This work was supported, in part, by National Institutes of Health R01 CA131990 (BFS and RRM). Live-cell imaging was performed in the Microscopy, Imaging and Cytometry Resources Core supported, in part, by NIH Center grant P30 CA22453 to the Karmanos Cancer Institute, Wayne State University and by the Perinatology Research Branch of the National Institutes of Child Health and Development, Wayne State University.

1. Prepare DQ-substrates
<ol>
	<li>Allow lyophilized DQ-substrates to warm to room temperature before opening vials, prepare stock solution of 1 mg/ml of DQ-substrate in deionized water, divide into 50 &mu;l aliquots and store at 4 &deg;C.</li>
</ol>
It may be necessary to agitate DQ-substrate in an ultrasonic water bath for ~5 min and heat to 50 &deg;C to facilitate dispersion.
<ol start="2">
	<li>Thaw rBM on ice overnight at 4 &deg;C; rBM should be handled on ice at all times.</li>
	<li>To make 10X phosphate buffered saline (PBS), dissolve 80 g NaCl (1.37 M), 2 g KCl (0.027 M), 14.4 g Na2HPO4 (1 M), and 2.4 g KH2PO4 ( 0.02 M) in 800 ml ultrapure water. Adjust pH of PBS buffer solution to 7.4 with 1.0 M HCl. Bring volume to 1 liter, sterilize by filtration.</li>
	<li>Prepare collagen I solution on ice: 8 parts of chilled collagen I to 1 part of chilled 10X PBS. Adjust pH of mixture to 7.2-7.6 using sterile 0.1 M NaOH. Check pH with pH strips and adjust the final volume to 10 parts with sterile water.</li>
	<li>Prepare DQ-collagen I:collagen I matrix by diluting DQ-collagen I in collagen I solution in a prechilled tube to a final concentration of 25 &mu;g/ml and 2.4 mg/ml, respectively.</li>
	<li>Prepare DQ-collagen IV:reconstituted basement membrane (rBM) matrix by diluting DQ-collagen IV in rBM (Cultrex or Matrigel) in a prechilled tube to a final concentration of 25 &mu;g/ml and 12-15 mg/ml, respectively. Mix on ice using gentle pipetting to avoid creating bubbles.</li>
</ol>
2. Prepare MAME cocultures
See Figure 1 for a schematic diagram of cocultures.
<ol>
	<li>Place two rectangular plastic coverslips (sterilized in 70% alcohol for 10 min and air-dried) in a 35-mm culture dish or use an IbiTreat 35-mm &mu;-Dish. Directions here are for both coverslips and &mu;-Dishes, which we use for studies on an upright microscope. For studies on an inverted microscope, we use only &mu;-Dishes.</li>
	<li>Mix the desired number of fibroblasts with DQ-collagen I:collagen I matrix. For the long-term cocultures illustrated in Figures 2 and 3, we use 500 fibroblasts in 10 &mu;l of media plus 60 &mu;l of DQ-collagen I:collagen I matrix.</li>
	<li>Using a 100-&mu;l micropipettor, carefully pipette and spread 70 &mu;l of fibroblast:DQ-collagen I:collagen I matrix over the entire surface of each coverslip and leave at 37 &deg;C in a humidified incubator without CO2 for 30 min to solidify.</li>
	<li>Transfer 35-mm dishes containing coverslips to 37 &deg;C incubator with 5% CO2 for 10 min to equilibrate.</li>
	<li>Remove from incubator and leave the 35-mm dishes under the hood until they come to room temperature.</li>
	<li>Add 60 &mu;l of DQ-collagen IV:rBM matrix on top of the solidified DQ-collagen I:collagen I matrix with embedded fibroblasts. With pipette tip, carefully spread DQ-collagen IV:rBM matrix evenly, avoiding scratching the fibroblast:DQ-collagen I:collagen I matrix.</li>
	<li>Transfer 35-mm dishes containing coverslips to 37 &deg;C incubator with 5% CO2 for 10 min to solidify. While the DQ-collagen IV:rBM matrix is solidifying, trypsinize and count epithelial cells.</li>
	<li>Place 50 &mu;l of an epithelial/tumor cell suspension onto coverslips coated with DQ-collagen IV:rBM matrix. Place 35-mm dishes containing the coverslips into a 37 &deg;C incubator. Allow 40 to 60 min for the cells to attach to DQ-collagen IV:rBM matrix. For the long-term cocultures illustrated in Figures 2 and 3, we use 2500 epithelial or tumor cells.</li>
	<li>Add 2 ml of culture medium containing 2% rBM to each 35-mm dish. If doing any drug treatments, the drugs should be added at this time.</li>
	<li>Incubate for desired period of time before imaging. For long-term cultures, media should be changed every 3-4 days. The cells can be maintained in their normal culture medium. For MAME cocultures of breast cell lines, we use Epithelial Basal Medium (MEBM-PRF) supplemented with MEGM SingleQuots.</li>
</ol>
3. Perform 4D (3D + time) imaging of live MAME cocultures by confocal microscopy
<ol>
	<li>For the proof-of-principle studies illustrated, MAME cocultures were labeled with a fluorescent cell tracking dye according to our published procedure1. After washing with PBS, cocultures were incubated with 5 &mu;M CellTracker Orange in MEGM medium for 45 min in a 37 &deg;C incubator, washed again with PBS and incubated with prewarmed MEGMmedium for 30 min in 37 &deg;C with 5% CO2 incubator before imaging.</li>
	<li>Image MAME cocultures live at a low magnification (10X, water dipping lens) on a Zeiss LSM-510 META confocal microscope.</li>
	<li>Optical sections are captured at intervals throughout the entire depth of the structures. For our studies, we capture optical sections at 10 &mu;m intervals.</li>
	<li>Use optical sections to reconstruct images in 3D. We use Volocity software to generate 3D reconstructions.</li>
