The authors gratefully acknowledge Dr Vasily Gurchenkov (Institut Curie) for image acquiring and processing in Figure 3 and PICT-IBISA Imaging Facility (Institute Curie). This work was supported by ANR-09-JCJC0023-01, ARC SFI20111203863 and PIC 3D &#8211; Complex in vitro cellular models.

1. TAMRA-collagen I Labeling
<ol>
	<li>Prepare a 10 mg/ml TAMRA solution by adding 2.5 ml DMSO to the supplied 25 mg TAMRA powder. Dissolve it by vortexing until complete dissolution. Store at -20 &#186;C and protect from light.</li>
	<li>Prepare 2 L of Labeling Buffer (0.25 M NaHCO3, 0.4 M NaCl). Adjust pH to 9.5 using 10 M solution of NaOH. Keep at 4 &#186;C. From this point, all operations are carried out at 4 &#186;C unless otherwise stated and fluorescent material is protected from light using aluminum foil.</li>
	<li>Fill a 1 ml disposable syringe with 1 ml of highly concentrated rat tail collagen I solution. High concentration collagen solutions are typically provided at concentrations around 10 mg/ml and are very viscous. Be sure to manipulate it slowly to avoid formation of air bubbles.</li>
	<li>Using a 21 G&#160;hypodermic needle, inject the collagen into a presoaked 3 ml dialysis cassette of 10,000 MWCO cut off. Use caution to avoid damaging the membrane with the needle. Remove all air from the dialysis cassette by pulling up the syringe piston. Dialyze it overnight against 1 L of Labeling Buffer.</li>
	<li>Mix 100 &#181;l of 10 mg/ml TAMRA solution with 900 &#181;l of Labeling Buffer. Note: This should be done with both TAMRA solution and Labeling Buffer at room temperature since DMSO freezes at 4 &#186;C. After mixing, bring the diluted TAMRA solution back to 4 &#186;C.</li>
	<li>Carefully remove the collagen from the dialysis cassette using a 2 ml disposable syringe with a 21 G&#160;hypodermic needle. Mix 1 ml of the dialyzed collagen solution with 1 ml of diluted TAMRA solution by pipetting.</li>
	<li>Transfer the collagen/TAMRA mix into a 2 ml microcentrifuge tube and incubate overnight with rotation.</li>
	<li>Transfer the 2 ml of TAMRA-labeled collagen into a presoaked 3 ml dialysis cassette and dialyze overnight against 1 L of Labeling Buffer to remove the excess of free dye.</li>
	<li>To restore the TAMRA-labeled collagen to the collagen original solution, place the dialysis cassette into 1 L of 0.2% (v/v) acetic acid solution, pH 4, and dialyze overnight.</li>
	<li>Measure the final volume of the TAMRA-labeled collagen and calculate its final concentration, considering the initial volume and concentration of the collagen solution used. Store at 4 &#186;C.</li>
</ol>
2. 3D TAMRA-collagen Matrices with Embedded Single Cells
<ol>
	<li>Calculate the volume of 2 mg/ml TAMRA-Collagen Mix necessary for the experiment. Always prepare 20% more to account for pipetting losses due to the collagen high viscosity.</li>
	<li>Prepare a stock solution of 10x PBS and 1 N NaOH. Filter-sterilize and maintain at 4 &#186;C. From this point, all operations are carried out on ice unless otherwise stated and under sterile conditions.</li>
	<li>Mix 10x PBS and 1 N NaOH in appropriate volumes to achieve the desired collagen concentration and pH 7.4. For example, for a final volume of 1 ml of TAMRA-Collagen Mix at pH 7.4, combine 100 &#181;l of 10x PBS with 5 &#181;l of 1N NaOH. Mix well.</li>
	<li>Add the appropriate volumes of both TAMRA-labeled collagen and unlabeled collagen in a 1:6 ratio to achieve a final total collagen concentration of 2 mg/ml. For example, for a final volume of 1 ml of 2 mg/ml TAMRA-Collagen Mix, add 90.48 &#181;l of 3.68 mg/ml TAMRA-labeled collagen and 415.71 &#181;l of 4.01 mg/ml of unlabeled collagen. Mix well and slowly by pipetting, avoiding formation of air bubbles.</li>
