestly, but meaningfully, accelerated via CS reconstructions with little error in CEST contrast quantification.

Effectiveness of fat suppression using a water-selective binomial-pulse excitation in chemical exchange saturation transfer (CEST) magnetic resonance imaging.

The purpose of this study was to characterize the individual contribution of multiple fat peaks to the measured chemical exchange saturation transfer (CEST) signal when using water-selective binomial-pulse excitation and to determine the effects of multiple fat peaks in the presence of B0 inhomogeneity. The excitation profiles of multiple binomial pulses were simulated. A CEST sequence with binomial-pulse excitation and modified point-resolved spectroscopy localization was then applied to the in vivo lumbar spinal vertebrae to determine the signal contributions of three distinct groups of lipid resonances. These confounding signal contributions were measured as a function of the irradiation frequency offset to determine the effect of the multi-peak nature of the fat signal on CEST imaging of exchange sites (at 1.0, 2.0 and 3.5ppm) and robustness in the presence of B0 inhomogeneity. Numerical simulations and in vivo experiments showed that water excitation (WE) using a 1-3-3-1 (WE-4) pulse provided the broadest signal suppression, which provided partial robustness against B0 inhomogeneity effects. Confounding fat signal contributions to the CEST contrasts at 1.0, 2.0 and 3.5ppm were unavoidable due to the multi-peak nature of the fat signal. However, these CEST sites only suffer from small lipid artifacts with ∆B0 spanning roughly from - 50 to 50Hz. Especially for the CEST site at 3.5ppm, the lipid artifacts are smaller than 1% with ∆B0 in this range. In WE-4-based CEST magnetic resonance imaging, B0 inhomogeneity is the limiting factor for fat suppression. The CEST sites at 1.0, 2.0ppm and 3.5ppm unavoidably suffer from lipid artifacts. However, when the ∆B0 is confined to a limited range, these CEST sites are only affected by small lipid artifacts, which may be ignorable in some cases of clinical applications.

Amide chemical exchange saturation transfer at 7\xa0T: a possible biomarker for detecting early response to neoadjuvant chemotherapy in breast cancer patients

BackgroundThe purpose of this work was to investigate noninvasive early detection of treatment response of breast cancer patients to neoadjuvant chemotherapy (NAC) using chemical exchange saturation transfer (CEST) measurements sensitive to amide proton transfer (APT) at 7 T.MethodsCEST images were acquired in 10 tumors of nine breast cancer patients treated with NAC. APT signals in the tumor, before and after the first cycle of NAC, were quantified using a three-pool Lorentzian fit of the z-spectra in the region of interest. The changes in APT were subsequently related to pathological response after surgery defined by the Miller-Payne system.ResultsSignificant differences (P < 0.05, unpaired Mann-Whitney test) were found in the APT signal before and after the first cycle of NAC in six out of 10 lesions, of which two showed a pathological complete response. Of the remaining four lesions, one showed a pathological complete response. No significant difference in changes of APT signal were found between the different pathological responses to NAC treatment (P > 0.05, Kruskal-Wallis test).ConclusionsThis preliminary study shows the feasibility of using APT CEST magnetic resonance imaging as a noninvasive biomarker to assess the effect of NAC in an early stage of NAC treatment of breast cancer patients.Trial registrationRegistration number, NL49333.041.14/NTR4980. Registered on 16 October 2014.

Chemical Exchange Saturation Transfer MR Imaging: Preliminary Results for Differentiation of Malignant and Benign Thoracic Lesions.

To prospectively evaluate the capability of amide proton transfer-weighted chemical exchange saturation transfer magnetic resonance (MR) imaging for characterization of thoracic lesions. The institutional review board approved this study, and written informed consent was obtained from 21 patients (13 men and eight women; mean age, 72 years) prior to enrollment. Each patient underwent chemical exchange saturation transfer MR imaging by using respiratory-synchronized half-Fourier fast spin-echo imaging after a series of magnetization transfer pulses. Next, a magnetization transfer ratio asymmetry at 3.5 ppm map was computationally generated. Pathology examinations resulted in a diagnosis of 13 malignant and eight benign thoracic lesions. The malignant lesions were further diagnosed as being nine lung cancers, comprising six adenocarcinomas, three squamous cell carcinomas, and four other thoracic malignancies. The Student t test was used to evaluate the capability of magnetization transfer ratio asymmetry (at 3.5 ppm), as assessed by means of region of interest measurements, for differentiating benign and malignant lesions, lung cancers and other thoracic lesions, and adenocarcinomas and squamous cell carcinomas. Magnetization transfer ratio asymmetry (at 3.5 ppm) was significantly higher for malignant tumors (mean ± standard deviation, 3.56% ± 3.01) than for benign lesions (0.33% ± 0.38, P = .008). It was also significantly higher for other thoracic malignancies (6.71% ± 3.46) than for lung cancer (2.16% ± 1.41, P = .005) and for adenocarcinoma (2.88% ± 1.13) than for squamous cell carcinoma (0.71% ± 0.17, P = .02). Amide proton transfer-weighted chemical exchange saturation transfer MR imaging allows characterization of thoracic lesions.

