Work partially funded by Prostate Cancer Canada (grant # D2014-4 to SG and CJD) and the Canadian Institutes of Health Research (grant # MOP-111069 to SG). We would like to thank Dr. Richard Poulin for editorial assistance and Mrs. Chanel Dupont for technical assistance.

NOTE: No ethics consideration since animal and human cancer cells were purchased or kindly provided to us.
1. Making Collagen Plugs (Pseudo-primary Tumor)
<ol>
	<li>Prepare a collagen dispersion. Type I collagen from rat tail tendons (RTT) can be either extracted and sterilized as previously reported17, or purchased. Disperse freeze-dried RTT collagen (3.25-3.50 mg/ml in 0.02 N acetic acid) using a blender (high-speed setting; five 2 min runs) for a uniform mixing.</li>
	<li>Harvest (trypsin-EDTA, usually) and use trypan blue exclusion for counting viable cells using a hemocytometer. Adjust to the desired cell density (5 x 104 cells per plug).</li>
	<li>Prepare all solutions (NaOH, fetal bovine serum, DMEM 5x, NaHCO3) separately (Table 1) under sterile conditions and keep chilled on ice. Note: The order of addition of the various solutions is important to prevent osmotic or acidic shocks in cells.</li>
	<li>Perform cell dispersion (1.25 x 106 cells) into the final collagen solution (5 ml) as quickly as possible. Mix well (by pipetting up and down) while avoiding air bubbles, and then quickly distribute 200 &#181;l of the ready-to-use solution in each well of a 96-well plate. Gently strike the multi-well plate on the work area surface of the cell culture hood to remove air bubbles and to spread the solution evenly inside the wells.</li>
	<li>After filling up all the wells (this step takes about 15-20 min per 96-well plate), store it into the incubator.</li>
	<li>Incubate the plate at 37 &#176;C from 2 hr to overnight. Collagen gelation (i.e., fibrillogenesis) occurs within 30 min. Add culture medium (100 &#181;l/well) to the culture to perform an overnight incubation.</li>
</ol>
2. First Layer of Fibrin Gel
<ol>
	<li>Fibrinogen Solution Preparation. 
	NOTE: The same batch of fibrin gel should ideally be used for more reproducible results, as fibrin gel formation may vary between different batches of commercial freeze-dried fibrinogen.
	<ol>
		<li>Always use a freshly prepared fibrinogen solution. Bring the freeze-dried fibrinogen to room temperature before opening the vial to avoid hydrate crystal formation.</li>
		<li>Progressively dissolve the fibrinogen in pre-warmed (37 &#176;C) Hank&#39;s Balanced Salt Solution (HBSS) with Ca2+/Mg2+ at a working concentration of 3 mg/ml (consider preparing a 15% excess of the minimum final volume required: e.g., 17.25 mg in 5.75 ml for a 5 ml solution).
		<ol>
			<li>Add pre-warmed HBSS dropwise at first to solubilize fibrinogen fragments. Break down larger fragments with a spatula in the beaker. Agitate the beaker from time to time to facilitate mixing. Do not use a stirrer plate during the procedure. Dissolve the remaining powder by pipetting the suspension up and down.</li>
		</ol>
		</li>
		<li>Keep the fibrinogen solution lukewarm while sterilizing the solution by passing it through a 0.22 &#181;m filter. Note: If the HBSS is not warm enough or the fibrinogen not fully dissolved, the solution may clog the filter. If applicable, replace filter once or twice, which may decrease the fibrinogen concentration, and thus fibrin clot stiffness.</li>
		<li>Suspend the cells of interest (e.g., endothelial cells) into ready-to-use fibrinogen solution while adjusting its final volume, as an alternative procedure.</li>
	</ol>
	</li>
	<li>Preparing the Thrombin Solutions.
	<ol>
		<li>Prepare a stock solution in ddH2O (50 NIH units/ml), then sterilize it using a 0.22 &#181;m filter.</li>
		<li>Use a fibrinogen/thrombin ratio &#8805;1:0.0075 (v/v) in order to generate the fibrin gel.</li>
