A Macrophage-Tumor Spheroid Co-Invasion Assay

We want to investigate the interplay of cancer and immune cells to better understand how they interact during metastasis. In most cases, in vivo models are the routine way to pursue research questions regarding the 3D invasion of cancer cells in the context of the immune system, or isolated components of the system are in focus. A method allowing the analysis of cellular behavior that mimics the complexity of the in vivo situation, but allows for a more direct manipulation of specific environmental characteristics under the microscope.

We demonstrate that unaltered cancer cells shall reduce invasion in the 3D collagen I matrix when they are co-cultivated together with macrophages which have been depleted for the motor protein KIF16B. This points to a role of KIF16B-driven recycling of macrophage surface proteins in the tumor microenvironment. We would like to continue to enhance our method in a way that allows the visualization of matrix degradation at the interface of macrophage cancer cell context, thus getting a better understanding of degradative and adhesive mechanisms.

To begin, cultivate H1299 GFP cells with a cancer cell medium in a 25 square centimeter cell culture flask at 37 degrees Celsius with 5%carbon dioxide until they reach 80%confluence. Wash the cells once with DPBS and add one milliliter of Trypsin-EDTA for two to three minutes until the cells are detached. Stop the enzymatic reaction by adding two milliliters of cancer cell media.

Next, centrifuge the detached cell suspension in a 15-milliliter tube at 245 G for five minutes. Remove the supernatant containing trypsin solution before washing the pellet with five milliliters of DPBS. After centrifugation, re-suspend the pellet in five milliliters of cancer cell medium.

Count the cells using a Neubauer chamber. Transfer 8, 000 cells into a 96-well ultra low adhesion plate in a final volume of 25 microliters of cancer cell medium. Incubate the cells for three days at 37 degrees Celsius and 5%carbon dioxide.

After three days, check the spheroids under the microscope for uniformity. After collecting the human blood sample, transfer 20 milliliters of blood from a transfusion bag into a 50-milliliter reaction tube. Add 15 milliliters of lymphocyte separation medium into a new 50-milliliter tube.

Carefully transfer 20 milliliters of blood to the lymphocyte separation medium-containing tube without mixing, and centrifuge the mixture at 450 G for 30 minutes at four degrees Celsius. During centrifugation, add 10 milliliters of cold RPMI 1640 Medium into a new 50-milliliter tube. After centrifugation, transfer the dense white phase from the blood-containing tube into the prepared RPMI-containing tube, and fill it up to 50 milliliters with cold RPMI.

Centrifuge the suspension at 450 G for 10 minutes at four degrees Celsius, and discard the supernatant. Re-suspend the pellet in 10 milliliters of cold RPMI and fill it up to 50 milliliters with RPMI 1640. Centrifuge the mix again, and re-suspend the pellet in 50 milliliters of RPMI.

Again, centrifuge the sample, and re-suspend the cell pellet in 1.5 milliliters of cold monocyte buffer. Next, add 250 microliters of anti CD14 MicroBeads in the cell suspension, and incubate for 15 minutes on ice. During incubation, prepare a separation column by attaching a filter to a magnetic separator rack.

Add 900 microliters of monocyte buffer into the column for equilibration. After incubation, pour the cell suspension onto the filter and allow it to flow through by gravity into a waste tube. Add one milliliter of monocyte buffer to wash the column containing the cells bound to magnetic beads.

Replace the waste tube under the column with a fresh 50-milliliter tube containing 20 milliliters of RPMI. Add three milliliters of monocyte buffer to the column. Remove the column from the magnetic rack and attach the stamp.

Press the cells into the prepared 50 milliliter tube and fill it up to 30 milliliters with RPMI. Count the cells using a Neubauer counting chamber under a light microscope, and adjust the cell number to two times 10 to the power of six cells per milliliter with RPMI. Seed one milliliter of cell suspension into each well of a six-well chamber, and incubate for two to four hours at 37 degrees Celsius with 5%carbon dioxide.

After incubation, check if the cells adhered properly and replace the RPMI with 1.5 milliliters of monomedium. Continue incubation for up to six days until monocytes differentiate into macrophages. After deriving primary human macrophages from the donor blood samples in a six-well plate, add 500 microliters of Accutase, and incubate for 40 minutes to detach the macrophages.

Then add 500 microliters of medium and transfer to a 15-milliliter centrifuge tube for centrifugation. Then wash the cells once with two milliliters of DPBS and count them with a Neubauer chamber. Dilute the desired number of macrophages in 40 microliters of collagen I mix, and briefly vortex the tube.

To wash the spheroids, add 300 microliters of DPBS into the wells of a 96-well plate. Use a one milliliter blue pipette tip with a cut end to collect the steroid with DPBS. Once the steroid settles into the tip, briefly push the pipette to the bottom of the imaging chamber to transfer the tumor steroid by surface tension.

Remove as much residual DPBS transferred with the steroid as possible. Swiftly add 40 microliters of collagen I macrophage mix onto the imaging chamber containing steroid. Incubate the plate for 30 minutes at 37 degrees Celsius and 5%carbon dioxide under humid conditions to fully polymerize the collagen mix.

After polymerization, carefully add 25 microliters of cancer cell media to each well, and continue incubation for three days. Image the live sample using a laser scanning microscope at desired time points. The H1299 GFP spheroid area was measured to be 40, 307 square micrometers on day three of invasion, whereas the spheroid perimeter was found to be 1700.17 micrometers, indicating growth over time.

A collective invasion of cancer cells from the spheroid was observed. 51 isolated particles were identified, serving as an indicator for individual cancer cell invasion. The spheroid maintained an aspect ratio of 1.10, and a circularity of 0.18, showing low deformation.