Perinatal exposure to the immune-suppressant di-n-octyltin dichloride affects brain development in rats.

Disruption of the immune system during embryonic brain development by environmental chemicals was proposed as a possible cause of neurodevelopmental disorders. We previously found adverse effects of di-n-octyltin dichloride (DOTC) on maternal and developing immune systems of rats in an extended one-generation reproductive toxicity study according to the OECD 443 test guideline. We hypothesize that the DOTC-induced changes in the immune system can affect neurodevelopment. Therefore, we used in-vivo MRI and PET imaging and genomics, in addition to behavioral testing and neuropathology as proposed in OECD test guideline 443, to investigate the effect of DOTC on structural and functional brain development. Male rats were exposed to DOTC (0, 3, 10, or 30 mg/kg of diet) from 2 weeks prior to mating of the F0-generation until sacrifice of F1-animals. The brains of rats, exposed to DOTC showed a transiently enlarged volume of specific brain regions (MRI), altered specific gravity, and transient hyper-metabolism ([18F]FDG PET). The alterations in brain development concurred with hyper-responsiveness in auditory startle response and slight hyperactivity in young adult animals. Genomics identified altered transcription of key regulators involved in neurodevelopment and neural function (e.g. Nrgrn, Shank3, Igf1r, Cck, Apba2, Foxp2); and regulators involved in cell size, cell proliferation, and organ development, especially immune system development and functioning (e.g. LOC679869, Itga11, Arhgap5, Cd47, Dlg1, Gas6, Cml5, Mef2c). The results suggest the involvement of immunotoxicity in the impairment of the nervous system by DOTC and support the hypothesis of a close connection between the immune and nervous systems in brain development.

Imatinib attenuates reperfusion injury in a rat model of acute myocardial infarction.

Following an acute myocardial infarction, reperfusion of an occluded coronary artery is often accompanied by microvascular injury, leading to worse long-term prognosis. Experimental studies have revealed the potential of tyrosine-kinase inhibitor imatinib to reduce vascular leakage in various organs. Here, we examined the potential of imatinib to attenuate microvascular injury in a rat model of myocardial reperfusion injury. Isolated male Wistar rat hearts (n = 20) in a Langendorff system and male Wistar rats (n = 37) in an in vivo model were randomly assigned to imatinib or placebo and subjected to ischaemia and reperfusion. Evans-blue/Thioflavin-S/TTC staining and Cardiac Magnetic Resonance Imaging were performed to assess the extent of reperfusion injury. Subsequently, in vivo hearts were perfused ex vivo with a vascular leakage tracer and fluorescence and electron microscopy were performed. In isolated rat hearts, imatinib reduced global infarct size, improved end-diastolic pressure, and improved rate pressure product recovery compared to placebo. In vivo, imatinib reduced no-reflow and infarct size with no difference between imatinib and placebo for global cardiac function. In addition, imatinib showed lower vascular resistance, higher coronary flow, and less microvascular leakage in the affected myocardium. At the ultrastructural level, imatinib showed higher preserved microvascular integrity compared to placebo. We provide evidence that low-dose imatinib can reduce microvascular injury and accompanying myocardial infarct size in a rat model of acute myocardial infarction. These data warrant future work to examine the potential of imatinib to reduce reperfusion injury in patients with acute myocardial infarction.

Partial Adrenalectomy Carries a Considerable Risk of Incomplete Cure in Primary Aldosteronism.

PURPOSE: Laparoscopic adrenalectomy is standard treatment for patients with unilateral aldosterone-producing adenomas, but surgeons are increasingly tempted to perform partial adrenalectomy, disregarding potential multinodularity of the adrenal. We assess the diagnostic value of endoscopic ultrasound for differentiating solitary adenomas from multinodularity by examining in-depth adrenal pathology with ex vivo 11.7 T magnetic resonance imaging and immunohistochemistry. MATERIALS AND METHODS: In 15 primary aldosteronism patients, we performed intraoperative endoscopic ultrasound, ex vivo magnetic resonance imaging and histopathological examination. Every adrenal was intraoperatively and postoperatively assessed for solitary adenomas or multinodular hyperplasia. After unblinding for ex vivo magnetic resonance imaging results a second detailed histopathological examination, including immunohistochemistry analysis with CYP11B2 (aldosterone synthase) and chemokine receptor 4 (CXCR4), a new marker for aldosterone-producing adenomas, was performed. Finally, presence of somatic mutations linked to aldosterone-producing adenomas was assessed. RESULTS: The sensitivity and specificity of endoscopic ultrasound to identify multinodularity were 46% and 50%, respectively. We found multinodular hyperplasia in 87% of adrenals with ex vivo magnetic resonance imaging combined with detailed histopathology, and 6 adrenals contained multiple CYP11B2-producing nodules. Every CYP11B2 positive nodule and 61% of CYP11B2 negative nodules showed CXCR4 staining. Finally, in 4 adrenals (27%) we found somatic mutations. In multinodular glands, only 1 nodule harbored this mutation. CONCLUSIONS: Intraoperative endoscopic ultrasound in primary aldosteronism patients has low accuracy to identify multinodularity. Ex vivo magnetic resonance imaging can serve as a tool to direct detailed histopathological examination, which frequently shows CYP11B2 production in multiple nodules. Therefore, partial adrenalectomy is inappropriate in primary aldosteronism as multiple aldosterone-producing nodules easily stay behind.

