Exchange Saturation Transfer Magnetic Resonance Imaging 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.

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.

1371-P: Dynamic Mapping of Skeletal Muscle Perfusion and Phosphocreatine with 1H Magnetic Resonance Imaging: A Rest-Exercise Study in Diabetes

Aim: Evaluating time and spatially resolved 1H magnetic resonance imaging (MRI) in quantifying muscle tissue phosphocreatine (PCr) kinetics (without extra hardware) and perfusion in healthy individuals and patients with type 2 diabetes mellitus (DM) . Methods: 18 subjects were imaged in 3 groups using a 3T MRI scanner; young healthy (26y, n= 6) , old healthy (62y, n = 6) , and DM patients (66y, n = 6) . For each subject, a chemical exchange saturation transfer (CEST) MRI scan was performed in the calf every 1 min at rest (3 min) , during exercise (4 min standardized isometric plantarflexion) , and a recovery period (6 min) . Subjects also underwent mapping of skeletal muscle perfusion in a separate exercise protocol. PCr concentration maps were quantified by Bloch-McConnell fitting, and values were obtained in the medial gastrocnemius and soleus muscles. Results: DM patients show a longer PCr recovery time τ during recovery compared to both healthy groups: young healthy τ = 47.1 ± 18.2s, old healthy τ = 41.9 ± 15.1s, and DM τ =122.2 ± 58.6s, p = 0.02 (Figure) . There is a negative correlation between PCr and perfusion during exercise in healthy groups (r = 0.38, p = 0.034) that is not significant in the DM group. Conclusion: Dynamic PCr mapping by proton MRI in calf muscle reveals impaired energetics that is uncoupled from muscle perfusion in DM compared to healthy controls. Disclosure R.Wahidi: None. R.Li: None. M.A.Zayed: None. M.Hastings: None. J.Zheng: None.

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.

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.

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.

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.

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.