This work was supported by a grant from the Jan Kornelis de Cock foundation.

1. Create Silicone Molds for Tumor-simulating Inclusions
<ol>
	<li>Collect solid items of the desired shape and size that can serve as models for tumor-simulating inclusions, e.g., beads or marbles.</li>
	<li>Thoroughly clean the tumor models. To ensure an easy removal from the silicone mold, the tumor models can be sprayed with anti-stick spray or covered with a thin layer of petroleum jelly or beeswax.</li>
	<li>Place each model in a separate thin walled square (plastic) box with a smooth surface. If necessary, fixate the model to the bottom of the box to keep it in position. Use a box that is slightly bigger than the tumor model itself to avoid wasting excessive amounts of silicone.</li>
	<li>Pour the required amount of silicone component A in a mixing bowl and add silicone component B in a 10:1 ratio by weight. Mix both components thoroughly. Optionally, a vacuum pump can be used to remove air bubbles from the silicone mixture.</li>
	<li>Gently pour the silicone mixture in the plastic box to prevent trapping air bubbles. The silicone mixture should be processed within 45 min to obtain optimal results.</li>
	<li>Let the silicone mixture solidify for at least 6 hr before cutting the mold and removing the tumor model. Optionally, the silicone mold can be cut in a zigzag pattern to allow it to fit back together cleanly. Maximum strength of the silicone is obtained after 3 days.</li>
</ol>
2. Create Tris-buffered Saline Solution
<ol>
	<li>Create a Tris-buffered saline (TBS) solution by adding 6.1 g (50 mM) Tris and 8.8 g (150 mM) NaCl to 800 ml deionized water.</li>
	<li>Add 1.0 g (15 mmol) of NaN3 to block oxygenation of hemoglobin (step 3.3 and 4.4) and to inhibit bacterial growth. CAUTION: NaN3 is a severe poison. It may be fatal in contact with skin or if swallowed. The toxicity of this compound is comparable to that of soluble alkali cyanides and the lethal dose for an adult human is about 0.7 g. Always follow the safety instructions as provided by the manufacturer.</li>
	<li>Adjust the pH to 7.4 and bring the volume to 1,000 ml with deionized water.</li>
</ol>
3. Create Fluorescent Inclusions
<ol>
	<li>Add 2 g agarose to 50 ml TBS from step 2. The higher melting point of agarose compared to gelatin (step 4.2) will prevent the inclusions from dissolving and leaking fluorescent dye when placed in melted gelatin. Optionally, the amount of added agarose can be altered to 1 or 3 g to obtain softer or palpable tumor inclusions, respectively.</li>
	<li>Heat the agarose slurry using a microwave until the boiling point is reached. Stir thoroughly until the agarose is completely dissolved.</li>
	<li>Add 1.1 g (17 &#181;mol) hemoglobin and 5 ml intralipid 20% dissolved in 50 ml of TBS to the agarose mixture under constant stirring to resemble the optical characteristics of the surrounding breast phantom tissue (step 4).</li>
	<li>Add 20.0 mg (25.8 &#181;mol) of the fluorescent dye indocyanine green to 83.8 ml deionized water. Make sure the dye is completely dissolved.</li>
	<li>Pipet 5.0 ml from this solution and add it to the agarose mixture to obtain a final concentration of 14 &#181;M. Optionally, other fluorescent dyes than ICG can be used if desired with their own optimum concentration.</li>
	<li>Gently fill the silicone molds created in step 1 with the hot agarose mixture using a syringe (Figure 1A). Repeat this process until all molds are filled.</li>
	<li>Let the fluorescent inclusions solidify at RT for approximately one hr. Protect the inclusions from light by covering the entire mold with aluminum foil.</li>
