Circulating MicroRNA-92b-3p as a Novel Biomarker for Monitoring of Synovial Sarcoma.

The lack of useful biomarkers is a crucial problem for patients with soft tissue sarcomas (STSs). Emerging evidence has suggested that circulating microRNAs (miRNAs) in body fluids have novel impact as biomarkers for patients with malignant diseases, but their significance in synovial sarcoma (SS) patients remains unknown. Initial global miRNA screening using SS patient serum and SS cell culture media identified a signature of four upregulated miRNAs. Among these candidates, miR-92b-3p secretion from SS cells was confirmed, which was embedded within tumour-derived exosomes rather than argonaute-2. Animal experiments revealed a close correlation between serum miR-92b-3p levels and tumour dynamics. Clinical relevance was validated in two independent clinical cohorts, and we subsequently identified that serum miR-92b-3p levels were significantly higher in SS patients in comparison to that in healthy individuals. Moreover, serum miR-92b-3p was robust in discriminating patients with SS from the other STS patients and reflected tumour burden in SS patients. Overall, liquid biopsy using serum miR-92b-3p expression levels may represent a novel approach for monitoring tumour dynamics of SS.

Sustained delivery of vincristine inside an orthotopic mouse sarcoma model decreases tumor growth.

BACKGROUND: Sarcoma accounts for 20% of solid tumors in children. Surgery has significant morbidity. We hypothesized that delivering chemotherapy directly into tumors through sustained release silk systems could slow tumor growth. METHODS: Human Ewing sarcoma cells A673 were cultured with vincristine and doxorubicin to determine half maximal inhibitory concentration (IC50). Cells were injected into mouse hind leg to create orthotopic tumors. Tumor volumes were measured using ultrasound. When volume reached >250mm3, interventions included: implantation of drug-free silk foam (Control-F), doxorubicin 400µg foam (Dox400-F), vincristine 50µg foam (Vin50-F), drug-free silk gel (Control-G), vincristine 50µg gel (Vin50-G), or single dose intravenous vincristine 50µg (Vin50-IV). End-point was volume>1000mm3. Kaplan Meier and ANOVA were used. RESULTS: IC50 for vincristine and doxorubicin was 0.5ng/mL and 200ng/mL, respectively. There was no difference between Dox400-F [6±1days to end point (DTEP)] and Control-F (5±1.3 DTEP). Vin50-F (12.4±3.5 DTEP) had slower growth compared to Control-F (p<0.001), and there was no difference between Vin50-F and Vin50-IV (14±0 DTEP). Growth was slowest with Vin50-G, 28±10.3 DTEP compared to all other treatment groups (p<0.05). CONCLUSION: Sustained delivery of vincristine inside the sarcoma tumor with silk gel decreased tumor growth. Applying this intratumoral treatment strategy may potentially decrease the extent of surgical excision.

Synthesis, characterisation and evaluation of a novel copper-64 complex with selective uptake in EMT-6 cells under hypoxic conditions.

The radiometal (64)Cu is now widely used in the development of diagnostic imaging agents for positron emission tomography (PET). The present study has led to the development and evaluation of a novel chelating agent for (64)Cu: the new monothiourea tripodal ligand 1-benzoyl-3-{6-[(bis-pyridin-2-ylmethyl-amino)-methyl]-pyridin-2-yl}-thiourea (MTUBo). X-ray crystallographic analysis has shown this ligand forms a mononuclear complex with copper(II) and co-ordinates via a trigonal bipyramidal N4S array of donor atoms. Promisingly, cell uptake studies revealed that (64)Cu-MTUBo selectively accumulates in EMT-6 cells incubated under hypoxic conditions which may result from its relatively high Cu(II/I) redox potential. Small-animal PET imaging and ex vivo biodistribution studies in EMT-6 tumor bearing BALB/c mice revealed significant tumor uptake after 1 h p.i., yielding tumor-to-muscle (T/M) and tumor-to-blood (T/B) ratios of 8.1 and 1.1, respectively. However, injection of (64)Cu-acetate resulted in similar uptake indicating that the observed uptake was most likely non-specific. Despite showing high in vitro stability, it is likely that in vivo the complex undergoes transchelation to proteins within the blood in a relatively short timeframe. For comparison, the hypoxia imaging agent (64)Cu-ATSM was also evaluated in the same murine tumor model and showed about 60% higher tumor uptake than (64)Cu-MTUBo.