Pyruvate-lactate exchange and glucose uptake in human prostate cancer cell models. A study in xenografts and suspensions by hyperpolarized [1-13 C]pyruvate MRS and [18 F]FDG-PET.

Reprogramming of energy metabolism in the development of prostate cancer can be exploited for a better diagnosis and treatment of the disease. The goal of this study was to determine whether differences in glucose and pyruvate metabolism of human prostate cancer cells with dissimilar aggressivenesses can be detected using hyperpolarized [1-13 C]pyruvate MRS and [18 F]FDG-PET imaging, and to evaluate whether these measures correlate. For this purpose, we compared murine xenografts of human prostate cancer LNCaP cells with those of more aggressive PC3 cells. [1-13 C]pyruvate was hyperpolarized by dissolution dynamic nuclear polarization (dDNP) and [1-13 C]pyruvate to lactate conversion was followed by 13 C MRS. Subsequently [18 F]FDG uptake was investigated by static and dynamic PET measurements. Standard uptake values (SUVs) for [18 F]FDG were significantly higher for xenografts of PC3 compared with those of LNCaP. However, we did not observe a difference in the average apparent rate constant kpl of 13 C label exchange from pyruvate to lactate between the tumor variants. A significant negative correlation was found between SUVs from [18 F]FDG PET measurements and kpl values for the xenografts of both tumor types. The kpl rate constant may be influenced by various factors, and studies with a range of prostate cancer cells in suspension suggest that LDH inhibition by pyruvate may be one of these. Our results indicate that glucose and pyruvate metabolism in the prostate cancer cell models differs from that in other tumor models and that [18 F]FDG-PET can serve as a valuable complementary tool in dDNP studies of aggressive prostate cancer with [1-13 C]pyruvate.

MRI evaluation of vulvar squamous-cell carcinoma in fresh radical local excision specimens for cancer localization and prediction of surgical tumor-free margins.

In the surgical treatment of vulvar squamous-cell carcinoma (VSCC), tumor-free margins of 8 mm or more are considered adequate. However, limited perioperative information on the tumor-free margins other than the surgeon's own estimation is available. The purpose of this study was therefore to investigate the feasibility of ex vivo MRI in localizing VSCC and to assess the surgical tumor-free margins in fresh radical local excision (RLE) specimens to guide the surgeon during resections. Nine patients with biopsy-proven VSCC scheduled for RLE were prospectively included. Intact fresh specimens were scanned using a 7 T preclinical MR-scanner. Whole mount H&E-stained slides were obtained every 3 mm and correlated with ex vivo MRI. A pathologist annotated VSCC and minimal tumor-free margins (3 o'clock, 9 o'clock, basal) on the digitalized histological slides. An observer with knowledge of histology (the non-blinded annotation) and a radiologist blinded to histology (the blinded annotation) separately performed annotation of the same features on ex vivo MRI. Linear correlation and agreement of the ex vivo MRI measurements with histology were assessed. Diagnostic performance for VSCC localization and identification of margins less than 8 mm was expressed as positive and negative predictive values (PPV, NPV). In 153 matched ex vivo MRI slices, the observer correctly identified 79/91 margins as less than 8 mm (PPV 87%) and 110/124 margins as 8 mm or greater (NPV 89%). The radiologist correctly annotated absence of VSCC in 73/81 (NPV 90%) and presence in 65/72 (PPV 90%) slices. Sixty-four of 90 margins were correctly identified as less than 8 mm (PPV 71%) and 83/102 margins as 8 mm or greater (NPV 81%). Both non-blinded and blinded annotations were linearly correlated and demonstrated good agreement with histology. Accurate localization of VSCC and measurements of the surgical tumor-free margins in fresh RLE specimens using ex vivo MRI seems feasible. High diagnostic performance in VSCC localization and identification of margins less than 8 mm suggest ex vivo MRI to be clinically applicable.