	<li>After solidification, gently open the mold and press out the inclusion (Figure 1B). Optionally, use the tip of the syringe to apply small drops of melted agarose mixture on the surface of the inclusion. By repeating this process several times on the same location, small tumor spurs can be created to simulate infiltrative tumors.</li>
	<li>Protect the agarose inclusions from light and dehydration by wrapping them in aluminum foil and store them in a humidified storage container at 4 &#176;C. 
	NOTE: The use of lower or higher fluorescent dye concentrations than the known concentration optimum will both result in diminished fluorescent signal intensity. The seemingly counterintuitive reduction in signal intensity with increasing dye concentrations above the optimum fluorescent dye concentration is due to a phenomenon known as quenching. When assessing the maximal depth penetration of a fluorescent dye in phantoms, using the optimal concentration is mandatory.</li>
</ol>
4. Create Breast Phantoms
<ol>
	<li>Obtain a cup-shaped mold to create breast phantoms of the desired size and volume, e.g., a glass or plastic bowl. The mold should have a smooth surface to prevent the gelatin form adhering to the mold. A mold volume of 500 ml will create breast phantoms of sufficient size.</li>
	<li>To create a breast phantom with a volume of 500 ml, add 50 g of gelatin 250 bloom to 500 ml TBS (step 2). Heat the gelatin slurry to 50 &#176;C under constant stirring.</li>
	<li>Once the gelatin is completely dissolved, let the gelatin mixture gradually cool down and maintain it at a constant temperature of 35 &#176;C using a hot water bath.</li>
	<li>Under constant stirring, add 5.5 g (85 mmol) bovine hemoglobin and 25 ml intralipid 20% to simulate absorption and scattering of photons in tissue, respectively.</li>
	<li>Prechill the cup-shaped mold at 4 &#176;C for at least 1 hr. Next, pour the gelatin mixture in the mold to a level that corresponds to the predefined depth of the agarose tumor-simulating inclusion (Figure 1C). Let the gelatin mixture solidify at 4 &#176;C for 30 min to one hr.</li>
	<li>After solidification, position a tumor-simulating fluorescent agarose inclusion on the surface of the phantom and temporarily fixate the inclusion with a small needle. Up to a maximum of three tumor-simulating fluorescent inclusions can be incorporated in a single breast phantom. Sufficient space (at least 5 cm) should be kept between individual tumor-simulating inclusions (Figure 1D).</li>
	<li>Pour the remainder of the warm gelatin mixture in the remaining mold volume, allowing for adherence of both layers without creating refraction artifacts. Mark the location of the fluorescent tumor-simulating inclusions on the mold. Let the phantom solidify O/N at 4 &#176;C.</li>
	<li>Once solidified, remove the needles used for temporary fixation of the inclusions and gently remove the breast phantom from its mold (Figure 1E). Protect the breast phantom from light and dehydration by wrapping it in aluminum foil and store it in a humidified storage container at 4 &#176;C.</li>
</ol>
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51776/51776fig1highres.jpg" width="500" /> 
Figure 1.&#160;Sequential steps of creating breast phantoms containing fluorescent tumor-simulating inclusions. After creating silicone molds of the desired shape and size, the molds are filled with melted agarose mixture using a syringe (A). Tumor-simulating inclusions of differing size and shape were produced in the current study (B). Next, a thin layer of melted gelatin mixture is poured in a customized coated wooden breast mold (C). After solidification, the tumor-simulating inclusions are positioned, temporarily fixated, and covered with another layer of melted gelatin mixture (D). After solidification, the breast phantom is gently removed from its mold (E). The phantom can then be applied for simulating various NIRF imaging applications (F). <a href="https://www.jove.com/files/ftp_upload/51776/51776fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Set the NIRF Camera System
