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

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.

1. 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 °C, 5% CO₂, 95% humidity) for 2-5 min.
2. Check that the cells have detached under a microscope. Then add complete Dulbecco's modified Eagle’s medium (DMEM) media (DMEM + 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 250 ng/mL amphotericin B, 0.4 μg/mL hydrocortisone, and 50 μg/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.
3. Centrifuge the cell suspension at 200 x g for 5 min.
4. Remove the supernatant and suspend the cell pellet in 2-5 mL of complete DMEM.
5. 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.
6. Dispense 50 μL 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.
7. Transfer the plate to the cell culture incubator (37 °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.

2. Three-dimensional Spheroid Invasion Assay

NOTE: Prepare 2x solution because the gel will be diluted 1:1 when added into the wells.

1. Thaw HMDM on ice and fibrinogen stock solution in a water bath maintained at 37 °C. Do not disturb the fibrinogen until it is completely solubilized and do not put the solution on ice; precipitation will occur.
2. 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.
3. Add 50 μL 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.
4. Return the plate back to the cell culture incubator and allow the HMDM/fibrin matrix to solidify for 30 min, and gently add 100 μL of complete DMEM into each well on top of the gel.

3. Imaging

1. Image the spheroids daily using an inverted light microscope. Alternatively, use automatic imaging systems.

4. Image Segmentation with Ilastik

1. 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.
2. Add images for analyses. Click Input Data and Add New and then choose images (Figure 1B).
3. 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.
   1. 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.
4. 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).
   1. Mark the background with one of the labels (Figure 1D, yellow) and the cells with the other label (Figure 1D, blue).
   2. Train the software for the first 10% of the images. Choose next image from Current View.
   3. 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.
5. 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).
6. Choose the desired output file format from Choose Export Image Settings… 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.

5. Area Analysis with Fiji ImageJ

1. Scale setting
   1. 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.
   2. 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).
2. Installing the macro
   1. If a plugin ilastik is not installed, follow these steps: click Help, Update…. 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.
   2. 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.
3. Area analysis
   1. 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).
   2. Press the a button from the keyboard (macro) and the area will appear in the Summary window as Total Area (Figure 3E).