Imaging primary mouse sarcomas after radiation therapy using cathepsin-activatable fluorescent imaging agents.

PURPOSE: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. METHODS AND MATERIALS: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. RESULTS: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. CONCLUSIONS: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.

Segmentation of dynamic contrast enhanced magnetic resonance imaging data.

INTRODUCTION: Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) allows in vivo characterization of tumour vasculature. As such, it is applicable for monitoring the effects of treatments targeting vasculature. The aims of this study were to evaluate the properties of tumour areas segmented-out by DCE-MRI parameters and to evaluate the changes induced by the vascular disrupting agent (VDA) combretastatin A-4 disodium phosphate (CA4DP), a leading VDA in clinical trials, in these areas. MATERIAL AND METHODS: Two tumour models previously shown to respond differently to CA4DP were chosen. The C3H mammary carcinoma and the KHT sarcoma were grown in the right rear foot of CDF(1) and C3H/km mice, respectively, and treated when at 200 or 800 mm(3) in size. DCE-MRI, using the contrast agent Gd-DTPA, was performed on a 7 T spectroscopy/imaging system before and 3 hours after i.p. CA4DP administration at a dose of 100 mg/kg. From the voxel concentration-time curves, the semiquantitative parameter of initial area under the curve (IAUC), the model parameters transfer constant K(trans), interstitial volume v(e), and blood plasma volume v(p), were calculated. Tumour images were segmented into three groups based on the DCE-MRI model parameters using the K-means algorithm, and the groups were ranked by IAUC. RESULTS: The resulting voxels of the tumour segments were mainly spatially connected structures. Initial DCE-MRI parameter values showed different dependencies on tumour model and size in the regions. For all regions in all tumour groups, the treatment reduced IAUC by 36-51%, whereas the model parameters showed more dependencies on tumour model and size. DISCUSSION: This segmentation technique identifies tumour regions with different microenvironmental characteristics responding differently to CA4DP and may be valuable in the optimization of combined VDA with radiotherapy or chemotherapy. The method may also prove useful for optimization and monitoring of local treatment such as radiotherapy.

Dynamic contrast-enhanced MRI using macromolecular contrast media for monitoring the response to isolated limb perfusion in experimental soft-tissue sarcomas.

The objective of this study was to evaluate the potential of dynamic contrast-enhanced MRI for quantitative characterization of tumor microvessels and to assess the microvascular changes in response to isolated limb perfusion with TNF-alpha and melphalan. Dynamic contrast-enhanced MRI was performed in an experimental cancer model, using a macromolecular contrast medium, albumin-(Gd-DTPA)45. Small fragments of BN 175, a soft-tissue sarcoma, were implanted in 11 brown Norway (BN) rats. Animals were assigned randomly to a control (Haemaccel) or drug-treated group (TNF-alpha/melphalan). MRI was performed at baseline and 24 h after ILP. The transendothelial permeability (K(PS)) and the fractional plasma volume (fPV) were estimated from the kinetic analysis of MR data using a two-compartment bi-directional model. K(PS) and fPV decreased significantly in the drug-treated group compared to baseline (p<0.05). In addition, K(PS) post therapy was significantly lower (p<0.05) in the drug-treated group than in the control group. There was no significant difference in fPV between the drug-treated and the control group after therapy. Tumor microvascular changes in response to isolated limb perfusion can be determined after 24 h by dynamic contrast-enhanced MRI. The data obtained in this experimental model suggest possible applications in the clinical setting, using the appropriate MR contrast agents.

MS-2 fibrosarcoma characterization by laser induced autofluorescence.

