mTOR inhibition enhances synaptic and mitochondrial function in Alzheimer's disease in an APOE genotype-dependent manner.

Apolipoprotein ε4 (APOE4) carriers develop brain metabolic dysfunctions decades before the onset of Alzheimer's disease (AD). A goal of the study is to identify if rapamycin, an inhibitor for the mammalian target of rapamycin (mTOR) inhibitor, would enhance synaptic and mitochondrial function in asymptomatic mice with human APOE4 gene (E4FAD) before they showed metabolic deficits. A second goal is to determine whether there may be genetic-dependent responses to rapamycin when compared to mice with human APOE3 alleles (E3FAD), a neutral AD genetic risk factor. We fed asymptomatic E4FAD and E3FAD mice with control or rapamycin diets for 16 weeks from starting from 3 months of age. Neuronal mitochondrial oxidative metabolism and excitatory neurotransmission rates were measured using in vivo 1H-[13C] proton-observed carbon-edited magnetic resonance spectroscopy, and isolated mitochondrial bioenergetic measurements using Seahorse. We found that rapamycin enhanced neuronal mitochondrial function, glutamate-glutamine cycling, and TCA cycle rates in the asymptomatic E4FAD mice. In contrast, rapamycin enhances glycolysis, non-neuronal activities, and inhibitory neurotransmission of the E3FAD mice. These findings indicate that rapamycin might be able to mitigate the risk for AD by enhancing brain metabolic functions for cognitively intact APOE4 carriers, and the responses to rapamycin are varied by APOE genotypes. Consideration of precision medicine may be needed for future rapamycin therapeutics.

High-resolution extracellular pH imaging of liver cancer with multiparametric MR using Deep Image Prior.

Noninvasive extracellular pH (pHe ) mapping with Biosensor Imaging of Redundant Deviation in Shifts (BIRDS) using MR spectroscopic imaging (MRSI) has been demonstrated on 3T clinical MR scanners at 8 × 8 × 10 $$ \times 8\times 10 $$ mm3 spatial resolution and applied to study various liver cancer treatments. Although pHe imaging at higher resolution can be achieved by extending the acquisition time, a postprocessing method to increase the resolution is preferable, to minimize the duration spent by the subject in the MR scanner. In this work, we propose to improve the spatial resolution of pHe mapping with BIRDS by incorporating anatomical information in the form of multiparametric MRI and using an unsupervised deep-learning technique, Deep Image Prior (DIP). Specifically, we used high-resolution T 1 $$ {\mathrm{T}}_1 $$ , T 2 $$ {\mathrm{T}}_2 $$ , and diffusion-weighted imaging (DWI) MR images of rabbits with VX2 liver tumors as inputs to a U-Net architecture to provide anatomical information. U-Net parameters were optimized to minimize the difference between the output super-resolution image and the experimentally acquired low-resolution pHe image using the mean-absolute error. In this way, the super-resolution pHe image would be consistent with both anatomical MR images and the low-resolution pHe measurement from the scanner. The method was developed based on data from 49 rabbits implanted with VX2 liver tumors. For evaluation, we also acquired high-resolution pHe images from two rabbits, which were used as ground truth. The results indicate a good match between the spatial characteristics of the super-resolution images and the high-resolution ground truth, supported by the low pixelwise absolute error.

Effect of Incomplete Cryoablation and Matrix Metalloproteinase Inhibition on Intratumoral CD8+ T-Cell Infiltration in Murine Hepatocellular Carcinoma.

