CTLA-4 blockade drives loss of Treg stability in glycolysis-low tumours.

Limiting metabolic competition in the tumour microenvironment may increase the effectiveness of immunotherapy. Owing to its crucial role in the glucose metabolism of activated T cells, CD28 signalling has been proposed as a metabolic biosensor of T cells1. By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis1. Here we investigate the effect of CTLA-4 blockade on the metabolic fitness of intra-tumour T cells in relation to the glycolytic capacity of tumour cells. We found that CTLA-4 blockade promotes metabolic fitness and the infiltration of immune cells, especially in glycolysis-low tumours. Accordingly, treatment with anti-CTLA-4 antibodies improved the therapeutic outcomes of mice bearing glycolysis-defective tumours. Notably, tumour-specific CD8+ T cell responses correlated with phenotypic and functional destabilization of tumour-infiltrating regulatory T (Treg) cells towards IFNγ- and TNF-producing cells in glycolysis-defective tumours. By mimicking the highly and poorly glycolytic tumour microenvironments in vitro, we show that the effect of CTLA-4 blockade on the destabilization of Treg cells is dependent on Treg cell glycolysis and CD28 signalling. These findings indicate that decreasing tumour competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumour glycolysis. Moreover, these results reveal a mechanism by which anti-CTLA-4 treatment interferes with Treg cell function in the presence of glucose.

First in-human evaluation of [1-13C]pyruvate in D2O for hyperpolarized MRI of the brain: A safety and feasibility study.

PURPOSE: To investigate the safety and value of hyperpolarized (HP) MRI of [1-13C]pyruvate in healthy volunteers using deuterium oxide (D2O) as a solvent. METHODS: Healthy volunteers (n = 5), were injected with HP [1-13C]pyruvate dissolved in D2O and imaged with a metabolite-specific 3D dual-echo dynamic EPI sequence at 3T at one site (Site 1). Volunteers were monitored following the procedure to assess safety. Image characteristics, including SNR, were compared to data acquired in a separate cohort using water as a solvent (n = 5) at another site (Site 2). The apparent spin-lattice relaxation time (T1) of [1-13C]pyruvate was determined both in vitro and in vivo from a mono-exponential fit to the image intensity at each time point of our dynamic data. RESULTS: All volunteers completed the study safely and reported no adverse effects. The use of D2O increased the T1 of [1-13C]pyruvate from 66.5 ± 1.6 s to 92.1 ± 5.1 s in vitro, which resulted in an increase in signal by a factor of 1.46 ± 0.03 at the time of injection (90 s after dissolution). The use of D2O also increased the apparent relaxation time of [1-13C]pyruvate by a factor of 1.4 ± 0.2 in vivo. After adjusting for inter-site SNR differences, the use of D2O was shown to increase image SNR by a factor of 2.6 ± 0.2 in humans. CONCLUSIONS: HP [1-13C]pyruvate in D2O is safe for human imaging and provides an increase in T1 and SNR that may improve image quality.

Multiparametric classification of sub-acute ischemic stroke recovery with ultrafast diffusion, 23 Na, and MPIO-labeled stem cell MRI at 21.1 T.

MRI leverages multiple modes of contrast to characterize stroke. High-magnetic-field systems enhance the performance of these MRI measurements. Previously, we have demonstrated that individually sodium and stem cell tracking metrics are enhanced at ultrahigh field in a rat model of stroke, and we have developed robust single-scan diffusion-weighted imaging approaches that utilize spatiotemporal encoding (SPEN) of the apparent diffusion coefficient (ADC) for these challenging field strengths. Here, we performed a multiparametric study of middle cerebral artery occlusion (MCAO) biomarker evolution focusing on comparison of these MRI biomarkers for stroke assessment during sub-acute recovery in rat MCAO models at 21.1 T. T2 -weighted MRI was used as the benchmark for identification of the ischemic lesion over the course of the study. The number of MPIO-induced voids measured by gradient-recalled echo, the SPEN measurement of ADC, and 23 Na MRI values were determined in the ischemic area and contralateral hemisphere, and relative performances for stroke classification were compared by receiver operator characteristic analysis. These measurements were associated with unique time-dependent trajectories during stroke recovery that changed the sensitivity and specificity for stroke monitoring during its evolution. Advantages and limitations of these contrasts, and the use of ultrahigh field for multiparametric stroke assessment, are discussed.

