Multiparametric chemical exchange saturation transfer MRI detects metabolic changes in breast cancer following immunotherapy.

BACKGROUND: With metabolic alterations of the tumor microenvironment (TME) contributing to cancer progression, metastatic spread and response to targeted therapies, non-invasive and repetitive imaging of tumor metabolism is of major importance. The purpose of this study was to investigate whether multiparametric chemical exchange saturation transfer magnetic resonance imaging (CEST-MRI) allows to detect differences in the metabolic profiles of the TME in murine breast cancer models with divergent degrees of malignancy and to assess their response to immunotherapy. METHODS: Tumor characteristics of highly malignant 4T1 and low malignant 67NR murine breast cancer models were investigated, and their changes during tumor progression and immune checkpoint inhibitor (ICI) treatment were evaluated. For simultaneous analysis of different metabolites, multiparametric CEST-MRI with calculation of asymmetric magnetization transfer ratio (MTRasym) at 1.2 to 2.0 ppm for glucose-weighted, 2.0 ppm for creatine-weighted and 3.2 to 3.6 ppm for amide proton transfer- (APT-) weighted CEST contrast was conducted. Ex vivo validation of MRI results was achieved by 1H nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry imaging with laser postionization and immunohistochemistry. RESULTS: During tumor progression, the two tumor models showed divergent trends for all examined CEST contrasts: While glucose- and APT-weighted CEST contrast decreased and creatine-weighted CEST contrast increased over time in the 4T1 model, 67NR tumors exhibited increased glucose- and APT-weighted CEST contrast during disease progression, accompanied by decreased creatine-weighted CEST contrast. Already three days after treatment initiation, CEST contrasts captured response to ICI therapy in both tumor models. CONCLUSION: Multiparametric CEST-MRI enables non-invasive assessment of metabolic signatures of the TME, allowing both for estimation of the degree of tumor malignancy and for assessment of early response to immune checkpoint inhibition.

EXERTION: a pilot trial on the effect of aerobic, smartwatch-controlled exercise on stroke recovery: effects on motor function, structural repair, cognition, mental well-being, and the immune system.

INTRODUCTION: Motor impairments are the objectively most striking sequelae after stroke, but non-motor consequences represent a high burden for stroke survivors as well. Depression is reported in one third of patients, the fatigue prevalence ranges from 23 to 75% due to heterogenous definitions and assessments. Cognitive impairment is found in one third of stroke patients 3-12 months after stroke and the risk for dementia is doubled by the event. Aerobic exercise has been shown to reduce depressive symptoms, counteract fatigue, and improve cognitive functions in non-stroke patients. Furthermore, exercise is known to strengthen the immune system. It is unknown, though, if aerobic exercise can counteract poststroke depression, fatigue, poststroke dementia and poststroke immunosuppression. Therefore, we aim to analyse the effect of aerobic exercise on functional recovery, cognition, emotional well-being, and the immune system. Reorganization of topological networks of the brain shall be visualized by diffusion MRI fibre tracking. METHODS: Adults with mild to moderate stroke impairment (initial NIHSS or NIHSS determined at the moment of maximal deterioration 1-18) are recruited within two weeks of stroke onset. Study participants must be able to walk independently without risk of falling. All patients are equipped with wearable devices (smartwatches) measuring the heart rate and daily step count. The optimal heart rate zone is determined by lactate ergometry at baseline. Patients are randomized to the control or the intervention group, the latter performing a heart rate-controlled walking training on own initiative 5 times a week for 45 min. All patients receive medical care and stroke rehabilitation to the usual standard of care. The following assessments are conducted at baseline and after 90 days: Fugl Meyer-assessment for the upper and lower extremity, 6 min-walk test, neuropsychological assessment (cognition: MoCA, SDMT; fatigue and depression: FSMC, HADS-D, participation: WHODAS 2.0 12-items), blood testing (i.e. immune profiling to obtain insights into phenotype and functional features of distinct immune-cell subsets) and cranial magnetic resonance imaging (MRI) with grid-sampled diffusion weighted imaging, white matter fibre tracking and MR spectroscopy. PERSPECTIVE: This study investigates the effect of smartwatch-controlled aerobic exercise on functional recovery, cognition, emotional well-being, the immune system, and neuronal network reorganization in stroke patients. Trial registration ClinicalTrials.gov NCT Number: NCT05690165. First posted19 January 2023. Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT05690165.

Vascular response patterns to targeted therapies in murine breast cancer models with divergent degrees of malignancy.