	<li>Add cells suspended in the appropriate volume of chilled cell medium without FBS to the TAMRA-Collagen Mix to obtain a final cell density of 105 cells/ml and a final collagen concentration of 2 mg/ml. For example, for 1 ml of 2 mg/ml TAMRA-Collagen Mix, add 388.81 &#181;l of media containing 105 cells. Mix well and slowly by pipetting, avoiding formation of air bubbles. Confirm pH by testing 10 &#181;l of the mix on a pH test strip.</li>
	<li>Pipette 100 &#181;l drops of TAMRA-Collagen Mix onto glass bottom dishes and allow it to polymerize at room temperature for 30-45 min. 100 &#181;l collagen drops have a typical diameter of 7 mm and are 2 mm high. When polymerized, the collagen turns into a white-ish gel. Note: Allowing the collagen to polymerize without closing the dish will avoid the formation of a water film around the collagen drop, reducing detachment from the glass. To increase adherence, glass dishes can be coated with poly-L-lysine at 100 &#181;g/ml.</li>
	<li>Carefully add sufficient culture medium to cover the collagen/cell drops. Avoid high fluxes of media or abrupt movements since collagen drops can easily detach. Keep at 37 &#186;C in 10% CO2 humidified air for enough time for cells to migrate in the matrix (typically 1-3 days).</li>
</ol>
3. 3D TAMRA-collagen Matrices with Embedded Cell Spheroids
<ol>
	<li>Calculate the volume of 2 mg/ml TAMRA-Collagen Mix necessary for the experiment. Always prepare 20% more to account for pipetting losses due to the collagen high viscosity.</li>
	<li>Prepare a stock solution of 10x PBS and 1 N NaOH.&#160; Filter-sterilize and maintain at 4 &#186;C. From this point, all operations are carried out on ice unless otherwise stated and under sterile conditions.</li>
	<li>Mix 10x PBS, 1 N NaOH and cell media without FBS in appropriate volumes to achieve the desired collagen concentration and pH 7.4. For example, for a final volume of 1 ml of TAMRA-Collagen Mix at pH 7.4, combine 100 &#181;l of 10x PBS, 5 &#181;l of 1 N NaOH and 388.81 &#181;l of media. Mix well.</li>
	<li>Add the appropriate volumes of both TAMRA-labeled collagen and unlabeled collagen in a 1:6 ratio to achieve a final total collagen concentration of 2 mg/ml. For example, for a final volume of 1 ml of 2 mg/ml TAMRA-Collagen Mix, add 90.48 &#181;l of 3.68 mg/ml TAMRA-labeled collagen and 415.71 &#181;l of 4.01 mg/ml of unlabeled collagen. Mix well and slowly by pipetting, avoiding formation of air bubbles. Confirm pH by testing 10 &#181;l of the mix on a pH test strip.</li>
	<li>Pipette 100 &#181;l drops of TAMRA-Collagen Mix onto glass bottom dishes and allow it to initiate polymerization for 2-5 min. This will help to prevent sinking of the spheroids by slightly increasing the gel viscosity. 100 &#181;l collagen drops have a typical diameter of 7 mm and are 2 mm high.</li>
	<li>Collect a cell spheroid and place it on a clean Petri dish. Remove any excess of liquid and resuspend it in 10 &#181;l of TAMRA-Collagen Mix. This is important to prevent dilution of the collagen with cell media.</li>
	<li>Using a P20 pipette, collect the collagen suspended spheroid and place it on the center top of the 100 &#181;l TAMRA-Collagen Mix drop. Note: Do not place more than one spheroid per collagen drop, since they can affect each other capacity to invade and/or migrate.</li>
	<li>Allow the TAMRA-Collagen Mix to polymerize at room temperature for 30-45 min. When polymerized, the collagen turns into a white-ish gel. Note: Allowing the collagen to polymerize without closing the dish will avoid the formation of a water film around the collagen drop, reducing detachment from the glass. To increase adherence, glass dishes can be coated with poly-L-lysine at 100 &#181;g/ml.</li>
	<li>Carefully add sufficient culture medium to cover the collagen/spheroids drops. Avoid high fluxes of media or abrupt movements since collagen drops can easily detach.</li>
	<li>Keep at 37 &#186;C in 10% CO2 humidified air for enough time for cells to invade/migrate in the matrix (typically 1-3 days).</li>
