This work was funded by The National Centre for the Replacement, Refinement and Reduction of Animals in Research (G1000121 ID no.94513) and the Oracle Cancer Trust. SE is supported by The Institute of Cancer Research and Cancer Research UK (grant number C309/A8274). We acknowledge NHS funding to the NIHR Biomedical Research Centre.

1. Generation of Reproducibly Sized Tumor Spheroids
<ol>
	<li>Wash tumor cell monolayers with phosphate buffered saline (PBS; 5 ml for a 25 cm2 or 8 - 10 ml for a 75 cm2 flask), add cell dissociation enzyme (1 ml for a 25 cm2 or 2 ml for a 75 cm2 flask) and incubate cells at 37 &#176;C for 2 - 5 min.</li>
	<li>Check cell detachment under a microscope and neutralize cell dissociation enzyme with complete growth medium (5 ml for a 25 cm2 or 8 ml for a 75 cm2 flask).</li>
	<li>Centrifuge cell suspension at 500 x g for 5 min.</li>
	<li>Remove supernatant, tap the tube and re-suspend cell pellet in 1 ml of complete growth medium using a P1000 pipette. This should yield a single cell suspension without cell clusters.</li>
	<li>Count cells using a hemocytometer and dilute the cell suspension to obtain 0.5 - 2 x 104 cells/ml (optimal cell density needs to be determined for each cell line in order to obtain tumor spheroids of 300 - 500 &#181;m diameter 4 days after cell seeding12,13).</li>
	<li>Transfer the cell suspension to a sterile reservoir and, using a multichannel pipette, dispense 200 &#181;l/well into ultra-low attachment (ULA) 96-well round bottom plates12.</li>
	<li>Transfer the plates to an incubator (37 &#176;C, 5% CO2, 95% humidity). Four days later, visually confirm tumor spheroid formation and proceed with the 3D invasion assay.</li>
</ol>
2. 3D Tumor Spheroid Invasion Assay
<ol>
	<li>Thaw BMM on ice.</li>
	<li>Keep a set of sterile filter tips for P10, P200 and P1000 pipettes and sterile tubes (1.5 ml volume or larger depending on total volume required) at -20 &#176;C.</li>
	<li>Place the ULA 96-well plates containing 4-day old spheroids on ice.</li>
	<li>Using a multichannel pipette, gently remove 100 &#181;l/well of growth medium from the spheroid plates. For this step angle the tips towards the inside wall of the U-bottom wells, avoiding contact with the bottom of the well and the location of the spheroids; minimize disturbance of the spheroids.</li>
	<li>Using ice-cold tips, transfer BMM to ice-cold tubes. For cytokine-induced invasion or for drug evaluation studies, add reagents (at 2x the final concentration) to the BMM using ice-cold tips. For example, use epidermal growth factor (EGF) to stimulate the invasion of CALS and CALR squamous carcinoma cells. Mix well by swirling gently, avoiding the formation of bubbles.</li>
	<li>Gently dispense 100 &#181;l of BMM into the U-bottom well, aiming the tip towards the inside wall of the well. This step is the most critical as, for optimal image analysis, spheroids must remain in the center of the well12.</li>
	<li>Repeat step 2.6 for all the required wells, allowing 5 - 6 replicates/condition. If necessary, change to a freshly-chilled tip. 
	NOTE: When more experienced, dispense BMM using a multichannel pipette.</li>
	<li>Using a sterile needle, remove bubbles, if present.
	<ol>
		<li>Using a microscope, visually check that spheroids are in a central position. If not, centrifuge the plate at 300 x g for 3 min at 4 &#176;C. This will ensure that spheroids are centrally located in each well.</li>
	</ol>
	</li>
	<li>Transfer the plate to an incubator at 37 &#176;C and allow the BMM to solidify.</li>
	<li>1 hr later, using a multichannel pipette, gently add 100 &#181;l/well of complete growth medium. For cytokine-induced invasion or drug evaluation studies, include cytokines or inhibitors in the medium (1x final concentration). Alternatively, if reagents were not added to the BMM in step 2.5, add them to the medium at this point (3x the desired final concentration).</li>
</ol>
3. Automated Image Acquisition
<ol>
	<li>Scan plates on the cytometer (for specifications see Table of Equipment and Materials) at intervals starting from time zero (t0 = day of the invasion assay set up, immediately after step 2.10 up to 72 hr, or 96 hr for slower invading tumor cell lines.</li>
	<li>Select the &#8216;Confluence&#8217; application.</li>
	<li>Check that the spheroid is in focus and, if needed, adjust manually and fix the &#8216;Focus off-set&#8217;.</li>