MRI as a tool to assess surgical margins and pseudocapsule features directly following partial nephrectomy for small renal masses.

PURPOSE: To evaluate the feasibility of ex vivo 7T MRI to assess surgical margins (SMs) and pseudocapsule (PC) features after partial nephrectomy (PN). MATERIALS AND METHODS: In this prospective, IRB-approved study, seven patients undergoing a PN for nine tumours between November 2014 and July 2015 were included for analysis after obtaining informed consent. MRI of the specimen was acquired using a 7T small bore scanner. The imaging protocol consisted of anatomical T1-, T2- and diffusion-weighted imaging. After formalin fixation, specimens were cut for pathology work-up in the same orientation as the MR images were obtained. The entire specimen was processed into H&E slides that were digitally scanned, annotated and correlated with radiological findings for negative SMs, PC presence, PC continuity and extra-PC-extension (EPCE). Sensitivity and specificity of MRI for assessment of these endpoints were calculated. RESULTS: The sensitivity and specificity for assessment of the SM were 100% and 75%, respectively. Two false-positive outcomes were reported, both in case of EPCE and a SM ≤0.5 mm. For the presence of a PC, sensitivity and specificity were 100% and 33%, respectively. Two false-positive scans with anatomical structures mimicking the presence of a PC occurred. If a PC was present, continuity and EPCE were assessed with a sensitivity and specificity of 75% and 100% and 67% and 100%, respectively. CONCLUSION: Ex vivo 7T MRI is a feasible tool for perioperative evaluation of SMs, and if present, PC features after PN. This may facilitate maximal sparing of renal parenchyma without compromising oncological outcomes. KEY POINTS: • Ex vivo MRI may contribute to improvement of negative surgical margins during partial nephrectomy. • Due to the assessment of surgical margins within a limited time span from obtaining the partial nephrectomy specimen, surgery for more complex tumours is possible with maximum sparing of healthy renal parenchyma without compromising oncological outcomes. • The intra operative assessment of pseudocapsule continuity along the resection margin enables maximal sparing of healthy renal parenchyma without delayed diagnosis of incomplete resection.

Visualization of calcium phosphate cement in teeth by zero echo time 1 H MRI at high field.

1 H magnetic resonance imaging (MRI) by a zero echo time (ZTE) sequence is an excellent method to image teeth. Calcium phosphate cement (CPC) materials are applied in the restoration of tooth lesions, but it has not yet been investigated whether they can be detected by computed tomography (CT) or MRI. The aim of this study was to optimize high-field ZTE imaging to enable the visualization of a new CPC formulation implanted in teeth and to apply this in the assessment of its decomposition in vivo. CPC was implanted in three human and three goat teeth ex vivo and in three goat teeth in vivo. An ultrashort echo time (UTE) sequence with multiple flip angles and echo times was applied at 11.7 T to measure T1 and T2 * values of CPC, enamel and dentin. Teeth with CPC were imaged with an optimized ZTE sequence. Goat teeth implanted with CPC in vivo were imaged after 7 weeks ex vivo. T2 * relaxation of implanted CPC, dentin and enamel was better fitted by a model assuming a Gaussian rather than a Lorentzian distribution. For CPC and human enamel and dentin, the average T2 * values were 273 ± 19, 562 ± 221 and 476 ± 147 µs, respectively, the average T2 values were 1234 ± 27, 963 ± 151 and 577 ± 41 µs, respectively, and the average T1 values were 1065 ± 45, 972 ± 40 and 903 ± 7 ms, respectively. In ZTE images, CPC had a higher signal-to-noise-ratio than dentin and enamel because of the higher water content. Seven weeks after in vivo implantation, the CPC-filled lesions showed less homogeneous structures, a lower T1 value and T2 * separated into two components. MRI by ZTE provides excellent contrast for CPC in teeth and allows its decomposition to be followed.

Ex vivo MRI evaluation of prostate cancer: Localization and margin status prediction of prostate cancer in fresh radical prostatectomy specimens.