</ol>
4. 3D Immunofluorescence Staining
<ol>
	<li>Carefully remove the cell media and rinse the collagen matrices containing cells/spheroids with PBS.</li>
	<li>Simultaneously fix and extract cells by incubating with extraction/fixation buffer (4% PFA, 0.3% Triton X-100, 5% sucrose in PBS) for 5 min. Supplement the buffer with 2 &#181;M Phalloidin and 2 &#181;M Taxol for visualization of cytoskeleton or cytoskeleton associated proteins.</li>
	<li>Further fix cells with 4% PFA, 5% sucrose in PBS for 30 min. Rinse with 0.05% Tween-20 in PBS.</li>
	<li>Prepare primary antibodies solutions in PBS. Incubate cells with primary antibodies for at least 1 hr at room temperature or overnight at 4 &#186;C. Make sure to add enough solution to cover the entire collagen drop. For a 35 mm glass bottom dish, 1.5-2 ml of solution will be necessary. Note: To reduce the volume of antibody solution, a water insoluble barrier, such as a circle line of silicone grease or a PDMS ring, can be placed around the collagen drop.</li>
	<li>Wash 3x 30 min with 0.05% Tween-20 in PBS.</li>
	<li>Prepare Alexa-conjugated secondary antibodies, Alexa-conjugated phalloidin and DAPI solutions in PBS. Incubate cells with the appropriate secondary antibodies for 2 hr at room temperature.</li>
	<li>Wash 3x 30 min with 0.05% Tween-20 in PBS.</li>
	<li>Remove excess of liquid, add enough mounting media to fill the glass bottom dish bottom (approx. 500 &#181;l) and place a 24 mm coverslip on top to seal. Do not press down the coverslip to avoid compressing the collagen drop and damaging the 3D organization.</li>
</ol>
5. Sample Imaging
Classic or spinning disk confocal microscopes can be used. An inverted system should preferentially be used to determine the distance of the imaged cells from the glass bottom. The system used for this work is an Inverted Confocal Spinning Disk equipped with a 40X/1.3NA and a 60X/1.4NA oil-immersion objectives (working distances 200 &#181;m and 130 &#181;m, respectively), a Photometrics CoolSNAP HQ2 camera, 1392 x 1040 imaging array, 6.45 x 6.45 &#956;m pixels and controlled by Metamorph imaging software. 405 nm, 491 nm, 561 nm, and 633 nm lasers are typically used at 30&#8211;50% power, gain 1 and binning of 2 x 2 to reduce exposure times. The microscope is also equipped with a Hg lamp and filter cubes for DAPI (exc 400-418 nm; em 450-465), FITC (exc 478-495; em 510-555) and Texas Red (exc 560-580; em 600-650) to use with the eye piece.
<ol>
	<li>Using the fluorescent lamp, place the 40X&#160;objective at the middle of the collagen drop. Get an overview of the sample, both in the x-y axis and in the z-axis. Make sure not to deviate more than 100 &#181;m from the gel edge on the x-y axis when acquiring the images to avoid edge effects. When imaging spheroids, make sure they are under the objective working distance. Change objective if appropriate.</li>
	<li>Switch to the confocal live imaging mode. By using the 561 nm laser to visualize the TAMRA-labeled collagen, starting from the glass bottom determine the z value at which collagen fibers start to appear. This is the substrate bottom and z = 0.</li>
	<li>Using the focus knob, go up 100 &#181;m from z = 0. Only cells at z = 100 &#181;m or above should be imaged to avoid tension effects from the rigid glass bottom.</li>
	<li>Select the cells to image. Optimize camera exposure times and laser power for each wavelength. Typical exposure times for DAPI and Alexa-488 Phalloidin are between 100-200 msec. Exposure times for antibody staining may vary.</li>
	<li>On confocal live mode using the appropriate laser to visualize the phalloidin staining and using the focus knob, define the z series interval by setting top and bottom z values to current.</li>
	<li>Define the z-step size. For 40X&#160;objectives, use 1 &#181;m. For 60X, use 0.5 &#181;m. Start acquisition.</li>
</ol>

