Imaging of Alkaline Phosphatase Activity in Bone Tissue

The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with 19Flourine magnetic resonance spectroscopic imaging (19FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using 19Fluorine magnetic resonance spectroscopy (19FMRS) and 19FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. 19FMRS and 19FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. 19FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized 19FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of 19FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, 19FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications

Definitive locoregional therapy (LRT) versus bridging LRT and liver transplantation with wait-and-not-treat approach for very early stage hepatocellular carcinoma

PURPOSE:Since the change in the United Network for Organ Sharing (UNOS) policy excluding patients with very early stage hepatocellular carcinoma (veHCC, single tumor nodule <2 cm) from receiving Model for End-stage Liver Disease (MELD) exception points, patients eligible to receive liver transplantation (LT) who fall in this category are commonly treated with locoregional therapy (LRT) after progression to UNOS T2 stage (1 nodule of 2–5 cm or up to 3 nodules, none above 3 cm). The aim of the current study is to compare the outcomes of patients treated with bridging LRT and LT with wait-and-not-treat approach with patients treated with definitive LRT.METHODS:A retrospective study has been performed on patients with veHCC evaluated in multidisciplinary liver tumor clinic of a large academic center between 2004–2011. Patients eligible for LT were assigned to the wait-and-not-treat group while patients who were not eligible were assigned to the definitive LRT group. Tumor size, time to treatment, severity of liver disease, recurrence and survival from time of detection were reviewed and recorded.RESULTS:A total of 19 patients were identified and treated with definitive LRT while 57 patients were treated with bridging LRT prior to LT after disease progression to T2 stage. Patients in the definitive LRT group were older (70.4±10.2 years vs. 58.7±5.9 years, P < 0.001) and had more comorbid conditions compared with the wait-and-not-treat group. Mean survival for definitive LRT group at the end of 5 years was 34.3±6.0 months with a median of 30.3 months (95% CI, 5.7–55.0 months) compared with 48.7±2.6 months for the wait-and-not-treat group, respectively (median not reached). The 3- and 5-year survival rates were 53.3% and 33.3% for the definitive LRT group compared with 78.9% and 68.4% for the patients in the wait-and-not-treat group. Survival rate at the end of 5 years was significantly better for the wait-and-not-treat group (P = 0.013).CONCLUSION:Based on the findings of current retrospective study, treating veHCC (UNOS T1 stage) patients listed for LT with bridging LRT after disease progression to T2 stage appears to be safe and effective with high 5-year survival rates

All data and code for analysis of presented data

This folder provides all data presented and analyzed within the manuscript. This includes R and Matlab scripts used to analyze the data and prepare the figures

Data from: Electrolytic ablation enables cancer cell targeting through pH modulation

Minimally invasive ablation strategies enable locoregional treatment of tumors. Electrolytic ablation functions through the local delivery of direct current, without thermal effects, facilitating enhanced precision. However, the clinical application of electrolytic ablation is limited by an incompletely characterized mechanism of action. Here we show that acid and base production at the electrodes precipitates local pH changes causing the rapid cell death that underlies macroscopic tumor necrosis at pH > 10.6 or < 4.8. The extent of cell death can be modulated by altering the local buffering capacity and antioxidant availability. These data demonstrate that electrolytic ablation is distinguished from other ablation strategies via its ability to induce cellular necrosis by directly altering the tumor microenvironment. These findings may enable further development of electrolytic ablation as a curative therapy for primary, early stage tumors

Non-invasive imaging of ALP activity in rat tibia cortical bone.

<p>A: RARE ¹H images of the bone sample anatomy including the rat tibia cortical bone core within the glass vial. B and C: ¹⁹FMRSI-derived parametric maps of regional DiFMUP and hydrolysis product concentrations overlaid onto RARE ¹H images of the bone sample (B and C, respectively). D: ¹⁹FMRSI-derived parametric maps of regional ALP concentration and activity overlaid onto RARE ¹H images of the bone sample.</p

ALP-dependent DiFMUP activation on the surface of ALP-positive osteoblastic bone cells and in the presence of rat tibial bone.

<p>A: Histochemistry detected ALP expression as violet-red staining on 7F2 cells as shown in low and high magnification images 1 and 2, respectively. In contrast, no ALP expression was detectable on the MC3T3-E1#4 cells shown also in low (3) and high magnification (4). B: Activation (hydrolysis) of DiFMUP occurred on 7F2 cells (closed squares), but not on ALP-negative MC3T3-E1#4 cells (open triangles). No-cell background (Bkg) measurements are also presented (open circles). C: Following the separation of cells (CE) and medium (M) and a single wash step (W), the vast majority of the hydrolysis product was found in the medium. Mean values and standard deviations are shown (n = 3). D: DiFMUP in physiological solution was activated in a time-dependent fashion in the presence of highly purified rat tibia bone (closed squares). Addition of the ALP inhibitor levamisole reduced DiFMUP activation (open squares). E: DiFMUP in alkaline solution was activated by a single highly purified tibia bone chip (closed squares). Presence of levamisole significantly suppressed the activation (open squares). Mean values and standard deviations are shown (n = 3).</p