PURPOSE: To investigate the ability of high field ex vivo magnetic resonance imaging (MRI) to localize prostate cancer (PCa) and to predict the margin status in fresh radical prostatectomy (RP) specimens using histology as the reference standard. MATERIALS AND METHODS: This Institutional Review Board (IRB)-approved study had written informed consent. Patients with biopsy-proved PCa and a diagnostic multiparametric 3T MRI examination of the prostate prior to undergoing RP were prospectively included. A custom-made container provided reference between the 7T ex vivo MRI obtained from fresh RP specimens and histological slicing. On ex vivo MRI, PCa was localized and the presence of positive surgical margins was determined in a double-reading session. These findings were compared with histological findings obtained from completely cut, whole-mount embedded, prostate specimens. RESULTS: In 12 RP specimens, histopathology revealed 36 PCa lesions, of which 17 (47%) and 20 (56%) were correlated with the ex vivo MRI in the first and second reading session, respectively. Nine of 12 (75%) index lesions were localized in the first session, in the second 10 of 12 (83%). Seven and 8 lesions of 11 lesions with Gleason score >6 and >0.5 cc were localized in the first and second session, respectively. In the first session none of the four histologically positive surgical margins (sensitivity 0%) and 9 of 13 negative margins (specificity 69%) were detected. In second session the sensitivity and specificity were 25% and 88%, respectively. CONCLUSION: Ex vivo MRI enabled accurate localization of PCa in fresh RP specimens, and the technique provided information on the margin status with high specificity. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018;47:439-448.

In vivo MR guided boiling histotripsy in a mouse tumor model evaluated by MRI and histopathology.

Boiling histotripsy (BH) is a new high intensity focused ultrasound (HIFU) ablation technique to mechanically fragmentize soft tissue into submicrometer fragments. So far, ultrasound has been used for BH treatment guidance and evaluation. The in vivo histopathological effects of this treatment are largely unknown. Here, we report on an MR guided BH method to treat subcutaneous tumors in a mouse model. The treatment effects of BH were evaluated one hour and four days later with MRI and histopathology, and compared with the effects of thermal HIFU (T-HIFU). The lesions caused by BH were easily detected with T2 w imaging as a hyper-intense signal area with a hypo-intense rim. Histopathological evaluation showed that the targeted tissue was completely disintegrated and that a narrow transition zone (<200 µm) containing many apoptotic cells was present between disintegrated and vital tumor tissue. A high level of agreement was found between T2 w imaging and H&E stained sections, making T2 w imaging a suitable method for treatment evaluation during or directly after BH. After T-HIFU, contrast enhanced imaging was required for adequate detection of the ablation zone. On histopathology, an ablation zone with concentric layers was seen after T-HIFU. In line with histopathology, contrast enhanced MRI revealed that after BH or T-HIFU perfusion within the lesion was absent, while after BH in the transition zone some micro-hemorrhaging appeared. Four days after BH, the transition zone with apoptotic cells was histologically no longer detectable, corresponding to the absence of a hypo-intense rim around the lesion in T2 w images. This study demonstrates the first results of in vivo BH on mouse tumor using MRI for treatment guidance and evaluation and opens the way for more detailed investigation of the in vivo effects of BH. Copyright © 2016 John Wiley & Sons, Ltd.

In vivo ³¹P magnetic resonance spectroscopic imaging (MRSI) for metabolic profiling of human breast cancer xenografts.

PURPOSE: To study cancer associated with abnormal metabolism of phospholipids, of which several have been proposed as biomarkers for malignancy or to monitor response to anticancer therapy. We explored 3D (31) P magnetic resonance spectroscopic imaging (MRSI) at high magnetic field for in vivo assessment of individual phospholipids in two patient-derived breast cancer xenografts representing good and poor prognosis (luminal- and basal-like tumors). MATERIALS AND METHODS: Metabolic profiles from luminal-like and basal-like xenograft tumors were obtained in vivo using 3D (31) P MRSI at 11.7T and from tissue extracts in vitro at 14.1T. Gene expression analysis was performed in order to support metabolic differences between the two xenografts. RESULTS: In vivo (31) P MR spectra were obtained in which the prominent resonances from phospholipid metabolites were detected at a high signal-to-noise ratio (SNR >7.5). Metabolic profiles obtained in vivo were in agreement with those obtained in vitro and could be used to discriminate between the two xenograft models, based on the levels of phosphocholine, phosphoethanolamine, glycerophosphocholine, and glycerophosphoethanolamine. The differences in phospholipid metabolite concentration could partly be explained by gene expression profiles. CONCLUSION: Noninvasive metabolic profiling by 3D (31) P MRSI can discriminate between subtypes of breast cancer based on different concentrations of choline- and ethanolamine-containing phospholipids.