Ultrafast in vivo diffusion imaging of stroke at 21.1 T by spatiotemporal encoding.

PURPOSE: This study quantifies in vivo ischemic stroke brain injuries in rats using ultrahigh-field single-scan MRI methods to assess variations in apparent diffusion coefficients (ADCs). METHODS: Magnitude and diffusion-weighted spatiotemporally encoded imaging sequences were implemented on a 21.1 T imaging system, and compared with spin-echo and echo-planar imaging diffusion-weighted imaging strategies. ADC maps were calculated and used to evaluate the sequences according to the statistical comparisons of the ipsilateral and contralateral ADC measurements at 24, 48, and 72 h poststroke. RESULTS: Susceptibility artifacts resulting from normative anatomy and pathological stroke conditions were particularly intense at 21.1 T. These artifacts strongly distorted single-shot diffusion-weighted echo-planar imaging experiments, but were reduced in four-segment interleaved echo-planar imaging acquisitions. By contrast, nonsegmented diffusion-weighted spatiotemporally encoded images were largely immune to field-dependent artifacts. Effects of stroke were apparent in both magnitude images and ADC maps of all sequences. When stroke recovery was followed by ADC variations, spatiotemporally encoded, echo-planar imaging, and spin-echo acquisitions revealed statistically significant increase in ADCs. CONCLUSIONS: Consideration of experiment duration, image quality, and mapped ADC values provided by spatiotemporally encoded demonstrates that this single-shot acquisition is a method of choice for high-throughput, ultrahigh-field in vivo stroke quantification.

Area per lipid and cholesterol interactions in membranes from separated local-field (13)C NMR spectroscopy.

Investigations of lipid membranes using NMR spectroscopy generally require isotopic labeling, often precluding structural studies of complex lipid systems. Solid-state (13)C magic-angle spinning NMR spectroscopy at natural isotopic abundance gives site-specific structural information that can aid in the characterization of complex biomembranes. Using the separated local-field experiment DROSS, we resolved (13)C-(1)H residual dipolar couplings that were interpreted with a statistical mean-torque model. Liquid-disordered and liquid-ordered phases were characterized according to membrane thickness and average cross-sectional area per lipid. Knowledge of such structural parameters is vital for molecular dynamics simulations, and provides information about the balance of forces in membrane lipid bilayers. Experiments were conducted with both phosphatidylcholine (dimyristoylphosphatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC)) and egg-yolk sphingomyelin (EYSM) lipids, and allowed us to extract segmental order parameters from the (13)C-(1)H residual dipolar couplings. Order parameters were used to calculate membrane structural quantities, including the area per lipid and bilayer thickness. Relative to POPC, EYSM is more ordered in the ld phase and experiences less structural perturbation upon adding 50% cholesterol to form the lo phase. The loss of configurational entropy is smaller for EYSM than for POPC, thus favoring its interaction with cholesterol in raftlike lipid systems. Our studies show that solid-state (13)C NMR spectroscopy is applicable to investigations of complex lipids and makes it possible to obtain structural parameters for biomembrane systems where isotope labeling may be prohibitive.

Referenceless reconstruction of spatiotemporally encoded imaging data: principles and applications to real-time MRI.

