Name: chemotherapy-induced vascular toxicity - real-time in vivo imaging of vessel impairment
Uploaded: 2015-01-07T13:00:00.000+00:00
Duration: 4 min 48 s
Description: Watch this Scientific Journal Video about Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment at JoVE.com

Ethics statement: All experiments were approved by the Institutional Animal Care and Use Committee. Animal care was according to institutional guidelines. ICR female mice (7 - 8 weeks old;&#160;25 - 30 g) were housed in air conditioned, light controlled animal facilities of the Sackler Faculty of Medicine in Tel-Aviv University. At term, animals were euthanized with anesthesia overdose.
1. Fibred Confocal Fluorescence Microscopy (FCFM) Calibration
<ol>
	<li>Turn the device ON.</li>
	<li>Connect the microprobe (mini0/30).</li>
	<li>Calibrate the device according to manufacturer&#8217;s instructions.</li>
</ol>
2. Mice Preparation for Imaging&#160;
<ol>
	<li>Anesthetize&#160;by a subcutaneous injection of both Ketaset (100 mg/kg) and&#160;XYL-M2 (6 mg/kg). Confirm proper anesthetization by unresponsiveness to toe-pinch.</li>
	<li>Incise the skin&#160;below&#160;the&#160;groin&#160;in order to&#160;reveal&#160;the femoral arterial vessels. Keep the incision site moist with saline following incision.</li>
	<li>Heat the tail by using a bag (or a glove) filled with warm water (not too hot to touch) for approximately 30 sec. Prepare an intravenous (IV) shunt for administration of FITC dextran (a contrast agent) and of either saline or chemotherapeutic agent, by inserting a needle (30 G, 1/2 inch) into&#160;the&#160;tail vein and attaching a 1 ml syringe to it. Ensure the vein is open by injecting saline. 
	NOTE: An IV administration of FITC dextran (high molecular weight; 100&#160;&#181;l; 10 mg/ml; 2,000 kDa)&#160;facilitates visualizing the femoral microvasculature by FCFM.&#160;Doxorubicin (100 &#181;l; 8 mg/kg, Adriamycin) or saline&#160;will also be later&#160;administered IV into the pre-heated tail vein.</li>
	<li>Position the mouse supinely on a polystyrene stage. Secure the mouse to the pad and maintain position using surgical duct tape.</li>
</ol>
3. Imaging of Femoral Blood Vessels by FCFM During and After Administration of Doxorubicin&#160;or Saline
NOTE: The fibered confocal microscope used in this study is composed of two units: (1) microprobe (mini0/30). (2) Laser scanning unit (LSU-488; 488 nm wavelength).
<ol>
	<li>Perform all time-laps analyses using the LSU 488 nm wavelength laser.&#160; 
	NOTE: The main unit detector detects the filtered (500 - 650 nm) emitted fluorescence. The acquired images&#160;are&#160;reconstructed afterwards and displayed at&#160;a&#160;rate of 12 frames/sec.</li>
	<li>Disconnect carefully the syringe from the needle and attach a new syringe containing FITC dextran. Administer (IV) 100 &#181;l of FITC dextran.</li>
	<li>Shift&#160;the microprobe (mini0/30) to a suitable field of view and fixate it, following adjustment to the z-axis, in order to obtain the corresponding image. Wait for the initial signal to fade until a clear and focused signal is visible.</li>
	<li>Record a baseline blood flow for a short stabilization period (~30 sec). Then connect to the needle another syringe, containing either doxorubicin or saline. IV administer 100 &#181;l doxorubicin or saline.</li>
	<li>Monitor the flow of injected FITC-dextran continuously for 20 min. On the FCFM-associated software, use the diameter button on the upper ruler in order to measure the blood vessels&#160;and&#160;categorize them as small (&#60;15 &#181;m) or large (&#62;15 &#181;m).</li>
	<li>Euthanize the animal with anesthesia overdose.</li>
</ol>