Background Image-guided tumor ablation is the first-line therapy for early-stage hepatocellular carcinoma (HCC), with ongoing investigations into its combination with immunotherapies. Matrix metalloproteinase (MMP) inhibition demonstrates immunomodulatory potential and reduces HCC tumor growth when combined with ablative treatment. Purpose To evaluate the effect of incomplete cryoablation with or without MMP inhibition on the local immune response in residual tumors in a murine HCC model. Materials and Methods Sixty 8- to 10-week-old female BALB/c mice underwent HCC induction with use of orthotopic implantation of syngeneic Tib-75 cells. After 7 days, mice with a single lesion were randomized into treatment groups: (a) no treatment, (b) MMP inhibitor, (c) incomplete cryoablation, and (d) incomplete cryoablation and MMP inhibitor. Macrophage and T-cell subsets were assessed in tissue samples with use of immunohistochemistry and immunofluorescence (cell averages calculated using five 1-µm2 fields of view [FOVs]). C-X-C motif chemokine receptor type 3 (CXCR3)- and interferon γ (IFNγ)-positive T cells were assessed using flow cytometry. Groups were compared using unpaired Student t tests, one-way analysis of variance with Tukey correction, and the Kruskal-Wallis test with Dunn correction. Results Mice treated with incomplete cryoablation (n = 6) showed greater infiltration of CD206+ tumor-associated macrophages (mean, 1.52 cells per FOV vs 0.64 cells per FOV; P = .03) and MMP9-expressing cells (mean, 0.89 cells per FOV vs 0.11 cells per FOV; P = .03) compared with untreated controls (n = 6). Incomplete cryoablation with MMP inhibition (n = 6) versus without (n = 6) led to greater CD8+ T-cell (mean, 15.8% vs 8.29%; P = .04), CXCR3+CD8+ T-cell (mean, 11.64% vs 8.47%; P = .004), and IFNγ+CD8+ T-cell infiltration (mean, 11.58% vs 5.18%; P = .02). Conclusion In a mouse model of HCC, incomplete cryoablation and systemic MMP inhibition showed increased cytotoxic CD8+ T-cell infiltration into the residual tumor compared with either treatment alone. © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Gemmete in this issue.

Transmembrane pH gradient imaging in rodent glioma models.

A unique feature of the tumor microenvironment is extracellular acidosis in relation to intracellular milieu. Metabolic reprogramming in tumors results in overproduction of H+ ions (and lactate), which are extruded from the cells to support tumor survival and progression. As a result, the transmembrane pH gradient (ΔpH), representing the difference between intracellular pH (pHi) and extracellular pH (pHe), is posited to be larger in tumors compared with normal tissue. Controlling the transmembrane pH difference has promise as a potential therapeutic target in cancer as it plays an important role in regulating drug delivery into cells. The current study shows successful development of an MRI/MRSI-based technique that provides ΔpH imaging at submillimeter resolution. We applied this technique to image ΔpH in rat brains with RG2 and U87 gliomas, as well as in mouse brains with GL261 gliomas. pHi was measured with Amine and Amide Concentration-Independent Detection (AACID), while pHe was measured with Biosensor Imaging of Redundant Deviation in Shifts (BIRDS). The results indicate that pHi was slightly higher in tumors (7.40-7.43 in rats, 7.39-7.47 in mice) compared with normal brain (7.30-7.38 in rats, 7.32-7.36 in mice), while pHe was significantly lower in tumors (6.62-6.76 in rats, 6.74-6.84 in mice) compared with normal tissue (7.17-7.22 in rats, 7.20-7.21 in mice). As a result, ΔpH was higher in tumors (0.64-0.81 in rats, 0.62-0.65 in mice) compared with normal brain (0.13-0.16 in rats, 0.13-0.16 in mice). This work establishes an MRI/MRSI-based platform for ΔpH imaging at submillimeter resolution in gliomas.

[WITHDRAWN] Activation of transsulfuration pathway to maintain cysteine is a thermogenic checkpoint for the conservation of energy.

The authors have requested that this preprint be removed from Research Square.

MR Imaging-Based In Vivo Macrophage Imaging to Monitor Immune Response after Radiofrequency Ablation of the Liver.

PURPOSE: To establish molecular magnetic resonance (MR) imaging instruments for in vivo characterization of the immune response to hepatic radiofrequency (RF) ablation using cell-specific immunoprobes. MATERIALS AND METHODS: Seventy-two C57BL/6 wild-type mice underwent standardized hepatic RF ablation (70 °C for 5 minutes) to generate a coagulation area measuring 6-7 mm in diameter. CD68+ macrophage periablational infiltration was characterized with immunohistochemistry 24 hours, 72 hours, 7 days, and 14 days after ablation (n = 24). Twenty-one mice were subjected to a dose-escalation study with either 10, 15, 30, or 60 mg/kg of rhodamine-labeled superparamagnetic iron oxide nanoparticles (SPIONs) or 2.4, 1.2, or 0.6 mg/kg of gadolinium-160 (160Gd)-labeled CD68 antibody for assessment of the optimal in vivo dose of contrast agent. MR imaging experiments included 9 mice, each receiving 10-mg/kg SPIONs to visualize phagocytes using T2∗-weighted imaging in a horizontal-bore 9.4-T MR imaging scanner, 160Gd-CD68 for T1-weighted MR imaging of macrophages, or 0.1-mmol/kg intravenous gadoterate (control group). Radiological-pathological correlation included Prussian blue staining, rhodamine immunofluorescence, imaging mass cytometry, and immunohistochemistry. RESULTS: RF ablation-induced periablational infiltration (206.92 µm ± 12.2) of CD68+ macrophages peaked at 7 days after ablation (P < .01) compared with the untreated lobe. T2∗-weighted MR imaging with SPION contrast demonstrated curvilinear T2∗ signal in the transitional zone (TZ) (186 µm ± 16.9), corresponsing to Iron Prussian blue staining. T1-weighted MR imaging with 160Gd-CD68 antibody showed curvilinear signal in the TZ (164 µm ± 3.6) corresponding to imaging mass cytometry. CONCLUSIONS: Both SPION-enhanced T2∗-weighted and 160Gd-enhanced T1-weighted MR imaging allow for in vivo monitoring of macrophages after RF ablation, demonstrating the feasibility of this model to investigate local immune responses.

