The authors thank the funders of this study: the Sigrid Jus&#233;lius Foundation, the Cancer Society of Finland, and Helsinki University Central Hospital research funds. The authors acknowledge the Biomedicum Imaging Unit at the University of Helsinki for technical help.

1. Generation of Multicellular Tumor Spheroids
NOTE: The protocol is demonstrated here with UT-SCC-42A and -42B cell lines but it could also be applied using other cell lines.
<ol>
	<li>Wash the UT-SCC-42A and -42B cells with 6 mL of phosphate-buffered saline (PBS), add 0.05% trypsin-EDTA (3 mL for 75 cm2 flask), and place the flask in a cell culture incubator (37 &#176;C, 5% CO2, 95% humidity) for 2-5 min.</li>
	<li>Check that the cells have detached under a microscope. Then add complete Dulbecco&#39;s modified Eagle&#8217;s medium (DMEM) media (DMEM + 10% fetal bovine serum, 100 U/mL penicillin, 100 &#956;g/mL streptomycin, 250 ng/mL amphotericin B, 0.4 &#956;g/mL hydrocortisone, and 50 &#956;g/mL ascorbic acid) to neutralize the enzyme (6 mL for 75 cm2 flask) and transfer the cell suspension into a 15 mL conical tube. 
	NOTE: Select the cell culture medium that suits the cells being studied.</li>
	<li>Centrifuge the cell suspension at 200 x g for 5 min.</li>
	<li>Remove the supernatant and suspend the cell pellet in 2-5 mL of complete DMEM.</li>
	<li>Count the cells and dilute the cell suspension with complete DMEM to a final concentration of 20,000 cells/mL. 
	NOTE: Optimal cell count must be determined for each cell line.</li>
	<li>Dispense 50 &#956;L of the cell suspension into each ultra-low attachment 96-well round bottom plate well for a final concentration of 1,000 cells per well.</li>
	<li>Transfer the plate to the cell culture incubator (37 &#176;C, 5% CO2, 95% humidity). Four days later, visually confirm tumor spheroid formation with an inverted microscope and proceed with the assay. Ensure that there is only one spheroid per well. 
	NOTE: The time taken to form a spheroid varies between different cell lines.</li>
</ol>
2. Three-dimensional Spheroid Invasion Assay
NOTE: Prepare 2x solution because the gel will be diluted 1:1 when added into the wells.
<ol>
	<li>Thaw HMDM on ice and fibrinogen stock solution in a water bath maintained at 37 &#176;C. Do not disturb the fibrinogen until it is completely solubilized and do not put the solution on ice; precipitation will occur.</li>
	<li>Mix together the appropriate volume of each reagent: 1 mg/mL HMDM (final concentration: 0.5 mg/mL), 0.6 U/mL thrombin (final concentration: 0.3 U/mL), 66.6 mg/mL aprotinin (final concentration: 33.3 ug/mL), and 1 mg/mL fibrinogen (final concentration: 0.5 mg/mL). 
	NOTE: Add fibrinogen just before dispensing the mixture into the wells and work quickly; it will form a gel in a few minutes. Treat just a few wells at a time.</li>
	<li>Add 50 &#956;L of the gel into each well. Direct the tip towards the inside wall of the well and pipet slowly. Avoid air bubbles (using the reverse pipetting technique) and try not to move the spheroid from the center of the well.</li>
	<li>Return the plate back to the cell culture incubator and allow the HMDM/fibrin matrix to solidify for 30 min, and gently add 100 &#956;L of complete DMEM into each well on top of the gel.</li>
</ol>
3. Imaging
<ol>
	<li>Image the spheroids daily using an inverted light microscope. Alternatively, use automatic imaging systems.</li>
</ol>
4. Image Segmentation with Ilastik
<ol>
	<li>Open ilastik and select Pixel Classification workflow (Figure 1A). It classifies the pixels based on annotations made by the user. Save ilastik project (.ilp) to computer.</li>
	<li>Add images for analyses. Click Input Data and Add New and then choose images (Figure 1B).</li>
	<li>For feature selection, click Feature Selection and Select Features (Figure 1C, red rectangle). 
	NOTE: The selected features should roughly correspond to the visual properties that separate the objects from the background, and they will be used for training the classifier.
	<ol>
		<li>Select features by clicking the boxes. The selected boxes will turn green (Figure 1C, blue rectangle). 
		NOTE: Here the user can select from several different feature types and scales. Color/Intensity should be selected to separate objects based on color or brightness. Edge should be selected to separate objects based on brightness or color gradients. Texture is an important feature if the objects in the image have a special textural appearance. For this assay, Color/Intensity (sigma 0) and Edge (sigma 6) are used.</li>
	</ol>
	</li>
	<li>For training, click Training and in the Training section there are two labels: Label 1 and Label 2 (Figure 1D, red rectangle). If there is only one label, add a new label by pressing Add label (Figure 1D, blue rectangle).
	<ol>
		<li>Mark the background with one of the labels (Figure 1D, yellow) and the cells with the other label (Figure 1D, blue).</li>
		<li>Train the software for the first 10% of the images. Choose next image from Current View.</li>
		<li>After the cells and the background are marked (of the first image), press Live Update (Figure 1D, purple rectangle). 
		NOTE: With the next image, ilastik will automatically perform the analysis according to the previous images.</li>
	</ol>
	</li>
	<li>After training is done and ilastik has analyzed all images, click Prediction Export. Choose Simple Segmentation from Source; Do not choose Probabilities or anything else (Figure 1E).</li>
