The authors wish to acknowledge Prostate Cancer UK for funding SH under the Prostate Cancer UK Centre of Excellence and Travelling Prize Fellowship (TLD-PF16-004) and HP under INNOVATE (PG14-018-TR2). This work was supported by researchers at the National Institute for Health Research University College London Hospitals Biomedical Research Centre.

The protocol adheres to local guidelines and is approved by the UCL/UCLC Biobank Research Ethics Committee (Reference 15/YH/0311).
NOTE: As this method involves the sampling of human tissue, all local procedures regarding ethics and consent must be observed in advance of beginning the protocol. Radical prostatectomy cases may be included if both MRI and biopsy data are available in advance of surgery, with tumor diameter &#8805;5 mm. Cases should be excluded if the index lesion is not well defined, i.e., only diffuse changes are visible by MRI.
1. Prostate Slicing Apparatus
<ol>
	<li>Purchase the prostate slicing apparatus (Table of Materials). Alternatively, print a blade handle using a 3D printer as previously published10. 
	NOTE: The device and disposable blades used here were purchased under material transfer agreement from the Institute of Cancer Research, London, UK.</li>
</ol>
2. Tumor Targeting
<ol>
	<li>Review clinical notes to identify the index lesion as indicated by diagnostic biopsy, e.g., left posterior.</li>
	<li>Review MRI images to measure location of the above tumor.
	<ol>
		<li>Find the sequence where the tumor is most visible in the axial plane, e.g., T2-weighted.</li>
		<li>Scroll through axial images to find the image where the tumor is largest and print image for reference.</li>
		<li>In the corresponding coronal image, measure the distance from the base of the prostate to the selected axial position, and the full length of the prostate from apex to base (mm), and print for reference.</li>
	</ol>
	</li>
</ol>
3. Collection of pProstate
<ol>
	<li>Check patient notes to ensure appropriate informed consent has been obtained for this procedure and any downstream research applications.</li>
	<li>Following radical prostatectomy, collect the prostate in a dry pot. Ensure no formalin or other fixative has been added to the prostate.</li>
	<li>Transfer to a suitable sterile location for sampling, e.g., a laminar flow hood in a pathology laboratory.</li>
	<li>Proceed to sampling as soon as possible if fresh tissue is required. 
	NOTE: For certain applications (e.g., assessment of DNA which should not degrade as quickly as RNA), it may be appropriate to refrigerate the specimen and take samples the next day.</li>
</ol>
4. Specimen Preparation
<ol>
	<li>Prepare laminar flow hood and prostate slicing apparatus according to local decontamination procedures, using sterile technique. Here, spray 70% ethanol and wipe across all surfaces. Use sterile single-use needles and scalpels. Use slicer blades up to three times; wash after each use in hot soapy water, then spray and wipe with 70% ethanol.</li>
	<li>Weigh the prostate (g) using a standard scale.</li>
	<li>Ink the prostate. Paint the left side with blue ink and right side with black ink. Cover the full capsule and seminal vesicles&#160;with ink to later denote the surgical margins. 
	NOTE: Inking procedures may vary locally and can be modified accordingly.</li>
</ol>
5. Prostate Slicing
<ol>
	<li>Assemble the slicing apparatus by inserting the walls perpendicularly into the base of the stand (Figure 1A).</li>
	<li>Place prostate so that the base and apex are facing opposite walls, with the posterior side down and anterior up. Place gold pins around prostate. Push prostate inwards slightly if necessary to get a snug fit, which will support the prostate during slicing.</li>
	<li>Measure prostate length from base to apex, using a ruler, and compare with prostate length as measured by MRI. If the prostate has shrunk, apply an ad hoc correction to the anticipated distance from base to target transverse slice. For example, if the full length of the prostate in the MRI image is 50 mm, but when measured with a ruler at this point it has shrunk to 45 mm, reduce the anticipated slicing position by 10%.</li>
	<li>Measure from the base to the desired transverse slice. Choose the pin that sits closest to this measurement to slice around.</li>
	<li>Wearing chainmail gloves to prevent injury, hold slicing device (Figure 1B), place blades either side of the identified pin and use the spacer to keep blades 5 mm apart. Take slice by slowly and firmly moving the blades downwards, forwards and backwards with long strokes (Figure 1C). Ensure a full slice has been separated before disassembling apparatus.</li>
	<li>Remove walls and pins and carefully take the slice out onto a sterile sheet of cork board using gloves.</li>
</ol>
6. Tissue Sampling
<ol>
	<li>Visually inspect the transverse slice and compare with the axial MRI image. In some cases, the tumor area may appear paler than surrounding tissue.</li>
	<li>Palpate the transverse slice gently. In some cases, the tumor may feel firmer than the surrounding tissue.</li>
	<li>Using the axial MRI image as a guide, select one or more areas for sampling.</li>
	<li>Take biopsy punches of desired area of tissue.
	<ol>
		<li>Using a 6 mm punch, push down on the desired area of tissue.</li>
		<li>Twist the tissue punch on the spot and down against the cork to ensure full separation and use a sharp scalpel to separate if necessary.</li>
		<li>Remove the punch and place into tubes/molds as necessary by ejecting using the plunger.</li>
		<li>Repeat for further tumor and benign samples as required, with separate sterile biopsy punches. Ink the holes where punches were taken in red.</li>
		<li>Note the location of each punch along with the weight of the prostate and any observations on tissue color/firmness.</li>
	</ol>
	</li>
</ol>
7. Submission of Prostate for Local Diagnostics
<ol>
	<li>Pin the prostate to cork with sterile single-use needles prior to fixation in order to prevent tissue shrinkage and warping, which could alter the appearance of the surgical margins.</li>
	<li>Following pinning to cork, submit the prostate to the histopathology department for standard clinical diagnostics.</li>
</ol>
8. Decontamination of Apparatus
<ol>
	<li>Discard all disposable equipment in biomedical waste streams and/or sharps containers as designated locally.</li>
	<li>Decontaminate the laminar flow hood and prostate slicing apparatus in accordance with local risk assessments appropriate to human tissue (e.g., by spraying with 70% EtOH and wiping).&#160;</li>
</ol>