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The authors declare that they have no competing financial interests.

The protocol described here to fluorescently label collagen I using TAMRA provides an excellent method to allow easy visualization of the collagen network organization, by using a standard confocal microscope equipped with a 561 nm laser. An advantage of this technique comparing to reflectance confocal microscopy is the ability to image collagen fibers deeper into the 3D matrix. The intensity and contrast of the fibers reflection diminishes substantially with depth due to laser light absorption and scattering. Also, the ...

The field of cell migration has been braving into the brand new third dimensional world. It is intuitive to study cell migration in an environment that most closely resembles the physiological one and, therefore, three-dimensional (3D). However, due to technical limitations, most cell migration studies have been done analyzing cell movement across rigid two-dimension (2D) substrates, either untreated or coated with appropriate extracellular matrix (ECM) proteins.
The first studies dedicated to cell migration in three dimensional collagen lattices go back over 20 years1-3. However, only over the past 5 years it has become clear that migratory cells could substantially differ in their morphology and mode of migration depending on the dimensionality of the substrate. In 2D, cells only contact the substrate with their ventral surface using focal adhesions, resulting in the formation of broad flat protrusions (lamellipodia) with embedded finger-like protrusions (filopodia) at their leading edge. These structures, together with stress fibers that connect the cell front to the trailing edge, are believed to be crucial for cell movement in 2D. In 3D matrices, cells are generally more elongated, with the entire cell surface contacting the ECM, causing considerable changes in the formation and functional relevance of many of these structures. Conversely, other cellular features gain relevance in 3D migration, such as nuclear deformation and structures involved in ECM remodeling4.
Despite these known morphological alterations, as well as differences in migration modes5-7, which can vary depending on the ECM and cell types, structural and functional analysis of cells embedded within 3D matrices still remains unusual. Working with thick and dense 3D matrices carries technical difficulties, such as high-resolution microscopy imaging, and incompatibilities with most standard protocols optimized for 2D cultures, like immunofluorescent labeling of endogenous proteins. Also, because the use of 3D matrices is a relatively new approach, researchers are still investigating the best conditions to closely resemble specific in vivo situations, such as the normal stromal architecture of different tissue organs or the ECM organization around a tumor. Discrepancies in results by different groups concerning, for instance, cancer cell modes of migration or the existence of focal adhesions, have generated some controversy8. A lot of effort has been recently dedicated to reach a consensus in terms of ECM chemical nature, pore size, fiber thickness, and matrix stiffness. Many different types of 3D ECMs are currently used, varying from cell derived matrices to commercially available matrigel, pepsinized bovine collagen I, or nonpepsinized rat tail collagen I. Each of these matrices has specific physical and chemical properties and one needs to relate the matrix of choice to the physiological process being studied. In addition, pore size and fiber thickness can depend on polymerization conditions, such as pH and temperature9,10. Binding to and distance from rigid substrates such as glass, can also change the elastic properties of the matrix10,11.
This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. Methods for making cancer cell spheroids have previously been described, the most popular ones being the hanging drop method12,13 and the agarose-coated plate method14. As an appropriate ECM substrate for cancer cell migration, nonpepsinized rat tail collagen I polymerized at room-temperature is used at 2 mg/ml. Nonpepsinized acid-extracted collagen I from rat tail retains both N- and C-terminal telopeptides, nonhelical portions of the collagen molecule responsible for native collagen intermolecular crosslinking and fibrilar stability15. Together, these conditions allow the formation of collagen networks that most closely resemble the ones observed in vivo10. To allow visualization of the collagen fibers, both in fixed and living cultures, a detailed protocol is &#160;provided to fluorescently label collagen in vitro10 using 5-(and-6)-carboxytetramethylrhodamine (TAMRA), succinimidyl ester. This protocol has been adapted from Baici&#160;et al.16,17, where fluorescein isothiocyanate is used to label soluble collagen molecules. As fluorescein, TAMRA is an amino-reactive fluorescent dye that reacts with nonprotonated aliphatic amino groups of proteins, such as the free amino group at the N-terminus and, more importantly, the side amino group of lysines. This reaction only occurs at basic pH, when the lysine amino group is in the nonprotonated form. In addition to TAMRA being more stable than fluorescein over time, its emission spectra falls on the orange/red range (ex/em = 555/518 nm), which can be usefully combined for live cell imaging of GFP-tagged proteins. Using soluble collagen labeled molecules with amino-reactive dyes does not affect the polymerization process nor the density, pore size and crosslinking status of the collagen matrix10,16,18,19.
This protocol also includes a method for 3D immunofluorescent labeling of endogenous proteins, which has been further optimized to label the cytoskeleton or cytoskeleton associated proteins. The final focus of this protocol is on methods to acquire high-resolution images of 3D cultures using confocal microscopy with reduced contribution from rigid glass coverslips on the collagen matrix tension.

<table border="0" cellpadding="0" cellspacing="0" width="603">
	<colgroup>
		<col span="2" />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">TAMRA, SE</td>
			<td style="width:195px;">Invitrogen</td>
			<td style="width:136px;">C-1171</td>
			<td style="width:79px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat tail Collagen High Concentration</td>
			<td>BD Biosciences</td>
			<td>354249</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat tail Collagen</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phalloidin</td>
			<td>Sigma</td>
			<td>P2141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taxol (Paxitel)</td>
			<td>Sigma</td>
			<td>T7402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-alpha Tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 488 Phalloidin</td>
			<td>Invitrogen</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 633 Goat anti-Mouse antibody</td>
			<td>Invitrogen</td>
			<td>A21052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting media</td>
			<td>Fisher Scientific</td>
			<td>106 226 89 </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name of Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dialysis Cassette</td>
			<td>Pierce</td>
			<td>66380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottom dishes</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MetaMorph Microscopy Automation &#38; Image Analysis Software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaris 7.2.3</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

revealing the cytoskeletal organization of invasive cancer cells in 3d

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

Labeling rat-tail collagen I with TAMRA allows an easy preparation and visualization of 3D collagen networks. Slow polymerization at room temperature results in the formation of collagen networks with comparable organization to the ones found in vivo10.
Here we present a protocol for immunofluorescence staining of the cytoskeleton of CT26 cancer cells, both as spheroids and as single cells. To better preserve the cytoskeleton, buffers are supplemented with unlabele...

Watch this Scientific Journal Video about Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D at JoVE.com

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

revealing-the-cytoskeletal-organization-of-invasive-cancer-cells-in-3d

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Medicine

15.6K Views.  Institut Curie. This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

Video: Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

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

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

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

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 ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...