A Proximal Culture Method to Study Paracrine Signaling Between Cells

This method can help answer key questions in the intercellular communications field about reciprocal paracrine versus juxtacrine interactions between two types of cells. The main advantage of this method is that it is a very simple and reproducible technique that accurately captures paracrine signaling under typical culture conditions while at the same time preventing effective juxtacrine signaling. The implications of this technique extend to a better understanding of the mechanism of the crosstalk between the cancer cells and the tumor microenvironment.

Though this method can provide insight into the interactions between the cancer cells and the tumor stroma, it can also be applied to other systems such as wound healing and angiogenesis. Begin by detaching the ovarian cancer cells from an 80%confluent mixture with one milliliter of 0.25%trypsin for 40 to 60 seconds, followed by neutralization of the reaction with six milliliters of complete growth medium. Collect the cells by centrifugation and resuspend the pellet in five milliliters of fresh complete growth medium.

After counting, dilute the cells to a 100, 000 ovarian cancer cells per milliliter of complete growth medium concentration and place three inverted 0.4 micrometer pore tissue culture treated polycarbonate membrane cell culture inserts per experimental condition in an appropriately sized sterile tissue culture dish. Label the inserts according to the experimental condition and carefully pipette the cancer cells onto the bottom of each insert in concentric circles, starting from the middle of the membrane and moving outward to form an 800 microliter dome. Be extremely careful when seeding the cells onto the bottom surface of the insert so that the dome of the medium containing cells does not run off the edges of the insert during the incubation.

When all of the inserts have been seeded, cover the dish and carefully place the inserts into the cell culture incubator. After about four hours, detach the HPMC with two milliliters of 0.25%trypsin for one to two minutes, followed by neutralization of the reaction with 12 milliliters of complete growth medium. Collect the HPMC by centrifugation and resuspend the pellet in 10 milliliters of complete growth medium for counting.

Dilute the cells to a 300, 000 HPMC per milliliter of complete growth medium and add 2.5 milliliters of fresh complete growth medium to each well of a six-well culture plate. Using sterile forceps, place each insert right side up into each well of the six-well plate so that the lower ovarian cancer cell attached surfaces are immersed in the media. Then seed 1.5 milliliters of HPMC into the well of each insert.

Then place the plate in the cell culture incubator for 72 hours. At the end of the incubation, carefully remove the inserts from each well and rinse both sides of the inserts with PBS. Place each insert into a new six-well plate and add 0.5 milliliters of trypsin to the insert wells and two milliliters of trypsin to the bottom wells.

After two minutes at 37 degrees Celsius, add one milliliter of complete growth medium to each insert and carefully pipette the solution a few times without tearing the membrane or spilling the cell suspension into the well below. While pipetting, care should be taken to prevent the cross-contamination of the cells from one chamber with the others. Transfer the resuspended cells into a labeled collection tube and further neutralize the trypsin with an additional two milliliters of complete growth medium.

To collect the cells on the bottom of the inserts, flush the bottoms of the membranes two to three times with the two milliliters of trypsin from the corresponding well of the six-well plate so that the detached cells are caught in the appropriate well bottoms. When all of the cells have been detached, neutralize the trypsin in each well with six milliliters of fresh growth medium and transfer the cell suspensions to labeled collection tubes. Then collect all of the cells by centrifugation and resuspend the pellets in 0.7 milliliters of phenol and guanidine thiocyanate-based lysis reagent per tube for RNA extraction and isolation according to standard protocols.

Proximal culture of HPMC with ovarian cancer cells results in an increased expression of fibronectin and Transforming Growth Factor or TGF beta one compared to control HPMC seeded on inserts in the absence of cancer cells. The expression of both fibronectin and TGF beta one also significantly increases in ovarian cancer cells upon proximal culture with HPMCs compared to control ovarian cell cultured in the absence of HPMC. Conversely, ovarian cancer cells treated with HPMC conditioned medium demonstrate a significantly reduced fibronectin expression with no change in TGF beta one expression.

Blocking of TGF beta one with a neutralizing antibody however results in a significant decrease in TGF beta one expression in ovarian cancer cells in proximal culture with HPMCs. Interestingly, a slight increase in TGF beta one expression is observed in control non-proximally cultured ovarian cancer cells likely as a compensatory response. Proximal culture with ovarian cancer cells slightly increases E-cadherin expression in HPMCs while a marked decrease in E-cadherin is observed in ovarian cancer cells grown in proximity with HPMCs compared to controls further demonstrating the effectiveness of the proximal culture system for studying paracrine signaling between ovarian cancer cells and normal cells at the site of metastasis.

While attempting this procedure, it's important to optimize the number of cells seeded and the duration of the proximal culture. Following this procedure, other methods like immunoblotting can be performed to answer additional questions about the changes in protein expression and the activation of downstream processing signaling pathways during the tumor cell mesothelial cell co-culture. After its development, this technique paved the way for researchers in the field of tumor microenvironment, immunology, developmental biology to explore reciprocal paracrine interactions between cells through secreted factors.