	<li>Under &#8216;Sampling settings&#8217;, select to scan one central field of view (FOV). Following BMM addition, tumor spheroids should have remained in a central position, thus there is no need to scan the entire well.</li>
	<li>In the software, define the wells to be scanned by highlighting them on the plate map. Click on &#8216;Start scan&#8217;. 
	NOTE: The scan is automatically saved and can be reviewed later off-line.</li>
</ol>
4. Automated Image Analysis
<ol>
	<li>Select &#8216;Confluence&#8217; application.</li>
	<li>In the &#8216;Analysis&#8217; tab, adjust application settings (e.g., &#8220;precision&#8221;: low, normal, high; &#8220;intensity threshold&#8221;: 1 - 15) to produce a precise segmentation around the spheroid, including cell processes and invadopodia extending into the BMM (see Figure 1). Adjust the settings for each tumor cell line and/or at various time points. The &#8216;Confluence&#8217; application measures the % of the well area covered by the invading cells, in the selected FOV.</li>
	<li>Check that the analysis settings are appropriate (i.e., segmentation is accurate) for other spheroids in the plate by clicking on a few different wells. If the segmentation does not accurately outline a spheroid, adjust the application settings further.</li>
	<li>Click on &#8216;Start analysis&#8217;.</li>
	<li>On the &#8216;Results&#8217; tab, check multiple wells to verify analysis quality. Export &#8216;Well level data&#8217; to a spreadsheet program.</li>
	<li>Repeat analysis for all the time points. Calculate the mean % confluence for replicate wells and plot invasion (% confluence) against time in a bar chart, using scientific graphing and statistical software of choice.</li>
</ol>
5. Manual Image Acquisition
<ol>
	<li>Record an image for each tumor spheroid at intervals starting from t = 0 up to 72 - 96 hr, depending on the speed of invasion of the cell line in question, using an inverted microscope (for a 96-well plate this takes approximately 20 - 30 min). Use a 10X objective. Use a 4X objective when an invaded area is too large to be completely captured within the 10X field of view.</li>
	<li>Save each individual image acquired.</li>
	<li>For calibration, take images of a stage graticule (1 mm) using both 10X and 4X objectives (and save images).</li>
</ol>
6. Manual Image Analysis
<ol>
	<li>Open the stage graticule images into the image analysis software of choice.</li>
	<li>Enter the graticule measurements, the units (&#181;m), the objectives used (10X or 4X) and perform the calibration. This step is required only once. For subsequent image analysis, simply reload the required calibration settings.</li>
	<li>Open the invasion assay images and select the calibration settings depending on the microscope objective (10X or 4X) used to obtain the images.
	<ol>
		<li>Alternatively, import images obtained on an imaging cytometer (step 3) and analyze manually, as described for images obtained using an inverted microscope (see results shown in Figure 3 and Figure 4).</li>
	</ol>
	</li>
	<li>Measure the area covered by the spheroids.
	<ol>
		<li>In the software used here for the Representative Results (for specifications see Table of Equipment and Materials), go to &#8216;Measure&#8217; and select &#8216;Count/size&#8217;. Chose &#8216;File&#8217; then &#8216;Load settings&#8217; and select pre-determined assay settings. Then choose &#8216;Count&#8217;. The spheroid should then be accurately segmented.</li>
		<li>Alternatively go to &#8216;Edit&#8217; then &#8216;Draw/Merge Objects&#8217; to open the &#8216;Magic Wand/Trace&#8217; window. Use the wand cursor to click on the spheroid image and obtain segmentation. Select &#8216;Trace&#8217; (in the &#8216;Magic Wand&#8217; window) to manually outline the spheroid using a mouse.</li>
	</ol>
	</li>
	<li>Export measurements of different parameters (area, diameter, perimeter etc.) to a spreadsheet. Record on the spreadsheet the relevant image information (e.g., well number, relative time point).</li>
	<li>Plot the mean area of replicate spheroids (invasion) versus time using scientific graphing and statistical software. Alternatively, calculate the change in spheroid area at each time point, relative to the area at t = 0. Then plot invasion (% t0) versus time as a linear graph.</li>
</ol>