	<li>Choose the desired output file format from Choose Export Image Settings&#8230; in the Prediction Export section (Figure 1E). Export the results of the labeling by clicking Export All (Figure 1F). The files will be exported to the same folder with the original images. 
	NOTE: In this experiment, .h5 format is used.</li>
</ol>
5. Area Analysis with Fiji ImageJ
<ol>
	<li>Scale setting
	<ol>
		<li>Open the original image with a scale bar in Fiji ImageJ (Figure 2A). Ensure that the image has the same size and dimension as the analyzed images. Use the Line Selection tool to draw a line of a known length (Figure 2B). 
		NOTE: This protocol is executed by using Fiji ImageJ 1.51 (64-bit) and ilastik -1.3.2rc.</li>
		<li>Click Analyze and Set Scale (Figure 2C). Set the known distance in the Known Distance field and set the proper unit and click Global (Figure 2D).</li>
	</ol>
	</li>
	<li>Installing the macro
	<ol>
		<li>If a plugin ilastik is not installed, follow these steps: click Help, Update&#8230;. On the open window ImageJ Updater, click Manage Update Sites. On the open window Manage Update Sites, click next to ilastik and then click Close. Then click Apply Changes in the window ImageJ Updater. This process can take some time. The next window will be Information with the text Updated Successfully. Click OK and restart ImageJ.</li>
		<li>Click Plugins, Macros, and Install (Figure 3A). Then choose the counter.ijm file (see Supplemental Information) and click Open. 
		NOTE: The macro was written especially for measuring the area. The macro should be installed every time the software is opened.</li>
	</ol>
	</li>
	<li>Area analysis
	<ol>
		<li>Analyze images when all plugins are installed. Click Plugins and scroll down to ilastik (Figure 3C). Choose Import HDF5, choose the file with .h5 format, click Open and Load and apply LUT (Figure 3C,D).</li>
		<li>Press the a button from the keyboard (macro) and the area will appear in the Summary window as Total Area (Figure 3E).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Jung, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Spheroids+with+Enhanced+Aggressiveness+to+Mimic+Human+Liver+Cancer+In+vitro+and+In+Vivo.">Cell Spheroids with Enhanced Aggressiveness to Mimic Human Liver Cancer In vitro and In Vivo.</a> Scientific Reports. 7, 10499-10514 (2017).</li><li>Vinci, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+establishment+and+analysis+of+three-dimensional+tumor+spheroid-based+functional+assays+for+target+validation+and+drug+evaluation.">Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation.</a> BMC Biology. 10, 29 (2012).</li><li>Nath, S., Devi, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+systems+in+cancer+research:+Focus+on+tumor+spheroid+model.">Three-dimensional culture systems in cancer research: Focus on tumor spheroid model.</a> Pharmacology and Therapeutics. , 94-108 (2016).</li><li>Albanese, A., Lam, A. K., Sykes, E. A., Rocheleau, J. V., Chan, W. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-on-a-chip+provides+an+optical+window+into+nanoparticle+tissue+transport.">Tumour-on-a-chip provides an optical window into nanoparticle tissue transport.</a> Nature Communications. 4, 2718 (2013).</li><li>Friedrich, J., Seidel, C., Ebner, R., Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid-based+drug+screen:+considerations+and+practical+approach.">Spheroid-based drug screen: considerations and practical approach.</a> Nature Protocols. 4, 309-324 (2009).</li><li>Berens, E. B., Holy, J. M., Riegel, A. T., Wellstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cancer+Cell+Spheroid+Assay+to+Assess+Invasion+in+a+3D+Setting.">A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting.</a> Journal of Visualized Experiments. (105), e53409 (2015).</li><li>Vinci, M., Box, C., Eccles, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+(3D)+Tumor+Spheroid+Invasion+Assay.">Three-Dimensional (3D) Tumor Spheroid Invasion Assay.</a> Journal of Visualized Experiments. (99), e52686 (2015).</li><li>Salo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+human+leiomyoma+tissue+derived+matrix+for+cell+culture+studies.">A novel human leiomyoma tissue derived matrix for cell culture studies.</a> BMC Cancer. 15, 981 (2015).</li><li>Nurmenniemi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+organotypic+model+mimics+the+tumor+microenvironment.">A novel organotypic model mimics the tumor microenvironment.</a> The American Journal of Pathology. 175, 1281-1291 (2009).</li><li>Salo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+three-dimensional+assays+based+on+human+leiomyoma-derived+matrices.">Organotypic three-dimensional assays based on human leiomyoma-derived matrices.</a> Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences. 373 (1737), 20160482 (2018).</li><li>Overall, C. M., Blobel, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+search+of+partners:+linking+extracellular+proteases+to+substrates.">In search of partners: linking extracellular proteases to substrates.</a> Nature Reviews Molecular Cell Biology. 8, 245-257 (2007).</li><li>Ahmed, T. A. E., Dare, E. V., Hincke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin:+a+versatile+scaffold+for+tissue+engineering+applications.">Fibrin: a versatile scaffold for tissue engineering applications.</a> Tissue Engineering Part B: Reviews. 14 (2), 199-215 (2008).</li><li>Hoornstra, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fermented+Lingonberry+Juice+Inhibits+Oral+Tongue+Squamous+Cell+Carcinoma+Invasion+In+vitro+Similarly+to+Curcumin.">Fermented Lingonberry Juice Inhibits Oral Tongue Squamous Cell Carcinoma Invasion In vitro Similarly to Curcumin.</a> In Vivo. 32, 1089-1095 (2018).</li></ol>

