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 border="0" cellpadding="0" cellspacing="0" style="width:791px;" width="790">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:256px;">Amphotericin B solution</td>
			<td style="width:93px;">Merck</td>
			<td style="width:119px;">A2942</td>
			<td style="width:323px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Aprotinin from bovine lung</td>
			<td style="width:93px;">Merck</td>
			<td style="width:119px;">A6279</td>
			<td>Measure the protein concentration before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ascorbic acid</td>
			<td style="width:93px;">Applichem</td>
			<td style="width:119px;">A1052</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">BioLite Cell Culture Treated Flasks</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td style="width:119px;">130190</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:256px;">DMEM/F-12</td>
			<td style="width:93px;">Gibco by Life Technologies</td>
			<td style="width:119px;">31330-038</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">DS-Fi2 Digital Camera</td>
			<td style="width:93px;">Nikon Instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">DS-U3 Digital Camera Controller</td>
			<td style="width:93px;">Nikon Instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Eclipse TS100 Inverted Microscope</td>
			<td style="width:93px;">Nikon Instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:256px;">Fetal bovine serum (FBS)</td>
			<td style="width:93px;">Gibco by Life Technologies</td>
			<td style="width:119px;">10270106</td>
			<td>Heat inactivate for 30 min at 56&#176;C in waterbath before use.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Fibrinogen, Plasminogen-Depleted, Human Plasma</td>
			<td style="width:93px;">Merck</td>
			<td style="width:119px;">341578</td>
			<td>Solubility: 25 mg/ml in H&#8322;O. Warm the H&#8322;O and the fibrinogen to 37&#176;C prior to dissolution. Do not disturb until the fibrinogen is completely solubilized. Stock solutions stored at -70&#176;C should be thawed in a water bath maintained at 37&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Fiji ImageJ 1.51 image processing program</td>
			<td style="width:93px;">Freeware, NIH</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrocortisone</td>
			<td style="width:93px;">Merck</td>
			<td style="width:119px;">H0888</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">Ilastik software for image classification and segmentation</td>
			<td style="width:93px;">Freeware</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">IncuCyte Zoom Live-Cell Analysis System</td>
			<td style="width:93px;">Essen BioScience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Myogel</td>
			<td style="width:93px;">Lab made</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Penicillin-Streptomycin</td>
			<td style="width:93px;">Gibco by Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Phosphate buffered saline (PBS)</td>
			<td style="width:93px;">Lab made</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Scepter 2.0 Cell Counter</td>
			<td style="width:93px;">Merck</td>
			<td style="width:119px;">PHCC20040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Scepter Cell Counter Sensors, 60 &#181;m</td>
			<td style="width:93px;">Merck</td>
			<td style="width:119px;">PHCC60050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Thrombin from human plasma</td>
			<td style="width:93px;">Merck</td>
			<td style="width:119px;">T6884-100UN</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:256px;">Trypsin-EDTA (0.5%) 10X, no phenol red</td>
			<td style="width:93px;">Gibco by Life Technologies</td>
			<td style="width:119px;">15400054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Ultra-Low Attachment 96-Well Plate</td>
			<td style="width:93px;">Corning</td>
			<td style="width:119px;">7007</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:256px;">UT-SCC-42A and UT-SCC-42-B cell lines</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></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

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models.
In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

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 ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

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

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