White matter abnormalities in the Hdc knockout mouse, a model of tic and OCD pathophysiology.

Introduction: An inactivating mutation in the histidine decarboxylase gene (Hdc) has been identified as a rare but high-penetrance genetic cause of Tourette syndrome (TS). TS is a neurodevelopmental syndrome characterized by recurrent motor and vocal tics; it is accompanied by structural and functional abnormalities in the cortico-basal ganglia circuitry. Hdc, which is expressed both in the posterior hypothalamus and peripherally, encodes an enzyme required for the biosynthesis of histamine. Hdc knockout mice (Hdc-KO) functionally recapitulate this mutation and exhibit behavioral and neurochemical abnormalities that parallel those seen in patients with TS. Materials and methods: We performed exploratory RNA-seq to identify pathological alterations in several brain regions in Hdc-KO mice. Findings were corroborated with RNA and protein quantification, immunohistochemistry, and ex vivo brain imaging using MRI. Results: Exploratory RNA-Seq analysis revealed, unexpectedly, that genes associated with oligodendrocytes and with myelin production are upregulated in the dorsal striatum of these mice. This was confirmed by qPCR, immunostaining, and immunoblotting. These results suggest an abnormality in myelination in the striatum. To test this in an intact mouse brain, we performed whole-brain ex vivo diffusion tensor imaging (DTI), which revealed reduced fractional anisotropy (FA) in the dorsal striatum. Discussion: While the DTI literature in individuals with TS is sparse, these results are consistent with findings of disrupted descending cortical projections in patients with tics. The Hdc-KO model may represent a powerful system in which to examine the developmental mechanisms underlying this abnormality.

Brain-targeting, acid-responsive antioxidant nanoparticles for stroke treatment and drug delivery.

Stroke is the leading cause of death and disability. Currently, there is no effective pharmacological treatment for this disease, which can be partially attributed to the inability to efficiently deliver therapeutics to the brain. Here we report the development of natural compound-derived nanoparticles (NPs), which function both as a potent therapeutic agent for stroke treatment and as an efficient carrier for drug delivery to the ischemic brain. First, we screened a collection of natural nanomaterials and identified betulinic acid (BA) as one of the most potent antioxidants for stroke treatment. Next, we engineered BA NPs for preferential drug release in acidic ischemic tissue through chemically converting BA to betulinic amine (BAM) and for targeted drug delivery through surface conjugation of AMD3100, a CXCR4 antagonist. The resulting AMD3100-conjugated BAM NPs, or A-BAM NPs, were then assessed as a therapeutic agent for stroke treatment and as a carrier for delivery of NA1, a neuroprotective peptide. We show that intravenous administration of A-BAM NPs effectively improved recovery from stroke and its efficacy was further enhanced when NA1 was encapsulated. Due to their multifunctionality and significant efficacy, we anticipate that A-BAM NPs have the potential to be translated both as a therapeutic agent and as a drug carrier to improve the treatment of stroke.

Comparison of Lanthanide Macrocyclic Complexes as 23Na NMR Sensors.