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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=Endothelial+cell+effects+of+cytotoxics:+balance+between+desired+and+unwanted+effects.">Endothelial cell effects of cytotoxics: balance between desired and unwanted effects.</a> Cancer Treat Rev. 30 (6), 495-513 (2004).</li><li>Kerbel, R. 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='Accidental'+anti-angiogenic+drugs.+anti-oncogene+directed+signal+transduction+inhibitors+and+conventional+chemotherapeutic+agents+as+examples.Eur.">'Accidental' anti-angiogenic drugs. anti-oncogene directed signal transduction inhibitors and conventional chemotherapeutic agents as examples.Eur.</a> J Cancer. 36 (10), 1248-1257 (2000).</li><li>Soultati, A., 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=Endothelial+vascular+toxicity+from+chemotherapeutic+agents:+preclinical+evidence+and+clinical+implications.">Endothelial vascular toxicity from chemotherapeutic agents: preclinical evidence and clinical implications.</a> Cancer Treat Rev. 38 (5), 473-483 (2012).</li><li>Tempelhoff, G. F., 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=Blood+coagulation+during+adjuvant+epirubicin/cyclophosphamide+chemotherapy+in+patients+with+primary+operable+breast+cancer.">Blood coagulation during adjuvant epirubicin/cyclophosphamide chemotherapy in patients with primary operable breast cancer.</a> J Clin Oncol. 14 (9), 2560-2568 (1996).</li><li>Ben Aharon, I., 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=Doxorubicin-induced+vascular+toxicity--targeting+potential+pathways+may+reduce+procoagulant+activity.">Doxorubicin-induced vascular toxicity--targeting potential pathways may reduce procoagulant activity.</a> PLoS One. 8 (9), e7515 (2013).</li><li>Laemmel, E., 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=Fibered+confocal+fluorescence+microscopy+(Cell-viZio)+facilitates+extended+imaging+in+the+field+of+microcirculation.+A+comparison+with+intravital+microscopy.">Fibered confocal fluorescence microscopy (Cell-viZio) facilitates extended imaging in the field of microcirculation. A comparison with intravital microscopy.</a> J Vasc Res. 41 (5), 400-411 (2004).</li><li>Al-Gubory, K. H., Houdebine, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+green+fluorescent+protein-expressing+cells+in+transgenic+animals+using+fibred+confocal+fluorescence+microscopy.">In vivo imaging of green fluorescent protein-expressing cells in transgenic animals using fibred confocal fluorescence microscopy.</a> Eur J Cell Biol. 85 (8), 837-845 (2006).</li><li>Bar-Joseph, 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=In+vivo+bioimaging+as+a+novel+strategy+to+detect+doxorubicin-induced+damage+to+gonadal+blood+vessels.">In vivo bioimaging as a novel strategy to detect doxorubicin-induced damage to gonadal blood vessels.</a> PLoS One. 6 (9), e23492 (2011).</li><li>Kaushal, V., Kaushal, G. P., Mehta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+toxicity+of+anthracyclines+on+cultured+endothelial+cells.">Differential toxicity of anthracyclines on cultured endothelial cells.</a> Endothelium. 11 (5-6), 253-258 (2004).</li><li>Kim, E. J., 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=Doxorubicin-induced+platelet+cytotoxicity:+a+new+contributory+factor+for+doxorubicin-mediated+thrombocytopenia.">Doxorubicin-induced platelet cytotoxicity: a new contributory factor for doxorubicin-mediated thrombocytopenia.</a> J Thromb Haemost. 7 (7), 1172-1183 (2009).</li><li>Walsh, J., Wheeler, H. R., Geczy, C. 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Evaluating chemotherapy-induced vascular toxicity is challenging due to the difficulty in visualizing the dynamics of vasculature in response to a stimuli in real-time. Numerous clinical studies have implicated that several chemotherapies cause direct vascular injury, yet the mechanism and characteristics of this toxicity remains to be elucidated. We have established a real-time, in vivo molecular imaging platform for evaluating the potential vascular toxicity of chemotherapy in mice comprising of fibered confocal fluorescent microscopy as described herein8-10. This high-resolution molecular imaging of mice is suitable for visualizing arterial blood flow and vessels&#8217; architecture. It enables real-time detection of treatment-induced complications in the same animal over an extended period of time. We evaluated two classes of chemotherapies: doxorubicin which is known to be toxic to endothelial cells in vitro as well as in tissues obtained from animals treated with doxorubicin10-15, and paclitaxel as a control chemotherapy for which the former evidence for any vascular effect is very limited.
The laser scanning confocal technology of FCFM facilitates tracing fluorescence-dyed deep tissues at real-time and produces time-lapse video images of blood vessels in vivo9. In our study FCFM was used to observe the acute vascular effect of doxorubicin, as a prototype vasculotoxic agent. This effect, which began shortly after doxorubicin administration, was dependent upon blood vessels size: the smaller the vessels&#39; diameter, the more prominent the damage. The fluorescence signal of small-diameter (&#60;15 &#181;m) vessels diminished gradually as a result of constant vessels constriction caused by doxorubicin. No apparent recovery was detected during the next 8 min of real-time imaging. Large diameter (&#62;15 &#181;m) vessels were less damaged; the integrity of their wall was compromised, exhibiting an irregular surface. These effects were unique to doxorubicin and were not evident when paclitaxel was utilized, indicating that this methodology delineates meticulously the specific effect of the drug.
Modifications and troubleshooting
Throughout the protocol, the laser power may be changed during the baseline recording in order to illustrate the vessels. However, laser power must not be changed during the experiment. In addition, a high laser power might bleach the fluorescence agent during time. Additionally, once recording is finished, it is possible to change the contrast of the movie and to edit its length and speed. Measurements of the vessels may then be made and changes that occur may be followed.
Limitations of the technique and critical steps
The FCMF device has few limitations. The region of interest (ROI) might change during the imaging time. Additionally, the imaged area might dry out; one should keep the area hydrated. The fluorescence agent might bleach and the signal will be lost. One critical step one should pay attention to is to keep the animal and the probe well fixed to avoid changes of the ROI.
Significance and future applications
The established experimental platform may serve as an immediate test for response to chemotherapy or alternatively as a potential biological marker to characterize the potential vascular toxicity profile. Based upon the studied mechanism of vascular impairment, the method can also be useful in the future for evaluating potential agents specified to reduce vascular toxicity induced by chemotherapy. The need to decrease the potential long term vascular complications in cancer survivors drives us to explore the mechanism behind chemotherapy-induced vascular toxicity.