	</ol>
	</li>
	<li>Generating the Fibrin Gel.
	<ol>
		<li>Keep the sterile fibrinogen and thrombin stock solutions on ice during all the next steps. The fibrin gels are allowed to form in 24-well plates.</li>
		<li>Promptly overlay the surface of each well with the fibrinogen solution (200 &#181;l/well) while avoiding air bubble formation. Process 6 wells at a time.</li>
		<li>Once the fibrinogen solution completely covers the surface of the wells, tilt the plate at a 45&#176; angle and add 1.5 &#181;l of thrombin solution to the first well by dropping the thrombin into the center of the well, and then gently swirl the plate horizontally for 1-2 sec.
		<ol>
			<li>Leave the plate in a stable position under the laminar flow hood (5-10 min) until the gelation/clotting process has completed (N.B.: the polymerization process must not be disturbed, e.g., by transporting the plate to the incubator).</li>
		</ol>
		</li>
		<li>Once the first six wells have polymerized, repeat the same sequence (i.e. the 3 previous steps) for the next six wells until all wells have been processed.</li>
	</ol>
	</li>
</ol>
3. Second Layer of Fibrin Gel and Sandwiched Collagen Plug
<ol>
	<li>Option A: (Using the Collagen Plug Immediately).
	<ol>
		<li>Make sure that the first layer of fibrin gel has polymerized in all wells by delicately tilting the plate. Place the 96-well plate containing the collagen gel plugs side by side with the 24-well plate (containing the fibrin gels) to ease transfer of the collagen plugs.</li>
		<li>Add one drop of HBSS into each well of the plate containing the collagen plugs.</li>
		<li>Remove each collagen plug from the well with a thin needle mounted on a syringe (used as a handle) or using a micro-spoon (see video). Transfer each collagen plug onto the first fibrin gel layer using one or two micro-spoons, while making sure that the collagen plug is well centered into the well and that sterility is well maintained.</li>
		<li>Overlay the previously formed fibrin gel with the second layer of fibrinogen solution (300 &#181;l/well) and introduce the thrombin as described in 2.3, keeping a minimal 1:0.0075 ratio and a sequence of six wells at a time.</li>
	</ol>
	</li>
	<li>Option B (Coating the Collagen Plug with a Thin Layer of Growth Factor-reduced Basement Membrane (GFRBM)).
	<ol>
		<li>Cool all the prepared solutions and instruments beforehand and keep them at 4 &#176;C or on ice (e.g., pipettes, tips, test tubes) during handling since frozen aliquots of GFRBM are very sensitive to excessive heating rate during thawing (follow the manufacturer&#39;s instructions).</li>
		<li>Following removal from the plate wells, soak each collagen plug for 2 min in a 1.5-ml centrifuge tube on ice containing 100 &#181;l of a pure GRFBM solution.</li>
		<li>Transfer each coated plug onto the first fibrin layer while ensuring it is well-centered, as described earlier. Incubate the plug-containing plates at 37 &#176;C for 5 min to allow the GRFBM to form a gel. Add the second fibrin layer as in step 3.1.4.</li>
	</ol>
	</li>
</ol>
4. Cell Culture Medium Conditions
<ol>
	<li>Fill each well with culture medium (400 &#181;l). The culture media and supplements will be selected based on the cell line and experimental conditions.</li>
	<li>Add aprotinin, an antifibrinolytic agent, to culture medium at a final concentration of 100 kallikrein inhibitor units (KIU)/ml. 
	NOTE: Store the plates in a cell culture incubator under the conditions used for the cell line tested.</li>
	<li>Replenish cultures with fresh medium every other day or according to the experimental schedule, and add aprotinin. Before adding fresh medium, slightly tilt the plate (at a 30-35&#176; angle) and incline the pipet against the side of the well while carefully suctioning the conditioned medium under constant observation.</li>
</ol>