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Open Access fees were supported by Nexcelom Bioscience, LLC.

The two tumor models described here (glioblastoma and SCCHN) were specifically selected to exemplify our 3D assay as they are clinically relevant locally invasive cancers with an unmet need for effective therapies12. Following drug treatment, assessment of in vitro pharmacodynamic (PD) biomarker changes can be performed easily by western blot or immunoassays of lysates following the use of specific commercially available reagents to allow cell recovery from BMM and/or immunofluorescent analysis of invading tumor spheroids by whole mount staining.
This invasion assay is a useful technique for rapid and reproducible assessment of tumor cell invasion into a semisolid medium and therefore particularly appropriate for future in vitro drug screening. Cancer cells invade the matrix in a 3D manner as they spread out from a &#8220;micro-tumor&#8221;, represented by the tumor spheroid, and extend into an extracellular matrix-like environment. The true three-dimensionality of the assay is evident in the cell morphology, which is different from the flat, adherent morphology cells assume when moving on a solid substrate. The degree of invasion is easily quantified using either an imaging cytometer, allowing an automated read-out, or by using a standard microscope in combination with imaging software. Moreover, this method is suitable for fluorescent imaging13 (for example with cell lines expressing fluorescent proteins or pre-labelled with fluorescent dyes).
The method is simple to perform with the only critical step represented by the addition of the BMM, when there is a risk of disturbing the central position of the spheroid in the U-shaped well. This can result in suboptimal image analysis, with spheroids in different focal planes. It is therefore critical to add the BMM carefully and slowly to each well. After BMM addition it is advisable to check the plate under a microscope and if the localization is considered unacceptable, this can be remedied by gentle centrifugation. With experience, this is rarely necessary. Whilst this method is robust and very reproducible, inter-experimental variation could occur with different batches of BMM. To avoid this, it is advisable to purchase sufficient BMM from a single batch to complete a series of studies.
A limitation of the method (as for any such assays) is the difficulty in distinguishing between invasion and proliferation, which the tumor cells likely undergo during the assay time frame. Although the doubling time of cells can be taken into account, or cell cycle inhibitors such as cytochalasin D introduced, it is not easy to control or clearly distinguish between these two different aspects of tumor progression, especially for fast growing tumor cells and for those that have a more &#8220;expansive&#8221;, rather than infiltrative, invasion pattern. For this reason it is suggested that a parallel 3D growth assay is performed to evaluate specific effects of any inhibitory or stimulatory agents. If careful dose response studies are performed, it may be possible to select concentrations that minimize effects on proliferation. For example we have shown that the HSP90 inhibitor 17-AAG inhibits U-87 MG 3D tumor spheroid invasion already at 24 hr and at concentrations below the concentration inhibiting 3D growth by 50% (GI50)12.
On the other hand, the significance of this assay compared to other standard invasion assays (e.g., filter based assays or invasion of single cells into 3D matrices1), is that tumor cells invade into the surrounding matrix from the spheroid, that resembles a &#8220;micro-tumor&#8221; or a &#8220;micro-metastasis&#8221;, and therefore takes into account important aspects of the pathophysiology of a tumor mass. The method we present provides information on the collective behavior of tumor cells, when initially leaving the spheroids, and also individually, when single cells reach more distant regions in the BMM. Additionally, cells within tumor spheroids may experience hypoxia and nutrient deprivation which, through changes in gene expression, can promote migration and invasion; such features are absent in 2D assays. Moreover, all of this is available in a high-throughput format through the use of specific 96-well plates and the latest imaging technology, allowing a more complex 3D assay to be easily used in target validation and drug discovery.
The method we present can accommodate further applications to address additional aspects of tumor cell invasion. In particular, extra complexity can be envisaged by the addition of host cells, such as fibroblasts and/or endothelial cells, into the spheroid itself or the surrounding matrix. This can also be modified, not only in terms of stiffness (by the use of different concentrations of BMM), but also in terms of composition (by the addition of other components dependent on the specific tissue/organ of the adopted tumor model: e.g., tenascin for brain cancers16 and collagen for breast carcinoma17).
A similar set-up (generation of spheroids and imaging) can also be adapted to assess tissue invasion in 3D, where tumor spheroids are co-cultured with embryoid bodies resembling a complex tissue12 or with other cell-specific organoids (e.g., astrocytes for glioma18 or crypt cultures for gastrointestinal cancers) but further work is required to enable automated image analysis.

