Time-Resolved Fluorescence Imaging and Analysis of Cancer Cell Invasion in the 3D Spheroid Model

This protocol can be used to study the mechanisms governing the invasion of cancer cells into the extracellular matrix in real-time. The main advantage of this technique is that it uses a simple spheroid imaging device, which makes the spheroid invasion assay easier to set up and improves its efficiency and cost. While we demonstrate using the spheroid invasion assay to model mammary carcinoma, this assay is an excellent model for the invasion of any solid tumor into the surrounding healthy tissue.

After 3D printing of the spacer, weight out a 10:1 ratio of base polymer to cross-linker in a plastic cup and use a disposable pipette to thoroughly mix the resulting PDMS solution in the cup. Place the cup into a vacuum chamber and quickly release the vacuum pressure to remove any air trapped at the surface of the mixture and to dissipate the remaining air bubbles. Incubate the 3D printed spacer at 100 degrees Celsius for five minutes to increase its flexibility and clean two glass plates with 100%isopropanol.

Place the spacer so that it is flush between the two cleaned plates and place two large binder clips on the bottom edge and one onto the top corner to seal the mold onto the outside edges of the plates. Inspect the top part of the mold to ensure that the spacer is flush with the glass plates and use a disposable pipette with approximately two centimeters cut from the tip to slowly but continuously add the PDMS mixture to the top left corner of the mold. When the entire mold has been filled, place the mold into the vacuum chamber to remove any air bubbles that have formed, before curing the PDMS for one hour at 100 degrees Celsius.

At the end of the incubation, place the mold at room temperature. When it is cool to the touch, remove the binder clips and glass plates from the spacer containing the cured PDMS and use a razor blade to cut through the seal that was created on all four sides of the mold between the spacer and the glass plate. Pull apart the mold to reveal the PDMS sheet in the spacer and use tweezers to carefully peel the PDMS sheet off of the spacer.

Place the sheet onto a cutting mat and use a biopsy punch to punch out a 17.5 millimeter diameter PDMS disc, then punch three evenly distributed 5.5 millimeter holes into the disc. Use tape to remove any dust particles from each insert and stick the cleaned inserts onto a piece of double-sided tape. Wrap the tape around the lid of a 10 centimeter Petri dish and place the lid into a plasma machine along with open 35 millimeter glass bottom dishes.

Activate the inserts and glass with one minute of plasma treatment at 300 millitorrs and use tweezers to quickly attach the treated side of each insert to the glass part of one treated glass bottom dish per insert. When all of the inserts have been applied, use the pointer finger and thumb to apply even pressure to each insert while rotating the dish to securely attach the inserts to the dishes. When all of the inserts have been secured, incubate the resulting spheroid imaging devices for 20 minutes at 60 degrees Celsius, before performing a second round of plasma treatment as demonstrated.

After the second treatment, add 35 microliters of freshly prepared coating solution to each insert hole. After one hour at room temperature, remove the coating solution and rinse the device three times with distilled water. After the last wash, add 35 microliters of cross-linking solution to each hole for a 30-minute incubation at room temperature.

At the end of the incubation, rinse the device three times with distilled water as demonstrated, before filling each device with 70%ethanol for a 30-minute incubation under UV light. At the end of the sterilization, rinse the devices three times with distilled water and add 2.5 milliliters of storage solution to each device. To embed the spheroids in collagen, wash the devices three times with three milliliters of PBS per wash.

After the last wash, allow the devices to completely dry, before adding one spheroid in 30 microliters of freshly prepared college one solution into one hole of the insert and starting a timer. After confirming the presence of the spheroid in the hole, add spheroids to the other two holes as demonstrated. Use a 10 microliter pipette tip to recenter any spheroids that are located close to the PDMS border, or to separate the spheroids if multiple spheroids are dispensed and stop the timer.

To vertically center the spheroid in the collagen layer, after seeding, flip the device upside down and incubate the device at 37 degrees until the collagen polymerizes, reversing the device orientation every two minutes for a total of 30 minutes, then add 2.5 milliliters of medium per device and acquire an image of the spheroids at the zero hour time point. Using a spheroid image device facilitates the efficient embedding and time-lapse recording of cancer cell invasion within the collagen or via longitudinal imaging. While this spheroid was imaged longitudinally over the course of six days requiring the stable expression of cytoplasmic and/or nuclear fluorescent proteins, similar longitudinal imaging can be performed over a shorter time period using dye labeling.

Spheroids can also be fixed and immunolabeled. For example, these spheroids were immunolabeled for epithelial-cadherin, cortactin, and F-actin. In these images, a step-by-step illustration of the image processing procedure using the Fiji macro to measure the area of the spheroid over time can be observed.

Be sure to dispense a single spheroid into each hole of the spheroid imaging device and to position the spheroid at the center of the hole in both XY and Z directions. The device design can easily be modified by changing the size and arrangement of the holes, inserts, and glass bottom dishes to accommodate a higher spheroid throughput.