The authors have nothing to disclose.

The presented method provides a 3D human tumor-based assay to evaluate the invasiveness of cancer cells within human tumor microenvironment mimicking matrix. Cancer cell invasion can be easily measured by daily imaging with a standard microscope and by using image analysis software. The method demonstrates cell invasion rather than merely proliferation.
Unlike MSDM, the HMDM/fibrin matrix does not require any temperature control. This method is easy to perform, in particular without the need to transfer the spheroids from one plate to another. It has only one technically sensitive step, the addition of fibrinogen to the matrix. Since fibrinogen starts to gel after a few minutes, adding fibrinogen requires robust pipetting and treatment of only a few wells at a time. There is also a risk of disturbing the spheroid and moving it from its central position in the U-shaped well if pipetting is done in a high-pressure manner. Dislocation of the spheroid can complicate imaging and eventually the analysis.
The assay can be modified by altering the concentration of HMDM or fibrinogen. The cells can also be fixed and stained for subsequent molecular studies. Even though this method can be modified to a fully automated form, it can also be carried out with a normal microscope and two open source software, with no need for costly equipment.
Compared to the traditional MSDM-based 3D assays, this assay could display better the cell invasion properties. Spheroids grown in a basement membrane-containing laminin-rich matrix, such as MSDM, might have more proliferation of cancer cells than actual invasion, rendering it unsuitable for invasion assays in several cancer cell lines due to their low invading capacity. Another frequently used matrix, type I collagen, tends to shrink from the edges during 3D cultivation, which affects spheroid localization and imaging of the wells. Additionally, the differences between the intrinsic invasive properties of the more (HSC-3) and less (SCC-25) aggressive tongue carcinoma cell lines have been demonstrated using human tumor microenvironment mimicking models (myoma discs and its soluble form matrix)10,13.
The assay is also more ethical than using MSDM since HMDM is extracted from the leftover material of human leiomyoma tumor. Currently, HMDM is the only available human tumor-derived ECM product that appears to be suitable for many cancer-related in vitro assays8,10. In the future, animal tissue-derived matrices could be replaced by human tumor-based matrices to reduce the need to sacrifice animals for matrix production.
Replacing 2D cell culture assays with 3D methods provides more accurate information on cancer cell behavior. Currently, there are several 3D models and commercial matrices, but, unfortunately, none are suitable for all cancer cell lines. Researchers should select the most suitable matrix for their particular assay. Optimal matching of assay and matrix can be challenging, but it could significantly increase the reliability of the results.