Classical anticancer drug development is largely focused on the use of in vitro cell-based assays to screen for cytotoxic agents that inhibit tumor cell proliferation and/or promote programmed cell death (apoptosis). More recently, efforts have moved towards targeted therapies with the development of inhibitors of the underlying molecular causes of malignant cell proliferation. In spite of some spectacular results, such agents are often associated with the rapid development of drug resistance. Given that it is the invasive and metastatic potential of tumor cells that is responsible for the majority of cancer deaths, and that relapsed disease often presents a more aggressive phenotype, it is logical also to consider complementary in vitro experimental models1,2 to identify novel agents that will inhibit these additional key &#8216;hallmarks&#8217; of cancer.
During malignant progression, tumor cells acquire the ability to invade the surrounding tissue and/or spread into distant organs (metastasis). Cancer cells penetrate the basement membrane by the formation of invadopodia3,4. These structures are enriched with actin filaments, specific adhesion proteins and proteinases and are collectively responsible for tumor cell motility and degradation of the extracellular matrix (ECM)5. Invadopodia extend into the ECM and are believed to be important for tumor cell invasion and also extravasation into vascular channels, facilitating hematogenous (or lymphatic) dissemination and metastasis.
Current standard methods to assess tumor cell invasion in vitro include the following. Transwell-based or Boyden chamber assays2,6 where single cell suspensions are seeded on top of a filter coated with a thick layer of ECM-derived proteins. Cells then invade and move into the lower chamber in response to a chemo-attractant. Commonly used ECM proteins are type I collagen, or a basement membrane-like matrix (BMM, e.g., Matrigel, derived from the EHS tumor7) that mimics the natural composition of basement membranes.
As an alternative to the filter-based Boyden chamber type assays8, cells can be seeded on top of an ECM gel (to which fibroblasts may be added) where they form a monolayer and then individually or collectively invade into the gel. Invasion can be measured in terms of number of invading cells and/or distance travelled from the gel surface9. Tumor cells can also be completely embedded into a matrix either as a single cell suspension or as spheroids - usually placed between two layers of ECM gel- allowing cells to invade out of the tumor mass into the surrounding matrix2,6,10,11.
The three-dimensional (3D) tumor spheroid invasion assay described here provides a rapid, automatable invasion system using a highly reproducible, standardized method12 in a 96-well plate format with one spheroid per well. BMM is added directly to each well to provide a semi-solid matrix into which tumor cells invade from the spheroid body. The extent of tumor cell invasion is monitored at intervals over a period of 72 - 96 hr. This method provides the following advantages over the current in vitro invasion assays mentioned above: the tumor cells are organized into a 3D structure mimicking a tumor micro-region or a micro-metastasis; the tumor spheroids are highly reproducible in size; the invasion assay is performed in situ in the same plate as tumor spheroid development, without the need to move them to secondary plates; the method, combined with the latest technologies of automated image analysis, enables both high content and high throughput analyses of tumor cell invasion.
The image analysis is performed using an imaging cytometer, which scans a 96-well plate within 10 min. By using the confluence application, the extent and rate of invasion achieved either by single cells or by cell clusters spreading out from the tumor spheroids and invading into the matrix can be measured in a dynamic fashion. For lower throughput, an alternative method for image analysis is presented, based on the use of an inverted microscope and standard imaging software.

