Synchrotron Radiation X-ray Fluorescence Elemental Mapping in Healthy versus Malignant Prostate Tissues Provides New Insights into the Glucose-Stimulated Zinc Trafficking in the Prostate As Discovered by MRI.

Prostatic zinc content is a known biomarker for discriminating normal healthy tissue from benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Given that zinc content is not readily measured without a tissue biopsy, we have been exploring noninvasive imaging methods to detect these diagnostic differences using a zinc-responsive MRI contrast agent. During imaging studies in mice, we observed that a bolus of glucose stimulates secretion of zinc from the prostate of fasted mice. This discovery allowed the use of a Gd-based zinc sensor to detect differential zinc secretion in regions of healthy versus malignant prostate tissue in a transgenic adenocarcinoma mouse model of PCa. Here, we used a zinc-responsive MRI agent to detect zinc release across the prostate during development of malignancy and confirm the loss of total tissue zinc by synchrotron radiation X-ray fluorescence (µSR-XRF). Quantitative µSR-XRF results show that the lateral lobe of the mouse prostate uniquely accumulates high concentrations of zinc, 1.06 ± 0.08 mM, and that the known loss of zinc content in the prostate is only observed in the lateral lobe during development of PCa. Additionally, we confirm that lesions identified by a loss of zinc secretion indeed represent malignant neoplasia and that the relative zinc concentration in the lesion is reduced to 0.370 ± 0.001 mM. The µSR-XRF data also provided insights into the mechanism of zinc secretion by showing that glucose promotes movement of zinc pools (∼1 mM) from the glandular lumen of the lateral lobe of the mouse prostate into the stromal/smooth muscle surrounding the glands. Co-localization of zinc and gadolinium in the stromal/smooth muscle areas as detected by µSR-XRF confirm that glucose initiates secretion of zinc from intracellular compartments into the extracellular spaces of the gland where it binds to the Gd-based agent and albumin promoting MR image enhancement.

Time-resolved fluorescence (TRF) and diffuse reflectance spectroscopy (DRS) for margin analysis in breast cancer.

PURPOSE: One of the major problems in breast cancer surgery is defining surgical margins and establishing complete tumor excision within a single surgical procedure. The goal of this work is to establish instrumentation that can differentiate between tumor and normal breast tissue with the potential to be implemented in vivo during a surgical procedure. METHODS: A time-resolved fluorescence and reflectance spectroscopy (tr-FRS) system is used to measure fluorescence intensity and lifetime as well as collect diffuse reflectance (DR) of breast tissue, which can subsequently be used to extract optical properties (absorption and reduced scatter coefficient) of the tissue. The tr-FRS data obtained from patients with Invasive Ductal Carcinoma (IDC) whom have undergone lumpectomy and mastectomy surgeries is presented. A preliminary study was conducted to determine the validity of using banked pre-frozen breast tissue samples to study the fluorescence response and optical properties. Once the validity was established, the tr-FRS system was used on a data-set of 40 pre-frozen matched pair cases to differentiate between tumor and normal breast tissue. All measurements have been conducted on excised normal and tumor breast samples post surgery. RESULTS: Our results showed the process of freezing and thawing did not cause any significant differences between fresh and pre-frozen normal or tumor breast tissue. The tr-FRS optical data obtained from 40 banked matched pairs showed significant differences between normal and tumor breast tissue. CONCLUSION: The work detailed in the main study showed the tr-FRS system has the potential to differentiate malignant from normal breast tissue in women undergoing surgery for known invasive ductal carcinoma. With further work, this successful outcome may result in the development of an accurate intraoperative real-time margin assessment system. Lasers Surg. Med. 50:236-245, 2018. © 2018 Wiley Periodicals, Inc.

Early regional cuprizone-induced demyelination in a rat model revealed with MRI.

The cuprizone model of demyelination is well established in the mouse as a tool for the study of the mechanisms of both demyelination and remyelination. It is often desirable, however, to have a larger model, such as the rat, especially for imaging-based studies, yet initial work has failed to show demyelination in cuprizone-fed rats. Several recent studies have demonstrated demyelination in the rat, but only in the corpus callosum. In this study, we acquired high-resolution, three-dimensional images of the whole brain every 2 weeks, using a T1 -weighted magnetization-prepared rapid acquisition gradient echo imaging sequence, optimized for myelin contrast, in order to assess myelination across the entire rat brain over a period of 8 weeks on a 1% cuprizone diet. We observed a consistent pattern of demyelination, beginning in the cerebellum by 4 weeks and involving more rostral regions of the brain by 8 weeks on the cuprizone diet, with validation using Luxol fast blue histology. This imaging technique permits the effects of cuprizone-induced demyelination to be followed longitudinally in a single animal, over the entire brain. In turn, this may facilitate the establishment of the cuprizone model of demyelination in the rat.