<ol>
	<li>Sooriakumaran, P., Henderson, A., Denham, P., Langley, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+of+obtaining+prostate+tissue+for+gene+expression+profiling.">A novel method of obtaining prostate tissue for gene expression profiling.</a> International Journal of Surgical Pathology. 17 (3), 238-243 (2009).</li><li>Jhavar, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+radical+prostatectomy+specimens+for+correlation+of+data+from+histopathological,+molecular+biological,+and+radiological+studies:+a+new+whole+organ+technique.">Processing of radical prostatectomy specimens for correlation of data from histopathological, molecular biological, and radiological studies: a new whole organ technique.</a> Journal of Clinical Pathology. 58 (5), 504-508 (2005).</li><li>Wheeler, T. M., Lebovitz, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fresh+tissue+harvest+for+research+from+prostatectomy+specimens.">Fresh tissue harvest for research from prostatectomy specimens.</a> The Prostate. 25 (5), 274-279 (1994).</li><li>Esgueva, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+prostate+cancer+biobanking:+toward+a+processing+protocol+amenable+for+the+International+Cancer+Genome+Consortium.">Next-generation prostate cancer biobanking: toward a processing protocol amenable for the International Cancer Genome Consortium.</a> Diagnostic Molecular Pathology. 21 (2), 61-68 (2012).</li><li>Dev, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biobanking+after+robotic-assisted+radical+prostatectomy:+a+quality+assessment+of+providing+prostate+tissue+for+RNA+studies.">Biobanking after robotic-assisted radical prostatectomy: a quality assessment of providing prostate tissue for RNA studies.</a> Journal of Translational Medicine. 9, 121 (2011).</li><li>Tolkach, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blind+Biobanking+of+the+Prostatectomy+Specimen:+Critical+Evaluation+of+the+Existing+Techniques+and+Development+of+the+New+4-Level+Tissue+Extraction+Model+With+High+Sampling+Efficacy.">Blind Biobanking of the Prostatectomy Specimen: Critical Evaluation of the Existing Techniques and Development of the New 4-Level Tissue Extraction Model With High Sampling Efficacy.</a> The Prostate. 77 (4), 396-405 (2017).</li><li>Warren, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+sampling+tissue+for+research+which+preserves+pathological+data+in+radical+prostatectomy.">Method for sampling tissue for research which preserves pathological data in radical prostatectomy.</a> The Prostate. 73 (2), 194-202 (2013).</li><li>Heavey, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PatiEnt+prOstate+samPLes+for+rEsearch,+a+tissue+collection+pathway+utilizing+magnetic+resonance+imaging+data+to+target+tumor+and+benign+tissue+in+fresh+radical+prostatectomy+specimens.">PatiEnt prOstate samPLes for rEsearch, a tissue collection pathway utilizing magnetic resonance imaging data to target tumor and benign tissue in fresh radical prostatectomy specimens.</a> The Prostate. 79 (7), 768-777 (2019).</li><li>Bailey, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VERDICT+MRI+validation+in+fresh+and+fixed+prostate+specimens+using+patient-specific+moulds+for+histological+and+MR+alignment.">VERDICT MRI validation in fresh and fixed prostate specimens using patient-specific moulds for histological and MR alignment.</a> NMR in Biomedicine. 32 (5), 4073 (2019).</li><li>Bourne, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparatus+for+Histological+Validation+of+In+Vivo+and+Ex+Vivo+Magnetic+Resonance+Imaging+of+the+Human+Prostate.">Apparatus for Histological Validation of In Vivo and Ex Vivo Magnetic Resonance Imaging of the Human Prostate.</a> Frontiers in Oncology. 7, 47 (2017).</li></ol>