BACKGROUND AND OBJECTIVE: MS-2 fibrosarcoma implanted in BALB-CDF1 mice was investigated by frequency and time domain measurements of the autofluorescence (AF) radiation emitted upon excitation by a N(2) laser beam (337.1 nm). STUDY DESIGN/MATERIALS AND METHODS: AF spectra were obtained by using a spectrograph, a multichannel plate and an optical multichannel analyzer for the steady state detection. Time-resolved spectra were performed by means of a monochromator, a photomultiplier, and a digital signal analyzer. RESULTS: Spectral measurements show that the autofluorescence intensity of pathologic tissue is lower than that of healthy one in the 400- to 500- spectral region. In the same spectral range, we found the fluorescence decay to be the sum of a fast and a slow component. The lifetime of the fast component of tumoral tissue is significantly lower than that of healthy samples. CONCLUSION: Frequency and time domain measurements used in combination show that MS2-fibrosarcoma is characterized by the probable presence of the free form of NADH.

Dynamic contrast-enhanced MRI and fractal characteristics of percolation clusters in two-dimensional tumor blood perfusion.

Dynamic contrast-enhanced magnetic resonance imaging (DE-MRI) of the tumor blood pool is used to study tumor tissue perfusion. The results are then analyzed using percolation models. Percolation cluster geometry is depicted using the wash-in component of MRI contrast signal intensity. Fractal characteristics are determined for each two-dimensional cluster. The invasion percolation model is used to describe the evolution of the tumor perfusion front. Although tumor perfusion can be depicted rigorously only in three dimensions, two-dimensional cases are used to validate the methodology. It is concluded that the blood perfusion in a two-dimensional tumor vessel network has a fractal structure and that the evolution of the perfusion front can be characterized using invasion percolation. For all the cases studied, the front starts to grow from the periphery of the tumor (where the feeding vessel was assumed to lie) and continues to grow toward the center of the tumor, accounting for the well-documented perfused periphery and necrotic core of the tumor tissue.

The importance of electric field distribution for effective in vivo electroporation of tissues.

Cells exposed to short and intense electric pulses become permeable to a number of various ionic molecules. This phenomenon was termed electroporation or electropermeabilization and is widely used for in vitro drug delivery into the cells and gene transfection. Tissues can also be permeabilized. These new approaches based on electroporation are used for cancer treatment, i.e., electrochemotherapy, and in vivo gene transfection. In vivo electroporation is thus gaining even wider interest. However, electrode geometry and distribution were not yet adequately addressed. Most of the electrodes used so far were determined empirically. In our study we 1) designed two electrode sets that produce notably different distribution of electric field in tumor, 2) qualitatively evaluated current density distribution for both electrode sets by means of magnetic resonance current density imaging, 3) used three-dimensional finite element model to calculate values of electric field for both electrode sets, and 4) demonstrated the difference in electrochemotherapy effectiveness in mouse tumor model between the two electrode sets. The results of our study clearly demonstrate that numerical model is reliable and can be very useful in the additional search for electrodes that would make electrochemotherapy and in vivo electroporation in general more efficient. Our study also shows that better coverage of tumors with sufficiently high electric field is necessary for improved effectiveness of electrochemotherapy.

Laser-induced fluorescence studies of the biodistribution of carotenoporphyrins in mice.

The biodistribution of two recently developed tumour markers, trimethylated (CP(Me)3) and trimethoxylated (CP(OMe)3) carotenoporphyrin, was investigated by means of laser-induced fluorescence (LIF) after i.v. injection into 38 tumour-bearing (MS-2 fibrosarcoma) female Balb/c mice. At 3, 24, 48 or 96 h after administration, the carotenoporphyrin fluorescence was measured in tumoral and peritumoral tissue, as well as in the abdominal, thoracic and cranial cavities. The fluorescence was induced by a nitrogen laser-pumped dye laser, emitting light at 425 nm, and analysed by a polychromator equipped with an image-intensified CCD camera. The fluorescence was evaluated at 490, 655 and 720 nm: the second and third wavelengths represent the carotenoporphyrin (CP)-related peaks, whereas the first one is close to the peak of the tissue autofluorescence. The tumour and the liver were the two tissue types showing the strongest carotenoporphyrin-related fluorescence, whereas the cerebral cortex and muscle consistently exhibited weak substance-related fluorescence. In most tissue types, the fluorescence intensities decreased over time. A few exceptions were observed, notably the liver, in which the intensity remained remarkably constant over the time period investigated.

Significance of serum laminin and type IV collagen levels for metastasis in murine RCT sarcoma.