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The authors have no disclosure.

As an important technical footnote, it is essential that no gap is present at the interface between the central and the peripheral gels. Otherwise, it might reduce the capacity of the cells to migrate/invade the fibrin gel. A space between the collagen and the fibrin gels may form during the first 24 hr of culture if thrombin has not been appropriately diluted. It is also possible that the cell line tested might lead the collagen gel to contract during culture, thereby causing a relatively large space to form between both gels. This is especially expected when stromal cells are mixed in the collagen gel along with cancer cells due to myofibroblastic-like activity18. In order to avoid this artifact, the collagen plug should be incubated at 37 &#176;C for a longer period (&#8805;48 hr) to induce gel contraction prior to assembling the fibrin sandwich.
A problem also encountered with the assay is the presence of discernible fibrin fibrils or agglomerates. This confers a cloudy instead of translucent aspect to the fibrin gel, which complicates data interpretation due to perturbation of the cancer cell migratory pathways. Such issues may arise from inadequate or excessive shaking of the fibrinogen solution during the polymerization process in the culture plates. It may also be due to the improper addition of thrombin into the fibrinogen solution (step 2.3). An improperly formed or cloudy fibrin gel cannot be fixed once solidified. Collagen or fibrin gels may contain air bubbles that, in most cases, will be spontaneously freed out after a few days in the incubator; nonetheless, proper pipetting minimizes their occurrence. It is recommended to introduce an anti-fibrinolytic agent at the onset of the cell culture period. Fibrinolysis may occur after a long period of cell culture due to cellular activity and the activation of proteolytic enzymes (i.e., plasminogen activator) within the collagen plug and the satellite tumors, depending on the cancer cell types and their aggressiveness19.The release of the collagen plug from the degraded fibrin gel and cell attachment to the bottom of the plates are two consequences of excessive fibrinolysis (see troubleshooting section in Table 2). A limitation of the present technique is the thickness of the fibrin gel, which can make it difficult to observe cells through the entire gel. However, the use a microscope with a long working distance condenser helps to circumvent this limitation and to monitor cells along the entire Z-axis. Confocal or intravital microscopy may also provide an alternative to visualize the cells in the fibrin gel.
One of the major objectives for developing this 3D cell culture model was to provide an extracellular matrix compartment through which cancer cells are able to freely migrate and develop. Fibrin was selected because of its presence in tumors, which owes to increasing vascular permeability induced by inflammation, necrosis and altered angiogenesis, and since fibrin is also known to facilitate tumor cell invasion20,21. The interface between the two compartments presents much interest as it mimics the production of new supportive matrix, such as fibrin, in tumors. Moreover, oxygen and nutrient diffusion, and in the longer run, pH stability, are possibly dysregulated in the collagen compartment compared to the peripheral fibrin compartment in which diffusion is facilitated. Due to the large number of cancer cells initially aggregated in a central collagen plug, signs of necrosis/apoptosis are observed within the primary tumor during culture, simulating the phenomenon observed during the expansion of primary tumors in patients22. Moreover, the formation of satellite tumors is remarkably related to the aggressiveness of cancer cells as well as their metastatic behavior. Depending on the cell line and type of experiment, the incubation period required for observing relevant metastatic processes may be as long as 30 days. This is usually longer than for other 3D assays, but the added delay brings the advantage of studying a model more representative of in situ tumors.
It is well recognized that cells grown in 3D culture models display differences in cell signaling and gene expression compared to monolayer cell cultures5,23. Growing cancer cells in 3D also affects their sensitivity to chemotherapeutic agents24-26. An important and attractive feature of this 3D cell culture model resides in the possibility to modify the ECM concentration, and thus the stiffness of the gel, which may be of interest to those working on the biomechanical properties of the cell-cell and cell-ECM interactions, as well as the effect of matrix properties on cell invasion/migration processes27. The 3D cell culture assay presented here can be adapted to suit different settings and/or experimental conditions. For example, the assay can be scaled up to accommodate studies requiring a large number of cells; by doubling the amount of material and the number of cells, collagen plugs can be prepared in a 48-well plate and layered onto a fibrin gel in a 6-well plate. On the other hand, it may be difficult to miniaturize the culture system for high-throughput studies and measurements because of inconsistent homogeneity of the fibrin gel.
The introduction of non-cancerous cells in one of the two gel compartments may modify the behavior of the cancer cells. For instance, the formation of satellite tumors is exacerbated when the prostate cancer cell line PC3 is mixed with fibroblasts and endothelial cells14. Alternatively, fluorescently-labeled cell lines can be introduced in the gel compartments to investigate cell-cell interactions and/or to track the cells during the culture period. Furthermore, histological sections can be prepared after gel fixation; however, the quality of the immunohistochemistry results may vary between antibodies since cross-reactions with fibrin may occur. Routine staining of histological sections such as with hematoxylin and eosin (H&#38;E) can reveal cell organization inside the gel matrices (Figure 3D).
It is relatively easy to remove the collagen plug from the fibrin gel (e.g., by pulling out the plug with a glass pipette). Thus, cells and satellite tumors present in the fibrin gel can be grown separately from those found in the collagen plug. Cells from the satellite colonies can be harvested by extracting the colonies with surgical scissors or a scalpel and growing them in culture medium without aprotinin to free cells from the fibrin gel. Collecting cells from the different compartments of the composite gel can allow various further studies, including gene and protein expression analysis. It can also be used to isolate cell populations with aggressive phenotypes for further in vitro or in vivo studies. For instance, this 3D culture model was used to identify genes involved in melanoma metastasis16. All those representative applications clearly demonstrate the flexibility allowed by a bi-compartmental 3D cell culture system in which cell co-culture can be carried out.
In conclusion, the design of our 3D culture system is flexible and can offer new avenues to investigate biological and molecular events during cancer cell apoptosis, migration and metastasis as well as for anticancer drug evaluation.