<table><tbody><tr><td>Corning Matrigel basement membrane matrix</td><td><a href="http://www.corning.com" target="_blank"><img src="/img/new_window.png"> Corning</a></td><td>354234</td><td>Keep at 4 &amp;deg;C to prevent solidification and dispense using cooled pipette tips</td></tr><tr><td>Cultrex basement membrane extract, PathClear</td><td><a href="http://www.trevigen.com" target="_blank"><img src="/img/new_window.png"> Trevigen</a></td><td>3432-005-01</td><td>Keep at 4 &amp;deg;C to prevent solidification and dispense using cooled pipette tips</td></tr><tr><td>Ultra-low attachment 96-well plate</td><td><a href="http://www.corning.com" target="_blank"><img src="/img/new_window.png"> Corning</a></td><td>7007</td><td></td></tr><tr><td>EGF</td><td><a href="http://www.miltenyibiotec.com" target="_blank"><img src="/img/new_window.png"> Miltenyi Biotec</a></td><td>130-093-825</td><td>Add 100 &amp;mu;l 1% BSA (w/v) in water to 100 &amp;mu;g EGF. To 25 &amp;mu;l of this, add 4,975 &amp;mu;l RHB-A medium to give a 5 &amp;mu;g/ml working stock. Aliquot and store at -20 &amp;deg;C</td></tr><tr><td>RHB-A medium</td><td><a href="http://www.stemcellsinc.com" target="_blank"><img src="/img/new_window.png"> Stem Cells Inc.</a></td><td>SCS-SF-NB-01</td><td>For diluting EGF</td></tr><tr><td>DMEM</td><td><a href="http://www.lifetechnologies.com/" target="_blank"><img src="/img/new_window.png"> GIBCO</a></td><td>31966-021</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Fetal bovine serum</td><td><a href="http://www.biosera.com" target="_blank"><img src="/img/new_window.png"> Biosera</a></td><td>1001/500</td><td>Heat at 56 &amp;deg;C to inactivate complement before use</td></tr><tr><td>TrypLE</td><td><a href="http://www.lifetechnologies.com/" target="_blank"><img src="/img/new_window.png"> GIBCO</a></td><td>12605</td><td>Cell dissociation solution</td></tr><tr><td>Celigo cytometer</td><td><a href="http://www.nexcelom.com" target="_blank"><img src="/img/new_window.png"> Nexcelom Bioscience</a></td><td>n/a</td><td>Specialist equipment for image capture and analysis. Manual image capture by microscopy and independent image analysis software is an alternative</td></tr><tr><td>IX70 inverted microscope</td><td><a href="http://www.olympus.co.uk/" target="_blank"><img src="/img/new_window.png"> Olympus</a></td><td>n/a</td><td>Used with camera for manual image capture</td></tr><tr><td>Retiga Exi Fast 1394 digital camera</td><td><a href="http://www.qimaging.com/" target="_blank"><img src="/img/new_window.png"> Qimaging</a></td><td>n/a</td><td>Attached to microscope for manual image capture</td></tr><tr><td>Image-Pro Plus 7.0</td><td><a href="http://www.mediacy.com/" target="_blank"><img src="/img/new_window.png"> Media Cybernetics</a></td><td>n/a</td><td>Software used to control camera and microscope for manual image capture</td></tr><tr><td>Image-Pro Analyzer 7.0</td><td><a href="http://www.mediacy.com/" target="_blank"><img src="/img/new_window.png"> Media Cybernetics</a></td><td>n/a</td><td>Stand-alone image analysis software for manual measurment of spheroid size</td></tr><tr><td>Excel 2010</td><td><a href="http://www.microsoft.com/" target="_blank"><img src="/img/new_window.png"> Microsoft</a></td><td>n/a</td><td>Scientific graphing and statistical software</td></tr><tr><td>Prism 6.0</td><td><a href="http://www.graphpad.com/" target="_blank"><img src="/img/new_window.png"> GraphPad</a></td><td>n/a</td><td>Scientific graphing and statistical software</td></tr></tbody></table>

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.