Dietary lipids do not contribute to the higher hepatic triglyceride levels of fructose- compared to glucose-fed mice.

Fructose consumption has been associated with the surge in obesity and dyslipidemia. This may be mediated by the fructose effects on hepatic lipids and ATP levels. Fructose metabolism provides carbons for de novo lipogenesis (DNL) and stimulates enterocyte secretion of apoB48. Thus, fructose-induced hepatic triglyceride (HTG) accumulation can be attributed to both DNL stimulation and dietary lipid absorption. The aim of this study was to assess the effects of fructose diet on HTG and ATP content and the contributions of dietary lipids and DNL to HTG. Measurements were performed in vivo in mice by magnetic resonance imaging (MRI) and novel magnetic resonance spectroscopy (MRS) approaches. Abdominal adipose tissue volume and intramyocellular lipid levels were comparable between 8-wk fructose- and glucose-fed mice. HTG levels were ∼1.5-fold higher in fructose-fed than in glucose-fed mice (P<0.05). Metabolic flux analysis by (13)C and (2)H MRS showed that this was not due to dietary lipid absorption, but due to DNL stimulation. The contribution of oral lipids to HTG was, after 5 h, 1.60 ± 0.23% for fructose and 2.16 ± 0.35% for glucose diets (P=0.26), whereas that of DNL was higher in fructose than in glucose diets (2.55±0.51 vs.1.13±0.24%, P=0.01). Hepatic energy status, assessed by (31)P MRS, was similar for fructose- and glucose-fed mice. Fructose-induced HTG accumulation is better explained by DNL and not by dietary lipid uptake, while not compromising ATP homeostasis.

Cell tracking using (19)F magnetic resonance imaging: technical aspects and challenges towards clinical applications.

(19)F MRI is emerging as a new imaging technique for cell tracking. It is particularly attractive because of its potential for direct and precise cell quantification. The most important challenge towards in vivo applications is the sensitivity of the technique, i.e. the detection limit in a reasonable imaging time. Optimal sensitivity can be achieved with dedicated (19)F compounds together with specifically adapted hardware and acquisition methods. In this paper we introduce the (19)F MRI technique focusing on these key sensitivity issues and review the state-of-the-art of (19)F MRI and developments towards its clinical use. We calculate (19)F detection limits reported in preclinical cell and clinical (19)F drug studies in terms of tissue concentration in a 1 cm(3) voxel, as an alternate way to compare detection limits. We estimate that a tissue concentration of a few millimoles per litre (mM) of (19)F is required for a human study at a resolution of 1 cm(3).

The Potential of Iron Oxide Nanoparticle-Enhanced MRI at 7 T Compared With 3 T for Detecting Small Suspicious Lymph Nodes in Patients With Prostate Cancer.

BACKGROUND: Accurate detection of lymph node (LN) metastases in prostate cancer (PCa) is a challenging but crucial step for disease staging. Ultrasmall superparamagnetic iron oxide (USPIO)-enhanced magnetic resonance imaging (MRI) enables distinction between healthy LNs and nodes suspicious for harboring metastases. When combined with MRI at an ultra-high magnetic field, an unprecedented spatial resolution can be exploited to visualize these LNs. PURPOSE: The aim of this study was to explore USPIO-enhanced MRI at 7 T in comparison to 3 T for the detection of small suspicious LNs in the same cohort of patients with PCa. MATERIALS AND METHODS: Twenty PCa patients with high-risk primary or recurrent disease were referred to our hospital for an investigational USPIO-enhanced 3 T MRI examination with ferumoxtran-10. With consent, they underwent a 7 T MRI on the same day. Three-dimensional anatomical and T2*-weighted images of both examinations were evaluated blinded, with an interval, by 2 readers who annotated LNs suspicious for metastases. Number, size, and level of suspicion (LoS) of LNs were paired within patients and compared between field strengths. RESULTS: At 7 T, both readers annotated significantly more LNs compared with 3 T (474 and 284 vs 344 and 162), with 116 suspicious LNs on 7 T (range, 1-34 per patient) and 79 suspicious LNs on 3 T (range, 1-14 per patient) in 17 patients. For suspicious LNs, the median short axis diameter was 2.6 mm on 7 T (1.3-9.5 mm) and 2.8 mm for 3 T (1.7-10.4 mm, P = 0.05), with large overlap in short axis of annotated LNs between LoS groups. At 7 T, significantly more suspicious LNs had a short axis <2.5 mm compared with 3 T (44% vs 27%). Magnetic resonance imaging at 7 T provided better image quality and structure delineation and a higher LoS score for suspicious nodes. CONCLUSIONS: In the same cohort of patients with PCa, more and more small LNs were detected on 7 T USPIO-enhanced MRI compared with 3 T MRI. Suspicious LNs are generally very small, and increased nodal size was not a good indication of suspicion for the presence of metastases. The high spatial resolution of USPIO-enhanced MRI at 7 T improves structure delineation and the visibility of very small suspicious LNs, potentially expanding the in vivo detection limits of pelvic LN metastases in PCa patients.