BACKGROUND: Response assessment of targeted cancer therapies is becoming increasingly challenging, as it is not adequately assessable with conventional morphological and volumetric analyses of tumor lesions. The tumor microenvironment is particularly constituted by tumor vasculature which is altered by various targeted therapies. The aim of this study was to noninvasively assess changes in tumor perfusion and vessel permeability after targeted therapy in murine models of breast cancer with divergent degrees of malignancy. METHODS: Low malignant 67NR or highly malignant 4T1 tumor-bearing mice were treated with either the multi-kinase inhibitor sorafenib or immune checkpoint inhibitors (ICI, combination of anti-PD1 and anti-CTLA4). Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with i.v. injection of albumin-binding gadofosveset was conducted on a 9.4 T small animal MRI. Ex vivo validation of MRI results was achieved by transmission electron microscopy, immunohistochemistry and laser ablation-inductively coupled plasma-mass spectrometry. RESULTS: Therapy-induced changes in tumor vasculature differed between low and highly malignant tumors. Sorafenib treatment led to decreased tumor perfusion and endothelial permeability in low malignant 67NR tumors. In contrast, highly malignant 4T1 tumors demonstrated characteristics of a transient window of vascular normalization with an increase in tumor perfusion and permeability early after therapy initiation, followed by decreased perfusion and permeability parameters. In the low malignant 67NR model, ICI treatment also mediated vessel-stabilizing effects with decreased tumor perfusion and permeability, while ICI-treated 4T1 tumors exhibited increasing tumor perfusion with excessive vascular leakage. CONCLUSION: DCE-MRI enables noninvasive assessment of early changes in tumor vasculature after targeted therapies, revealing different response patterns between tumors with divergent degrees of malignancy. DCE-derived tumor perfusion and permeability parameters may serve as vascular biomarkers that allow for repetitive examination of response to antiangiogenic treatment or immunotherapy.

Profiling specific cell populations within the inflammatory tumor microenvironment by oscillating-gradient diffusion-weighted MRI.

BACKGROUND: The inflammatory tumor microenvironment (TME) is formed by various immune cells, being closely associated with tumorigenesis. Especially, the interaction between tumor-infiltrating T-cells and macrophages has a crucial impact on tumor progression and metastatic spread. The purpose of this study was to investigate whether oscillating-gradient diffusion-weighted MRI (OGSE-DWI) enables a cell size-based discrimination between different cell populations of the TME. METHODS: Sine-shaped OGSE-DWI was combined with the Imaging Microstructural Parameters Using Limited Spectrally Edited Diffusion (IMPULSED) approach to measure microscale diffusion distances, here relating to cell sizes. The accuracy of IMPULSED-derived cell radii was evaluated using in vitro spheroid models, consisting of either pure cancer cells, macrophages, or T-cells. Subsequently, in vivo experiments aimed to assess changes within the TME and its specific immune cell composition in syngeneic murine breast cancer models with divergent degrees of malignancy (4T1, 67NR) during tumor progression, clodronate liposome-mediated depletion of macrophages, and immune checkpoint inhibitor (ICI) treatment. Ex vivo analysis of IMPULSED-derived cell radii was conducted by immunohistochemical wheat germ agglutinin staining of cell membranes, while intratumoral immune cell composition was analyzed by CD3 and F4/80 co-staining. RESULTS: OGSE-DWI detected mean cell radii of 8.8±1.3 µm for 4T1, 8.2±1.4 µm for 67NR, 13.0±1.7 for macrophage, and 3.8±1.8 µm for T-cell spheroids. While T-cell infiltration during progression of 4T1 tumors was observed by decreasing mean cell radii from 9.7±1.0 to 5.0±1.5 µm, increasing amount of intratumoral macrophages during progression of 67NR tumors resulted in increasing mean cell radii from 8.9±1.2 to 12.5±1.1 µm. After macrophage depletion, mean cell radii decreased from 6.3±1.7 to 4.4±0.5 µm. T-cell infiltration after ICI treatment was captured by decreasing mean cell radii in both tumor models, with more pronounced effects in the 67NR tumor model. CONCLUSIONS: OGSE-DWI provides a versatile tool for non-invasive profiling of the inflammatory TME by assessing the dominating cell type T-cells or macrophages.

Multiparametric MRI enables for differentiation of different degrees of malignancy in two murine models of breast cancer.