We investigated in vivo the correlation between pulmonary metastasis and serum concentrations of laminin and type IV collagen in high-metastatic RCT(+) and low-metastatic RCT(-) clones established from poorly differentiated RCT sarcoma in C3H/He mice. The in vitro invasiveness of these cell clones through the extracellular matrix was also studied. In the in vitro invasion assay, high-metastatic RCT(+) cells showed significantly more invasiveness than low-metastatic RCT(-) cells. The different invasiveness of these cloned cells was associated with their different ability to attach to and degrade the matrix. In mice inoculated with RCT(+) cells, serum levels of laminin and type IV collagen rose with the increase in the number of pulmonary metastatic nodules. Serum levels of these extracellular matrix components started to rise when pulmonary metastatic nodules were macroscopically observed in the RCT(+) group. In mice bearing RCT(-) cells with lower metastatic ability, there were no significant increases in serum extracellular matrix levels. These findings suggested that serum concentrations of laminin and type IV collagen might be useful markers for the early detection of metastasis.

Time-gated fluorescence imaging for the diagnosis of tumors in a murine model.

A system for time-gated fluorescence imaging was used to perform measurements on tumor-bearing mice treated with hematoporphyrin derivative (HpD). The aim of the study was to define the potential of this technique in the diagnosis of tumors by taking advantage of the long fluorescence lifetime of the exogenous dye with respect to the decay times of the natural fluorescence. After the administration of three different drug doses (5, 10 and 25 mg/kg body weight), fluorescence images were acquired at various uptake times (from 2 h to 10 d), to determine the best instrumental conditions and experimental procedure for the detection of tumors in the murine model considered. The optimal fluorescence contrast between the tumor area and the surrounding healthy tissue was found at 12 h after the administration of either 5 or 10 mg/kg HpD and was anticipated at 8 h for the highest drug dose. In this optimum condition, the tumor region could be identified even after the injection of 5 mg/kg HpD. A better fluorescence contrast was always obtained in 15 ns-delayed images with respect to synchronous ones.

Application of charge-coupled device technology for measurement of laser light and fluorescence distribution in tumors for photodynamic therapy.

Laser and fluorescence light distributions with applications for photodynamic therapy were measured in mouse tumors using a non-invasive electronic optical imaging system. The system consists of a liquid-nitrogen-cooled, charge-coupled-device (CCD) array camera under computer control with 576 x 384 detection elements having dimensions of 23 microns x 23 microns. The available dynamic range of the array is approx. 10(3), and the effective wavelength range is 400-1000 nm. An interstitially placed cylindrical diffusing optical fiber was used to provide tumor illumination. The light distribution pattern from the fiber was determined by immersing the cylindrical diffusing tip in a fluorescing solution and recording the emission image. Fluorescence imaging facilitates an accurate measurement of light intensity distribution while avoiding problems associated with the directional nature of other detection methods used with diffusing fibers. Radiation-induced fibrosarcoma tumors on C3H mice were grown to about 1 cm diameter for in vivo recording of light distribution from the tumor volume and for determination of effective light penetration distance at 18 wavelengths in the range 458-995 nm. Endogenous tumor fluorescence and Photofrin II fluorescence intensity were measured over the wavelength range 585-725 nm to investigate the possible application of CCD imaging technology for drug distribution measurements. Model experiments were begun to evaluate the relative importance of potential distortions of light distribution measurements using this approach.

Hyaline droplet accumulation in rodent kidney proximal tubules: an association with histiocytic sarcoma.

