Name: spatial measurements of perfusion, interstitial fluid pressure and liposomes accumulation in solid tumors
Uploaded: 2016-08-18T13:00:00
Description: Watch this Scientific Journal Video about Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors at JoVE.com

The authors would like to thank Dr. Javed Mahmood for assistance with culturing MDA-MB-231 cells and implanting the MDA-MB-231 xenografts, Linyu Fan for preparing the CT-liposomes. Shawn Stapleton is grateful for funding from the Natural Sciences and Engineering Research Postgraduate Scholarships Program and the Terry Fox Foundation Strategic Initiative for Excellence in Radiation Research for the 21st Century (EIRR21) at CIHR. This study was supported by grants from the Terry Fox New Frontiers Program (020005) and the Canadian Institutes of Health Research (102569).

All in vivo experiments were performed under a protocol approved by the University Health Network Institutional Animal Care and Use Committee.
1. Animal Model
<ol>
	<li>Culture between 5 to 7 x 106 MDA-MB-231 breast adenocarcinoma tumor cells in DMEM together with 10% Fetal Bovine Serum (FBS) and 100x dilution of penicillin-streptomycin.</li>
	<li>Harvest cells when they are 80% confluent using a 0.05% trypsin-EDTA solution. After 3-5 min neutralize trypsin-EDTA ...

<ol>
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The methods for image-based measurement presented herein enable determination of the spatial distribution of tumor microcirculation properties, IFP, and CT-liposome accumulation. Previous attempts to relate these properties have relied on performing bulk measurements across multiple tumor-bearing animals and therefore lack the sensitivity to elucidate mechanisms responsible for heterogeneity in intra-tumoral accumulation that has commonly been observed for nano-sized drug delivery systems15. DCE-CT provides a ...

Measuring the intra-tumoral accumulation of nanoparticle drug delivery systems may provide an important tool to determine if an adequate concentration of cytotoxic drug has been achieved within the tumor. The development of &#34;image-able&#34; liposomal systems allows for non-invasive and quantitative in vivo detection of the drug delivery vehicle using imaging modalities such as positron emission tomography (PET)1, optical fluorescence2, and computed tomography (CT)3,4 and magnetic resonance imaging (MRI)5. Imaging has been used to determine the pharmacokinetics and biodistribution of liposome delivery systems and to reveal the extent of inter-subject and intra-tumoral heterogeneity in nanoparticle accumulation6,7. However, imaging of nanoparticles alone does not identify the biological barriers that have contributed to their poor accumulation and distribution. This knowledge is paramount to the rational development of more efficacious formulations, and strategies to improve intra-tumoral accumulation8. It has been demonstrated that therapeutic strategies can be applied to modulate specific biological barriers resulting in improved nanoparticle transport9. Additionally, nanoparticle formulations have been developed to specifically overcome specific biological transport barrier10. In both scenarios, measurements of biological barriers could be used to guide the use of an appropriate nanoparticle drug delivery strategy.
Tumor microcirculation and elevated IFP are believed to be two key determinants of the intra-tumoral accumulation of nanoparticles, such as liposomes, in solid tumors9,11. However, other barriers that to contribute to poor liposome accumulation include a dense extracellular matrix, impermeable vasculature, and solid tissue pressure12. These barriers are related in a spatio-temporal manner, with abnormal blood flow and elevated interstitial fluid pressure being two important factors driving the initial delivery and extravasation of nanoparticles. As previously discussed, establishing the relationship between the tumor microcirculation, elevated IFP, and the intra-tumoral accumulation of liposomes is imperative for proper interpretation of liposome imaging data. Herein quantitative methods to measure the relationship between the tumor microcirculation, elevated IFP, and nanoparticle accumulation in a solid tumor are presented. This is accomplished by performing co-localized measurements of the intra-tumoral distribution of a CT liposome contrast agent using volumetric CT imaging, tumor microcirculation using dynamic contrast enhanced computed tomography imaging, and tumor IFP using an image-guided robotic needle positioning system, termed the CT-IFP robot13.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">MDA-MB-231 metastatic breast adenocarcinoma tumor cells&#160;</td>
			<td style="width:471px;">ATCC</td>
			<td style="width:119px;">HTB-26</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)&#160;</td>
			<td style="width:471px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:471px;">Sigma-Aldrich</td>
			<td style="width:119px;">F1051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HyClone Penicillin-Streptomycin 100x Solution</td>
			<td>GE Healthcare Life Sciences</td>
			<td style="width:119px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)</td>
			<td>Avanti Lipids Inc., USA</td>
			<td>850355P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol (CH)</td>
			<td>Avanti Lipids Inc., USA</td>
			<td style="width:119px;">700000P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol) 2000 (DSPE-PEG2000)</td>
			<td>Avanti Lipids Inc., USA</td>
			<td style="width:119px;">880128P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnipaque (Iohexol) 300 mg of iodine/mL&#160;</td>
			<td>GE Healthcare, CA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">80 nm pore size Track-Etch polycarbonate membranes</td>
			<td style="width:471px;">Whatman Inc., USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 nm pore size Track-Etch polycarbonate membranes</td>
			<td style="width:471px;">Whatman Inc., USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL Lipex Extruder&#160;</td>
			<td>Nothern Lipids Inc, CA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Dialysis Bag Molecular Weight Cut Off (MWCO) of 8 kDa</td>
			<td>Spectrum Labs, USA&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">750,000 Nomical Molecular Weight Cut Off (NMWC) Tangential flow column&#160;</td>
			<td>MidGee ultrafiltration cartridge, GE Healthcare, CA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic pump&#160;</td>
			<td>Watson Marlow Inc., USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV spectrometer</td>
			<td>Helios &#947;, Spectronic Unicam,&#160; USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">90Plus particle size analyzer&#160;</td>
			<td>Brookhaven, Holtsville, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">eXplore Locus Ultra micro-CT system&#160;</td>
			<td>GE Healthcare, CA</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">Manipulated using CT-Console Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">AxRecon GPU-based Reconstruction</td>
			<td>&#160;Acceleware Corp. CA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">27G Catheter SURFLO Winged Infusion Set</td>
			<td>Terumo Medical Products, USA</td>
			<td style="width:119px;">SV*27EL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">PE20 polyethylyne tubing</td>
			<td>Becton Dickinson, USA</td>
			<td style="width:119px;">427406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen tip 25G &#215; 3.5&#8242;&#8242; Whitacre spinal needle&#160;</td>
			<td>Becton Dickinson, USA</td>
			<td style="width:119px;">405140</td>
			<td>IFP needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P23XL&#160; pressure transducer&#160;</td>
			<td>Harvard Apparatus, CA</td>
			<td style="width:119px;">P23XL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">PowerLab 4/35, Bridge Amp, with LabChart Pro 7.0</td>
			<td>ADInstruments Pty Ltd., USA</td>
			<td style="width:119px;">PL3504, FE221</td>
			<td>IFP acquisition system and acquisition software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:632px;">CT-Sabre Small Animall Intervention system (CT-IFP Robot)</td>
			<td style="width:471px;">Parallax Innovations, CA</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">Manipulated using CT-IFP robot Control Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">CT-IFP robot alignment software</td>
			<td style="width:471px;"></td>
			<td style="width:119px;"></td>
			<td style="width:299px;">Custom Matlab software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">DCE-CT Analysis Software</td>
			<td style="width:471px;"></td>
			<td style="width:119px;"></td>
			<td style="width:299px;">Custom Matlab software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:632px;">Matlab 2013b</td>
			<td style="width:471px;">Mathworks, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

