This study is supported in part by DOD Breast Cancer IDEA Award W81XWH-08-1-0583 and NIH/NCI CA141348-01A1 (DZ) and FAMRI clinical scientist award (DS). Imaging infrastructure is provided by Southwestern Small Animal Imaging Research Program supported in part by U24 CA126608 and Simmons Cancer Center (P30 CA142543) and NIH 1S10RR024757-01.

All animal procedures were approved by the Institutional Animal Care and Use Committee of University of Texas Southwestern Medical Center.
1. In vitro HIF-1&alpha; bioluminescence assay
<ol>
 <li>Materials and Methods:
 <ul>
 <li>Human metastatic breast cancer cell line MDA-MB231 transfected with a novel HIF-1-dependent reporter gene, 5HRE-ODD-luc was generated by Dr. Harada.</li>
 <li>In hypoxic condition, the enhanced expression of oxygen-dependent degradation domain (ODD)-Luciferase fusion protein is driven by 5 copies of hypoxia-response element (5HRE). The presence of ODD causes the degradation of ODD-Luc protein resulting in extremely low background luciferase activity in normoxic conditions. Therefore, this novel system can be used to detect hypoxic regions in a tumor by real time imaging. The construction of this 5HRE-ODD-luc expression vector has been reported by Harada et al1,2.</li>
 </ul>
 </li>
 <li>Culture cells under normoxia or hypoxia:
 <ul>
 <li>Maintain the recombinant MDA-MB231 cells in 10% fetal bovine serum (FBS)-DMEM medium containing 1% glutamine, antibiotics of 400 &mu;g/ml of G418 and 1% penicillin/streptomycin.</li>
 <li>For the in vitro HIF-1&alpha; bioluminescence assay, plate 3 x 105 MDA-MB231 cells expressing 5HRE-ODD-luc vector in each well of two six well dish.</li>
 <li>Allow cells to attach the dish wall after overnight incubation and then transfer one dish into a hypoxia chamber (Billups-Rothenberg, Inc. Del Mar, CA) for hypoxia studies, while keep the other dish under normoxic condition (21% O2).</li>
 <li>Reassemble the chamber and gas the chamber with 0.1% O2 by connecting the inlet port tubing with a gas cylinder. 
 Note: Both inlet and outlet port clamps must be open during this procedure.</li>
 <li>Disconnect gas source after chamber has been purged and seal chamber by closing plastic clamps.</li>
 <li>Place the chamber in a 37 &deg;C incubator with 5% CO2. 
 Note: The chamber must be humidified to prevent excessive evaporation of cultures. This can be accomplished by placing 10 - 20 ml sterile water in the chamber.</li>
 </ul>
 </li>
 <li>Bioluminescence assay:
 <ul>
 <li>After 24 hr incubation, remove the medium and wash the cells quickly with ice cold PBS (2X).</li>
 <li>Add 1 ml of cold PBS with 100 &mu;l of Luciferin (Gold Biotechnology, St Louis, MO).</li>
 <li>Acquire BL imaging (IVIS Spectrum system, Caliper Life Sciences, Hopkinton, MA) with various exposure times (1, 30, 60, 180 s).</li>
 <li>Measure light intensity in each well using the Living Image software (Caliper Life Sciences).</li>
 </ul>
 </li>
</ol>
2. Establishment of a breast cancer brain metastasis model
<ol>
 <li>Preparation of the MDA-MB231/5HRE-ODD-luc cells
 <ul>
 <li>Retrieve and culture the MDA-MB231/5HRE-ODD-luc cells in DEME medium containing 10% FBS, 1% glutamine and 1% penicillin/streptomycin.</li>
 <li>Replace medium every 2-3 days. Tripsinize and wash the cells when they reach 80% confluence.</li>
 <li>Count appropriate number of cells and resuspend them in serum free DEME medium with 25% Matrigel (BD Biosciences, San Jose, CA) with a final concentration of 105 cells in 4 &mu;l volume.</li>
 <li>Place cells on ice prior to intracranial injection.</li>
 </ul>
 </li>
 <li>Surgical implantation
 <ul>
 <li>Female nude mice (BALB/c nu/nu; National Cancer Institute, Bethesda, MD) at 4-6 weeks old are used in this study.</li>
 <li>Anesthetize (3% isoflurane/ O2 in an induction chamber; isoflurane from Baxter International Inc., Deerfield, IL, USA) and maintain the animals with isoflurane (1%) in oxygen (1 dm3/min) during the surgical procedure3.</li>
 <li>The right parietal skin should be prepped with betadine and then 70% alcohol prior to incision.</li>
 <li>Using a high-speed drill, burr a 1 mm hole in the right hemisphere of the skull, 1mm anterior to the coronal suture and 2 mm lateral to saggital suture.</li>
 <li>Draw 4 &mu;l cell mixture (105 cells) using a 10 &mu;l Hamilton syringe (Hamilton Company, Reno, NV). Inject the cells directly into right caudal diencephalon, 1.5 mm beneath the dura mater using a custom-made 32G Hamilton needle. Keep the needle in place for about 30 s before withdrawal. Usage of a 32G fine needle minimizes tissue damage.</li>