PURPOSE: Ultrafast sequences based on "Hybrid" spatiotemporal encoding (SPEN) replace echo-planar imaging's phase encoding "blips," while retaining a k-space readout acquisition. Hardware imperfections during acquisition may lead to ghosts and striped artifacts along the SPEN dimension; akin to echo-planar imaging's Nyquist ghosts, but weaker. A referenceless method to eliminate these artifacts in Hybrid SPEN is demonstrated. THEORY AND METHODS: Owing to its encoding in direct space, rather than reciprocal space, undersampling in SPEN does not generate an echo-planar-imaging-like aliasing, but instead lowers the spatial resolution. Hybrid SPEN data can be split into two undersampled signals: a reference one comprised of the odd-echos, and an even-echo set that has to be "corrected" for consistency with the former. A simple way of implementing such a correction that enables a joint high-resolution reconstruction is proposed. RESULTS: The referenceless algorithm is demonstrated with various examples, including oblique scans, large in vivo datasets from real-time dynamic contrast-enhanced perfusion experiments, and human brain imaging. CONCLUSIONS: The referenceless correction enables robust single-scan imaging under changing conditions-such as patient motion and changes in shimming over time-without the need of ancillary navigators. This opens new options for real-time MRI and interactive scanning.

In vivo magnetic resonance of hyperpolarized [(13)C1]pyruvate: metabolic dynamics in stimulated muscle.

The metabolic status of muscle changes according to the energetic demands of the organism. Two key regulators of these changes include exercise and insulin, with exercise eliciting catabolic expenditure within seconds and insulin enabling anabolic energy investment over minutes to hours. This study explores the potential of time-resolved hyperpolarized dynamic (13)C spectroscopy to characterize the in vivo metabolic phenotype of muscle during functional and biochemical insulin-induced stimulation of muscle. Using [(13)C1]pyruvic acid as a tracer, we find that despite the different time scales of these forms of stimulation, increases in pyruvate label transport and consumption and concomitant increases in initial rates of the tracer metabolism to lactate were observed for both stimuli. By contrast, rates of tracer metabolism to labeled alanine increased incrementally for insulin but remained unchanged following exercise-like muscle stimulation. Kinetic analysis revealed that branching of the hyperpolarized [(13)C]pyruvate tracer between lactate and alanine provides significant tissue-specific biomarkers that distinguish between anabolic and catabolic fates in vivo according to the routing of metabolites between glycolytic and amino acid pathways.

An NMR database for simulations of membrane dynamics.

Computational methods are powerful in capturing the results of experimental studies in terms of force fields that both explain and predict biological structures. Validation of molecular simulations requires comparison with experimental data to test and confirm computational predictions. Here we report a comprehensive database of NMR results for membrane phospholipids with interpretations intended to be accessible by non-NMR specialists. Experimental ¹³C-¹H and ²H NMR segmental order parameters (S(CH) or S(CD)) and spin-lattice (Zeeman) relaxation times (T(1Z)) are summarized in convenient tabular form for various saturated, unsaturated, and biological membrane phospholipids. Segmental order parameters give direct information about bilayer structural properties, including the area per lipid and volumetric hydrocarbon thickness. In addition, relaxation rates provide complementary information about molecular dynamics. Particular attention is paid to the magnetic field dependence (frequency dispersion) of the NMR relaxation rates in terms of various simplified power laws. Model-free reduction of the T(1Z) studies in terms of a power-law formalism shows that the relaxation rates for saturated phosphatidylcholines follow a single frequency-dispersive trend within the MHz regime. We show how analytical models can guide the continued development of atomistic and coarse-grained force fields. Our interpretation suggests that lipid diffusion and collective order fluctuations are implicitly governed by the viscoelastic nature of the liquid-crystalline ensemble. Collective bilayer excitations are emergent over mesoscopic length scales that fall between the molecular and bilayer dimensions, and are important for lipid organization and lipid-protein interactions. Future conceptual advances and theoretical reductions will foster understanding of biomembrane structural dynamics through a synergy of NMR measurements and molecular simulations.

Solid-state ²H NMR shows equivalence of dehydration and osmotic pressures in lipid membrane deformation.

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state ²H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our ²H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S(CD)) of DMPC approach very large values of ≈ 0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state ²H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state ²H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.