Exceptionally Inert Lanthanide(III) PARACEST MRI Contrast Agents Based on an 18-Membered Macrocyclic Platform.

We report a macrocyclic ligand based on a 3,6,10,13-tetraaza-1,8(2,6)-dipyridinacyclotetradecaphane platform containing four hydroxyethyl pendant arms (L(1)) that forms extraordinary inert complexes with Ln(3+) ions. The [EuL(1)](3+) complex does not undergo dissociation in 1 M HCl over a period of months at room temperature. Furthermore, high concentrations of phosphate and Zn(2+) ions at room temperature do not provoke metal-complex dissociation. The X-ray crystal structures of six Ln(3+) complexes reveal ten coordination of the ligand to the metal ions through the six nitrogen atoms of the macrocycle and the four oxygen atoms of the hydroxyethyl pendant arms. The analysis of the Yb(3+)- and Pr(3+)-induced paramagnetic (1)H NMR shifts show that the solid-state structures are retained in aqueous solution. The intensity of the (1)H NMR signal of bulk water can be modulated by saturation of the signals of the hydroxy protons of Pr(3+), Eu(3+), and Yb(3+) complexes following chemical-exchange saturation transfer (CEST). The ability of these complexes to provide large CEST effects at 25 and 37 °C and pH 7.4 was confirmed by using CEST magnetic resonance imaging experiments.

Science to Practice: Monitoring Oncolytic Virus Therapy with Chemical Exchange Saturation Transfer MR Imaging—Wishful Thinking?

Farrar et al demonstrate that modifying an oncolytic virus (OV) so that it produces excess protein when it infects a cancer cell is a process that can be detected both in vitro and in vivo in infected cancer cells by using chemical exchange saturation transfer (CEST) magnetic resonance (MR) imaging. The effect is at the limits of MR imaging detection (approximately 1%), but experience with functional MR imaging of the brain, with comparably small effects, should give pause to anyone who immediately writes this observation off as an exercise in wishful thinking. OVs are improving in their specificity, virulence, and ability to induce immune responses. Now, they have been modified to express proteins that are detectable with CEST MR imaging early after delivery into a tumor. This is clearly a surprising and hopeful development in the long road of OVs from the laboratory to the clinic.

Establishing the Lysine-rich Protein CEST Reporter Gene as a CEST MR Imaging Detector for Oncolytic Virotherapy.

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.

Optimization of 7-T chemical exchange saturation transfer parameters for validation of glycosaminoglycan and amide proton transfer of fibroglandular breast tissue.

To (a) implement simulation-optimized chemical exchange saturation transfer (CEST) measurements sensitive to amide proton transfer (APT) and glycosaminoglycan (GAG) hydroxyl proton transfer effects in the human breast at 7 T and (b) determine the reliability of these techniques for evaluation of fibroglandular tissue in the healthy breast as a benchmark for future studies of pathologic findings. All human studies were institutional review board approved, were HIPAA compliant, and included informed consent. The CEST parameters of saturation duration (25 msec) and amplitude (1 μT) were chosen on the basis of simulation-driven optimization for APT contrast enhancement with the CEST effect quantified by using residuals of a Lorentzian fit. Optimized parameters were implemented at 7 T in 10 healthy women in two separate examinations to evaluate the reliability of CEST magnetic resonance (MR) imaging measurements in the breast. CEST z-spectra were acquired over saturation offset frequencies ranging between ±40 ppm by using a quadrature unilateral breast coil. The imaging-repeat imaging reliability was assessed in terms of the intraclass correlation coefficient, which indicates the ratio of between-subject variation to total variation. Simulations were performed of the Bloch equations with chemical exchange-guided selection of optimal values for pulse duration and amplitude, 25 msec and 1 μT, respectively. Reliability was evaluated by using intraclass correlation coefficients (95% confidence intervals), with acceptable results: 0.963 (95% confidence interval: 0.852, 0.991) and 0.903 (95% confidence interval: 0.609, 0.976) for APT and GAG, respectively. Simulations were used to derive optimal CEST preparation parameters to elicit maximal CEST contrast enhancement in healthy fibroglandular breast tissue due to APT at 7 T. By using these parameters, reproducible values were obtained for both the amide and hydroxyl protons from CEST MR imaging at 7 T and are feasible in the human breast.

