Funding for this research is from Cancer Research UK.

1. Retroviral labeling of cells with fluorescent proteins
<ol>
	<li>Plate retroviral packaging cells (e.g. Phoenix) out at 0.25 x 106 cells per well of a 6-well dish in 10% fetal bovine serum (FBS)/DMEM.</li>
	<li>Transfect cells with retroviral DNA 48 hours later cells using Effectene according to the manufacturers' instructions.</li>
	<li>Rinse wells twice with medium 24 hours later, then add 1.5 ml of 10% FBS/DMEM per well.</li>
	<li>Collect packaged virus in tissue culture medium 48 hours later by pipetting and transferring to 2 mL microcentrifuge tubes.</li>
	<li>Centrifuge at 1600 rpm for 5 min to pellet any cells.</li>
	<li>Remove supernatant to a clean tube and store at -80&deg;C.</li>
	<li>Cells to be retrovirally transduced are plated out at 1.5 x 105 cells per well of a 6 well dish and placed in a humidified incubator at 37&deg;C overnight.</li>
	<li>The next day, remove the media from the cells and add 1 ml of virus stock supplemented with 4 &mu;l polybrene (2 mg/mL) to each well. Replace plates in incubator.</li>
	<li>Following incubation for 5-6 hours, add 2 ml 10% FBS/DMEM to each well and plates are replaced in 37&deg;C inbubator overnight.</li>
	<li>The next day, replace the media and allow cells 24 hours before adding appropriate selective media.</li>
	<li>When confluent (which depends on the growth rate of the cells, and efficiency of the retroviral transduction, but typically takes from 4-14 days), trypsinize cells and sort for fluorescence using non-transduced cells as a reference, collect and pool cells with fluorescence greater than non-transduced cells and freeze aliquots at -80&deg; C for future use.</li>
</ol>
2. Inverse Matrigel invasion assay
<ol>
	<li>Slowly thaw an aliquot of complete Matrigel (i.e. containing growth factors) on ice (Figure 1A).</li>
	<li>Once defrosted, dilute Matrigel 1:1 in ice cold PBS (along with any other additional treatments at 2x concentration in the PBS prior to dilution; Figure 1B). To make it easier to handle Matrigel before polymerization, all plasticware (e.g. tips, tubes, etc.) should be ice cold.</li>
	<li>Insert as many 8 micron pore 6.5 mm diameter uncoated Transwells as required into the wells of a 24 well tissue culture plate, then carefully pipette 100 &mu;l of the diluted Matrigel into the wells and leave to incubate for &tilde;30 mins at 37&deg;C to solidify (Figure 1C).</li>
	<li>During this time, prepare one or more fluorescently-labeled cell suspensions at between 1 to 4 x 105 cells per ml, depending on cell line, from each pre-treated condition (e.g. siRNA, drug treatment) in their normal growth medium (Figure 1D).</li>
	<li>When the Matrigel has solidified, invert the Transwells and pipette 100 &mu;l of the cell suspension onto the upward facing underside of the filter (Figure 1E).</li>
	<li>Carefully cover the Transwells with the base of a 24 well tissue culture plate, making contact with each droplet of cell suspension (Figure 1F).</li>
	<li>Incubate the plate in the inverted state for 4 hours to allow for cell attachment (Figure 1G).</li>
	<li>Following this time, turn the plates right-side-up and wash each Transwell by sequential dipping into 2 x 1 ml serum free medium (Figure 2A).</li>
	<li>Leave the Transwells in a third well containing any drugs or treatments required (Figure 2B).</li>
	<li>Gently pipette 100 &mu;l of 10% FBS/DMEM plus chemoattractant (e.g. EGF at 25 ng/ml) into the Transwell on top of the solidified Matrigel/PBS mixture, replace the lid and incubate for 3-5 days at 37&deg;C with 5% CO2 (Figure 2C).</li>
</ol>
3. Staining and visualization
<ol>
	<li>Cells expressing fluorescent proteins can skip the steps below prior to confocal microscopy</li>
	<li>To image non-fluorescent cells invading Matrigel , place Transwells in fresh 24 well dishes and pipette 1 ml of 4 &mu;M Calcein AM stain solution on top of each Matrigel plug, allowing it to spill over the sides and stain from the top and bottom. Calcein AM (acetoxymethyl ester of calcein) is a live cell dye that stains the entire cells green and requires no fixation.</li>
	<li>Incubate 1 hour at 37&deg;C in 5% CO2 humidified atmosphere, at which point cells are fully stained and ready to be imaged by confocal microscopy.</li>
	<li>Alternatively, non-fluorescent cells invading Matrigel may be fixed and stained as described below in 3.5 to 3.9.</li>
	<li>Transfer each Transwell to a fresh 24-well plate. Overlay 1 ml of 4% para-formaldehyde/0.2% Triton-X 100.</li>
	<li>Incubate at room temperature for 0.5 hour.</li>
	<li>Remove fixative and wash 3 times with 1 ml PBS.</li>
	<li>Remove cytoplasmic RNA with 30 min RNase treatment using 100 &mu;g/ml RNase. Wash 2 times with PBS.</li>
	<li>For visualization add 0.01 mg/ml Propidium Iodide (PI) diluted in PBS and leave at room temperature in the dark for 0.5 hour. Wash 3 times with PBS. At this stage, PI stained transwell can be kept at room temperature in the dark for at least 1 month.</li>
	<li>Precise imaging methods by confocal microscopy are dependent upon available resources. Using an inverted confocal microscope, place Transwell with a small amount of residual PBS onto large coverslip (ensure no bubbles are present) over non-immersion 20 X objective and capture optical sections every 10-15 &mu;m from the bottom of the matrigel plug. Individual optical slices can be used to quantify the extent of invasion (Figure 3A) or to build into 3-dimensional objects using appropriate software such as Volocity (Figure 3B).</li>
</ol>
4. Representative Results
An example of a Z-series of optical slices is shown in Figure 3A. In this instance, cells were stained with Calcein AM and the number of cells invading up from the filter can be seen to decrease with distance. Quantification of invasion can be done by analyzing the ratio of Calcein AM positive pixels to negative pixels at each interval, or by using the fixation/staining method detailed above and counting PI positive nuclei at each position. One advantage of Calcein AM staining is that 3-dimensional reconstructions of cell invasion can be assembled using software such as Volocity, giving a visual depiction of the mode of invasion (Figure 3B). If cells are labeled by expression of fluorescent proteins, then the positions of each color cell can be visualized in 3-dimensional reconstructions, either viewed from the side (Figure 3C) or by making slices through the reconstruction (Figure 3D).
<img alt="Figure 1" src="/files/ftp_upload/3525/3525fig1.jpg" /> 
Figure 1. Schematic diagram of steps involved in setting up inverse invasion assay. A) Matrigel ECM thawed on ice. B) Matrigel is diluted 1:1 with PBS containing any drug treatments at twice final concentration. C) Transwell inserts are placed into multiwell plates, and Matrigel pipetted into each. D) Cell suspensions made at desired concentration. E) Once the Matrigel has set, the plate is inverted and removed, cells are plated onto the underside filter of the Transwell inserts. F) In the inverted position, the multiwall plate is carefully placed over Transwell inserts, making contact with the cell suspension. G) Cells are allowed to adhere to the filter for 4 hours.
<img alt="Figure 2" src="/files/ftp_upload/3525/3525fig2.jpg" /> 
Figure 2. Continuation of the schematic diagram for the inverse invasion assay. A) Once cells have adhered, dip each Transwell into serum-free media twice to remove loose cells. B) Place washed Transwell into a final well containing media plus treatments as required. C) Media containing chemoattractant (e.g. 10% fetal bovine serum) with treatments as required is carefully layered onto Matrigel.
<img alt="Figure 3" src="/files/ftp_upload/3525/3525fig3.jpg" /> 
Figure 3. Representative images of results of inverse invasion assay. A) Optical sections of cells stained with Calcein AM invading Matrigel, taken at 15 &mu;m intervals by confocal microscopy. B) Reconstruction of a 3-dimensional reconstruction of cell invasion from a stack of confocal Z-series images, viewed from the side. Reprinted from reference 10. C) Three-dimensional reconstruction of GFP and RFP-labeled cells invading Matrigel viewed from the side. D) Optical slice through the 3-dimensional reconstruction of GFP and RFP-labeled cells. Reprinted from reference 10.