Clinical evidence indicates that several classes of chemotherapies elicit a variety of vascular pathologies manifested by Raynaud phenomenon, hypertension, myocardialinfarction, cerebrovascular attack, and hepatic veno-occlusivedisease1,2. &#8220;&#8217;Accidental&#8217; anti-angiogenic drugs&#8221; is a fairly new term, which describes conventional chemotherapeutic agents that act as possible angiogenesis inhibitors, although they not originally developed for this purpose3-5 but designed to eliminate tumor cells by imposing as little &#8220;collateral damage&#8221; to normal cells as possible3. Several chemotherapies have been implied as vasculo-toxicants as observed in clinical studies using serum biomarkers. Among these are alkylating agents (such as cyclophosphamide), platinum compounds (such as cisplatin) and anthracyclines1,2,5-7.
Acute cardiovascular complications may occur as a result of vascular toxicity induced by chemotherapy. They may progress into chronic conditions like atherosclerosis and account for increased risk of late vascular morbidity. Yet, despite mounting clinical evidence, there is a paucity of designated studies emphasizing the mechanism of vascular toxicity and therefore, further elucidation of the exact pathogenesis they inflict is warranted.
A major challenge in revealing the mechanism of chemotherapy-induced vascular toxicity derives from the complexity of investigating vascular function in vivo. We describe herein a platform of high-resolution in vivo molecular imaging in mice that enables to capture blood flow and vessels&#8217; characteristics. This platform facilitates the detection of direct treatment-induced vascular effects: in real-time, as well as following them over a period of time within the same individuals.

<table><tbody><tr><td>general anesthesia</td><td>Fort Dodge Animal Health, IA, USA and Biove Laboratories, France</td><td></td><td>100 mg/kg ketaset and 6 mg/kg XYL-M2</td></tr><tr><td>depilatory cream (Veet)</td><td>ReckittBenckiser, Bristol, UK</td><td></td><td></td></tr><tr><td>30 G, 1/2 inch needle attached to 1 ml syringe</td><td></td><td></td><td></td></tr><tr><td>FITC dextran (10 mg/ml; MW 2,000 kDa)</td><td>Sigma</td><td>FD2000S</td><td>100 &amp;mu;l volume</td></tr><tr><td>Doxorubicin</td><td>Teva, Israel</td><td></td><td>8 mg/kg, Adriamycin</td></tr><tr><td>paclitaxel</td><td>Taro, Israel</td><td></td><td>1.2 mg/kg, Medexel</td></tr><tr><td>saline</td><td></td><td></td><td></td></tr></tbody></table>

chemotherapy-induced vascular toxicity - real-time in vivo imaging of vessel impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