spatial measurements of perfusion, interstitial fluid pressure and liposomes accumulation in solid tumors

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and elevated IFP are linked to the heterogeneous intra-tumoral distribution of nanotechnology-based drug delivery systems such as liposomes. In the present study, the relationship between tumor microcirculation, elevated IFP, and accumulation of nanoparticles was investigated through in vivo experimentation. This was accomplished by evaluation of the tumor microcirculation using dynamic contrast enhanced computed tomography (DCE-CT) and measurement of tumor IFP using a novel image-guided robotic needle placement system connected to the micro-CT scanner. The intra-tumoral accumulation of liposomes was determined by CT image-based assessment of a nanoparticle liposomal formulation that stably encapsulate the contrast agent iohexol (CT-liposomes). CT imaging allowed for co-localization of the spatial distribution of tumor hemodynamics, IFP and CT-liposome accumulation in an individual subcutaneous xenograft mouse model of breast cancer. Measurements led to the discovery that perfusion and plasma volume fraction are strong mediators of the intra-tumoral distribution of liposomes. Furthermore, the results suggest that IFP plays an indirect role in mediating liposome distribution through modulating blood flow.

The aforementioned protocol should yield CT-liposomes with an encapsulated concentration of iohexol, mean liposome diameter, and zeta potential of 55 mg ml-1, 91.8 &#177; 0.3 nm and -45.5 &#177; 2.5 mV, respectively. Figure 1a includes representative DCE-CT imaging results, yielding a time series of volumetric data that show the temporal changes in intra-tumoral accumulation of iohexol. Selecting a ROI within the tumor yields a TIC that can be quantified using ...

Watch this Scientific Journal Video about Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors at JoVE.com

Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors

The heterogeneous intra-tumoral accumulation of liposomes has been linked to an abnormal tumor microenvironment. Herein methods are presented to measure tumor microcirculation by perfusion imaging and elevated interstitial fluid pressure (IFP) using an image-guided robotic system. Measurements are compared to the intra-tumoral accumulation of liposomes, determined using volumetric micro-CT imaging.