The Iron Curtain: Macrophages at the Interface of Systemic and Microenvironmental Iron Metabolism and Immune Response in Cancer.

Macrophages fulfill central functions in systemic iron metabolism and immune response. Infiltration and polarization of macrophages in the tumor microenvironment is associated with differential cancer prognosis. Distinct metabolic iron and immune phenotypes in tumor associated macrophages have been observed in most cancers. While this prompts the hypothesis that macroenvironmental manifestations of dysfunctional iron metabolism have direct associations with microenvironmental tumor immune response, these functional connections are still emerging. We review our current understanding of the role of macrophages in systemic and microenvironmental immune response and iron metabolism and discuss these functions in the context of cancer and immunometabolic precision therapy approaches. Accumulation of tumor associated macrophages with distinct iron pathologies at the invasive tumor front suggests an "Iron Curtain" presenting as an innate functional interface between systemic and microenvironmental iron metabolism and immune response that can be harnessed therapeutically to further our goal of treating and eliminating cancer.

The N-terminus of the intrinsically disordered protein α-synuclein triggers membrane binding and helix folding.

Alpha-synuclein (αS) is a 140-amino-acid protein that is involved in a number of neurodegenerative diseases. In Parkinson's disease, the protein is typically encountered in intracellular, high-molecular-weight aggregates. Although αS is abundant in the presynaptic terminals of the central nervous system, its physiological function is still unknown. There is strong evidence for the membrane affinity of the protein. One hypothesis is that lipid-induced binding and helix folding may modulate the fusion of synaptic vesicles with the presynaptic membrane and the ensuing transmitter release. Here we show that membrane recognition of the N-terminus is essential for the cooperative formation of helical domains in the protein. We used circular dichroism spectroscopy and isothermal titration calorimetry to investigate synthetic peptide fragments from different domains of the full-length αS protein. Site-specific truncation and partial cleavage of the full-length protein were employed to further characterize the structural motifs responsible for helix formation and lipid-protein interaction. Unilamellar vesicles of varying net charge and lipid compositions undergoing lateral phase separation or chain melting phase transitions in the vicinity of physiological temperatures served as model membranes. The results suggest that the membrane-induced helical folding of the first 25 residues may be driven simultaneously by electrostatic attraction and by a change in lipid ordering. Our findings highlight the significance of the αS N-terminus for folding nucleation, and provide a framework for elucidating the role of lipid-induced conformational transitions of the protein within its intracellular milieu.

Imaging endogenous macrophage iron deposits reveals a metabolic biomarker of polarized tumor macrophage infiltration and response to CSF1R breast cancer immunotherapy.

Iron deposits are a phenotypic trait of tumor-associated macrophages (TAMs). Histological iron imaging and contrast-agent free magnetic resonance imaging (MRI) can detect these deposits, but their presence  in human cancer, and correlation with immunotherapeutic response is largely untested. Here, primarily using these iron imaging approaches, we evaluated the spatial distribution of polarized macrophage populations containing high endogenous levels of iron in preclinical murine models and human breast cancer, and used them as metabolic biomarkers to correlate TAM infiltration with response to immunotherapy in preclinical trials. Macrophage-targeted inhibition of the colony stimulating factor 1 receptor (CSF1R) by immunotherapy was confirmed to inhibit macrophage accumulation and slow mammary tumor growth in mouse models while also reducing hemosiderin iron-laden TAM accumulation as measured by both iron histology and in vivo iron MRI (FeMRI). Spatial profiling of TAM iron deposit infiltration defined regions of maximal accumulation and response to the CSF1R inhibitor, and revealed differences between microenvironments of human cancer according to levels of polarized macrophage iron accumulation in stromal margins. We therefore demonstrate that iron deposition serves as an endogenous metabolic imaging biomarker of TAM infiltration in breast cancer that has high translational potential for evaluation of immunotherapeutic response.

Author Correction: Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche.