Conventional two-dimensional (2D) cell culture studies have considerably contributed to cancer research. Currently, researchers are shifting more towards three-dimensional (3D) cell culture assays to better mimic the in vivo conditions1. The 3D cancer cell culture more accurately reflects the complex tumor microenvironment in terms of cell-cell and cell-matrix interactions, gene expression profiles, drug sensitivity, and signaling pathway activity2,3.
Several 3D cell culture models are used in cancer research such as tumor tissue explant, tumor on a chip, and multicellular tumor spheroids3,4. Multicellular tumor spheroids are now widely used, as they mimic several features of the in vivo conditions in human tumors1,5. When the spheroid diameter is greater than 500 &#956;m, it even has hypoxic regions and a necrotic center, representing thus the in vivo tumor situation2.
Many synthetic (e.g., polydimethylsiloxane) and animal-derived (e.g., rat tail type I collagen and mouse sarcoma-derived matrix, Matrigel, referred to as MSDM) matrices have been developed for 3D cell culture assays3,6,7,8. Thus far, none of the commercially available matrices have originated from human tumor tissue. Therefore, they lack the features of the human tumor microenvironment, which has significant effects on cancer cell invasion processes8.
Myogel (human myoma-derived matrix, referred to as HMDM) is extracted from human uterus leiomyoma tumor tissue9. It has been shown that the protein content of HMDM differs significantly from that of MSDM. In fact, 66% of HMDM proteins are different from MSDM proteins. On the other hand, some proteins, such as laminin, type IV collagen, heparan sulfate proteoglycans, nidogen, and epidermal growth factor, are present in both matrices10. Additionally, the mouse differs from the human in enzyme contents, with humans having 78 fewer proteases than mice11.
Fibrin has been widely used alone or in combination with other materials as a scaffold material12. In 3D cell culture assays, commercially available human fibrinogen and thrombin are combined to form a fibrin hydrogel12.
This protocol describes an improvement of the previously introduced 3D tumor spheroid invasion assay7.This new protocol applies human tumor-derived matrix instead of mouse-derived tumor matrix. It also involves imaging and analysis techniques using ilastik and Fiji ImageJ software. This protocol could be used for spheroid assay of several different solid cancer cell lines. It offers a biologically relevant tool to develop novel anti-cancer therapies and to study the effects of specific molecules on cancer cell invasion.

<table><tbody><tr><td>Amphotericin B solution</td><td>Merck</td><td>A2942</td><td></td></tr><tr><td>Aprotinin from bovine lung</td><td>Merck</td><td>A6279</td><td>Measure the protein concentration before use.</td></tr><tr><td>Ascorbic acid</td><td>Applichem</td><td>A1052</td><td></td></tr><tr><td>BioLite Cell Culture Treated Flasks</td><td>Thermo Scientific</td><td>130190</td><td></td></tr><tr><td>DMEM/F-12</td><td>Gibco by Life Technologies</td><td>31330-038</td><td></td></tr><tr><td>DS-Fi2 Digital Camera</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>DS-U3 Digital Camera Controller</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Eclipse TS100 Inverted Microscope</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco by Life Technologies</td><td>10270106</td><td>Heat inactivate for 30 min at 56 &amp;deg;C in waterbath before use.</td></tr><tr><td>Fibrinogen, Plasminogen-Depleted, Human Plasma</td><td>Merck</td><td>341578</td><td>Solubility: 25 mg/ml in H&lt;sub&gt;2&lt;/sub&gt;O. Warm the H&lt;sub&gt;2&lt;/sub&gt;O and the fibrinogen to 37 &amp;deg;C prior to dissolution. Do not disturb until the fibrinogen is completely solubilized. Stock solutions stored at -70 &amp;deg;C should be thawed in a water bath maintained at 37 &amp;deg;C.</td></tr><tr><td>Fiji ImageJ 1.51 image processing program</td><td>Freeware, NIH</td><td></td><td></td></tr><tr><td>Hydrocortisone</td><td>Merck</td><td>H0888</td><td></td></tr><tr><td>Ilastik software for image classification and segmentation</td><td>Freeware</td><td></td><td></td></tr><tr><td>IncuCyte Zoom Live-Cell Analysis System</td><td>Essen BioScience</td><td></td><td></td></tr><tr><td>Myogel</td><td>Lab made</td><td></td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco by Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Lab made</td><td></td><td></td></tr><tr><td>Scepter 2.0 Cell Counter</td><td>Merck</td><td>PHCC20040</td><td></td></tr><tr><td>Scepter Cell Counter Sensors, 60 &amp;micro;m</td><td>Merck</td><td>PHCC60050</td><td></td></tr><tr><td>Thrombin from human plasma</td><td>Merck</td><td>T6884-100UN</td><td></td></tr><tr><td>Trypsin-EDTA (0.5%) 10x, no phenol red</td><td>Gibco by Life Technologies</td><td>15400054</td><td></td></tr><tr><td>Ultra-Low Attachment 96-Well Plate</td><td>Corning</td><td>7007</td><td></td></tr><tr><td>UT-SCC-42A and UT-SCC-42-B cell lines</td><td></td><td></td><td>The cell lines were established at the Department of Otorhinolaryngology-Head and Neck Surgery, Turku University Hospital (Turku, Finland).</td></tr></tbody></table>