Glucose versus fructose metabolism in the liver measured with deuterium metabolic imaging.

Chronic intake of high amounts of fructose has been linked to the development of metabolic disorders, which has been attributed to the almost complete clearance of fructose by the liver. However, direct measurement of hepatic fructose uptake is complicated by the fact that the portal vein is difficult to access. Here we present a new, non-invasive method to measure hepatic fructose uptake and metabolism with the use of deuterium metabolic imaging (DMI) upon administration of [6,6'-2H2]fructose. Using both [6,6'-2H2]glucose and [6,6'-2H2]fructose, we determined differences in the uptake and metabolism of glucose and fructose in the mouse liver with dynamic DMI. The deuterated compounds were administered either by fast intravenous (IV) bolus injection or by slow IV infusion. Directly after IV bolus injection of [6,6'-2H2]fructose, a more than two-fold higher initial uptake and subsequent 2.5-fold faster decay of fructose was observed in the mouse liver as compared to that of glucose after bolus injection of [6,6'-2H2]glucose. In contrast, after slow IV infusion of fructose, the 2H fructose/glucose signal maximum in liver spectra was lower compared to the 2H glucose signal maximum after slow infusion of glucose. With both bolus injection and slow infusion protocols, deuterium labeling of water was faster with fructose than with glucose. These observations are in line with a higher extraction and faster turnover of fructose in the liver, as compared with glucose. DMI with [6,6'-2H2]glucose and [6,6'-2H2]fructose could potentially contribute to a better understanding of healthy human liver metabolism and aberrations in metabolic diseases.

Subnodal Correspondence of PSMA Expression and USPIO-MRI in Metastatic Pelvic Lymph Nodes in Prostate Cancer.

OBJECTIVES: Two advanced imaging modalities used to detect lymph node (LN) metastases in prostate cancer patients are prostate-specific membrane antigen (PSMA) positron emission tomography/computed tomography and ultrasmall superparamagnetic iron oxide (USPIO)-enhanced magnetic resonance imaging (MRI). As these modalities use different targets, a subnodal comparison is needed to interpret both their correspondence and their differences. The aim of this explorative study was to compare ex vivo 111In-PSMA µSPECT images with high-resolution 7 T USPIO µMR images and histopathology of resected LN specimens from prostate cancer patients to assess the degree of correspondence at subnodal level. MATERIALS AND METHODS: Twenty primary prostate cancer patients who underwent pelvic LN dissection were included and received USPIO contrast and 111In-PSMA. A total of 41 LNs of interest (LNOIs) were selected for ex vivo imaging based on γ-probe detection or palpation. µSPECT and µMRI acquisition were performed immediately after resection. Overlay of µSPECT images on MR images was performed, and the level of correspondence (LoC) between µSPECT and µMR findings was assessed according to a 4-point Likert classification scheme. RESULTS: Forty-one LNOIs could be matched to an LN on ex vivo µMRI. Coregistration of µSPECT and USPIO-enhanced water-selective multigradient echo MR images was successful for all 41 LNOIs. Ninety percent of the lesions showed excellent correspondence regarding the presence of metastatic tissue and affected subnodal site (LoC 4; 37/41). In only 1 of 41 LNOIs, a small metastasis was misclassified by both techniques. Three LNOIs were classified as LoC 3 (7%) and 1 LNOI as LoC 2. All LoC 2 and LoC 3 lesions had PSMA-expressing metastases on final histopathology. CONCLUSIONS: Coregistration of µSPECT and USPIO-µMRI showed excellent subnodal correspondence in the majority (90%) of LNs. Ex vivo imaging may thus help localize small cancer deposits within resected LNs and could contribute to improved interpretation of in vivo imaging of LNs.