Nuclear magnetic resonance (NMR) agents, composed of paramagnetic lanthanide ions (Ln3+) complexed with negatively charged cyclic chelating agents (Che(n+3)-) forming polyanionic lanthanide complexes (LnChen-), perturb sodium-23 (23Na) signals, a phenomenon which depends sodium ions (Na+) exchanging with LnChen-. We analyzed 23Na shiftability and broadening due to hyperfine and bulk magnetic susceptibility (BMS) effects that arise from LnChen- designs using selective Ln3+ ions (i.e., thulium, Tm3+; gadolinium, Gd3+; and europium, Eu3+) and macrocyclics derived from 1,4,7,10-tetraazacyclododecane (cyclen) [i.e., with phosphonate (DOTP8-) and carboxylate (DOTMA4-) arms] and 1,4,7-triazacyclononane (TACN) [i.e., with phosphonate (NOTP6-) arms]. All LnChen- complexes showed downfield shifts, but Gd3+ and Tm3+ agents, respectively, were dominated by BMS and hyperfine effects, in good agreement with theory. While 23Na shiftability and broadening were minimally affected by pH and competing cations (K+, Ca2+, and Mg2+) within physiological ranges, the 23Na shiftability and broadening were most sensitive to LnChen- concentration in relation to the interstitial Na+ level in vivo. Greatest 23Na shiftability and broadening were obtained with Tm3+ and Gd3+ agents, respectively. While BMS contribution to shiftability was most impacted by the number of unpaired electrons on Ln3+, negative charge on LnChen- regulated Na+ exchange for line broadening. In brain tumor models, TmDOTP5- with 23Na-NMR has been used previously to separate Na+ in intracellular, blood, and interstitial pools, while evidence here shows that GdDOTP5- can distinguish Na+ within intracellular and extracellular (i.e., blood and interstitial) pools. Given the biological importance of Na+ in vivo, future macrocyclic designs of LnChen- should be sought for 23Na-NMR biomedical applications.

Comparison of metabolic and immunologic responses to transarterial chemoembolization with different chemoembolic regimens in a rabbit VX2 liver tumor model.

OBJECTIVES: The goal of this study was to investigate the effects of TACE using Lipiodol, Oncozene™ drug-eluting embolics (DEEs), or LUMI™-DEEs alone, or combined with bicarbonate on the metabolic and immunological tumor microenvironment in a rabbit VX2 tumor model. METHODS: VX2 liver tumor-bearing rabbits were assigned to five groups. MRI and extracellular pH (pHe) mapping using Biosensor Imaging of Redundant Deviation in Shifts (BIRDS) were performed before and after intra-arterial therapy with conventional TACE (cTACE), DEE-TACE with Idarubicin-eluting Oncozene™-DEEs, or Doxorubicin-eluting LUMI™-DEEs, each with or without prior bicarbonate infusion, and in untreated rabbits or treated with intra-arterial bicarbonate only. Imaging results were validated with immunohistochemistry (IHC) staining of cell viability (PCNA, TUNEL) and immune response (HLA-DR, CD3). Statistical analysis was performed using Mann-Whitney U test. RESULTS: pHe mapping revealed that combining cTACE with prior bicarbonate infusion significantly increased tumor pHe compared to control (p = 0.0175) and cTACE alone (p = 0.0025). IHC staining revealed peritumoral accumulation of HLA-DR+ antigen-presenting cells and CD3 + T-lymphocytes in controls. cTACE-treated tumors showed reduced immune infiltration, which was restored through combination with bicarbonate. DEE-TACE with Oncozene™-DEEs induced moderate intratumoral and marked peritumoral infiltration, which was slightly reduced with bicarbonate. Addition of bicarbonate prior to LUMI™-beads enhanced peritumoral immune cell infiltration compared to LUMI™-beads alone and resulted in the strongest intratumoral immune cell infiltration across all treated groups. CONCLUSIONS: The choice of chemoembolic regimen for TACE strongly affects post-treatment TME pHe and the ability of immune cells to accumulate and infiltrate the tumor tissue. KEY POINTS: • Combining conventional transarterial chemotherapy with prior bicarbonate infusion increases the pHe towards a more physiological value (p = 0.0025). • Peritumoral infiltration and intratumoral accumulation patterns of antigen-presenting cells and T-lymphocytes after transarterial chemotherapy were dependent on the choice of the chemoembolic regimen. • Combination of intra-arterial treatment with Doxorubicin-eluting LUMI™-beads and bicarbonate infusion resulted in the strongest intratumoral presence of immune cells (positivity index of 0.47 for HLADR+-cells and 0.62 for CD3+-cells).

High-resolution pH imaging using ratiometric chemical exchange saturation transfer combined with biosensor imaging of redundant deviation in shifts featuring paramagnetic DOTA-tetraglycinate agents.

