A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

3D tumor spheroids have replaced the conventional 2D monolayer technique as the gold standard for in vitro cancer research. These models permit the co-culture of multiple cell types which reflect the heterogeneity of the tumor microenvironment and allow the exploitation of spatial interactions. Advantages of using the hanging droplets method to generate 3D spheroids is the lack of cell-plastic interaction and the ease of studying the interaction between the tumor cells and the non-tumor cells.

This technique also provides a reproducible and cost-effective way to model tumor-stromal interaction. 3D tumoroids are valuable preclinical models and are often used as a drug screening tool. This method can be applied to study the pharmacological effects of multiple compounds on tumor growth and the surrounding microenvironment.

The process of spheroid formation is user-friendly and requires conventional cell culture lab equipment. Spheroids can be imaged using any bench top inverted light microscope, and the analysis of spheroid size and shape can be performed using widely-available online image analysis software. To begin, remove Huh7 and Hep3B HCC tumor cell lines and COS-7 and LX2 fibroblast cell lines from their storage rack in liquid nitrogen and rapidly defrost them.

After defrosting, dilute the thawed cells with two milliliters of fresh culture medium. Centrifuge the cells. Discard the supernatant and resuspend the cell pellet in one milliliter of fresh warm culture medium.

Then seed the cells in T75 cell culture flasks and incubate them in a cell culture incubator until cells reach 60 to 70&confluency. For cell collection, aspirate the culture media and wash the cells three times with PBS. Then to detach the adherent cells from the bottom of the flasks, add two milliliters of pre-warmed trypsin and incubate at 37 degrees Celsius.

After four minutes, inactivate the trypsin by adding four milliliters of complete culture medium and collect the cell suspension, then centrifuge the cells and discard the supernatant before resuspending the cells in one milliliter of fresh culture medium. Finally, add an additional three milliliters of fresh culture medium. To count the cells, gently vortex the cell suspension.

Then using a 10 microliter pipette, mix 10 microliters of the cell suspension with 10 microliters of trypan blue and gently pipette the mixture up and down four times to ensure complete staining of the outer cell surface with the dye. Next, place a coverslip over the hemocytometer counting area. Then place the pipette tip containing the cell mixture next to the edge of the coverslip and gently expel the tip content into the counting slide.

After waiting for a few minutes for the slurry to settle, fix the hemocytometer on the microscope stage and count the cells overlapping the top or the right ruling while avoiding those overlapping the bottom or the left ruling. Finally, calculate the total number of cells using this formula. After aspirating the culture medium, wash the LX2 cells three times with PBS.

Detach the cells using trypsin as demonstrated previously and centrifuge them. After counting the cells as demonstrated previously, seed one times 10 to the sixth LX2 cells in 10 cubic centimeter dishes and incubate at 37 degrees Celsius. After 48 hours, collect the fibroblast-conditioned medium and centrifuge to pellet any floating cells.

Filter sterilize the conditioned medium using a 0.22 micron filter attached to a 20 milliliter syringe. Then aliquot the media into two milliliter tubes for storage at minus 80 degrees Celsius. Add 10 milliliters of sterile PBS to the bottom of a 10 cubic centimeter dish to provide humid conditions for the spheroids.

Then suspend 1, 500 HuH7 HCC cells with 1, 500 COS-7 mammalian fibroblast cells in hanging droplets to form spheres. Invert the lid of the dish to allow the media, including cell suspension, to hang over a humid environment. After three days, to take images of the spheroids, put the dish on the stage of an inverted microscope and adjust the magnification to five times.

Next, open the microscope software on the attached computer and adjust its focus to have a clear image of every spheroid. Then use the snap tool on the microscope software to acquire the images and save the acquired pictures. Add 10 milliliters of sterile PBS to the bottom of a 10 cubic centimeter dish.

Then suspend 3, 000 Hep3B HCC cells in the hanging droplets to form spheres and invert the lid of the dish allowing the droplets to hang over a humid environment for three days. Next, transfer the Hep3B spheroids into 20 microliters of fresh condition media from LX2 cells in hanging droplets. Then invert the lid of the dish on which the spheroids are formed and fix the lid on the stage of a light microscope.

Adjust the fine focus of the microscope to make each spheroid visible as demonstrated previously. After pressing the plunger button to carefully empty the air from the micropipette, insert the pipette tip in the droplet containing the spheroid to be transferred. Get very close to the spheroid without touching it with the tip.

Next, gently release the pressure on the plunger button to allow the suction of the spheroid into the micropipette tip in two microliters of media. Then transfer the spheroid into a new droplet hanging on a new 10 cubic centimeter dish. Take images of the spheroids at five times magnification using an inverted microscope from the day of transfer until day seven of culture in the LX2 conditioned media.

To analyze the images of the growing spheroids, open each spheroid image in an image analysis software and use the freehand selection tool to outline each spheroid. Then from the analysis dropdown button, select set measurement, followed by area and press OK.Next, manually draw a circle around each spheroid and once the sphere is circled, press Control M to allow the program to calculate the spheroid area in pixels. Then convert the area of the spheroid into a volume using this formula.

Finally, calculate the change in spheroid volume relative to its volume on the first day of image capture. During optimization of the cell density for spheroid formation, higher seeding densities of 12, 000 and 6, 000 cells yielded spheroids with an asymmetric shape, while a seeding density of 3, 000 cells gave a perfect rounded 3D spheroid. Thus, 3, 000 cell density spheroids were adapted for further experiments.

Longitudinal assessment of the proliferative impact from co-culturing tumor and fibroblast cell lines showed that from day four onwards, heterotypic spheroids grew in an ideal round-like shape compared to homotypic spheroids. Heterotypic spheroids initially showed a rapid growth phase starting from day four to day seven, followed by a slower phase at day eight. The spheroid volume then decreased on days nine and 10, possibly reflecting the depletion of nutrients or a hypoxic core and cell death.

In contrast, the homotypic spheroids exhibited a relatively static growth curve until day five, followed by a gradual increase in their growth curves from day six onward. The higher growth rate of heterotypic spheroids suggests that the direct contact between tumor and fibroblasts increases the size of the tumor spheroids. When three-day-old homotypic Hep3B spheroids were grown in fresh media, the cells formed perfectly rounded spheroids after three days and showed continued proliferation until day seven.

The growth rate was enhanced when spheroids were maintained in LX2 conditioned media, suggesting a fibroblast-driven proliferation of tumor spheroids. It is crucial to make sure that the sterile BBS is added to the bottom of the dishes to maintain suitable humid condition for spheroid formation. Also, be careful while inverting the lid so that the spheroids and the droplets are not disrupted.

3D tumor spheroids can be fixed or frozen and stored for future applications such as immunochemistry or immunofluorescence, as well as RNA extraction for transcriptomic analysis. Heterotypic spheroids with pre-labeled cells can be imaged and tracked to provide insights into cell-cell interactions within the tumor microenvironment.