3D tumor spheroid invasion is assessed using a simple, reproducible, automated method schematically illustrated in Figure 1: reproducibly sized tumor spheroids are obtained by plating cells into ULA 96-well round bottom plates. 4 days post-initiation spheroids are embedded into BMM. This provides a semi-solid structure into which tumor cells invade and spread out of the spheroid. Spheroids are located centrally at the base of each well and invasion into the BMM is easily monitored at intervals starting from t = 0 up to 72 - 96 hr using an imaging cytometer. This can provide fully automated image analysis.
Examples of different types of cancer cell invasion are illustrated in Figures 2 - 4. The highly malignant human glioblastoma multiforme (GBM) cell line U-87 MG, forms a tight, spherical 3D structure and is used here to exemplify brain tumors in which local invasion is a key life-threatening feature. Once embedded in BMM, cells from U-87 MG spheroids spread with a typical “starburst” invasion pattern12. Tumor cells radially extend into the matrix in which they maintain a 3-dimensional shape. The process is followed over a period of 72 hr as shown in Figure 2 for U-87 MG spheroids, when the extent of tumor cell invasion, with details of the cellular processes and invadopodia extending into the BMM is clearly evident. Fully automated image analysis using an image cytometer enables precise quantitation of the tumor cell invasion over time. The quantification in Figure 2 is obtained as illustrated in Figure 1, where the automated analysis produces segmentation around the cell protrusions extending into the BMM from the U-87 MG spheroid body. Note that for this cell line, the tumor spheroid body is excluded from the measurement of the invaded area.
A different pattern of invasion was displayed by human squamous head and neck cancer cell lines. CALS and CALR are isogenic but CALS is sensitive to, and CALR resistant to EGFR tyrosine kinase inhibitors14. In the absence of EGF, neither cell line invaded into the BMM (Figure 3) however, as the concentration of EGF was increased (to 2.5 ng/ml), the spheroids appear to ‘bud’. At the higher concentrations of EGF (40 and 80 ng/ml) the degree of invasion was reduced. This is consistent with the classical bell-shaped dose response to EGF in terms of invasion that has been previously reported15. In 20 ng/ml EGF, finger-like protrusions extruded from the main body of CALR spheroids whilst CALS cells were less invasive after 72 hr (Figure 3). This differential invasion was evident 48 hr after spheroids were placed into BMM (containing 20 ng/ml EGF for maximal distinction between cell lines) and greatest after 72 hr (Figure 4). These images were acquired rapidly using an imaging cytometer, as for U-87 MG. However, in this case, to exemplify an alternative method, the degree of invasion was quantified manually using stand-alone image analysis software and is presented graphically (Figures 3 and 4). These results illustrate a phenotypic difference between drug-sensitive and -resistant cells that could be utilised to screen for compounds that will specifically antagonize the drug resistant, invasive phenotype.
<img alt="Tumor spheroid formation in ULA 96-well plate, Matrigel embedding, Celigo cytometer analysis." src="/files/ftp_upload/52686/52686fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Schematic overview of 3D tumor spheroid invasion assay. The workflow shows the steps the method involves including representative images of U-87 MG glioblastoma spheroids just after embedding into BMM (top panel; t = 0) and after invasion into BMM (bottom panel; t = 72 hr) with and without the segmentation that measures the area covered by the invading cells. Bar = 100 μm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52686/52686fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell invasion assay over 72 hours; microscope images, bar graph shows invasion percentage increase." src="/files/ftp_upload/52686/52686fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Time-course of U-87 MG glioblastoma spheroid invasion into BMM. U-87 MG spheroid 3D invasion into BMM was monitored and quantified up to 72 hr using an imaging cytometer. (A) Representative images and (B) quantification of tumor cell invasion are shown. Bar = 500 μm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52686/52686fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="EGF-induced spheroid invasion; microscopy images and graph; CAL cell line response analysis." src="/files/ftp_upload/52686/52686fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. CALR (resistant) and CALS (sensitive) tumor spheroid invasion. (A) Representative images and (B) manual quantification of EGF dose-dependent CALR and CALS tumor spheroid invasion are shown. Bar = 500 µm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52686/52686fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Tumor invasion assay; CALS vs CALR at 0, 24, 48, 72h; phase contrast images, invasion graph." src="/files/ftp_upload/52686/52686fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Cells fromCALR (resistant) spheroids are more invasive than CALS (sensitive) tumor spheroids when placed in BMM. Upon EGF stimulation CALR tumor spheroids show a more pronounced invasion than CALS. (A) Representative images and (B) manual quantification of invasion are shown. Bar = 500 µm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52686/52686fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Three-Dimensional 3D Tumor Spheroid Invasion Assay at JoVE.com