	<li>Make movies of the 3D reconstructions so that structures, cell:cell interactions and interactions with the surrounding matrices can be visualized. We made movies with QuikTime Player 7.</li>
</ol>
4. Representative Results
The MAME cocultures that we developed are a tractable experimental model for live-cell imaging in real-time of interactions among the various cellular constituents that comprise a breast tumor and its microenvironment. Here we demonstrate the capability of MAME models to recapitulate cell:cell interactions during breast cancer progression and of live-cell imaging to follow those interactions over time. We illustrate the ability to image interactions between a preinvasive MCF10.DCIS human breast cell line and a human breast cancer-associated fibroblast line, WS-12Ti, over a period of more than three weeks in culture. The cocultures were imaged at 3, 16 and 23 days. Optical sections through the entire volume of the cocultures were reconstructed in 3D and representative examples are shown for each of the three timepoints in Figure 2. The red fluorescence in Figure 2 represents the MCF10.DCIS and WS-12Ti cells. To discriminate between the two cell types and for long-term cocultures, one can transfect or transduce individual cell types with fluorescent proteins prior to establishing the cultures. A MAME coculture of MCF10.DCIS and WS-12Ti cells that are differentially labeled with fluorescent proteins for identification is illustrated in Figure 3.
By incorporating substrates, in this case DQ-collagens, into the MAME cocultures, we can assess and quantify changes over time in proteolysis associated with transition to an invasive phenotype. We have established methods for quantifying the fluorescent cleavage products that result from DQ-collagen degradation2,3. We normalize degradation products throughout the entire 3D volume of the MAME cocultures on a per cell basis by labeling and counting cell nuclei. Furthermore, we can localize the degradation to extracellular and intracellular compartments and quantify the total, intracellular and extracellular degradation products as we have described previously2. The green fluorescence in Figures 2 and 3 represents cleavage products of the two DQ-collagen substrates. At this time, differentially-labeled collagens are not available that would allow us to distinguish between degradation of type IV collagen and degradation of type I collagen.
The interactions in the MAME cocultures are dynamic and can change over time. To obtain temporal and dynamic information, the same field of view can be imaged and optical sections taken through the entire specimen of the volume repeatedly over a period of time from minutes to weeks, thus collecting data in 4-D. In Figure 2, we illustrate rather dramatic changes that occur over 23 days: changes in cell number, cell migration and cell interactions, structure volumes, structure shapes, deviations of the structures from sphericity, invasive outgrowths and proteolysis. Furthermore, we also demonstrate an overall change in volume due to the MAME cocultures degrading their surrounding extracellular matrix over this time period. At three days as illustrated in Figure 2A, the volume is 110,000 &mu;m3. In contrast, the volume of the MAME cocultures at 16 and 23 days is only 46,250 &mu;m3, as illustrated in Figures 2B and C.
<img alt="Figure 1" src="/files/ftp_upload/3661/3661fig1.jpg" /> 
Figure 1. Schematic diagram of MAME cocultures. Plastic coverslip is coated with collagen I, containing DQ-collagen I and fibroblasts (magenta). A 2nd layer of reconstituted basement membrane (rBM) containing DQ-collagen IV is added. Tumor cells (red) are plated on top and 2% rBM in culture media is overlaid (white dots) with every change of media. Green represents the fluorescent cleavage products of DQ-collagen I and IV.
<img alt="Figure 2" src="/files/ftp_upload/3661/3661fig2.jpg" /> 
Figure 2. MAME cocultures of MCF10.DCIS human breast cells and WS-12Ti human breast fibroblasts. The fibroblasts were embedded in collagen I/DQ-collagen I and overlaid with rBM containing DQ-collagen IV. The DCIS cells were then seeded on the rBM and overlaid with media containing 2% rBM. Over the 23 days in coculture illustrated here, there was cell proliferation, degradation of DQ-collagen IV and I (green) and collagen matrices as indicated by the reduction in volume of the cocultures. Cells were localized by labeling with CellTracker Orange in situ before imaging (red). The same live cocultures were observed over the 23 days with images captured by confocal microscopy at 3, 16 and 23 days of coculture. The resulting optical slices were reconstructed in 3D with Volocity software. Magnification, 10X.
<img alt="Figure 3" src="/files/ftp_upload/3661/3661fig3.jpg" /> 
Figure 3. MAME 16 day cocultures of MCF10.DCIS human breast cells and WS-12Ti human breast fibroblasts that express RFP (red) and YFP (white), respectively. Cocultures were established and analyzed as in Figure 2. The two cell types shown here had, however, been differentially labeled so that they could be distinguished from one another. The higher magnification images in this figure, as compared to those in Figure 2, illustrate proteolysis of DQ-collagen IV at the surface of the DCIS cells and diffuse proteolysis of DQ-collagen I in areas near the fibroblasts. Magnification, 20X.