Chemical exchange saturation transfer (CEST) and biosensor imaging of redundant deviation in shifts (BIRDS) methods differ respectively by detecting exchangeable and nonexchangeable proton signals by magnetic resonance. Because CEST contrast depends on both temperature and pH, simultaneous CEST and BIRDS imaging can be employed to separate these contributions. Here, we test if high-resolution pH imaging in vivo is possible with ratiometric CEST calibrated for temperature variations measured by BIRDS. Thulium- and europium-based DOTA-tetraglycinate agents, TmDOTA-(gly)4- and EuDOTA-(gly)4- , were used for high-resolution pH mapping in vitro and in vivo, using BIRDS for temperature adjustments needed for a more accurate ratiometric CEST approach. Although neither agent showed pH dependence with BIRDS in vitro in the pH range 6 to 8, each one's temperature sensitivity was enhanced when mixed because of increased redundancy. By contrast, the CEST signal of each agent was affected by the presence of the other agent in vitro. However, pH could be measured more accurately when temperature from BIRDS was detected. These in vitro calibrations with TmDOTA-(gly)4- and EuDOTA-(gly)4- enabled high-resolution pH imaging of glioblastoma in rat brains. It was concluded that temperature mapping with BIRDS can calibrate the ratiometric CEST signal from a cocktail of TmDOTA-(gly)4- and EuDOTA-(gly)4- agents to provide temperature-independent absolute pH imaging in vivo.

Methylated tetra-amide derivatives of paramagnetic complexes for magnetic resonance biosensing with both BIRDS and CEST.

Paramagnetic agents that utilize two mechanisms to provide physiological information by magnetic resonance imaging (MRI) and magnetic resonance spectroscopic imaging (MRSI) are described. MRI with chemical exchange saturation transfer (CEST) takes advantage of the agent's exchangeable protons (e.g., -OH or -NHx , where 2 ≥ x ≥ 1) to create pH contrast. The agent's incorporation of non-exchangeable protons (e.g., -CHy , where 3 ≥ y ≥ 1) makes it possible to map tissue temperature and/or pH using an MRSI method called biosensor imaging of redundant deviation in shifts (BIRDS). Hybrid probes based upon 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate chelate (DOTA4- ) and its methylated analog (1,4,7,10-tetraazacyclododecane-α, α', α″, α‴-tetramethyl-1,4,7,10-tetraacetate, DOTMA4- ) were synthesized, and modified to create new tetra-amide chelates. Addition of several methyl groups per pendent arm of the symmetrical chelates, positioned proximally and distally to thulium ions (Tm3+ ), gave rise to favorable BIRDS properties (i.e., high signal-to-noise ratio (SNR) from non-exchangeable methyl proton peaks) and CEST responsiveness (i.e., from amide exchangeable protons). Structures of the Tm3+ probes elucidate the influence of methyl group placement on sensor performance. An eight-coordinate geometry with high symmetry was observed for the complexes: Tm-L1 was based on DOTA4- , whereas Tm-L2 and Tm-L3 were based on DOTMA4- , where the latter contained an additional carboxylate at the distal end of each arm. The distance of Tm3+ from terminal methyl carbons is a key determinant for sustaining BIRDS temperature sensitivity without compromising CEST pH contrast; however, water solubility was influenced by introduction of hydrophobic methyl groups and hydrophilic carboxylate. Combined BIRDS and CEST detection of Tm-L2, which features two high-SNR methyl peaks and a strong amide CEST peak, should enable simultaneous temperature and pH measurements for high-resolution molecular imaging in vivo.

Renal plasticity revealed through reversal of polycystic kidney disease in mice.

Initiation of cyst formation in autosomal dominant polycystic kidney disease (ADPKD) occurs when kidney tubule cells are rendered null for either PKD1 or PKD2 by somatic 'second hit' mutations. Subsequent cyst progression remodels the organ through changes in tubule cell shape, proliferation and secretion. The kidney develops inflammation and fibrosis. We constructed a mouse model in which adult inactivation of either Pkd gene can be followed by reactivation of the gene at a later time. Using this model, we show that re-expression of Pkd genes in cystic kidneys results in rapid reversal of ADPKD. Cyst cell proliferation is reduced, autophagy is activated and cystic tubules with expanded lumina lined by squamoid cells revert to normal lumina lined by cuboidal cells. Increases in inflammation, extracellular matrix deposition and myofibroblast activation are reversed, and the kidneys become smaller. We conclude that phenotypic features of ADPKD are reversible and that the kidney has an unexpected capacity for plasticity controlled at least in part by ADPKD gene function.