Objective: The objective of this study was to non-invasively differentiate the degree of malignancy in two murine breast cancer models based on identification of distinct tissue characteristics in a metastatic and non-metastatic tumor model using a multiparametric Magnetic Resonance Imaging (MRI) approach. Methods: The highly metastatic 4T1 breast cancer model was compared to the non-metastatic 67NR model. Imaging was conducted on a 9.4 T small animal MRI. The protocol was used to characterize tumors regarding their structural composition, including heterogeneity, intratumoral edema and hemorrhage, as well as endothelial permeability using apparent diffusion coefficient (ADC), T1/T2 mapping and dynamic contrast-enhanced (DCE) imaging. Mice were assessed on either day three, six or nine, with an i.v. injection of the albumin-binding contrast agent gadofosveset. Ex vivo validation of the results was performed with laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), histology, immunhistochemistry and electron microscopy. Results: Significant differences in tumor composition were observed over time and between 4T1 and 67NR tumors. 4T1 tumors showed distorted blood vessels with a thin endothelial layer, resulting in a slower increase in signal intensity after injection of the contrast agent. Higher permeability was further reflected in higher Ktrans values, with consecutive retention of gadolinium in the tumor interstitium visible in MRI. 67NR tumors exhibited blood vessels with a thicker and more intact endothelial layer, resulting in higher peak enhancement, as well as higher maximum slope and area under the curve, but also a visible wash-out of the contrast agent and thus lower Ktrans values. A decreasing accumulation of gadolinium during tumor progression was also visible in both models in LA-ICP-MS. Tissue composition of 4T1 tumors was more heterogeneous, with intratumoral hemorrhage and necrosis and corresponding higher T1 and T2 relaxation times, while 67NR tumors mainly consisted of densely packed tumor cells. Histogram analysis of ADC showed higher values of mean ADC, histogram kurtosis, range and the 90th percentile (p90), as markers for the heterogenous structural composition of 4T1 tumors. Principal component analysis (PCA) discriminated well between the two tumor models. Conclusions: Multiparametric MRI as presented in this study enables for the estimation of malignant potential in the two studied tumor models via the assessment of certain tumor features over time.

Non-invasive MRI Studies of Ventilatory and Cardiovascular Performance in Edible Crabs Cancer pagurus During Warming Under Elevated CO2 Levels.

The thermal tolerance of marine decapod crustacea is defined through their capacities for oxygen uptake and distribution. High ambient CO2 levels were previously shown to reduce hemolymph oxygen levels at enhanced cardiac performance during warming. This study investigated the impacts of warming under two CO2 levels on ventilation and hemolymph circulation in edible crabs Cancer pagurus. It also highlights changes in the ventilatory and cardiac pauses displayed by Decapoda under routine metabolism. Animals were exposed to step-wise, sub-critical warming (12-20°C over 5 days) under control (470 µatm) and high (1,350 µatm) water PCO2. Flow-through respirometry was combined with magnetic resonance imaging and infra-red photoplethysmography to allow for simultaneous, non-invasive measurements of metabolic rates ( M ˙ O 2 ), ventilation and cardiovascular performance. Crabs spent significantly more time in a low M ˙ O 2 state (metabolic pause), when experiencing high CO2 conditions above 16°C, compared to normocapnic warming. Heart rates leveled off beyond 18°C at any CO2 level. Cardiac output continued to increase with high-CO2-warming, due to elevated cardiac stroke volumes. Consequently, temperature-dependent branchial hemolymph flow remained unaffected by CO2. Instead, a suppressing effect of CO2 on ventilation was found beyond 16°C. These results indicate constrained oxygen uptake at stable cardiovascular performance in a decapod crustacean. Cancer pagurus: urn:lsid:zoobank.org:act:B750F89A-84B5-448B-8D80-EBD724A1C9D4.

Studying the cardiovascular system of a marine crustacean with magnetic resonance imaging at 9.4 T.

OBJECTIVES: An approach is presented for high-field MRI studies of the cardiovascular system (CVS) of a marine crustacean, the edible crab Cancer pagurus, submerged in highly conductive seawater. MATERIALS AND METHODS: Structure and function of the CVS were investigated at 9.4 T. Cardiac motion was studied using self-gated CINE MRI. Imaging protocols and radio-frequency coil arrangements were tested for anatomical imaging. Haemolymph flow was quantified using phase-contrast angiography. Signal-to-noise-ratios and flow velocities in afferent and efferent branchial veins were compared with Student's t test (n = 5). RESULTS: Seawater induced signal losses were dependent on imaging protocols and RF coil setup. Internal cardiac structures could be visualized with high spatial resolution within 8 min using a gradient-echo technique. Variations in haemolymph flow in different vessels could be determined over time. Maximum flow was similar within individual vessels and corresponded to literature values from Doppler measurements. Heart contractions were more pronounced in lateral and dorso-ventral directions than in the anterior-posterior direction. DISCUSSION: Choosing adequate imaging protocols in combination with a specific RF coil arrangement allows to monitor various parts of the crustacean CVS with exceptionally high spatial resolution despite the adverse effects of seawater at 9.4 T.

Non-invasive quantification of cardiac stroke volume in the edible crab Cancer pagurus.