 <li>Fill the burr hole with bone wax and close the scalp with absorbable sutures.</li>
 <li>Prepare the incision region with 70% alcohol.</li>
 <li>Apply buprenorphine analgesia every 12 hrs for two days.</li>
 </ul>
 </li>
</ol>
3. In vivo bioluminescence imaging of tumor hypoxia dynamics in breast cancer brain metastasis
<ul>
 <li>Initiate longitudinal bioluminescence imaging (IVIS Spectrum system) two weeks after intracranial implantation and repeat once a week for 8-10 weeks.</li>
 <li>Anesthetize three mice at a time (3% isoflurane/ O2 in an induction chamber)</li>
 <li>Administer a solution of D-luciferin (120 mg/kg in PBS in a total volume of 80 &mu;l; Gold Biotechnology) subcutaneously in the neck region of each mouse as described in detail previously4.</li>
</ul>
D-luciferin is non-toxic and has been shown to be capable of penetrating intact blood-brain barrier (BBB) and cell membranes3,5.
<ul>
 <li>Place the 3 mice in the imaging chamber and maintained with isoflurane (1%) in oxygen (1 dm3/min) during imaging.</li>
 <li>Five minutes after luciferin injection, acquire BL imaging with an array of various exposure time (1, 30, 60, 180 s).</li>
</ul>
Our observations show that the peak light emission time is about 5 mins after subcutaneous administration of luciferin in the neck region3,4.
<ul>
 <li>Analyze data with the Living Imaging software (Caliper Life Sciences) by using absolute photon counts (photons/s) in a region of interest (ROI), manually drawn to outline the BLI signal of the brain.</li>
 <li>Plot time course curve of photon counts to indicate tumor hypoxia dynamics.</li>
 <li>Immediately after the last BLI, administer pimonidazole, the hypoxia marker and 1 hr later, sacrifice the mice and dissect the brains. Embed the whole brains in Optimal Cutting Temperature (O.C.T.) medium and freeze in -80 &deg;C freezer. Subsequent histological Cresyl violet staining and immunohistochemical staining against luciferase, hypoxia marker pimonidazole, and HIF-1&alpha; performed to validate imaging observations.</li>
</ul>
4. Representative results:
As shown in Fig.1, significantly higher BLI signal was observed after the transfected cells incubated in the hypoxic chamber (1% O2) for 24 hrs. Weak light emission was observed from the control cells under normoxia. This may result from the overcrowded cell population after 24 hr culture of 3 x 105 cells, which induced a stress signal to the cells to overexpress HIF-1&alpha;. Nonetheless, in vivo BLI results were validated by immunohistochemical staining showing colocalization between tumor hypoxia and luciferase expression.
An automated array of exposure times enables continuous acquisitions of a series of images, which facilitates capture of a weak signal with longer exposure time, and strong signals using a shorter time without saturating the CCD.
<img alt="Figure 1" src="/files/ftp_upload/3175/3175fig1.jpg" /> 
Figure 1 In vitro HIF-1&alpha; bioluminescence assay. Top row: 3 x 105 MDA-MB231/5HRE-ODD-luc cells incubated in each well of a 6-well-dish in a hypoxia chamber (0.1% O2) for 24 hr before the medium was removed, washed and replaced with 1 ml PBS. Immediately after 100 &mu;l luciferin was added into each well, BLI was acquired with an array of exposure times (1, 30, 60, 180 s). Strong luminescence was observed from representative wells. Bottom row: As a control, 3 x 105 cells incubated under normoxia (21% O2) emitted weak light.
<img alt="Figure 2" src="/files/ftp_upload/3175/3175fig2.jpg" /> 
Figure 2 In vivo bioluminescence imaging of tumor hypoxia dynamics. A) A weak light signal from right side of mouse brain was first visualized 5 weeks after intracranial implantation of MDA-MB231/5HRE-ODD-luc breast cancer cells. Increased optical signal was observed over additional 6 weeks, indicating increased tumor hypoxia. B) The plot showed the time course curve of quantitative photon counts of light signal.
<img alt="Figure 3" src="/files/ftp_upload/3175/3175fig3.jpg" /> 
Figure 3 Colocaliztion of luciferase and hypoxia detected by immunohisto-chemical staining. A frozen mouse brain bearing a metastasis of breast cancer MDA-MB231/5HRE-ODD-luc embedded in O.C.T. was sectioned. A 10 &mu;m section immunostained with hypoxic marker, pimonidazole, revealed intratumoral heterogeneity of hypoxia, which was found to correlate spatially with luciferase expression detected by anti-luciferase staining. Scale bar, 100 &mu;m.