<ol>
	<li>A NIRF camera system for intraoperative application is required for simulating targeted NIRF imaging in breast cancer surgery. Several NIRF imaging systems for real-time intraoperative NIRF imaging are currently available for investigational use. Although some differences between these devices exist, they all contain an excitation light source (for excitation of the fluorescent tumor inclusions) and a highly sensitive imaging device for detection of emitted photons.</li>
	<li>Make sure to use an excitation light source of a sufficient wavelength. For tumor-simulating inclusions containing ICG, use an excitation light source (e.g., laser) that emits photons between 750 and 800 nm. If an alternative fluorescent dye is used, the excitation wavelength should be adjusted conform the manufacturer&#8217;s instructions.</li>
	<li>In case the NIRF camera system contains an emission filter to filter out unwanted background signals, make sure that the correct filter is used. For tumor-simulating inclusions containing ICG, use an emission filter between 800 and 850 nm. Alternative fluorescent dyes may require different emission filters, depending on the manufacturers&#8217; instructions. 
	NOTE: Make sure that there is zero overlap between the excitation and the emission wavelengths to prevent oversaturated images. In addition, the image acquisition time might have to be adjusted to obtain optimal fluorescent images. In the case of deep seated fluorescent inclusions or weak fluorescent signals, image acquisition time can be increased for up to several sec to min. In the case of superficial inclusions or strong fluorescent signals, acquisition time can be decreased to several msec to allow for video-rate fluorescence imaging in real-time.</li>
</ol>
6. Simulation of NIRF Imaging Applications in Breast Cancer Surgery
<ol>
	<li>Take the tissue-simulating breast phantom from its container and place it on a flat nonfluorescent surface. Next, position the NIRF imaging device above the breast phantom, leaving a sufficient working distance for excision of the tumor-simulating inclusions.</li>
	<li>Localize the tumor-simulating fluorescent inclusion using NIRF imaging and/or palpation of the phantom breast. In case no fluorescent signal can be detected, the inclusion is either positioned too deep in the phantom for detection or the image acquisition time should be increased.</li>
	<li>Once the inclusion is localized, incise the phantom breast and remove the tumor-simulating inclusion under real-time NIRF-guidance using conventional surgical instruments. Alternatively, the inclusion can be excised guided solely by visual inspection and palpation of the breast phantom to simulate the standard-of-care.</li>
	<li>Directly after removal of the tumor-simulating inclusion, image the surgical cavity for any remaining fluorescent activity indicating inadequate excision.</li>
	<li>In case of any remaining fluorescent activity, excise the inclusion remnant under direct NIRF guidance until no fluorescent signal is left.</li>
	<li>Image the excised phantom fragments to simulate NIRF-guided macroscopic margin status assessment. Hereto, slice the phantom tissue in 3 - 5 mm plaques and image the plaques accordingly. Fluorescence signal reaching into the surgical margins indicates the existence of positive surgical margins.</li>
</ol>