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.

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 ...

three-dimensional-3d-tumor-spheroid-invasion-assay

Nanyang Technological University

Research

JoVE Journal

Medicine

57.1K Views.  The Institute of Cancer Research. 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.

Video: Three-Dimensional 3D Tumor Spheroid Invasion Assay

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

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

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

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

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Cancer Research

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

3D Models in Cancer Biology: Invasion Dynamics in Heterospheroids

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

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

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

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

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

Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major clinical challenge because no efficient method exist for their elimination once their dissemination is underway. A better understanding of the mechanisms regulating cancer cell invasion may lead to the development of novel potent therapies. Due to their physiological resemblance to tumors, spheroids embedded in collagen I have been extensively utilized by researchers to study the mechanisms governing cancer cell invasion into the extracellular matrix (ECM). However, this assay is limited by (1) a lack of control over the embedding of spheroids into the ECM; (2) high cost of collagen I and glass bottom dishes, (3) unreliable immunofluorescent labeling, due to the inefficient penetration of antibodies and fluorescent dyes and (4) time-consuming image processing and quantification of the data. To address these challenges, we optimized the three-dimensional (3D) spheroid protocol to image fluorescently labeled cancer cells embedded in collagen I, either using time-lapse videos or longitudinal imaging, and analyze cancer cell invasion. First, we describe the fabrication of a spheroid imaging device (SID) to embed spheroids reliably and in a minimal collagen I volume, reducing the assay cost. Next, we delineate the steps for robust fluorescence labeling of live and fixed spheroids. Finally, we offer an easy-to-use Fiji macro for image processing and data quantification. Altogether, this simple methodology provides a reliable and affordable platform to monitor cancer cell invasion in collagen I. Furthermore, this protocol can be easily modified to fit the users&#8217; needs.

Presented here is a protocol for the fabrication of a spheroid imaging device. This device enables dynamic or longitudinal fluorescence imaging of cancer cell spheroids. The protocol also offers a simple image processing procedure for the analysis of cancer cell invasion.

The invasion of cancer cells from the primary tumor into the adjacent healthy tissues is an early step in metastasis. Invasive cancer cells pose a major ...

Biology

Generation of a Three-Dimensional Spheroid Model of Tumor Cell Invasion

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/60998/a-3d-spheroid-model-for-glioblastoma'>Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma.&#160;J. Vis. Exp.&#160;(2020)</a>The video demonstrates the generation of a three-dimensional spheroid model of tumor cell invasion. Spheroids are formed from human brain tumor cells and embedded in a collagen matrix. The tumor cells at the spheroid periphery adopt an invasive phenotype, secreting proteases to degrade the collagen and advancing to invade the surrounding matrix.

Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma.&#160;J. Vis. Exp.&#160;(2020)The video demonstrates the generation of a three-dimensional ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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