The authors have nothing to disclose.

Critical steps within this protocol include identification of tumor region for sampling, measurement of prostate, and tissue sampling. Firstly, measurement of the MRI to identify the correct area of the sampling is key. We demonstrate this method in the accompanying video; however, we also recommend confirming measurements with a radiologist in the first instance. Clear clinical notes which point the researcher towards the area of the MRI images that contains the index lesion are ideal. Secondly, measurement of the prostate should be carried out with care, ensuring that the ruler is held at an angle to measure the full length from base to apex, parallel to the anterior of the prostate. Thirdly, tumor areas should be confirmed prior to sampling by visually inspecting the tissue slice in relation to the original MRI image, palpating the tissue (in some cases the tumor area can feel more dense), and visually assessing the color of the tissue (in some cases the tumor will appear more pale than surrounding benign tissue).
This protocol has been carried out in full at UCL/UCLH by non-clinical postdoctoral researchers, a pathology fellow, pathology consultants, and research technicians. In our experience all steps of the protocol can be learned in under ten cases regardless of technical background. However, we recommend training from a radiologist regarding MRI measurement and training from a pathologist regarding slicing in the first instance. The protocol can be modified by using a 3D printed slicing handle, as previously published10.
Potential limitations of the technique include the risk of impeding on diagnosis. Slicing the prostate is a key step, which could impede on grading or positive margin rates if done incorrectly. There are two potential issues here. Firstly, if all of the index lesion is removed and used for fresh tissue experimentation immediately, routine clinical diagnostics will not be carried out for this lesion and the patient may be misdiagnosed as having a lower grade cancer. To avoid this, the researcher should discuss the sampling plan with the consultant pathologist who will routinely review the case, prior to sampling, and agree on the number, and location, of samples to be taken. Small tumors may be excluded locally for this reason. Secondly, if the prostatic capsule is not pinned down correctly to cork board prior to fixation, this could allow the inner tissue to bulge outwards during fixation, altering the surgical margins. This could lead to a false positive margin, where the remaining tumor appears to reside at the capsule purely due to tissue warping.
The significance of this technique with respect to existing methods lies mainly with tumor targeting. A range of methods for sampling radical prostatectomy specimens has been published to date; however, these all rely on a fully or partially random sampling approach1,2,3,4,5,6,7. The use of biopsy and in particular MRI data here has improved efficiency, allowing for reduced sampling with increased confidence of obtaining tumor tissue8.
Future applications of this method allow for the adoption of a wider range of downstream techniques than with previous sampling methods. For example, the availability of fresh tissue that has a high probability of being tumor means that more expensive and/or labor-intensive fresh tissue techniques can be utilized, as many samples are not required to ensure the presence of tumor. This can include and is not limited to, ex vivo culture, ex vivo MRI, advanced imaging and transcriptomics.&#160;