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and ...

The overall goal of this experiment is to relate the intratumoral accumulation of nanotherapeutics to the properties of the tumor microenvironment, including the tumor microcirculation and the elevated interstitial fluid pressure, or IFP. This method lets us answer important questions about nanomedicines. Questions like what drives heterogeneous uptake of nanoparticles inside a tumor?The main advantage of this technique is that allows for co-localized spatial mapping of the properties of the tumor microenvironment and the intratumoral distribution of nanoparticles. After confirming the appropriate level of anesthesia by topenge, apply ointment to the mouse's eyes and tape the animal's limbs in the prone position on a thin plastic board. Next, insert a custom 27-gauge catheter connected to a 20-centimeter piece of PE10 tubing into the lateral tail vein and secure the tubing with several pieces of tape.Now, fill one one milliliter syringe with at least 200 microliters of computed tomography, or CT liposomes and one one milliliter syringe with at least 150 microliters of a 9-1 ratio by volume of free iohexol mixed with saline. Place the CT liposome syringe in a syringe pump and attach the catheter to the syringe, setting a pump rate of 600 microliters per minute, equivalent to 10 microliters per second. Then place the mouse on the micro CT scanner bed and use the laser positioning system to adjust the tumor so that it will be approximately the same orientation for each scan.Using the CT scanner console software for each imaging protocol of interest, select bright dark from the drop-down menu and click the scan button to initiate the calibration and to initialize the system. To obtain a volumetric anatomical micro CT of the tumor before any contrast agent is injected, first check the CT scanner console software indicator to confirm that the CT scanner safety interlocks have been cleared. Then select the scan that uses an x-ray energy of 80 kilovolts, a tube current of 70 milliamps and captures 1, 000 image projections.Then, initiate the scan. When the scan is finished, set the pump to inject approximately 150 microliters of the liposome solution and press the start button to inject the bolus of CT liposomes at a 55 milligram of iodine per milliliter of solution concentration. Manually flush the catheter with 50 microliters of saline to ensure the entire amount of liposome agent was injected and that the catheter has been cleared.After 10 minutes, perform a second anatomical scan of the tumor, as just demonstrated. To perform a DCE-CT, place a syringe of free iohexol solution into the syringe pump and set the pump to inject 100 microliters of the iohexol at the same injection rate. These injection volumes are higher than the standard of 200 microliters, but the animals are monitored during recovery and no adverse events have been observed.Then, on the CT scanner console, select a five minute dynamic scan using an x-ray energy of 80 kilovolts and a tube current of 90 milliamps, as just demonstrated, that captures 416 image projections every one second for the first 30 seconds followed by 416 image projections every 10 seconds. Capture five seconds of DCE-CT data, then start the injection pump. At the end of the scan, perform a third volumetric anatomical micro CT scan.48-70 hours later, capture anatomical CT images of the liposomes using the same volumetric settings as just demonstrated. To measure the IFP, tape the animal on the CT IFP robot platform such that the tumor is immobilized and accessible to the CT IFP robot system. Obtain an anatomic micro CT scan as just demonstrated.Then load the pre-needle insertion data into the CT IFP robot alignment software and adjust the window and level to visualize the tumor. Click on the rim of the tumor in any image followed by the selection of a second rim location on the adjacent side of the tumor. The software will calculate a series of positions along a linear line between the two points.Then, select the X, Y and Z coordinates for a series of five to eight evenly-spaced positions from the list. Next, flush the IFP system needle with a saline heparin solution and enter the first predetermined needle positions into the X, Y, Z coordinate windows of the CT IFP robot control software. Press the go button to move the robot to the desired location.Then, for each needle position in turn, click the insert needle button to insert the needle into the tissue. Pinch and release the PE20 tubing to confirm good fluid communication between the IFP needle and the tissue and observe that the IFP measurement increases and returns to the pre-pinching value on the IFP acquisition software. Finally, acquire an anatomic CT scan with the needle inserted, clicking the retract needle button at the end of the scan to remove the needle from the tissue.Selecting a region of interest within the tumor yields a time/intensity curve that can be used to yield quantitative estimates of select hemodynamic parameters in the tumor. Segmenting the tumor volume into equally-sized multiple regions of interest allows the quantification of the spatial distribution of these parameters within the tumor volume. The biodistribution of the CT liposomes at 48 hours post-injection can also be observed.As indicated, the agent is still circulating in the vascular system with a substantial uptake observed in the spleen and liver. The intratumoral accumulation of these CT liposomes is heterogeneous with a predominantly peripheral accumulation compared to the center of the tumor tissue. The needle can clearly be identified by high resolution micro CT, allowing the spatial localization of the IFP measurements within the tumor volume.Spatially co-localized measurement of the profusion and the plasma volume fraction demonstrates a significant correlation with the intratumoral accumulation of CT liposomes in subcutaneous tumors. Further, the radial distribution of the IFP correlates with the other hemodynamic measurements, suggesting a complex spatial/temporal relationship between the tumor microcirculation, the IFP and the intratumoral accumulation of the liposomes. While attempting this procedure, it's important to secure the animal on the scanner bed, ensuring minimal tumor motion between IFP measurements.The implications of this work extend to the development of new therapies. The transport of nanoparticles is a key first step in building efficacious therapeutics based on nanomedicine. After watching this video, you should have a good idea of how to spatially map tumor interstitial fluid pressure, the tumor microcirculation, and nanoparticle distribution.