In vivo continuous imaging at real-time
The imaging apparatus used here is a high definition, fibered confocal microscope, equipped with a probe that enables visualization of vasculature and its response to various stimuli as chemotherapy. This method is minimally invasive since although it may facilitate imaging of deep vessels or organ, it requires a small incision for the probe. The probe bundles consist of tens of thousands of fibers, microscope optics and a proprietary precision connector. A schematic representation is shown in Figure 1.
Imaging of femoral microvasculature
We have classified the network of femoral blood vasculature according to vessels diameter (small &#60; 15 &#181;m; large &#62; 15 &#181;m) by FCFM in mice injected with FITC-dextran. A rapid vasoconstriction (2 - 5 min) of small vessels was induced by doxorubicin. A complete disappearance of the FITC-dextran fluorescent, 8 min post doxorubicin treatment (Figure 2A e-g, arrows; Video 1). In several mice, reduction of the fluorescent signal in the blood vessel and increase signal of its surrounding was observed in the perivascular region, a few seconds after doxorubicin administration (Figure 2B g, arrows). These results indicate that increase in vessel permeability and leakage of the high molecular weight dextran from the blood vessel to the surrounding tissues. No fluorescence signal was evident following administration of doxorubicin in mice that were not injected previously with FITC-dextran. Paclitaxel-treated mice demonstrated similar blood vessels structure and flow rate to those observed in saline-injected mice (not shown), throughout the measurement period.
<img alt="Figure 1" src="/files/ftp_upload/51650/51650fig1highres.jpg" width="300" /> 
Figure 1. The FCFM. The confocal microscope used here is composed of two units: the microprobe (mini0/30) and the laser scanning unit (LSU-488; 488 nm wavelength).
<img alt="Figure 2" src="/files/ftp_upload/51650/51650fig2highres.jpg" /> 
Figure 2. Images of FITC-dextran fluorescence signal in femoral microvasculature. Vessels were dichotomously classified to minor (&#60;15 &#181;m) or major (&#62;15 &#181;m) according to their caliber. Femoral microvasculature of FITC dextran (100 &#181;l; 10 mg/ml) injected mice was imaged before and during IV administration of either doxorubicin or saline. (A) The minor vessels, imaged by FCFM, started vasoconstricting acutely 2 min after doxorubicin administration (f, arrow), showing a continuous narrowing of the fluorescent signal until its complete disappearance, with no recovery apparent recovery of the signal during the next eight minutes of the real-time imaging (g, arrow). (B) Appearance, in some snapshots, of a &#34;hazy&#34; area around the blood vessels walls of chemotherapy-injected mice (g arrow) immediately after injection, indicates potential Dextran-FITC leakage. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51650/51650fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1. Femoral microvasculature imaging. A representative photographed film of FITC-dextran fluorescent minor (&#60; 15 &#181;m in diameter) blood vessels. Ovarian and femoral microvasculature time laps photography: mice injected with 100 &#181;l FITC-dextran (10 mg/ml) were imaged and photographed from the moment of IV administration of doxorubicin. 2 - 5 min after doxorubicin injection, minor vessels showed dramatic vasoconstriction followed by complete abolishment of the fluorescent signal, already 8 min after treatment. (AVI) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51650/video1.mpeg" target="_blank">Please click here to view this video.</a>

Watch this Scientific Journal Video about Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment at JoVE.com

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

chemotherapy-induced-vascular-toxicity-real-time-vivo-imaging-vessel

Nanyang Technological University

Research

JoVE Journal

Medicine

7.3K Views.  Tel Aviv University. We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Video: Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

Cancer Research

Guided Tumor Inoculation and Multimodal Imaging in Mice

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Author Spotlight: Integrating High-Resolution Intravital Imaging and MRI to Enhance Stereotactic Body Radiation Therapy Planning

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

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