Access to high quality human prostate cancer tissue is a key requirement for driving effective research in the field. There are a number of existing methods to sample prostate tissue following radical prostatectomy for research. Typically these involve using punch biopsies to take random samples from a fresh, frozen or fixed slice of prostate tissue, and retrospectively confirming whether or not tumor is present in each sample by hematoxylin &#38; eosin (H&#38;E) as assessed by a uropathologist1,2,3,4,5. A recent review has compiled an overview of these existing methods6. These methods are useful for certain downstream applications, where tissue can be stored and assessed for tumor content at a later date, such as large scale genomic analyses like the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)4,7. However, these methods could be improved upon in if we were to use magnetic resonance imaging (MRI) and/or biopsy data to target specific areas of the prostate for sampling. This would improve the methodology in two ways; firstly, by reducing the number of tissue samples collected, increasing efficiency and reducing pressure on pathology departments and cost of storage, and secondly, by allowing fresh tissue to be used immediately without the need for immediate confirmation of tumor content, for new state of the art downstream technologies such as live tissue imaging, organoid generation or ex vivo culture. This research need has led to the development of the PEOPLE (PatiEnt prOstate samPLes for rEsearch) method, and the results from the first 84 cases biobanked using PEOPLE were recently published8. A variation of this method has also been published with a three-dimensional (3D) printed slicing apparatus and patient-specific mold, in order to facilitate ex vivo MRI on pre- and post-fixation tissue9,10.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:1040px;" width="1039">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">6 mm biopsy punch</td>
			<td style="width:247px;">Fisher Scientific</td>
			<td style="width:115px;">13404607</td>
			<td style="width:471px;">Disposable biopsy punches for removing 6 mm tissue samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Black Ink</td>
			<td style="width:247px;">Leica Biosystems</td>
			<td style="width:115px;">3801753</td>
			<td>Tissue marking &#38; margin dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Blue Ink</td>
			<td style="width:247px;">Leica Biosystems</td>
			<td style="width:115px;">3801751</td>
			<td>Tissue marking &#38; margin dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Chainmail hand glove</td>
			<td style="width:247px;">Arco</td>
			<td>1456803</td>
			<td>Chainmail gloves to protect hand during slicing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Cork board</td>
			<td style="width:247px;">Fisher Scientific</td>
			<td>12396447</td>
			<td>Cork board for pinning prostate to following sampling procedure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">Needles</td>
			<td style="width:247px;">SLS (Scientific Laboratory supplies)</td>
			<td>SYR6112</td>
			<td>Sterile needles to use to pin tissue to cork board following sampling</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:208px;">Prostate slicing aparatus</td>
			<td style="width:247px;">Insitute of Cancer Research, London</td>
			<td style="width:115px;">NA - must be obtained under MTA</td>
			<td>A kit containing the slicer handle, blades, spacer, base, walls and pins</td>
		</tr>
	</tbody>
</table>

use of magnetic resonance imaging and biopsy data to guide sampling procedures for prostate cancer biobanking

Previous methods for biobanking prostate tissue, following radical prostatectomy, generally involved random sampling. In order to increase efficiency, and enable a greater range of downstream applications, a more targeted method of sampling prostate tissue was developed. Here we use both magnetic resonance imaging (MRI) and biopsy data to target specific areas of the organ for sampling. The method involves use of a previously published prostate slicing device which removes a 5 mm transverse slice from a predetermined region of the prostate, followed by the removal of 6 mm punch biopsies from predetermined areas of this slice. These samples can be stored frozen or fixed for biobanking purposes, or used fresh immediately with 70% confidence of tumor content, as compared with 10% confidence from the random sampling approach. This enables the use of all standard downstream techniques such as genomics, proteomics or histological work, but also work that requires fresh tissue such as live tissue imaging or ex vivo culture.