Investigating the fundamental and biomedical characteristics of cancer cell invasion/migration and subsequent metastasis establishment is the subject of an intense research1,2. Metastasis is the ultimate stage of cancer and its clinical management remains elusive. A better understanding of metastasis at the cellular and molecular levels will enable the development of more efficient therapies3.
Several properties of metastatic cells can be explored in vitro4 including their stemness and potential to acquire a transition state (e.g., epithelioid-mesenchymal transition) to migrate and invade within and from the primary tumor5. However, the in vitro assessment of invasion/metastasis processes has been a challenge since it virtually excludes the contribution of the blood/lymphatic circulation. Organotypic cultures that embed tumor fragments in collagen gels have previously been used to monitor cancer aggressiveness. Although the complexity of tumors is preserved (e.g., the presence of non-cancerous cells), tumor fragments are exposed to limited medium diffusion, to sampling variation, and to an overgrowth of stromal cells6. An alternative method consists in growing cancer cells within components of the extracellular matrix (ECM), which mimics the three-dimensional (3D) cell environment. The proliferation of breast cancer cell lines in a collagen gel and/or a basement membrane-derived matrix is amongst the best-characterized examples of 3D cell culture. By using specific 3D cell culture environments, the disorganized assembly observed for breast cancer cells grown under standard conditions can be reversed to the spontaneous formation of mammary acini and tubular structures7-10. Furthermore, the formation of multicellular tumor spheroids derived from adenocarcinoma cancer cells congregated using different techniques (e.g., hanging drops, floating spheroids, agar embedment) now constitutes the most commonly used 3D cell culture assay11-13. However, this assay is limited by the restricted set of cancer cell lines that can form spheroids and by the short period available to study cells in these conditions.
In this visualized technique, we herein introduce a sophisticated 3D cell culture assay where cancer cells of interest are embedded in a collagen gel to allow the in vitro formation of a pseudo-primary tumor that can be alternatively coated with a basement membrane-derived matrix. Once formed, the pseudo-primary tumor is then sandwiched in an acellular matrix (fibrin gel in the present case), which allows the cancer cells to cross the interface between the two matrix compartments (see Figure 1). Interestingly, secondary tumor-like structures originating from the pseudo-primary tumor along with aggressive cancer cells appear in the fibrin gel. Such a 3D culture system offers the flexibility required to investigate, for example, anticancer drugs, gene expression and cell-cell and/or cell-ECM interactions14-16.
<img alt="Figure 1" src="/files/ftp_upload/54322/54322fig1.jpg" /> 
Figure 1: Overview of the Method. Schematic summary of the method to generate the 3D cell culture system as a model for cancer studies. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54322/54322fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Freeze-dried collagen</td>
			<td>Sigma-Aldrich</td>
			<td>C7661</td>
			<td>from rat tail tendon (soluble dispersion) or home-made (see Rajan et al., ref.#14)</td>
		</tr>
		<tr>
			<td>Fibrinogen (freeze-dried)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F8630</td>
			<td>Type I-S, 65-85% protein with &#8805;75% of protein is clottable</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>EMD Chemicals Inc.</td>
			<td>605157</td>
			<td>&#160;Gibbstown, NJ; NIH units/mg dry weight&#160;</td>
		</tr>
		<tr>
			<td>Growth factor-reduced Matrigel&#160;</td>
			<td>Corning</td>
			<td>356234</td>
			<td>Previously from BD Biosciences</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma-Aldrich</td>
			<td>A6279&#160;</td>
			<td>&#160;solution at 5-10TIU/ml (Trypsin Inhibitor Unit)&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Micro-spoons</td>
			<td>Fisher Scientific</td>
			<td>2140115</td>
			<td>Fisherbrand Handi-Hold Microspatula</td>
		</tr>
		<tr>
			<td>96 well plate, round base</td>
			<td>Sarstedt</td>
			<td>3925500</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Sarstedt</td>
			<td>3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s Medium</td>
			<td>Sigma Chemical, Co.</td>
			<td>D5546</td>
			<td>DMEM</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>CAA15-701</td>
			<td>FBS, Canadian origin.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma Chemical, Co.</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution&#160;</td>
			<td>Sigma Chemical, Co.</td>
			<td>H8264</td>
			<td>HBSS</td>
		</tr>
	</tbody>
</table>

