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

As we already know, the tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, we need a biologically relevant human tumor matrix. This is the only human tumor matrix based in vitro invasion assay.

Compared to the classical spheroid assay, this method is not as temperature sensitive and doesn't require spheroid transfer. This technique is suitable for patient-derived solid cancer samples such as head and neck carcinoma and can extended toward personalized medicine including chemoradiation therapies. This human tumor-based matrix has already been used for several applications including high-throughput drug testing, live-cell analysis, wound healing, and transfer assays.

The most technically sensitive step is the method's handling of the fibrinogen is added so you may want first to practice this step with empty wells. It is important to show the proper handling of the matrix and it's easy to analyze by showing the method visually. For multicellular tumor spheroid generation, dispense one times 10 to the third cells from the cell line of interest in 50 microliters of complete medium into each well of a 96-well ultra-low attachment round-bottom plate.

Then place the plate into a 37 degrees Celsius and 5%carbon dioxide cell culture incubator for four days before visually confirming tumor spheroid formation with an inverted microscope. To set up a three-dimensional spheroid invasion assay, thaw human myoma-derived matrix on ice and thaw fibrinogen stock solution in a 37 degrees Celsius water bath. When the reagents have warmed, mix together the appropriate volumes of complete medium, human myoma-derived matrix, thrombin, aprotinin, and fibrinogen, adding the fibrinogen just before dispensing the matrix mixture into the cells.

Immediately add 50 microliters of the resulting gel into each well of the spheroid culture plate, directing the tip toward the inside wall of each well and pipetting slowing without moving the spheroid from the center of the well. Since the fibrinogen starts to gel after a few minutes, it requires a robust but precise reverse pipetting and the treatment of only a few wells at a time. Return the plate to the cell culture incubator for 30 minutes to allow the human myoma-derived matrix fibrin gel to solidify before gently adding 100 microliters of complete medium to the top of each gel.

Then image the spheroid invasion daily. For image segmentation with ilastik, open the open-source image classification and segmentation software and select Pixel Classification workflow. Save the ilastik project to the computer.

The pixels will be classified based on annotations made by the user. Click Input Data and Add New to select images for the analysis. For feature selection, click Feature Selection and Select Features.

Click the appropriate boxes to select the features of interest. The selected boxes will turn green. For a training, click Training.

In the Training section, there should be two labels, Label 1 and Label 2. Mark the background with one of the labels and mark the cells with the other label. When all of the cells and background within the first image have been labeled, select next image from current view to continue to mark the cells and background until 10%of the images have been marked.

Then select Live Update to analyze all of the images. When the training has been completed and ilastik has analyzed all of the images, click Prediction Export and select Simple Segmentation from Source. Then select the desired output file format from Choose Export Image Settings and click Export All to export the results of the labeling.

The files will be exported to the same folder with the original images. For area analysis, first open the original image with a scale bar in Fiji ImageJ, taking care 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 and click Analyze and Set Scale.

Then set the known distance in the Known distance field. Set the proper unit and click Global. To install the ilastik plugin, click Help and Update and click Manage update sites in the ImageJ updater window.

Click next to ilastik, Close, and Apply changes. After several minutes, an information window will appear saying updated successfully. Click OK and restart ImageJ.

Next, click Plugins, Macros, and Install and select the counter. ijm file. Then click Open.

To complete the area analysis, click Plugins and scroll to select ilastik. Select Import HDF5 and select the file with the h5 format. Click Open and Load and apply lookup table.

Then click the A button on the keyboard and the area will appear in the summary window as the total area. In this representative experiment, four days after metastatic laryngeal squamous cell carcinoma cell line seeding, matrix was added onto the formed spheroids as demonstrated and the invasion was monitored daily on an inverted microscope. Once embedded, cells in the human myoma-derived fibrin matrix began to invade rapidly after one day and extended into the matrix over the next two days.

Cells in the mouse sarcoma-derived matrix did not invade into the surrounding matrix, instead forming an asymmetrical structure. Cells in the collagen invaded slightly but due to matrix shrinkage the analysis became difficult. In some wells, the spheroids even disappeared after three days.

Here, quantification of the invasion of a primary and a metastatic laryngeal squamous cell carcinoma cell in human myoma-derived fibrin matrix is shown. Live-cell analysis of human myoma-derived fibrin matrix spheroid invasion reveals cell movement in strands throughout the matrix that is followed by other cells over time. Remember to be careful not to move the spheroid from the center position when adding the matrix to the wells.

This procedure can be used to study how irradiation and specific molecules such as cancer drugs affect invasion. The cells can be fixed and stained for subsequent studies. In the classical mouse sarcoma model, the cells mainly proliferate and the invasion aspect is minimal.

This human tumor-based matrix induces invasion, making the inhibition studies more efficient.