Fresh prostate tissue sampled using the PEOPLE method can be used for a variety of downstream techniques, including genomic sequencing and ex vivo culture. The first 59 cases sampled using this method have been previously published in comparison with an earlier version of the method, along with initial downstream data8. The time from first slicing the prostate to freezing/fixing the punch biopsies here was approximately 1 min, which was kept to a minimum to avoid degradation of RNA. Time from removal of the prostate to prostate slicing should also be kept to a minimum, though here this took approximately 20 min due to our theatre and pathology labs being in different locations.
Depending on the downstream application, typically at least two samples are taken: one from an area of anticipated tumor tissue and one from an area of anticipated benign tissue. The key measure of success for the sampling method itself is to assess the tumor content in a given sample.
For entry into the 100,000 genomes project, an H&#38;E stained tissue section must be assessed by a uropathologist, and the sample must contain at least 40% tumor cells. Samples that contain less than 40% tumor may still be included in the project if they are successfully macrodissected. Of the first 92 cases sampled in this manner, 64% contained at least 40% tumor and were submitted to the 100,000 Genomes Project without macrodissection. DNA was extracted and was of sufficient yield and quality in all cases (Table 1). An initial subset of 59 of these samples was previously published in comparison with an earlier method8.
For ex vivo culture, matched tumor and benign tissue must be of sufficient quality to withstand 72 h culture without significant degradation. Multiple tissue samples from a total of three patients were cultured successfully8.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60216/60216fig1.jpg" /> 
Figure 1: Prostate slicing apparatus. This apparatus was obtained under material transfer agreement from the Institute of Cancer Research. (A) The walls are inserted perpendicular to the base, and gold pins are inserted into the base surrounding the prostate (prostate not pictured). (B) The replaceable parallel blades are inserted into the blade handle. (C) The blades pass between the gold pins in order to slice a 5 mm section of the prostate. <a href="https://www.jove.com/files/ftp_upload/60216/60216fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td></td>
			<td>n (%)</td>
		</tr>
		<tr>
			<td>Hit (&#62;40% tumor)</td>
			<td>59 (64%)</td>
		</tr>
		<tr>
			<td>Partial hit (5-30% tumor)</td>
			<td>6 (7%)</td>
		</tr>
		<tr>
			<td>Miss (0% tumor)</td>
			<td>27 (29%)</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>92 (100%)</td>
		</tr>
	</tbody>
</table>
Table 1: Tumor hit rate. Tumor hit rate was determined by a consultant pathologist specializing in prostate cancer, following review of H&#38;E stained tissue. Tumor cell content of &#62;40% was determined to be suitable for inclusion in the 100,000 Genomes Project, as per Genomics England guidelines.

Watch this Scientific Journal Video about Use of Magnetic Resonance Imaging and Biopsy Data to Guide Sampling Procedures for Prostate Cancer Biobanking at JoVE.com

Use of Magnetic Resonance Imaging and Biopsy Data to Guide Sampling Procedures for Prostate Cancer Biobanking

This method involves utilization of clinical diagnostic data for prostate cancer patients in order to guide sampling procedures, when biobanking tissue following radical prostatectomy. This overcomes issues with previously published methods around efficiency and availability of fresh tissue for a wider range of downstream applications.

Previous methods for biobanking prostate tissue, following radical prostatectomy, generally involved random sampling. In order to increase efficiency, and ...

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6.1K Views.  University College London. This method involves utilization of clinical diagnostic data for prostate cancer patients in order to guide sampling procedures, when biobanking tissue following radical prostatectomy. This overcomes issues with previously published methods around efficiency and availability of fresh tissue for a wider range of downstream applications.

Video: Use of Magnetic Resonance Imaging and Biopsy Data to Guide Sampling Procedures for Prostate Cancer Biobanking

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). 
The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. 
The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

Cardiac Magnetic Resonance Imaging at 7 Tesla

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher magnetic field strengths and potentially provides improved signal contrast and spatial resolution. While promising results have been achieved, ultra-high field CMR is challenging due to energy deposition constraints and physical phenomena such as transmission field non-uniformities and magnetic field inhomogeneities. In addition, the magneto-hydrodynamic effect renders the synchronization of the data acquisition with the cardiac motion difficult. The challenges are currently addressed by explorations into novel magnetic resonance technology. If all impediments can be overcome, ultra-high field CMR may generate new opportunities for functional CMR, myocardial tissue characterization, microstructure imaging or metabolic imaging. Recognizing this potential, we show that multi-channel radio frequency (RF) coil technology tailored for CMR at 7 Tesla together with higher order B0 shimming and a backup signal for cardiac triggering facilitates high fidelity functional CMR. With the proposed setup, cardiac chamber quantification can be accomplished in examination times similar to those achieved at lower field strengths. To share this experience and to support the dissemination of this expertise, this work describes our setup and protocol tailored for functional CMR at 7 Tesla.