three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

As previously mentioned, an interesting feature of this 3D cell culture assay is that cancer cells can not only migrate from the collagen plug to the adjacent fibrin gel, but also establish secondary tumors (e.g., satellite tumor-like structures). This can be directly observed with an inverted phase contrast microscope at low and high magnifications through the gel thickness, especially with a long working distance condenser (Figure 2). Using this 3D cell culture method, the behavior of known metastatic cells can be readily compared to that found in non-metastatic cells, as demonstrated repeatedly with different experimental setups15. For example, numerous satellite tumors are found randomly dispersed in the fibrin gel with the metastatic cell line B16F10 while only very few tiny satellite tumors can so be detected with its non-metastatic counterpart, B16F0 cell line (Figure 2).
<img alt="Figure 2" src="/files/ftp_upload/54322/54322fig2.jpg" /> 
Figure 2: Behavior of Mouse Melanoma B16F10 (A) and B16F0 (B) Cells in the 3D Cell Culture System. The B16F10 and B16F0 cell lines are known to be metastatic and weakly metastatic, respectively. An upper view of the composite gel is shown after 15 days in culture in 24-well plate. The collagen plug (*) is adjacent to a fibrin gel layer in which cells have migrated (arrows) and formed satellite tumors as seen at higher magnification. Quite conveniently, these cell lines express a high level of melanin that allows for direct visualization (i.e. without staining). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54322/54322fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The shape of the migration front and satellite tumors may vary as a function of cell line characteristics. For instance, the murine mammary gland carcinoma 4T07 cells, which are poorly metastatic but are very invasive locally, form a migration front that extends radially into the fibrin gel (Figure 3A). After 14 days of culture, those cells have pulled away from the migratory front to form stellar-shape structures closely resembling mammary gland structures (Figure 3B-D).
<img alt="Figure 3" src="/files/ftp_upload/54322/54322fig3.jpg" /> 
Figure 3: Mouse Mammary Gland Carcinoma 4T07 Cells. These cells demonstrate a strong capacity to migrate in the fibrin gel (A). The dashed line delineates the collagen plug and the arrows show the direction of the migration front. The cells evenly invade the fibrin matrix (A), and well-organized, stellar-shape tubular structures can be observed in 3D (B and C). Views under phase contrast microscopy (A-C) and histological sections stained by hematoxylin and eosin (D) are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54322/54322fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The cell structures observed with this 3D culture system, particularly the satellite tumors, can be quantified by planar projection after staining the gels with a methylene blue solution as shown previously16. Staining gel samples with Hoechst 33258 at various stages of the cell culture enables to visualize the cell nuclei under epifluorescence inverted microscopy (Figure 4).
<img alt="Figure 4" src="/files/ftp_upload/54322/54322fig4.jpg" /> 
Figure 4: Hormone-Responsive Breast Cancer Cells. Human Breast Cancer MCF-7 Cells were Exposed to Different Concentrations of Tamoxifen. Satellite tumors in fibrin gels (upper row) were observed by phase contrast microscopy. DNA from the collagen gels (indicating the presence of cells in pseudo-primary tumors) (lower row), was stained with Hoechst 33258 and visualized using epifluorescence microscopy. As expected, tamoxifen induced DNA fragmentation as a result of apoptosis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54322/54322fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>DMEM 5x (no bicarbonate) Prepare from powder</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>0.26 M NaHCO3</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>1 N NaOH</td>
			<td>20 &#181;l</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>100 &#181;l</td>
		</tr>
		<tr>
			<td colspan="2">Keep this solution on ice</td>
		</tr>
		<tr>
			<td>Cell suspension</td>
			<td>880 &#181;l</td>
		</tr>
		<tr>