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Medicine

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Imaging Features of Systemic Sclerosis-Associated Interstitial Lung Disease

Early diagnosis of systemic sclerosis-related interstitial lung disease (SSc-ILD) is important to enable treatment to be administered with minimal delay. However, diagnosing SSc-ILD is challenging because key symptoms are non-specific. High-resolution computed tomography (HRCT) of the chest is recognized as a sensitive imaging method for diagnosing and assessing SSc-ILD. Exposure of patients to ionizing radiation may be considered as a limitation, although methodological steps may be taken to moderate this. We present practical recommendations for performing HRCT scans and interpreting the results. Key features of SSc-ILD on HRCT include a non-specific interstitial pneumonia (NSIP) pattern with peripheral ground-glass opacities and extensive traction bronchiectasis. Despite similarities between SSc-ILD and idiopathic pulmonary fibrosis (IPF), HRCT can be used to differentiate between these conditions: in SSc-ILD compared with IPF, there is a greater proportion of ground-glass opacity and fibrosis is less coarse. A dilated, air-filled esophagus with diameter &#62;10 mm, suggestive of esophageal dysmotility is commonly seen in SSc-ILD. Pulmonary artery size greater than the adjacent ascending aorta suggests coexistent pulmonary hypertension. Nodules must be monitored due to the increased risk of lung cancer. A large extent of disease on HRCT (&#8805;20%) or a high fibrosis score suggests an increased risk of mortality. HRCT is central to diagnosing SSc-ILD, and serial assessments can be helpful in monitoring disease progression or treatment response.

Here, we present practical recommendations for performing thoracic high-resolution computed tomography for diagnosing and assessing systemic sclerosis-related interstitial lung disease.

Early diagnosis of systemic sclerosis-related interstitial lung disease (SSc-ILD) is important to enable treatment to be administered with minimal delay. ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Application of Lucilia sericata Larvae in Debridement of Pressure Wounds in Outpatient Settings

Biological therapy using Lucilia sericata larvae has numerous advocates worldwide, yet it is still fairly unknown and not commonly applied in daily practice because of the limited awareness and insufficient experience of medical and nursing personnel. There are case reports suggesting that maggot therapy can be applied and supported by lay caregivers, provided they are supervised and informed by physicians/nurses. The foregoing observation suggests that the method should be considered for implementation by a wider group of caregivers if accepted and meticulously supervised by trained and experienced medical staff. The concerns related to the therapeutic use of maggots in certain regions seem understandable, but are not supported by scientific facts. It should be noted that many therapeutic agents (including brood) used in medicine are of natural origin, and are associated with low production costs and high possibilities of implementation in the course of therapy. By analyzing the literature and using our own clinical and research experience, we have come to conclusions related to using larvae therapy, as a quick and safe method providing cleaning and revitalization in the process of treating wounds of various etiologies, especially pressure ulcers. In the current study, medical-grade Lucilia sericata maggots were applied to remove necrotic tissue from deep pressure sores. The treatment is mostly accepted by both caregivers and patients. In most cases, it is conducted by trained and experienced medical personnel in home and outpatient settings. Over the course of the conducted analyzes involving the collected specimens, there were no statistically significant relationships (p&gt; 0.05) confirmed between the wound surface successfully cleared by brood and variables, such as time from wound formation, location, surface size, and the depth of damage to the tissue structure. The lack of statistical dependence may result from the small size of the studied group. Based on the current findings, we have formulated the following conclusions: Maggot Debridement Therapy (MDT) is a fast and effective method enabling the preparation of the wound bed. The use of MDT in outpatient settings is safe and acceptable for patients and their caregivers.

Application of Lucilia sericata Larvae in Debridement of Pressure Wounds in Outpatient Settings

Maggots of Lucilia sericata were used in the debridement of deep pressure sores in outpatient settings for the treatment of pressure sores involving the complete skin thickness. The technique presented in the article does not pose risks for the patient.

Biological therapy using Lucilia sericata larvae has numerous advocates worldwide, yet it is still fairly unknown and not commonly applied in daily ...