Since recognition during the last decade that certain renal carcinogens can initially cause an accumulation of hyaline (protein) droplets in proximal tubules of male rats, it has become appropriate to establish whether this phenomenon of protein overload can also occur in rodent kidneys unrelated to chemical treatment. Kidney tissue from a number of selected rodent studies held in the National Toxicology Program (NTP) or Food and Drug Administration (FDA) archives were evaluated for hyaline droplet accumulation in proximal tubules. The survey concentrated on rats and mice of both sexes bearing hematopoietic tumors, as our preliminary observations had suggested this direction of study. The tissues of 101 Sprague-Dawley, 25 Osborne-Mendel, and 70 Fischer 344 rats and 96 B6C3F1 mice were examined. These animals provided an assortment of tumors including histiocytic sarcoma, lymphocytic lymphoma, mononuclear cell leukemia, and sarcoma. Hyaline droplet accumulation, primarily involving the P2 segment of proximal tubules, was diagnosed in 96% of rats with histiocytic sarcoma (74/77 cases in Sprague-Dawley, 17/18 in Osborne-Mendels, 7/7 in Fischers) and in 55% of B6C3F1 mice with histiocytic sarcoma (18/33 cases). There appeared to be a qualitative correlation between hyaline droplet accumulation and degree of tumor burden. Thus, in cases negative for hyaline droplets, the tumor was often confined to a single location, while increasing involvement of proximal segments beyond P2 occurred with more extensive multi-organ dissemination of the tumor. By immunohistochemistry on 11 cases of rat and 8 cases of mouse histiocytic sarcoma, the protein in hyaline droplets was identified as lysozyme, a known major secretory product of monocytes and macrophages. The hyaline droplets were negative for alpha 1-antitrypsin, alpha 2u-globulin, rat or mouse immunoglobulin, and albumin. More sparsely scattered droplets and granules present in proximal tubules of Fischer rats with mononuclear cell leukemia were negative for lysozyme but positive for either iron or lipofuscin pigment. The study establishes a clear association between renal tubule hyaline droplet and lysozyme accumulation in rats and mice with histiocytic sarcoma. Hyaline droplets secondary to neoplasia should be distinguished from chemically-induced hyaline droplet nephropathy in the male rat involving alpha 2u-globulin.

Soft-tissue sarcomas: detection of metabolic heterogeneity with P-31 MR spectroscopy.

Regional variations in metabolic parameters derived with multivoxel, localized phosphorus-31 magnetic resonance (MR) spectroscopy in spontaneous human-soft-tissue sarcomas were compared with variations in the same parameters in normal human legs. In addition, multivoxel P-31 MR spectroscopy of transplanted rodent sarcomas and microelectrode measurement of pH and PO2 in several locations within them were performed, and, when appropriate, results were compared with the clinical data. Striking voxel-voxel variability in parameters derived with P-31 spectroscopy was found in tumors, with less marked variability seen in normal legs. In the rodent tumors, spatial variations also were found in pH and PO2 measured by means of microelectrodes. These data are consistent with the results of regional measurements in tumors by means of other methods and suggest that clinical MR spectroscopic studies of tumors may need to consider the variability within malignant lesions.

Fibre-laser IR luminescence diagnostics of malignant tumours using rare earth porphyrins.

To eliminate the masking effect of the background luminescence of endogenic substances in biological tissues and to increase the luminescence contrast values of malignant tumours, sarcoma-implanted mice were administered with ytterbium porphyrins instead of free porphyrin radicals. This was followed by in vivo luminescence localization of tumours using fibre-laser spectrofluorometry. The luminescence contrast values obtained reached 10-45 which is evidently not a limit.

Improvement of the rotating frame experiment by detection of residual Z magnetization: a 31P MRS study of metabolite levels in a Meth-A sarcoma.

The radio frequency field (B1) gradient of a surface coil can be used to obtain spectra from a series of sample regions which experience different B1 field strengths. We previously reported that the sensitivity of this method, known as the surface coil rotating frame experiment (SCRFE), can be enhanced by applying a composite pulse immediately after signal acquisition to sample residual Z magnetization which is normally undetected. Initially this modified SCRFE was used to obtain spatially resolved spectra across a B1 gradient of a factor of 2.5. Here we demonstrate the extension of this method to map phosphorylated metabolites across a B1 gradient of a factor of close to 5. Computer simulations were used to evaluate the performance of the composite pulse, and to assist in analyzing the data. The method was used to obtain 31P spectra in vivo from various tissue layers within and beneath a murine Meth-A tumor. The spectra differentiated between metabolite levels in tumor tissue and underlying skeletal muscle. Metabolic heterogeneity within the tumor itself was also evident.

The effects of angiotensin II on Doppler signals from a soft-tissue tumour.

A 10 mHz continuous-wave Doppler system has been used to detect blood flow within a sarcoma implanted in a rabbit's ear. The effects of vasoconstriction produced by intravenous angiotensin II were studied. Criteria are described that enabled differentiation of the Doppler signals from the tumour from those of the adjacent central artery. The effect of vasoconstriction upon these criteria is also described.