			<td colspan="2">Well mix on ice and at last quickly add:</td>
		</tr>
		<tr>
			<td>RTT Collagen (3.5 mg/ml)</td>
			<td>2 ml</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>5 ml</td>
		</tr>
	</tbody>
</table>
Table 1: Collagen Plugs. Reagents required and preparation of a collagen solution for 18-19 plugs. Mix well immediately after addition of the collagen (by inverting the container up and down), while avoiding air bubbles, and then promptly distribute 200 &#181;l to each well.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Steps</td>
			<td>Problems</td>
			<td>Possible reasons</td>
			<td>Solutions</td>
		</tr>
		<tr>
			<td rowspan="4">Step 1. 
			Collagen plug</td>
			<td rowspan="2">Air bubbles</td>
			<td rowspan="2">Inappropriate pipetting*</td>
			<td>Mix well while avoiding excessive bubbles.</td>
		</tr>
		<tr>
			<td>Draw each 200 &#181;l with pipette piston all way down and release only 200 &#181;l (reverse pipetting).*&#160;</td>
		</tr>
		<tr>
			<td>Collagen plug contraction</td>
			<td>Collagen plug shrinks with certain cell lines, particularly when incubated overnight.</td>
			<td>Check if cells induce collagen contraction before starting experiment. It is recommended to monitor contraction over a few days. Otherwise, plugs may contract in the sandwiched gel.</td>
		</tr>
		<tr>
			<td>Gelation does not occur</td>
			<td>Check reagents for freshness, molarity, pH, etc.); make sure no reagent has been omitted.</td>
			<td>Start again from beginning.</td>
		</tr>
		<tr>
			<td rowspan="5">Step 2. 
			First layer of fibrin gel</td>
			<td>Air bubbles</td>
			<td>Same comments*; need practice.</td>
			<td>Same solution.* Air bubbles may remain in the fibrin gel but they usually disappear over time at 37 &#176;C.</td>
		</tr>
		<tr>
			<td rowspan="2">Gelation does not occur</td>
			<td rowspan="2">Wrong quantity of fibrinogen used; check thrombin concentration or degradation.</td>
			<td>Start again from beginning.&#160;</td>
		</tr>
		<tr>
			<td>Assume the thrombin solution is inadequate; slightly increase the concentration and/or volume of thrombin (e.g., twice).</td>
		</tr>
		<tr>
			<td>Residual liquid phase after gelation</td>
			<td>Fibrinogen solution and thrombin not well mixed.</td>
			<td>Start again from beginning.</td>
		</tr>
		<tr>
			<td>Fibrin is not homogenous (i.e. fibrillar structures, agglomerates)&#160;</td>
			<td>Thrombin has inadequately diffused in the gel; excessive or insufficient agitation</td>
			<td>Need practice; start again from beginning.</td>
		</tr>
		<tr>
			<td rowspan="4">Step 3. 
			Sandwich/2nd fibrin layer</td>
			<td colspan="3">Same comments as in step 2.</td>
		</tr>
		<tr>
			<td>Collagen plug is rapidly (few hours) surrounded by empty areas in fibrin&#160;&#160;</td>
			<td>Fibrin has not polymerized adequately in contact with collagen gel</td>
			<td>Start again from beginning</td>
		</tr>
		<tr>
			<td rowspan="2">Collagen plugs may be progressively (days-weeks) surrounded by empty areas in fibrin&#160;</td>
			<td>Local lysis;</td>
			<td rowspan="2">If it is restricted to a few places around the plug, the experiment can go on. Otherwise consider to start from beginning. Could be due to cells secreting excessive amount of plasminogen activator.</td>
		</tr>
		<tr>
			<td>Do not forget the antifibrinolytic agent!</td>
		</tr>
		<tr>
			<td rowspan="2">Step 4 and 5. 
			Cell culture and follow-up</td>
			<td>Fibrinolysis</td>
			<td>Problem with the antifibrinolytic agent (degradation, suboptimal dose, etc.).&#160;</td>
			<td>If extensive amount of satellite tumors or invasion, increase the dose of antifibrinolytic agent.&#160;</td>
		</tr>
		<tr>
			<td>Acidification</td>
			<td>Excessive cell growth.</td>
			<td>Change cell culture medium more frequently.</td>
		</tr>
	</tbody>
</table>
Table 2: Troubleshooting. Possible issues and proposed solutions.

Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

three-dimensional-culture-assay-to-explore-cancer-cell-invasiveness

Nanyang Technological University

Research

JoVE Journal

Medicine

10.3K Views.  CHU de Québec Research Centre. Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Video: Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

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 ...