<ol>
	<li>Olson, M. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin+cytoskeleton+in+cancer+cell+motility.">The actin cytoskeleton in cancer cell motility.</a> Clin. Exp. Metastasis. 26, 273-287 (2008).</li><li>Hoelzinger, D. B., Demuth, T., Berens, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+factors+that+sustain+glioma+invasion+and+paracrine+biology+in+the+brain+microenvironment.">Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment.</a> J. Natl. Cancer. Inst. 99, 1583-1593 (2007).</li><li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331, 1559-1564 (2011).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hallmarks+of+Cancer.">The Hallmarks of Cancer.</a> Cell. 100, 57-70 (2000).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nat. Rev. Cancer. 11, 135-141 (2011).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Methods. Enzymol. 406, 625-643 (2006).</li><li>Croft, D. R., Olson, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+conversion+between+rounded+and+elongated+modes+of+cancer+cell+movement.">Regulating the conversion between rounded and elongated modes of cancer cell movement.</a> Cancer Cell. 14, 349-351 (2008).</li><li>Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-step+pericellular+proteolysis+controls+the+transition+from+individual+to+collective+cancer+cell+invasion.">Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion.</a> Nat. Cell. Biol. 9, 893-904 (2007).</li><li>Hennigan, R. F., Hawker, K. L., Ozanne, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fos-transformation+activates+genes+associated+with+invasion.">Fos-transformation activates genes associated with invasion.</a> Oncogene. 9, 3591-3600 (1994).</li><li>Scott, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIM+kinases+are+required+for+invasive+path+generation+by+tumor+and+tumor-associated+stromal+cells.">LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.</a> J. Cell. Biol. 191, 169-185 (2010).</li></ol>