In the version of this article originally published, the key for Fig. 4c was incorrect. The symbols for 'Sham' and 'Den' were reversed. The error has been corrected in the PDF and HTML versions of the manuscript.

Nestin+NG2+ Cells Form a Reserve Stem Cell Population in the Mouse Prostate.

In the prostate, stem and progenitor cell regenerative capacities have been ascribed to both basal and luminal epithelial cells. Here, we show that a rare subset of mesenchymal cells in the prostate are epithelial-primed Nestin-expressing cells (EPNECs) that can generate self-renewing prostate organoids with bipotential capacity. Upon transplantation, these EPNECs can form prostate gland tissue grafts at the clonal level. Lineage-tracing analyses show that cells marked by Nestin or NG2 transgenic mice contribute to prostate epithelium during organogenesis. In the adult, modest contributions in repeated rounds of regression and regeneration are observed, whereas prostate epithelial cells derived from Nestin/NG2-marked cells are dramatically increased after severe irradiation-induced organ damage. These results indicate that Nestin/NG2 expression marks a novel radioresistant prostate stem cell that is active during development and displays reserve stem cell activity for tissue maintenance.

Quantification of Nanoparticle Enhancement in Polarized Breast Tumor Macrophage Deposits by Spatial Analysis of MRI and Histological Iron Contrast Using Computer Vision.

Magnetic resonance imaging applications utilizing nanoparticle agents for polarized macrophage detection are conventionally analyzed according to iron-dependent parameters averaged over large regions of interest (ROI). However, contributions from macrophage iron deposits are usually obscured in these analyses due to their lower spatial frequency and smaller population size compared with the bulk of the tumor tissue. We hypothesized that, by addressing MRI and histological pixel contrast heterogeneity using computer vision image analysis approaches rather than statistical ROI distribution averages, we could enhance our ability to characterize deposits of polarized tumor-associated macrophages (TAMs). We tested this approach using in vivo iron MRI (FeMRI) and histological detection of macrophage iron in control and ultrasmall superparamagnetic iron oxide (USPIO) enhanced mouse models of breast cancer. Automated spatial profiling of the number and size of iron-containing macrophage deposits according to localized high-iron FeMRI or Prussian blue pixel clustering performed better than using distribution averages to evaluate the effects of contrast agent injections. This analysis was extended to characterize subpixel contributions to the localized FeMRI measurements with histology that confirmed the association of endogenous and nanoparticle-enhanced iron deposits with macrophages in vascular regions and further allowed us to define the polarization status of the macrophage iron deposits detected by MRI. These imaging studies demonstrate that characterization of TAMs in breast cancer models can be improved by focusing on spatial distributions of iron deposits rather than ROI averages and indicate that nanoparticle uptake is dependent on the polarization status of the macrophage populations. These findings have broad implications for nanoparticle-enhanced biomedical imaging especially in cancer.

Adrenergic nerve degeneration in bone marrow drives aging of the hematopoietic stem cell niche.

Aging of hematopoietic stem cells (HSCs) is associated with a decline in their regenerative capacity and multilineage differentiation potential, contributing to the development of blood disorders. The bone marrow microenvironment has recently been suggested to influence HSC aging, but the underlying mechanisms remain largely unknown. Here we show that HSC aging critically depends on bone marrow innervation by the sympathetic nervous system (SNS), as loss of SNS nerves or adrenoreceptor ß3 signaling in the bone marrow microenvironment of young mice led to premature HSC aging, as evidenced by appearance of HSC phenotypes reminiscent of physiological aging. Strikingly, supplementation of a sympathomimetic acting selectively on adrenoreceptor ß3 to old mice significantly rejuvenated the in vivo function of aged HSCs, suggesting that the preservation or restitution of bone marrow SNS innervation during aging may hold the potential for new HSC rejuvenation strategies.

Iron imaging reveals tumor and metastasis macrophage hemosiderin deposits in breast cancer.