Differences in iron and manganese concentration may confound the measurement of myelin from R1 and R2 relaxation rates in studies of dysmyelination.

A model of dysmyelination, the Long Evans Shaker (les) rat, was used to study the contribution of myelin to MR tissue properties in white matter. A large region of white matter was identified in the deep cerebellum and was used for measurements of the MR relaxation rate constants, R1 = 1/T1 and R2 = 1/T2 , at 7 T. In this study, R1 of the les deep cerebellar white matter was found to be 0.55 ± 0.08 s (-1) and R2 was found to be 15 ± 1 s(-1) , revealing significantly lower R1 and R2 in les white matter relative to wild-type (wt: R1 = 0.69 ± 0.05 s(-1) and R2 = 18 ± 1 s(-1) ). These deviated from the expected ΔR1 and ΔR2 values, given a complete lack of myelin in the les white matter, derived from the literature using values of myelin relaxivity, and we suspect that metals could play a significant role. The absolute concentrations of the paramagnetic transition metals iron (Fe) and manganese (Mn) were measured by a micro-synchrotron radiation X-ray fluorescence (µSRXRF) technique, with significantly greater Fe and Mn in les white matter than in wt (in units of µg [metal]/g [wet weight tissue]: les: Fe concentration,19 ± 1; Mn concentration, 0.71 ± 0.04; wt: Fe concentration,10 ± 1; Mn concentration, 0.47 ± 0.04). These changes in Fe and Mn could explain the deviations in R1 and R2 from the expected values in white matter. Although it was found that the influence of myelin still dominates R1 and R2 in wt rats, there were non-negligible changes in the contribution of the metals to relaxation. Although there are already problems with the estimation of myelin from R1 and R2 changes in disease models with pathology that also affects the relaxation rate constants, this study points to a specific pitfall in the estimation of changes in myelin in diseases or models with disrupted concentrations of paramagnetic transition metals. Copyright © 2016 John Wiley & Sons, Ltd.

Altered transition metal homeostasis in the cuprizone model of demyelination.

In the cuprizone model of demyelination, the neurotoxin cuprizone is fed to mice to induce a reproducible pattern of demyelination in the brain. Cuprizone is a copper chelator and it has been hypothesized that it induces a copper deficiency in the brain, which leads to demyelination. To test this hypothesis and investigate the possible role of other transition metals in the model, we fed C57Bl/6 mice a standard dose of cuprizone (0.2% dry chemical to dry food weight) for 6 weeks then measured levels of copper, manganese, iron, and zinc in regions of the brain and visceral organs. As expected, this treatment induced demyelination in the mice. We found, however, that while the treatment significantly reduced copper concentrations in the blood and liver in treated animals, there was no significant difference in concentrations in brain regions relative to control. Interestingly, cuprizone disrupted concentrations of the other transition metals in the visceral organs, with the most notable changes being decreased manganese and increased iron in the liver. In the brain, manganese concentrations were also significantly reduced in the cerebellum and striatum. These data suggest a possible role of manganese deficiency in the brain in the cuprizone model.

The identification and differentiation of secondary colorectal cancer in human liver tissue using X-ray fluorescence, coherent scatter spectroscopy, and multivariate analysis.

Secondary colorectal liver cancer is the most widespread malignancy in patients with colorectal cancer. The aim of this study is to identify and differentiate between normal liver tissue and malignant secondary colorectal liver cancer tissue using X-ray scattering and X-ray fluorescence spectroscopy to investigate the best combination of data that can be used to enable classification of these two tissue types. X-ray fluorescence (XRF) and coherent scatter data were collected for 24 normal and 24 tumor matched pair tissue samples. The levels of 12 elements (P, S, K, Ca, Cr, Fe, Cu, Zn, As, Se, Br, and Rb) were measured in all samples. When comparisons were made between normal and tumor tissues, statistically significant differences were determined for K (p = 0.046), Ca (p = 0.040), Cr (p = 0.011), Fe, Cu, Zn, Br, and Rb (p < 0.01). However, for P, S, As, and Se, no statistically significant differences were found (p > 0.05). For the coherent scatter spectra collected, three peaks due to adipose, fibrous content, and water content of tissue were observed. The amplitude, full width half-maximum, and area under both fibrous content and water content peaks were found to be significantly higher in secondary colorectal liver tumors compared with surrounding normal liver tissue (p < 0.05). However, no significant differences were found for the adipose peak parameters (p > 0.05). Soft independent modeling of class analogy was performed using the XRF, coherent scatter, and elemental ratio data separately, and the accuracy of the classification of 20 unknown samples was found to be 50, 30, and 80%, respectively. Further analysis has shown that using a combination of the XRF and coherent scatter data in a single combined model gave improved normal and tumor liver tissue classification, with an accuracy that was found to be 85%.