19F MRI Imaging Strategies to Reduce Isoflurane Artifacts in In Vivo Images.

PURPOSE: Isoflurane (ISO) is the most commonly used preclinical inhalation anesthetic. This is a problem in 19F MRI of fluorine contrast agents, as ISO signals cause artifacts that interfere with unambiguous image interpretation and quantification; the two most attractive properties of heteronuclear MRI. We aimed to avoid these artifacts using MRI strategies that can be applied by any pre-clinical researcher. PROCEDURES: Three strategies to avoid ISO chemical shift displacement artifacts (CSDA) in 19F MRI are described and demonstrated with measurements of 19F-containing agents in phantoms and in vivo (n = 3 for all strategies). The success of these strategies is compared to a standard Rapid Acquisition with Relaxation Enhancement (RARE) sequence, with phantom and in vivo validation. ISO artifacts can successfully be avoided by (1) shifting them outside the region of interest using a narrow signal acquisition bandwidth, (2) suppression of ISO by planning a frequency-selective suppression pulse before signal acquisition or by (3) preventing ISO excitation with a 3D sequence with a narrow excitation bandwidth. RESULTS: All three strategies result in complete ISO signal avoidance (p < 0.0001 for all methods). Using a narrow acquisition bandwidth can result in loss of signal to noise ratio and distortion of the image, and a frequency-selective suppression pulse can be incomplete when B1-inhomogeneities are present. Preventing ISO excitation with a narrow excitation pulse in a 3D sequence yields the most robust results (relative SNR 151 ± 28% compared to 2D multislice methods, p = 0.006). CONCLUSION: We optimized three easily implementable methods to avoid ISO signal artifacts and validated their performance in phantoms and in vivo. We make recommendation on the parameters that pre-clinical studies should report in their method section to make the used approach insightful.

Mechanical high-intensity focused ultrasound creates unique tumor debris enhancing dendritic cell-induced T cell activation.

Introduction: In situ tumor ablation releases a unique repertoire of antigens from a heterogeneous population of tumor cells. High-intensity focused ultrasound (HIFU) is a completely noninvasive ablation therapy that can be used to ablate tumors either by heating (thermal (T)-HIFU) or by mechanical disruption (mechanical (M)-HIFU). How different HIFU ablation techniques compare with respect to their antigen release profile, their activation of responder T cells, and their ability to synergize with immune stimuli remains to be elucidated. Methods and results: Here, we compare the immunomodulatory effects of T-HIFU and M-HIFU ablation with or without the TLR9 agonist CpG in the ovalbumin-expressing lymphoma model EG7. M-HIFU ablation alone, but much less so T-HIFU, significantly increased dendritic cell (DC) activation in draining lymph nodes (LNs). Administration of CpG following T- or M-HIFU ablation increased DC activation in draining LNs to a similar extend. Interestingly, ex vivo co-cultures of draining LN suspensions from HIFU plus CpG treated mice with CD8+ OT-I T cells demonstrate that LN cells from M-HIFU treated mice most potently induced OT-I proliferation. To delineate the mechanism for the enhanced anti-tumor immune response induced by M-HIFU, we characterized the RNA, DNA and protein content of tumor debris generated by both HIFU methods. M-HIFU induced a uniquely altered RNA, DNA and protein profile, all showing clear signs of fragmentation, whereas T-HIFU did not. Moreover, western blot analysis showed decreased levels of the immunosuppressive cytokines IL-10 and TGF-ß in M-HIFU generated tumor debris compared to untreated tumor tissue or T-HIFU. Conclusion: Collectively, these results imply that M-HIFU induces a unique context of the ablated tumor material, enhancing DC-mediated T cell responses when combined with CpG.

Validation of In Vivo Nodal Assessment of Solid Malignancies with USPIO-Enhanced MRI: A Workflow Protocol.

