Our culture system captures the phenotypic plasticity of lung squamous cell carcinoma cells in response to tumor-stroma interactions and how these interactions regulate tumor morphology during lung squamous cell carcinoma progression. The main advantage of our system is its ability to model lung squamous cell biology in a 3D context while preserving the plasticity of the malignant cells. This 3D coculture provides a unique system to investigate tumor-stroma cell interactions and could be adapted to monitor responses of lung squamous cell carcinoma cells and cancer-associated fibroblasts to drug treatment.
To begin, thaw vials of basement membrane matrix in a four-degrees Celsius refrigerator overnight. Cool down two-milliliter plastic pipettes and tips at minus 20 degrees Celsius overnight. The next day, warm the reagents used to dissociate TUM622 cells, HEPES buffer, trypsin/EDTA, and trypsin neutralization buffer in a 37-degrees Celsius water bath.
Take the thawed basement membrane matrix out of the refrigerator and put the vial on ice. Cool down the tissue culture plates on a metal platform cooler placed on ice. Place centrifuge tubes on a metal cooling rack on ice.
Calculate the amount of cells needed based on the number of wells and the concentration of cells in each well to be prepared. Transfer TUM622 cell suspension into a cooled centrifuge tube and spin down at 300 times g in a hanging bucket centrifuge at four degrees Celsius for five minutes. With an aspirating pipette attached to an unfiltered tip, aspirate the supernatant carefully, leaving approximately 200 microliters of the medium in the tube.
Gently tap on the side of the tube to dislodge and dissociate the pellet and then return it to the cooling rack. Using the two-milliliter precooled pipettes, gently mix the matrix on ice by pipetting up and down a few times. Pipette at an even and moderate speed so that no bubbles are introduced into the matrix during this procedure.
Transfer 1.1 milliliters of the matrix into each centrifuge tube. Using precooled tips, pipette the matrix in each tube up and down about 10 times to make a uniform cell suspension. Transfer 310 microliters of the cell matrix suspension into each well of a precooled 24-well plate.
Place the pipette at a 90-degree angle to the plate surface and add the suspension to the center of the well. The suspension spreads and covers the entire well. To facilitate downstream immunofluorescence analysis, transfer 60 microliters of cell matrix suspension into the well center of a two-well chamber slide.
This allows the matrix to form a dome-like structure with much smaller volume. Return the plate and the chamber slide back into the tissue culture incubator and incubate for 30 minutes to allow the matrix to solidify. After that, examine the plate and slide under a light microscope to ensure that single cells are evenly distributed within the matrix.
Add one milliliter of pre-warmed 3D culture complete medium into each well of the plate and 1.5 milliliters of 3D culture medium into each well of the chamber slide, then return them to the incubator. After preparing cell suspensions of TUM622 and CAFs as previously described, count the CAF cell density by mixing 10 microliters of cell suspension with 10 microliters of trypan blue. Add 10 microliters of the mixture to each of the two chambers on the hemocytometer to count and calculate cell density.
To co-embed TUM622 cells and CAFs in basement membrane matrix, first calculate the desired number of cells used for plating based on the cell density information. CAFs are seeded at a 2:1 ratio of TUM622 cells. Transfer the calculated volume of TUM622s as well as CAF cell suspension into the same centrifuge tube.
Spin down and aspirate all medium. Resuspend in basement membrane matrix and plate 310 microliters of the mixture into each well of a 24-well plate. For immunofluorescence, transfer 60 microliters of TUM622 and CAF mixtures to chamber slides as described previously.
To coculture TUM622 with overlaid CAFs in basement membrane matrix, first set up TUM622 monoculture as previously described, transfer twice the number of CAF suspension into a centrifuge tube, and spin down at 300 times g for five minutes at room temperature. Aspirate the supernatant and resuspend the CAFs in culture medium, transfer twice the number of CAF cells that were resuspended in culture medium into each of the wells containing the embedded TUM622 cells. Typical TUM622 cells in 2D culture are rounded with large nuclei while CAFs are flat and elongated.
Seeded in 3D culture, single TUM622 cells were capable of forming organoids with acinarlike morphologies when embedded. Between days five and seven, a lumen became apparent in the acinarlike structures and remained hollow thereafter. Each acinus, composed of a monolayer of cells surrounding the hollow lumen, displayed proper apical basal polarity similar to that of lung epithelium in vivo.
These acinarlike structures were hyperplastic and continued to grow until 24 days before the extracellular matrix completely disintegrated. In the TUM622-CAF cocultures, either overlaying or co-embedding, the presence of CAFs greatly enhanced the number and size of the spheroids formed. Interestingly, when TUM622 acini came into close proximity with CAFs, they induced the acini to become invasive and migrate toward the CAFs, forming teardrop-like structures.
To ensure robust formation of acinarlike structures, it is critical to maintain the basement membrane matrix in its liquid form during the entire process of embedding the cells. TUM622 organoids can be used for a variety of downstream analyses, including but not limited to immunofluorescent, immunohistochemistry, flow cytometry, RNA and protein extraction, and can be also modified for drug screening. Using this system as a platform, one can investigate how tumor cell intrinsic as well as cell extrinsic changes in the tumor microenvironment can influence tumor epithelial architecture and carcinoma formation.