The authors have nothing to disclose.

Matrigel invasion assays have traditionally been set up with cells placed onto a layer of extracellular matrix protein with chemoattractant-induced motility towards and through a filter at the bottom. Invasiveness was scored as a function of how many cells could be counted on the underside of the filter. Although practically there is little difference with the &ldquo;inverse&rdquo; invasion assay described above, there is considerably more information about the process of invasion that can be determined by visualizing in...

<table border="1">
<tbody>
<tr>
<th>Name of the reagent</th>
<th>Company</th>
<th>Catalogue number</th>
</tr>
<tr>
<td>DMEM (Dulbecco&rsquo;s Modified Eagle&rsquo;s Medium)</td>
<td>GIBCO</td>
<td>21969</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>PAA</td>
<td>A15-101</td>
</tr>
<tr>
<td>Penicillin Streptomycin</td>
<td>GIBCO</td>
<td>15140</td>
</tr>
<tr>
<td>200 mM L-Glutamine (100x)</td>
<td>GIBCO</td>
<td>25050-032</td>
</tr>
<tr>
<td>Puromycin</td>
<td>Sigma-Aldrich</td>
<td>P8833</td>
</tr>
<tr>
<td>0.05&#37; Trypsin EDTA</td>
<td>GIBCO</td>
<td>25300</td>
</tr>
<tr>
<td>Polybrene</td>
<td>Sigma-Aldrich</td>
<td>AL-118</td>
</tr>
<tr>
<td>Lipofectamine 2000 Reagent</td>
<td>Invitrogen</td>
<td>11668019</td>
</tr>
<tr>
<td>6.5mm Transwells, 8.0 &micro;m pore size </td>
<td>Corning</td>
<td>3422</td>
</tr>
<tr>
<td>Complete Matrigel</td>
<td>BD Biosciences</td>
<td>354234</td>
</tr>
<tr>
<td>Calcein AM</td>
<td>Invitrogen</td>
<td>C1430</td>
</tr>
<tr>
<td>RNase</td>
<td>Qiagen</td>
<td>19101</td>
</tr>
<tr>
<td>Propidium Iodide</td>
<td>Sigma-Aldrich</td>
<td>P4864</td>
</tr>
<tr>
<td>Confocal microcope</td>
<td>Leica</td>
<td>SP2MP</td>
</tr>
</tbody>
</table>

modeling and imaging 3-dimensional collective cell invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Watch this Scientific Journal Video about Modeling and Imaging 3-Dimensional Collective Cell Invasion at JoVE.com

Modeling and Imaging 3-Dimensional Collective Cell Invasion

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

modeling-and-imaging-3-dimensional-collective-cell-invasion

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16.6K Views.  University of Strathclyde. Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Video: Modeling and Imaging 3-Dimensional Collective Cell Invasion

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 ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...