<ol>
	<li>Harada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combination+of+hypoxia-response+enhancers+and+an+oxygen-dependent+proteolytic+motif+enables+real-time+imaging+of+absolute+HIF-1+activity+in+tumor+xenografts.">The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts.</a> Biochem Biophys Res Commun. 360, 791-796 (2007).</li><li>Ou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+HIF-1+imaging+for+determining+optimal+timing+of+combining+bevacizumab+and+radiotherapy.">Usefulness of HIF-1 imaging for determining optimal timing of combining bevacizumab and radiotherapy.</a> Int J Radiat Oncol Biol Phys. 75, 463-467 (2009).</li><li>Zhou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+near-infrared+optical+imaging+of+2-deoxyglucose+uptake+by+intracranial+glioma+of+athymic+mice.">Dynamic near-infrared optical imaging of 2-deoxyglucose uptake by intracranial glioma of athymic mice.</a> PLoS One. 4, e8051-e8051 (2009).</li><li>Contero, A., Richer, E., Gondim, A., Mason, R. 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No conflicts of interest declared.

Breast cancer brain metastasis occurs in 30% of breast cancer patients at stage IV. It is associated with high morbidity and mortality and has a median survival of 13 months6. There is a need to have appropriate animal models to mimic this clinically devastating disease in order to facilitate our understanding of its intracranial initiation and progression as well as pathophysiological profiles. Here, we have developed an orthotopic breast cancer brain metastasis model by injecting human breast cancer cells, d...

<table border="1">
<tbody>
<tr>
<th>Name of the reagent</th>
<th>Company</th>
<th>Catalogue number</th>
<th>Comments (optional)</th>
</tr>
<tr>
<td>D-luciferin</td>
<td>Gold Biotechnology </td>
<td>L-123</td>
<td>120 mg/kg in PBS in a total volume of 80 &mu;l for in vivo study</td>
</tr>
<tr>
<td>Isoflurane</td>
<td>Baxter International Inc.</td>
<td>1001936060</td>
<td></td>
</tr>
<tr>
<td>Matrigel</td>
<td>BD Biosciences</td>
<td>354234</td>
<td></td>
</tr>
<tr>
<td>Hamilton syringe</td>
<td>Hamilton Company</td>
<td>1701</td>
<td></td>
</tr>
<tr>
<td>32G Hamilton needle</td>
<td>Hamilton Company</td>
<td>7803-04</td>
<td></td>
</tr>
<tr>
<td>Hypoxia chamber</td>
<td>Billups-Rothenberg, Inc.</td>
<td>MIC-101 </td>
<td></td>
</tr>
<tr>
<td>Bioluminescence imaging system</td>
<td>Caliper Life Sciences</td>
<td>IVIS Spectrum system</td>
<td></td>
</tr>
<tr>
<td>G418</td>
<td>Fisher scientific</td>
<td>SV3006901</td>
<td></td>
</tr>
</tbody>
</table>

in vivo bioluminescence imaging of tumor hypoxia dynamics of breast cancer brain metastasis in a mouse model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

Watch this Scientific Journal Video about In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model at JoVE.com

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

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19.5K Views.  University of Texas Southwestern Medical Center. Bioluminescence imaging of hypoxia inducible factor-1α activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Video: In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

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 ...

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 ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

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 ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

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

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

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

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

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