Preimplantation factor modulates oligodendrocytes by H19-induced demethylation of NCOR2.

Failed or altered gliogenesis is a major characteristic of diffuse white matter injury in survivors of premature birth. The developmentally regulated long noncoding RNA (lncRNA) H19 inhibits S-adenosylhomocysteine hydrolase (SAHH) and contributes to methylation of diverse cellular components, such as DNA, RNA, proteins, lipids, and neurotransmitters. We showed that the pregnancy-derived synthetic PreImplantation Factor (sPIF) induces expression of the nuclear receptor corepressor 2 (NCOR2) via H19/SAHH-mediated DNA demethylation. In turn, NCOR2 affects oligodendrocyte differentiation markers. Accordingly, after hypoxic-ischemic brain injury in rodents, myelin protection and oligodendrocytes' fate are in part modulated by sPIF and H19. Our results revealed an unexpected mechanism of the H19/SAHH axis underlying myelin preservation during brain recovery and its use in treating neurodegenerative diseases can be envisioned.

Imaging Hallmarks of the Tumor Microenvironment in Glioblastoma Progression.

Glioblastoma progression involves multifaceted changes in vascularity, cellularity, and metabolism. Capturing such complexities of the tumor niche, from the tumor core to the periphery, by magnetic resonance imaging (MRI) and spectroscopic imaging (MRSI) methods has translational impact. In human-derived glioblastoma models (U87, U251) we made simultaneous and longitudinal measurements of tumor perfusion (Fp), permeability (Ktrans), and volume fractions of extracellular (ve) and blood (vp) spaces from dynamic contrast enhanced (DCE) MRI, cellularity from apparent diffusion coefficient (ADC) MRI, and extracellular pH (pHe) from an MRSI method called Biosensor Imaging of Redundant Deviation in Shifts (BIRDS). Spatiotemporal patterns of these parameters during tumorigenesis were unique for each tumor. While U87 tumors grew faster, Fp, Ktrans, and vp increased with tumor growth in both tumors but these trends were more pronounced for U251 tumors. Perfused regions between tumor periphery and core with U87 tumors exhibited higher Fp, but Ktrans of U251 tumors remained lowest at the tumor margin, suggesting primitive vascularization. Tumor growth was uncorrelated with ve, ADC, and pHe. U87 tumors showed correlated regions of reduced ve and lower ADC (higher cellularity), suggesting ongoing proliferation. U251 tumors revealed that the tumor core had higher ve and elevated ADC (lower cellularity), suggesting necrosis development. The entire tumor was uniformly acidic (pHe 6.1-6.8) early and throughout progression, but U251 tumors were more acidic, suggesting lower aerobic glycolysis in U87 tumors. Characterizing these cancer hallmarks with DCE-MRI, ADC-MRI, and BIRDS-MRSI will be useful for exploring tumorigenesis as well as timely therapies targeted to specific vascular and metabolic aspects of the tumor microenvironment.

Diffusion weighted imaging as a biomarker of retinoic acid induced myelomeningocele.

Neural tube defects are a common congenital anomaly involving incomplete closure of the spinal cord. Myelomeningocele (MMC) is a severe form in which there is complete exposure of neural tissue with a lack of skin, soft tissue, or bony covering to protect the spinal cord. The all-trans retinoic acid (ATRA) induced rat model of (MMC) is a reproducible, cost-effective means of studying this disease; however, there are limited modalities to objectively quantify disease severity, or potential benefits from experimental therapies. We sought to determine the feasibility of detecting differences between MMC and wild type (WT) rat fetuses using diffusion magnetic resonance imaging techniques (MRI). Rat dams were gavage-fed ATRA to produce MMC defects in fetuses, which were surgically delivered prior to term. Average diffusion coefficient (ADC) and fractional anisotropy (FA) maps were obtained for each fetus. Brain volumes and two anatomically defined brain length measurements (D1 and D2) were significantly decreased in MMC compared to WT. Mean ADC signal was significantly increased in MMC compared to WT, but no difference was found for FA signal. In summary, ADC and brain measurements were significantly different between WT and MMC rat fetuses. ADC could be a useful complementary imaging biomarker to current histopathologic analysis of MMC models, and potentially expedite therapeutic research for this disease.