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The authors have nothing to disclose.

We simulated potential clinical applications of NIRF-guided BCS through the use of breast-shaped phantoms with integrated tumor-simulating inclusions. Intraoperative tumor localization, NIRF-guided tumor resection, evaluation on the extent of surgery, and postoperative assessment of surgical margins were all found feasible using a custom-build NIRF camera system. Noninvasive detection of fluorescent tumor-simulating inclusions was only feasible for inclusions positioned in the phantom tissue at a depth of 2 cm or less. I...

Breast-conserving surgery (BCS) followed by radiotherapy is the standard treatment for breast cancer patients with T1-T2 breast carcinoma1,2. Inaccuracies in intraoperative assessment of the extent of surgery result in positive surgical margins in 20 to 40% of the patients who underwent BCS, necessitating additional surgical intervention or radiotherapy3,4,5. Although extensive resection of adjacent healthy breast tissue might reduce the frequency of positive surgical margins, this will also hamper cosmetic outcome and increase comorbidity6,7. Novel techniques are therefore needed that provide intraoperative feedback on the location of the primary tumor and the extent of surgery. Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. Recently, our group reported on the first in-human trial of tumor-targeted fluorescence imaging in ovarian cancer patients, showing the feasibility of this technique to detect primary tumors and intraperitoneal metastases with high sensitivity8. Before proceeding to clinical studies in breast cancer patients, however, the feasibility of various tumor-targeted NIRF imaging applications in BCS can already be evaluated preclinically using phantoms.
The following research protocol describes the use of NIRF imaging in tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions9. The phantoms provide an inexpensive and versatile tool to simulate pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, assessment of the surgical margin status, and detection of residual disease. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. During the simulated surgical procedure, the surgeon is guided by tactile information (in the case of palpable inclusions) and visual inspection of the operative field. In addition, NIRF imaging is applied to provide the surgeon with real-time intraoperative feedback on the extent of surgery.
It should be emphasized that NIRF imaging requires the use of fluorescent dyes. Ideally, fluorescent dyes should be used that emit photons in the near-infrared spectral range (650 - 900 nm) to minimize absorption and scattering of photons by molecules physiologically abundant in tissue (e.g., hemoglobin, lipids, elastin, collagen, and water)10,11. Moreover, autofluorescence (i.e., the intrinsic fluorescence activity in tissues due to biochemical reactions in living cells) is minimized in the near-infrared spectral range, resulting in optimal tumor-to-background ratios11. By conjugating NIRF dyes to tumor-targeted moieties (e.g., monoclonal antibodies), targeted delivery of fluorescent dyes can be obtained for intraoperative imaging applications.
As the human eye is insensitive to light in the near-infrared spectral range, a highly sensitive camera device is required for NIRF imaging. Several NIRF imaging systems for intraoperative use have been developed so far12. In the current study, we used a custom build NIRF imaging system that was developed for intraoperative application in collaboration with the Technical University of Munich. The system allows for simultaneous acquisition of color images and fluorescence images. To improve the accuracy of the fluorescence images, a correction scheme is implemented for variations in light intensity in tissue. A detailed description is provided by Themelis et al.13

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bovine hemoglobin</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>H2500</td>
			<td>Simulates absorption of photons in tissue&#160;</td>
		</tr>
		<tr>
			<td>Intralipid 20%</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>I141</td>
			<td>Simulates scattering of photons in tissue</td>
		</tr>
		<tr>
			<td>Silicone A translucent 40 (2-components poly-addition silicone)</td>
			<td>NedForm, Geleen, The Netherlands</td>
			<td>N/A</td>
			<td>Package consists of components A and B, that should be mixed one on one (A:B=10:1).&#160; Link to manufacturers page: http://tinyurl.com/ncjq7jx</td>
		</tr>
		<tr>
			<td>Gelatine 250 Bloom</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>48724</td>
			<td>Construction of breast-shaped phantoms</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Hispanagar, Burgos, Spain</td>
			<td>N/A</td>
			<td>Construction of tumor-simulating inclusions</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>T1503&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hcl</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>258148</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH3</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>822335</td>
			<td>CAUTION: severe poison. The toxicity of this compound is comparable to that of soluble alkali cyanides and the lethal dose for an adult human is about 0.7 grams.</td>
		</tr>
		<tr>
			<td colspan="2">Examples of NIRF imaging devices for intraoperative application:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T2 NIRF imaging platform&#160;</td>
			<td>SurgVision BV, Heerenveen, The Netherlands</td>
			<td>N/A</td>
			<td>Customized NIRF imaging system used in the current study. More details available at www.surgvision.com</td>
		</tr>
		<tr>
			<td>Photodynamic Eye</td>
			<td>Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany</td>
			<td>PC6100</td>
			<td>www.iht-ltd.com</td>
		</tr>
		<tr>
			<td>FLARE imaging system kit</td>
			<td>The FLARE Foundation Inc, Wayland, MA, USA</td>
			<td>N/A</td>
			<td>www.theflarefoundation.org</td>
		</tr>
		<tr>
			<td>Fluobeam</td>
			<td>Fluoptics, Grenoble, France</td>
			<td>N/A</td>
			<td>www.fluoptics.com</td>
		</tr>
		<tr>
			<td>Artemis handheld camera</td>
			<td>Quest Medical Imaging BV, Middenmeer, the Netherlands</td>
			<td>N/A</td>
			<td>www.quest-mi.com</td>
		</tr>
		<tr>
			<td colspan="2">Examples of NIRF fluorescent dyes for intraoperative application:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Indocyanine green</td>
			<td>ICG-PULSION,&#160; Feldkirchen, Germany</td>
			<td>PICG0025DE&#160;&#160;</td>
			<td>Clinical grade fluorescent dye for NIRF imaging used in the current study. More details available at www.pulsion.com</td>
		</tr>
		<tr>
			<td>IRDye 800CW NHS Ester</td>
			<td>LI-COR Biosciences, Lincoln, NE, USA</td>
			<td>929-70021</td>
			<td>www.licor.com</td>
		</tr>
	</tbody>
</table>