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.

Four days after UT-SCC-42B (metastatic laryngeal SCC cell line) cell seeding, the matrix (HMDM/fibrin, MSDM, or collagen) was added on the formed spheroids (day 0) and the invasion was monitored for three days. Images here were obtained daily using an inverted microscope (Figure 4).
Once embedded, cells in the HMDM/fibrin matrix started to invade rapidly after one day and extended into the matrix (Figure 4A). Cells in the MSDM did not invade into the surrounding matrix, instead forming an asymmetrical structure (Figure 4B). On the other hand, cells in the collagen invaded slightly, but due to the matrix shrinkage the analysis was difficult (Figure 4C). In some collagen wells, the spheroids even disappeared after three days (Figure 4C).
Figure 5 illustrates the quantification of the cell invasion in HMDM/fibrin of the primary laryngeal SCC cell line UT-SCC-42A and the corresponding metastatic cell line UT-SCC-42B. The video (Movie 1) taken using a live-cell analysis system of the HMDM/fibrin spheroid shows the movement of the SCC cells within the matrix where they form strands followed by the other cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59567/59567fig1v2.jpg" /> 
Figure 1: Image segmentation. Selected steps of image segmentation using ilastik. <a href="https://www.jove.com/files/ftp_upload/59567/59567fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59567/59567fig2.jpg" /> 
Figure 2: Scale setting. Selected steps of scale setting using Fiji ImageJ. <a href="https://www.jove.com/files/ftp_upload/59567/59567fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59567/59567fig3.jpg" /> 
Figure 3: Invasion analysis using ImageJ. Selected steps of image analysis using Fiji ImageJ. <a href="https://www.jove.com/files/ftp_upload/59567/59567fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59567/59567fig4.jpg" /> 
Figure 4: UT-SCC-42B cell invasion in three different matrices. Representative images of SCC cell invasion in the 3D spheroids. Invasion was followed for three days and images were taken daily using an inverted microscope. (A) HMDM/fibrin, (B) MSDM, (C) collagen. The last column represents clarification of day 3 invasion. Scale bar = 200 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59567/59567fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59567/59567fig5.jpg" /> 
Figure 5: Quantification of SCC cell invasion in HMDM/fibrin. Primary laryngeal UT-SCC-42A and corresponding metastatic laryngeal UT-SCC-42B SCC cell line invasion was analyzed using ilastik and Fiji ImageJ software. Results represent mean &#177; standard deviation of three independent experiments, each in triplicate. <a href="https://www.jove.com/files/ftp_upload/59567/59567fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/59567/59567video1.jpg" /> 
Video 1: SCC cell movement within the HMDM/fibrin for three days. <a href="https://www.jove.com/files/ftp_upload/59567/Video_1.wmv" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay at JoVE.com

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

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 ...

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Cancer Research

15.4K Views.  University of Helsinki. 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.

Video: Fully Human Tumor-based Matrix in Three-dimensional 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 ...

Medicine

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 ...

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) ...

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 ...

Examining Macrophage-Tumor Spheroid Interactions in a 3D Collagen Matrix Setup

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

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 ...

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

<a href='https://app.jove.com/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 ...

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

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