Imaging Extracellular Acidification and Immune Activation in Cancer.

Metabolism reveals pathways by which cells, in healthy and disease tissues, use nutrients to fuel their function and (re)growth. However, gene-centric views have dominated cancer hallmarks, relegating metabolic reprogramming that all cells in the tumor niche undergo as an incidental phenomenon. Aerobic glycolysis in cancer is well known, but recent evidence suggests that diverse symbolic traits of cancer cells are derived from oncogene-directed metabolism required for their sustenance and evolution. Cells in the tumor milieu actively metabolize different nutrients, but proficiently secrete acidic by-products using diverse mechanisms to create a hostile ecosystem for host cells, and where local immune cells suffer collateral damage. Since metabolic interactions between cancer and immune cells hold promise for future cancer therapies, here we focus on translational magnetic resonance methods enabling in vivo and simultaneous detection of tumor habitat acidification and immune activation - innovations for monitoring personalized treatments.

Imaging the transmembrane and transendothelial sodium gradients in gliomas.

Under normal conditions, high sodium (Na+) in extracellular (Na+e) and blood (Na+b) compartments and low Na+ in intracellular milieu (Na+i) produce strong transmembrane (ΔNa+mem) and weak transendothelial (ΔNa+end) gradients respectively, and these manifest the cell membrane potential (Vm) as well as blood-brain barrier (BBB) integrity. We developed a sodium (23Na) magnetic resonance spectroscopic imaging (MRSI) method using an intravenously-administered paramagnetic polyanionic agent to measure ΔNa+mem and ΔNa+end. In vitro 23Na-MRSI established that the 23Na signal is intensely shifted by the agent compared to other biological factors (e.g., pH and temperature). In vivo 23Na-MRSI showed Na+i remained unshifted and Na+b was more shifted than Na+e, and these together revealed weakened ΔNa+mem and enhanced ΔNa+end in rat gliomas (vs. normal tissue). Compared to normal tissue, RG2 and U87 tumors maintained weakened ΔNa+mem (i.e., depolarized Vm) implying an aggressive state for proliferation, whereas RG2 tumors displayed elevated ∆Na+end suggesting altered BBB integrity. We anticipate that 23Na-MRSI will allow biomedical explorations of perturbed Na+ homeostasis in vivo.

A high-throughput imaging platform to characterize extracellular pH in organotypic three-dimensional in vitro models of liver cancer.

Given the extraordinary nature of tumor metabolism in hepatocellular carcinoma and its impact on oncologic treatment response, this study introduces a novel high-throughput extracellular pH (pHe ) mapping platform using magnetic resonance spectroscopic imaging in a three-dimensional (3D) in vitro model of liver cancer. pHe mapping was performed using biosensor imaging of redundant deviation in shifts (BIRDS) on 9.4 T and 11.7 T MR scanners for validation purposes. 3D cultures of four liver cancer (HepG2, Huh7, SNU475, VX2) and one hepatocyte (THLE2) cell line were simultaneously analyzed (a) without treatment, (b) supplemented with 4.5 g/L d-glucose, and (c) treated with anti-glycolytic 3-bromopyruvate (6.25, 25, 50, 75, and 100 µM). The MR results were correlated with immunohistochemistry (GLUT-1, LAMP-2) and luminescence-based viability assays. Statistics included the unpaired t-test and ANOVA test. High-throughput pHe imaging with BIRDS for in vitro 3D liver cancer models proved feasible. Compared with non-tumorous hepatocytes (pHe = 7.1 ± 0.1), acidic pHe was revealed in liver cancer (VX2, pHe = 6.7 ± 0.1; HuH7, pHe = 6.8 ± 0.1; HepG2, pHe = 6.9 ± 0.1; SNU475, pHe = 6.9 ± 0.1), in agreement with GLUT-1 upregulation. Glucose addition significantly further decreased pHe in hyperglycolytic cell lines (VX2, HepG2, and Huh7, by 0.28, 0.06, and 0.11, respectively, all p < 0.001), whereas 3-bromopyruvate normalized tumor pHe in a dose-dependent manner without affecting viability. In summary, this study introduces a non-invasive pHe imaging platform for high-yield screening using a translational 3D liver cancer model, which may help reveal and target mechanisms of therapy resistance and inform personalized treatment of patients with hepatocellular carcinoma.