Iron-deposition is a metabolic biomarker of macrophages in both normal and pathological situations, but the presence of iron in tumor and metastasis-associated macrophages is not known. Here we mapped and quantified hemosiderin-laden macrophage (HLM) deposits in murine models of metastatic breast cancer using iron and macrophage histology, and in vivo MRI. Iron MRI detected high-iron pixel clusters in mammary tumors, lung metastasis, and brain metastasis as well as liver and spleen tissue known to contain the HLMs. Iron histology showed these regions to contain clustered macrophages identified by their common iron status and tissue-intrinsic association with other phenotypic macrophage markers. The in vivo MRI and ex vivo histological images were further processed to determine the frequencies and sizes of the iron deposits, and measure the number of HLMs in each deposit to estimate the in vivo MRI sensitivity for these cells. Hemosiderin accumulation is a macrophage biomarker and intrinsic contrast source for cellular MRI associated with the innate function of macrophages in iron metabolism systemically, and in metastatic cancer.

Iron deposition is associated with differential macrophage infiltration and therapeutic response to iron chelation in prostate cancer.

Immune cells such as macrophages are drivers and biomarkers of most cancers. Scoring macrophage infiltration in tumor tissue provides a prognostic assessment that is correlated with disease outcome and therapeutic response, but generally requires invasive biopsy. Routine detection of hemosiderin iron aggregates in macrophages in other settings histologically and in vivo by MRI suggests that similar assessments in cancer can bridge a gap in our ability to assess tumor macrophage infiltration. Quantitative histological and in vivo MRI assessments of non-heme cellular iron revealed that preclinical prostate tumor models could be differentiated according to hemosiderin iron accumulation-both in tumors and systemically. Monitoring cellular iron levels during "off-label" administration of the FDA-approved iron chelator deferiprone evidenced significant reductions in tumor size without extensive perturbation to these iron deposits. Spatial profiling of the iron-laden infiltrates further demonstrated that higher numbers of infiltrating macrophage iron deposits was associated with lower anti-tumor chelation therapy response. Imaging macrophages according to their innate iron status provides a new phenotypic window into the immune tumor landscape and reveals a prognostic biomarker associated with macrophage infiltration and therapeutic outcome.

Cardio-chemical exchange saturation transfer magnetic resonance imaging reveals molecular signatures of endogenous fibrosis and exogenous contrast media.

BACKGROUND: Application of emerging molecular MRI techniques, including chemical exchange saturation transfer (CEST)-MRI, to cardiac imaging is desirable; however, conventional methods are poorly suited for cardiac imaging, particularly in small animals with rapid heart rates. We developed a CEST-encoded steady state and retrospectively gated cardiac cine imaging sequence in which the presence of fibrosis or paraCEST contrast agents was directly encoded into the steady-state myocardial signal intensity (cardioCEST). METHODS AND RESULTS: Development of cardioCEST: A CEST-encoded cardiac cine MRI sequence was implemented on a 9.4T small animal scanner. CardioCEST of fibrosis was serially performed by acquisition of a series of CEST-encoded cine images at multiple offset frequencies in mice (n=7) after surgically induced myocardial infarction. Scar formation was quantified using a spectral modeling approach and confirmed with histological staining. Separately, circulatory redistribution kinetics of the paramagnetic CEST agent Eu-HPDO3A were probed in mice using cardioCEST imaging, revealing rapid myocardial redistribution, and washout within 30 minutes (n=6). Manipulation of vascular tone resulted in heightened peak CEST contrast in the heart, but did not alter redistribution kinetics (n=6). At 28 days after myocardial infarction (n=3), CEST contrast kinetics in infarct zone tissue were altered, demonstrating gradual accumulation of Eu-HPDO3A in the increased extracellular space. CONCLUSIONS: cardioCEST MRI enables in vivo imaging of myocardial fibrosis using endogenous contrast mechanisms, and of exogenously delivered paraCEST agents, and can enable multiplexed imaging of multiple molecular targets at high-resolution coupled with conventional cardiac MRI scans.