BACKGROUND: In various cancer types, the first step towards extended metastatic disease is the presence of lymph node metastases. Imaging methods with sufficient diagnostic accuracy are required to personalize treatment. Lymph node metastases can be detected with ultrasmall superparamagnetic iron oxide (USPIO)-enhanced magnetic resonance imaging (MRI), but this method needs validation. Here, a workflow is presented, which is designed to compare MRI-visible lymph nodes on a node-to-node basis with histopathology. METHODS: In patients with prostate, rectal, periampullary, esophageal, and head-and-neck cancer, in vivo USPIO-enhanced MRI was performed to detect lymph nodes suspicious of harboring metastases. After lymphadenectomy, but before histopathological assessment, a 7 Tesla preclinical ex vivo MRI of the surgical specimen was performed, and in vivo MR images were radiologically matched to ex vivo MR images. Lymph nodes were annotated on the ex vivo MRI for an MR-guided pathological examination of the specimens. RESULTS: Matching lymph nodes of ex vivo MRI to pathology was feasible in all cancer types. The annotated ex vivo MR images enabled a comparison between USPIO-enhanced in vivo MRI and histopathology, which allowed for analyses on a nodal, or at least on a nodal station, basis. CONCLUSIONS: A workflow was developed to validate in vivo USPIO-enhanced MRI with histopathology. Guiding the pathologist towards lymph nodes in the resection specimens during histopathological work-up allowed for the analysis at a nodal basis, or at least nodal station basis, of in vivo suspicious lymph nodes with corresponding histopathology, providing direct information for validation of in vivo USPIO-enhanced, MRI-detected lymph nodes.

Simultaneous Recording of the Uptake and Conversion of Glucose and Choline in Tumors by Deuterium Metabolic Imaging.

Increased glucose and choline uptake are hallmarks of cancer. We investigated whether the uptake and conversion of [2H9]choline alone and together with that of [6,6'-2H2]glucose can be assessed in tumors via deuterium metabolic imaging (DMI) after administering these compounds. Therefore, tumors with human renal carcinoma cells were grown subcutaneously in mice. Isoflurane anesthetized mice were IV infused in the MR magnet for ~20 s with ~0.2 mL solutions containing either [2H9]choline (0.05 g/kg) alone or together with [6,6'-2H2]glucose (1.3 g/kg). 2H MR was performed on a 11.7T MR system with a home-built 2H/1H coil using a 90° excitation pulse and 400 ms repetition time. 3D DMI was recorded at high resolution (2 × 2 × 2 mm) in 37 min or at low resolution (3.7 × 3.7 × 3.7 mm) in 2:24 min. Absolute tissue concentrations were calculated assuming natural deuterated water [HOD] = 13.7 mM. Within 5 min after [2H9]choline infusion, its signal appeared in tumor spectra representing a concentration increase to 0.3-1.2 mM, which then slowly decreased or remained constant over 100 min. In plasma, [2H9]choline disappeared within 15 min post-infusion, implying that its signal arises from tumor tissue and not from blood. After infusing a mixture of [2H9]choline and [6,6'-2H2]glucose, their signals were observed separately in tumor 2H spectra. Over time, the [2H9]choline signal broadened, possibly due to conversion to other choline compounds, [[6,6'-2H2]glucose] declined, [HOD] increased and a lactate signal appeared, reflecting glycolysis. Metabolic maps of 2H compounds, reconstructed from high resolution DMIs, showed their spatial tumor accumulation. As choline infusion and glucose DMI is feasible in patients, their simultaneous detection has clinical potential for tumor characterization.

Novel Fluorinated Poly (Lactic-Co-Glycolic acid) (PLGA) and Polyethylene Glycol (PEG) Nanoparticles for Monitoring and Imaging in Osteoarthritis.

Polymeric nanoparticles (NPs) find many uses in nanomedicine, from drug delivery to imaging. In this regard, poly (lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) particles are the most widely applied types of nano-systems due to their biocompatibility and biodegradability. Here we developed novel fluorinated polymeric NPs as vectors for multi-modal nanoprobes. This approach involved modifying polymeric NPs with trifluoroacetamide (TFA) and loading them with a near-infrared (NIR) dye for different imaging modalities, such as magnetic resonance imaging (MRI) and optical imaging. The PLGA-PEG-TFA NPs generated were characterized in vitro using the C28/I2 human chondrocyte cell line and in vivo in a mouse model of osteoarthritis (OA). The NPs were well absorbed, as confirmed by confocal microscopy, and were non-toxic to cells. To test the NPs as a drug delivery system for contrast agents of OA, the nanomaterial was administered via the intra-articular (IA) administration method. The dye-loaded NPs were injected in the knee joint and then visualized and tracked in vivo by fluorine-19 nuclear magnetic resonance and fluorescence imaging. Here, we describe the development of novel intrinsically fluorinated polymeric NPs modality that can be used in various molecular imaging techniques to visualize and track OA treatments and their potential use in clinical trials.