Observation of true and pseudo NOE signals using CEST-MRI and CEST-MRS sequences with and without lipid suppression.

To investigate the characteristics of nuclear Overhauser enhancement (NOE) imaging signals in the brain at 7T. Fresh hen eggs, as well as six healthy, and six C6 glioma-bearing Wistar rats were scanned using chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) and chemical exchange saturation transfer-magnetic resonance spectroscopy (CEST-MRS) sequences (saturation duration 3 s, power 1.47 µT) with and without lipid suppression. CEST data were acquired over an offset range of -6 to +6 ppm relative to the water resonance in 0.5 ppm steps. The water signals were not disrupted by other protons during the CEST-MRS sequences, and true NOE signals could be observed. Using the CEST-MRI sequence without lipid suppression, pseudo NOE imaging signals were observed in the lipid-containing regions (egg yolk, scalp, and even white matter). These pseudo NOE signals were almost (but incompletely) removed with the lipid suppression. Egg yolk results indicated the presence of the NOE to olefinic protons overlapping with the water signal. In vivo experiments showed that the amide proton transfer signal was larger in the tumor, whereas the NOE signal was larger in the normal white matter. True NOE signals can be detected using MRS sequences, and considerable pseudo NOE imaging signals may be observed using MRI sequences.

Detection of the Prodrug-Activating Enzyme Carboxypeptidase G2 Activity with Chemical Exchange Saturation Transfer Magnetic Resonance

The purpose of this study is to evaluate if the differential exchange rates with bulk water between amine and amide protons can be exploited using chemical exchange saturation transfer magnetic resonance (CEST-MR) to monitor the release of glutamate induced by carboxypeptidase G2 (CPG2), an enzyme utilized in cancer gene therapy. Z spectra of solutions of the CPG2 substrate, 3,5-difluorobenzoyl-L-glutamate (amide), and glutamate (amine) were acquired at 11.7 T, 37 °C, across different pH (5-8). The ability of CEST-MR to monitor CPG2-mediated release of glutamate was assessed in extracts of CPG2-expressing cancer cells and purified solution of CPG2. The addition of CPG2 to a solution containing 3,5-difluorobenzoyl-L-glutamate led to a marked and progressively increasing CEST effect (+3 ppm), concomitant with the time-dependent release of glutamate induced by CPG2. CEST-MR allows the detection of CPG2 activity in vitro and supports the translation of CEST-MRI to assess CPG2-based gene therapy in vivo.

MRI biosensor for protein kinase A encoded by a single synthetic gene

Protein kinases including protein kinase A (PKA) underlie myriad important signaling pathways. The ability to monitor kinase activity in vivo and in real-time with high spatial resolution in genetically specified cellular populations is a yet unmet need, crucial for understanding complex biological systems as well as for preclinical development and screening of novel therapeutics. Using the hypothesis that the natural recognition sequences of protein kinases may be detected using chemical exchange saturation transfer magnetic resonance imaging, we designed a genetically encoded biosensor composed of eight tandem repeats of the peptide LRRASLG, a natural target of PKA. This sensor displays a measurable change in chemical exchange saturation transfer signal following phosphorylation by PKA. The natural PKA substrate LRRASLG exhibits a chemical exchange saturation transfer-magnetic resonance imaging contrast at +1.8 and +3.6 ppm, with a >50% change after phosphorylation with minutes-scale temporal resolution. Expression of a synthetic gene encoding eight monomers of LRRASLG yielded two peaks at these chemical exchange saturation transfer frequencies. Taken together, these results suggest that this gene may be used to assay PKA levels in a biologically relevant system. Importantly, the design strategy used for this specific sensor may be adapted for a host of clinically interesting protein kinases.