<ol>
	<li>Sameni, M., Dosescu, J., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+proteolysis:+stromal+and+inflammatory+cells+increase+tumor+proteolysis.">Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis.</a> Mol. Imaging. 2, 159-175 (2003).</li><li>Jedeszko, C., Sameni, M., Olive, M. B., Moin, K., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+protease+activity+in+living+cells:+from+two+dimensions+to+four+dimensions.">Visualizing protease activity in living cells: from two dimensions to four dimensions.</a> Curr. Protoc. Cell Biol. 39, (2008).</li><li>Jedeszko, C., Victor, B. C., Podgorski, I., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+hepatocyte+growth+factor+promotes+invasion+of+human+mammary+ductal+carcinoma+in+situ.">Fibroblast hepatocyte growth factor promotes invasion of human mammary ductal carcinoma in situ.</a> Cancer Res. 69, 9148-9155 (2009).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+live-cell+imaging+demonstrates+that+beta1-integrin+promotes+type+IV+collagen+degradation+by+breast+and+prostate+cancer+cells.">Functional live-cell imaging demonstrates that beta1-integrin promotes type IV collagen degradation by breast and prostate cancer cells.</a> Mol. Imaging. 7, 199-213 (2008).</li><li>Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p21-activated+kinase+1+coordinates+aberrant+cell+survival+and+pericellular+proteolysis+in+a+three-dimensional+culture+model+for+premalignant+progression+of+human+breast+cancer.">p21-activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer.</a> Neoplasia. 10, 314-328 (2008).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+quantifying+the+dynamics+of+tumor+associated+proteolysis.">Imaging and quantifying the dynamics of tumor associated proteolysis.</a> Clin. Exp. Metastasis. 26, 299-309 (2009).</li><li>Cavallo-Medved, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+demonstrates+that+proteases+in+caveolae+of+endothelial+cells+degrade+extracellular+matrix.">Live-cell imaging demonstrates that proteases in caveolae of endothelial cells degrade extracellular matrix.</a> Exp. Cell. Res. 315, 1234-1246 (2009).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li></ol>

We have nothing to disclose.

As demonstrated, the MAME cocultures can be used for live-cell imaging in real-time of interactions among the various cellular constituents that comprise a breast tumor and its microenvironment. In ongoing studies in our laboratory, we have used MAME cocultures to identify proteolytic pathways associated with transition from DCIS to invasive ductal carcinoma, as well as the interactions between proteolytic pathways and other pathways involved in this transition such as chemokine/cytokine/growth factor pathways. We furthe...

<table border="1">
<tbody>
 <tr>
 <td>Reagent/Equipment</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Reconstituted basement membrane (Cultrex)</td>
 <td>Trevigen</td>
 <td>3445-005-01</td>
 <td>Comparable to Matrigel (BD Biosciences)</td>
 </tr>
 <tr>
 <td>Collagen I</td>
 <td>Cohesion Laboratories</td>
 <td>5005-B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DQ-substrates 
 (collagen I, IV)</td>
 <td>Invitrogen</td>
 <td>DQ-col I-D12060 
 DQ-col&nbsp;IV&nbsp;D12052</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>22-mm plastic coverslips</td>
 <td>Fisher Scientific Co. </td>
 <td>12-547</td>
 <td>Cut in half, leave in 70% ethanol for 10 min and air-dry before use.</td>
 </tr>
 <tr>
 <td>Cell culture medium</td>
 <td>Lonza</td>
 <td>MEBM-PRF&nbsp;CC-3153 
 MEGM CC-4136</td>
 <td>Phenol red&ndash;free</td>
 </tr>
 <tr>
 <td>ibiTreat &mu;-Dish</td>
 <td>Ibidi</td>
 <td>80136</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Volocity software</td>
 <td>Perkin-Elmer</td>
 <td>Version 5.5</td>
 <td>Other brands of imaging software can be used to generate 3D reconstructions and movies.</td>
 </tr>
 </tbody>
</table>

mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

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14.3K Views.  Wayne State University. We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

Video: MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

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 ...

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 ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...