tissue-simulating phantoms for assessing potential near-infrared fluorescence imaging applications in breast cancer surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.

Results from this study have been previously reported elsewhere9.
Our data show that NIRF imaging can be applied to detect fluorescent tumor-simulating inclusions in tissue-simulating breast phantoms, simulating NIRF-guided breast-conserving surgery in breast cancer patients. Using our phantom model, we found intraoperative tumor localization, NIRF-guided tumor resection, intraoperative assessment of surgical cavity margins, and detection of residual disease to be feasible (...

Watch this Scientific Journal Video about Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery at JoVE.com

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

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12.0K Views.  University Medical Center Groningen. Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes. 

Video: Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

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 ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Voluntary Breath-hold Technique for Reducing Heart Dose in Left Breast Radiotherapy

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these techniques, patients hold their breath while radiotherapy is delivered, pushing the heart down and away from the radiotherapy field. Despite clear dosimetric benefits, these techniques are not yet in widespread use. One reason for this is that commercially available solutions require specialist equipment, necessitating not only significant capital investment, but often also incurring ongoing costs such as a need for daily disposable mouthpieces. The voluntary breath-hold technique described here does not require any additional specialist equipment. All breath-holding techniques require a surrogate to monitor breath-hold consistency and whether breath-hold is maintained. Voluntary breath-hold uses the distance moved by the anterior and lateral reference marks (tattoos) away from the treatment room lasers in breath-hold to monitor consistency at CT-planning and treatment setup. Light fields are then used to monitor breath-hold consistency prior to and during radiotherapy delivery.

The current priority in breast cancer radiotherapy is to reduce cardiac doses without compromising target tissue coverage. The voluntary breath-hold technique described here is a simple, inexpensive solution to this problem and capable of being instituted widely without the need for specialized equipment.

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

A Quantitative Sensory Testing Paradigm to Obtain Measures of Pain Processing in Patients Undergoing Breast Cancer Surgery

Chronic pain following surgery, persistent postsurgical pain, is an important highly prevalent condition contributing to significant symptom burden and lower quality of life. Persistent postsurgical pain is relatively refractory to treatment hence generating a high need for preventive strategies and treatments. Therefore, the identification of patients at risk of developing persistent pain is an area of active ongoing research. Recently it was demonstrated that peri-operative disruptions in central pain processing may be able to predict persistent postsurgical pain at long term follow-up in breast cancer patients. The aim of the current report is to present a short protocol to obtain pain thresholds to different stimuli at multiple sites and a measure of endogenous analgesia in breast cancer patients. We have used this method successfully in a clinical context and detail some representative results from a clinical study.

Persistent postsurgical pain may be related to changes in pain processing. By assessing pain in response to standardized stimuli, changes in pain processing can be elucidated. We present methods to obtain pain thresholds to different stimuli and a measure of endogenous analgesia in patients undergoing breast cancer surgery.

Chronic pain following surgery, persistent postsurgical pain, is an important highly prevalent condition contributing to significant symptom burden and ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with &#039;One Stop Shop&#039; Near-infrared Spectroscopy

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...