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.

The overall goal of this procedure is to provide an in vitro, semi-automated, three-dimensional microplate assay of tumor cell invasion. This is accomplished by first generating reproducibly sized tumor steroids in suspension in ultralow attachment round bottom 96, well plates at once steroid in each well. The second step is to remove parts of the medium and add basement membrane like matrix or BMM directly to each well to provide a semi-solid matrix into which tumor cells invade from the steroid body.

Next, the tumor steroid invasion is monitored at intervals over a period of 72 to 96 hours during which image acquisition is performed either automatically on a cytometer or manually on a microscope. The final step is to analyze tumor cell invasion through either an automated cytometer or imaging software. Ultimately, the 3D tumor steroid invasion assay is used to model cancer invasion in vitro in a format that is more physiologically relevant and amenable to target validation and drug screening studies.

We believe that this technique is complimentary to the simple two dimensional cell proliferation assays normally used for target validation and drug screening in cancer research because it also enables us to investigate invasion, which is a key aspect of cancer progression in a physiologically relevant three-dimensional format. I first had the idea for this method when I optimized the 3D tumor phe, the growth on microplate. I thought it would be good to use the same setup and the look also at the invasion, and the result was easy just to remove a part of the medium from each well and replace it with the basement membrane alike matrix.

Visual demonstration of this method is essential as keeping the steroids in a central position of each well after tradition of the BMM can be difficult at the beginning. This can result in suboptimal image analysis due due to the steroids being in different focal planes. With experience.

This rarely happens, but if necessary, the process can be facilitated by gentle centrifugation of the plate. To begin all the BMM on ICE overnight, keep a set of sterile filter tips for P 10 E 200 and P 1000 pipettes and sterile tubes are minus 20 degrees Celsius. Also place the ultra low attachment 96 well plates containing four day old steroids on ice.

Using a multichannel pipette, gently remove 100 microliters per well of growth medium from the sparrow plates. For this step angle the tip towards the inside wall of the U bottom. Well avoiding contact with the bottom of the well and the location of the sphe.

In order to minimize disturbance of the steroids using ice cold tips, transfer the BMM to ice cold tubes. For cytokine induced invasion or for drug evaluation studies, add reagents to the BMM using ice cold tips. For example, use epidermal growth factor or EGF to stimulate the invasion of Cal S and cal r squamous carcinoma cells.

Then gently dispense 100 microliters of the BMM into the ubo. Well aiming the tip towards the inside wall of the well. This step is the most critical.

As for optical image analysis, steroids must remain in the center of the, well. Repeat this step for all the required wells, allowing five to six replicates per condition. If necessary, change to a freshly chilled tip using a sterile needle.

Remove bubbles if present. Use a microscope to visually check the steroid are in a central position if they are not centrifuge the plate at 300 times G for three minutes of four degrees Celsius. This will ensure that steroids are centrally located in each well.

Next, transfer the plate to an incubator at 37 degrees Celsius and allow the BMM to solidify one hour later. Use a multichannel pipette to gently add 100 microliters per well of complete growth. Medium or cytokine induced invasion or drug evaluation studies.

Include cytokines or inhibitors in the medium for automated image acquisition. Scan plates on the cytometer at intervals. Starting from time zero, select the confluence application.

Then check that the steroid is in focus. If needed, adjust the focus manually and fix the focus offset. Under sampling settings.

Selector scan one central field of view. Following BMM addition, tumor steroids should have remained in a central position. Thus there is no need to scan the entire well in the software.

Define the wells to be scanned by highlighting them on the plate map. Then click on start scan. For automated image analysis, select confluence application.

In the analysis tab, adjust application settings to produce a precise segmentation around the steroid, including cell processes and invader podia extending into the BMM. Adjust the settings for each tumor cell line and or at various time points. Check that the analysis settings are appropriate for other steroids in the plate by clicking on a few different wells.

If the segmentation does not accurately outline a steroid, adjust the application settings further. Click on start analysis on the results tab. Check multiple wells to verify analysis quality before exporting well level data to a spreadsheet program.

Repeat the analysis for all the time points, then calculate the mean percent confluence for replicate wells and plus invasion against time in a bar chart. Using scientific graphing and statistical software of choice for manual image acquisition, use an inverted microscope equipped with the 10 x objective to record an image for each tumor steroid at intervals. Starting from T equals zero after T equals between 72 and 96 hours.

Depending on the speed of invasion of the cell lining in question, use a Forex objective when an invaded area is too large to be completely captured within the 10 x field of view. Save each individual image acquired for manual image analysis. Open the stage graphe images into the imaging analysis.

Software of choice for calibration is images of a stage graphe taking. Using both 10 x and four x objectives. Enter the graphic measurements, the units, and the objectives used and perform the calibration.

This step is required only once for subsequent image analysis. Simply reload the required calibration settings. Next, open the invasion assay images and select the calibration settings depending on the microscope objective used to obtain the images.

Measure the area covered by the steroids in the software used here. Go to measure and select count size. Choose while then load settings and select predetermined assay settings.

Then choose count. The steroid should then be accurately segmented. Export measurements are different parameters to a spreadsheet and record the relevant image information on the spreadsheet.

Finally, plot the mean area of replicate steroids or invasion versus time using scientific graphing and statistical software. Shown here as an example of cancer cell invasion observed in the U 87 MG glioblastoma cell line, which can be used to exemplify brain tumor invasion. Once embedded into BMM cells spread with a typical starburst invasion pattern, the process is followed over a period of 72 hours.

Fully automated image analysis. Using an image cytometer produces a segmentation around the cell protrusions and enables precise quantification of the tumor cell invasion. Note that for this cell line, the steroid body is excluded from the measurement of the invaded area.

Fully automated image analysis is easily performed and provides quantification of tumor cell invasion over time. A different pattern of cell invasion is displayed by human squamous, head and neck isogenic. Cancer cell lines Cal S is sensitive to, and CAL R is resistant to EG FFR tyrosine kinase inhibitors.

In the absence of EGF, neither cell line invaded into the BMM in the presence of EGF finger-like protrusions, extruded from the main body of Cal ars steroids. Whilst Cal s cells were less invasive after 72 hours, images in this case were acquired rapidly using an image cytometer and to exemplify an alternative method, the degree of invasion was quantified manually using standalone image analysis software Whilst attempting this procedure, it's important to remember that each cell line needs to be optimized for its ability to form steroids, the cell seeding density, the matrix composition, and the image and analysis settings Following this procedure. The same method that could also be adapted to assess a 3D tissue invasion using, for example, the tumors in co culture with embryo bodies to resemble a complex tissue or other organoids like astrocytes for glioma or crypt cultures for gastrointestinal cancers, or liver for hepatocellular carcinoma and colon cancer metastasis.