Characterization of the depth distribution of Ca, Fe and Zn in skin samples, using synchrotron micro-x-ray fluorescence (SµXRF) to help quantify in-vivo measurements of elements in the skin.

In vivo monitoring of trace and biometals in skin is normally quantified using phantoms that assume a constant elemental distribution within the skin. Layered calibration skin phantoms could potentially improve the reliability of in vivo calibration skin phantoms by better representing the actual in vivo distribution. This work investigates the micro-distribution of iron, calcium and zinc in prepared human skin samples taken from a number of locations on the body. Slices (orientation running from the skin surface into the dermis) were extracted from 18 formalin-fixed necropsy samples and scanned using the micro-XRF setup at the VESPERS beamline (Canadian Light Source). Elemental surface maps were produced using a 6×6 µm(2) beam in steps of 10 µm. Microscope images of histology slides were obtained for comparison. Statistically significant differences (p<0.01) were noted between the epidermal and dermal layers of skin for the elements examined (Ca, Fe and Zn), demonstrating the ability to clearly distinguish elemental content in each layer. Iron was consistently noted at the epidermal/dermal boundary. These results would indicate that when using phantoms to quantify elemental levels measured in the skin, note should be taken of the appropriate depth distribution.

Altered transition metal homeostasis in mice following manganese injections for manganese-enhanced magnetic resonance imaging.

In manganese-enhanced magnetic resonance imaging (MEMRI), the paramagnetic divalent ion of manganese (Mn(2+)) is injected into animals to generate tissue contrast, typically at much higher exposures than have been previously used in studies of Mn toxicity. Here we investigate the effect of these injections on the homeostasis of the transition metals iron and copper in mice to see if there are disruptions which should be considered in MEMRI studies. Manganese shares transport proteins with other transition metals including iron and copper, so it is possible that changes in manganese levels in tissue following injections of the metal may affect other metal levels too. This in turn may affect MRI contrast or the investigation of disease processes in the animal models being imaged. In this study, we measured manganese, iron, and copper concentrations in the blood, kidney, liver and in brain regions in mice treated with four injections of 30 mg/kg MnCl(2) 4H(2)O (dry chemical weight/body weight)-a common dose used in MEMRI. In addition to the expected increases in manganese in tissues, we noted a statistically significant reduction in copper in the kidney and liver. Also, we noted a statistically significant decrease in concentration of iron in the thalamus of the brain. These findings suggest that the high doses of manganese injected in MEMRI studies can disrupt the homeostasis of other transition metals in mice.

Efficient and rapid labeling of transplanted cell populations with superparamagnetic iron oxide nanoparticles using cell surface chemical biotinylation for in vivo monitoring by MRI.

Determination of the dynamics of specific cell populations in vivo is essential for the development of cell-based therapies. For cell tracking by magnetic resonance imaging (MRI), cells need to internalize, or be surface labeled with a MRI contrast agent, such as superparamagnetic iron oxide nanoparticles (SPIOs): SPIOs give rise to signal loss by gradient-echo and T(2)-weighted MRI techniques. In this study, cancer cells were chemically tagged with biotin and then magnetically labeled with anti-biotin SPIOs. No significant detrimental effects on cell viability or death were observed following cell biotinylation. SPIO-labeled cells exhibited signal loss compared to non-SPIO-labeled cells by MRI in vitro. Consistent with the in vitro MRI data, signal attenuation was observed in vivo from SPIO-labeled cells injected into the muscle of the hind legs, or implanted subcutaneously into the flanks of mice, correlating with iron detection by histochemical and X-ray fluorescence (XRF) methods. To further validate this approach, human mesenchymal stem cells (hMSCs) were also employed. Chemical biotinylation and SPIO labeling of hMSCs were confirmed by fluorescence microscopy and flow cytometry. The procedure did not affect proliferation and multipotentiality, or lead to increased cell death. The SPIO-labeled hMSCs were shown to exhibit MRI signal reduction in vitro and was detectable in an in vivo model. In this study, we demonstrate a rapid, robust, and generic methodology that may be a useful and practical adjuvant to existing methods of cell labeling for in vivo monitoring by MRI. Further, we have shown the first application of XRF to provide iron maps to validate MRI data in SPIO-labeled cell tracking studies.