BACKGROUND: Brachyuran crabs can effectively modulate cardiac stroke volume independently of heart rate in response to abiotic drivers. Non-invasive techniques can help to improve the understanding of cardiac performance parameters of these animals. This study demonstrates the in vivo quantification of cardiac performance parameters through magnetic resonance imaging (MRI) on the edible crab Cancer pagurus. Furthermore, the suitability of signal integrals of infra-red photoplethysmographs as a qualitative tool is assessed under severe hypoxia. RESULTS: Multi-slice self-gated cardiac cinematic (CINE) MRI revealed the structure and motion of the ventricle to quantify heart rates, end-diastolic volume, end-systolic volume, stroke volume and ejection fraction. CINE MRI showed that stroke volumes increased under hypoxia because of a reduction of end-systolic volumes at constant end-diastolic volumes. Plethysmograph recordings allowed for automated heart rate measurements but determination of a qualitative stroke volume proxy strongly depended on the position of the sensor on the animal. Both techniques revealed a doubling in stroke volumes after 6 h under severe hypoxia (water PO2 = 15% air saturation). CONCLUSIONS: MRI has allowed for detailed descriptions of cardiac performance in intact animals under hypoxia. The temporal resolution of quantitative non-invasive CINE MRI is limited but should encourage further refining. The stroke volume proxy based on plethysmograph recordings is feasible to complement other cardiac measurements over time. The presented methods allow for non-destructive in vivo determinations of multiple cardiac performance parameters, with the possibility to study neuro-hormonal or environmental effects on decapod cardio physiology.

CO2 induced pHi changes in the brain of polar fish: a TauCEST application.

Chemical exchange saturation transfer (CEST) from taurine to water (TauCEST) can be used for in vivo mapping of taurine concentrations as well as for measurements of relative changes in intracellular pH (pHi ) at temperatures below 37°C. Therefore, TauCEST offers the opportunity to investigate acid-base regulation and neurological disturbances of ectothermic animals living at low temperatures, and in particular to study the impact of ocean acidification (OA) on neurophysiological changes of fish. Here, we report the first in vivo application of TauCEST imaging. Thus, the study aimed to investigate the TauCEST effect in a broad range of temperatures (1-37°C) and pH (5.5-8.0), motivated by the high taurine concentration measured in the brains of polar fish. The in vitro data show that the TauCEST effect is especially detectable in the low temperature range and strictly monotonic for the relevant pH range (6.8-7.5). To investigate the specificity of TauCEST imaging for the brain of polar cod (Boreogadus saida) at 1.5°C simulations were carried out, indicating a taurine contribution of about 65% to the in vivo expected CEST effect, if experimental parameters are optimized. B. saida was acutely exposed to three different CO2 concentrations in the sea water (control normocapnia; comparatively moderate hypercapnia OAm  = 3300 µatm; high hypercapnia OAh  = 4900 µatm). TauCEST imaging of the brain showed a significant increase in the TauCEST effect under the different CO2 concentrations of about 1.5-3% in comparison with control measurements, indicative of changes in pHi or metabolite concentration. Consecutive recordings of 1 H MR spectra gave no support for a concentration induced change of the in vivo observed TauCEST effect. Thus, the in vivo application of TauCEST offers the possibility of mapping relative changes in pHi in the brain of polar cod during exposure to CO2 .

Water bicarbonate modulates the response of the shore crab Carcinus maenas to ocean acidification.

Ocean acidification causes an accumulation of CO2 in marine organisms and leads to shifts in acid-base parameters. Acid-base regulation in gill breathers involves a net increase of internal bicarbonate levels through transmembrane ion exchange with the surrounding water. Successful maintenance of body fluid pH depends on the functional capacity of ion-exchange mechanisms and associated energy budget. For a detailed understanding of the dependence of acid-base regulation on water parameters, we investigated the physiological responses of the shore crab Carcinus maenas to 4 weeks of ocean acidification [OA, P(CO2)w = 1800 µatm], at variable water bicarbonate levels, paralleled by changes in water pH. Cardiovascular performance was determined together with extra-(pHe) and intracellular pH (pHi), oxygen consumption, haemolymph CO2 parameters, and ion composition. High water P(CO2) caused haemolymph P(CO2) to rise, but pHe and pHi remained constant due to increased haemolymph and cellular [HCO3-]. This process was effective even under reduced seawater pH and bicarbonate concentrations. While extracellular cation concentrations increased throughout, anion levels remained constant or decreased. Despite similar levels of haemolymph pH and ion concentrations under OA, metabolic rates, and haemolymph flow were significantly depressed by 40 and 30%, respectively, when OA was combined with reduced seawater [HCO3-] and pH. Our findings suggest an influence of water bicarbonate levels on metabolic rates as well as on correlations between blood flow and pHe. This previously unknown phenomenon should direct attention to pathways of acid-base regulation and their potential feedback on whole-animal energy demand, in relation with changing seawater carbonate parameters.