The sensitivity gain inherent to ultrahigh field magnetic resonance holds promise for high spatial resolution imaging of the heart. Here, we describe a protocol customized for functional cardiovascular magnetic resonance (CMR) at 7 Tesla using an advanced multi-channel radio-frequency coil, magnetic field shimming and a triggering concept.

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

Fat-Water Phantoms for Magnetic Resonance Imaging Validation: A Flexible and Scalable Protocol

As new techniques are developed to image adipose tissue, methods to validate such protocols are becoming increasingly important. Phantoms, experimental replicas of a tissue or organ of interest, provide a low cost, flexible solution. However, without access to expensive and specialized equipment, constructing stable phantoms with high fat fractions (e.g., &#62;50% fat fraction levels such as those seen in brown adipose tissue) can be difficult due to the hydrophobic nature of lipids. This work presents a detailed, low cost protocol for creating 5x 100 mL&#160;phantoms with fat fractions of 0%, 25%, 50%, 75%, and 100% using basic lab supplies (hotplate, beakers, etc.) and easily accessible components (distilled water, agar, water-soluble surfactant, sodium benzoate, gadolinium-diethylenetriaminepentacetate (DTPA) contrast agent, peanut oil, and oil-soluble surfactant). The protocol was designed to be flexible; it can be used to create phantoms with different fat fractions and a wide range of volumes. Phantoms created with this technique were evaluated in the feasibility study that compared the fat fraction values from fat-water magnetic resonance imaging to the target values in the constructed phantoms. This study yielded a concordance correlation coefficient of 0.998 (95% confidence interval: 0.972-1.00). In summary, these studies demonstrate the utility of&#160;fat phantoms for validating adipose tissue imaging techniques across a range of clinically relevant tissues and organs.

The purpose of this work is to describe a protocol for creating a practical fat-water phantom that can be customized to produce phantoms with varying fat percentages and volumes.

As new techniques are developed to image adipose tissue, methods to validate such protocols are becoming increasingly important. Phantoms, experimental ...

Quantification of Levator Ani Hiatus Enlargement by Magnetic Resonance Imaging in Males and Females with Pelvic Organ Prolapse

Here we present a protocol to examine the levator ani hiatus in males and females with pelvic organ prolapse, during the Valsalva maneuver and while evacuating acoustic gel, using a horizontally oriented 1.5 T magnetic resonance (MR) scanner. On midsagittal images, the vertical distance of pelvic organs is measured in millimeters relative to the hymen plane (female) and to the lower border of the symphysis pubis (male), preceded by - (above) or + ( below) signs. On axial images, the levator ani area is calculated in square centimeters with a free-hand tracing method from three key images, passing through the midsymphysis (level I), tangential to the lower border of the symphysis (level II), and at the maximal anterior rectal wall bulging (level III). Areas at rest and strained are compared to find evidence of a percentage of increase. The purpose is to provide objective evidence of the maximal extent of pelvic organs descent and hiatus enlargement without the interference of foreign objects or the examiner&#39;s proximity, so as to overcome the limitations of pelvic examination and transperineal sonography (i.e., subjectivity and sex-related limitations [female only]).

Here we present a protocol to standardize the measurement of the levator ani hiatus size&#160;by magnetic resonance imaging. The purpose is to extract biomechanical inferences from image analysis by comparing resting and straining values in patients of both sexes with pelvic prolapse, using consistent anatomical bony landmarks.

Here we present a protocol to examine the levator ani hiatus in males and females with pelvic organ prolapse, during the Valsalva maneuver and while ...