Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Our research group primarily focuses on the crosstalk between cancer and the immune cells. We aim to investigate the impact of various biological mechanisms on the evolution of head and neck cancer through the modulation of tumor immunity. The purpose of this protocol is to assess invasion within extracellular matrix and a spatial colocalization of different cell types in a multi-layered context.

The advantage of this protocol is the use of heterospheroids incorporating two relevant non-neoplastic cell types alongside cancer cells to analyze invasion with extracellular matrix towards both a microporous membrane and chemotaxis stimuli. To begin add magnetic nanoparticles to the cell suspension and mix 10 times. Centrifuge the cells at 400G for five minutes at room temperature, and mix 10 times to resuspend the cells.

Then transfer and mix all the cells in a new tube for co-culturing, and dispense 100 microliters of co-cultured cells into each well of an ultra-low attachment plate. Insert the spheroid drive magnetic field under the plate to induce aggregation and spheroid formation and place it in the incubator for three hours. Using a 200-microliter tip, add 50 microliters of ECM to the center of a 24-well microporous membrane.

Remove bubbles with a sterile needle, ensuring that the matrix evenly covers the membrane surface. Incubate the membrane for one hour at 37 degrees Celsius for gel formation. To begin, prepare the 3D cell culture and the extracellular matrix-coated microporous membrane.

Add 500 microliters of culture medium, supplemented with 10%fetal bovine serum to the lower chamber of the microporous membrane as a chemo attractant. Then carefully remove the medium from the formed heterospheroids, and gently add 50 microliters of fresh non-supplemented medium per well over the spheroid. Using sterile scissors, cut the edge of a 200 microliter low retention tip and collect the spheroid, along with 50 microliters of medium.

Gently place the spheroid on top of the extracellular matrix. Next carefully add 150 microliters of non-supplemented medium drop by drop to reach a total volume of 200 microliters. Place the plate in the incubator for 48 hours.

Click on file in the analysis software. Select open, then choose the image and click on okay. Then click on analyze, followed by set measurements to select area, integrated density, and display label.

Click on okay. Finally click on analyze, and then measure. The bright field images represents spheroid or cell localization, identified by dark or black conglomerates.

Images of the different cells exhibited variation in fluorescence intensity and quantity across different images along the Z axis, indicating different patterns of proliferation and invasion among cells. Using confocal microscopy, different channels were overlaid for visualizing colocalization patterns during invasion. Image analysis of the representative results presented indicated that monocytes invade first, followed by neoplastic cells, and fibroblasts are the last cells to invade the extracellular matrix.