In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

The overall goal of this procedure is to examine the interactions between rare prostate cancer cells and e selectin expressing endothelial cells using a micro slide flow assembly. This is accomplished by first coating a micro slide with fibronectin. The second step is to culture human umbilical vein endothelial cells or VE in a dynamic flow system on a micro slide with a small channel width.

Next, the prostate cancer cells are labeled with an anti prostate specific membrane antigen or PSMA humanized monoclonal antibody, which gets internalized. The final step is to perfuse the anti PSMA labeled prostate cancer cells over the e selectin expressing ve. Ultimately, video microscopy is used to show the interactions between prostate cancer cells and the endothelial cells.

The main advantage of this technique over existing methods, such as parallel plate flow chamber and other dynamic assemblies, is that with this technique you can examine the interactions between rare prostate cancer cells, such as circulating tumor cells and cultured endothelial cells in a dynamic flow system. This method can help answer key questions in the field of metastasis, such as how do prostate tumor cells they metastasize through the blood vascular system? Do they use the same mechanism as being used by leukocytes during their ization?

Generally, individuals who are new to this technique will struggle because it involves culturing a mono layer of endothelial cells in a narrow channel under perfusion Under the tissue culture hood. First, rinse the micro slide with phosphate buffered saline or PBS. Then gently coat the micro slide with 200 microliters of 50 micrograms per milliliter fibronectin using a one milliliter lure lock syringe.

Cover the micro slide with the lid and keep it inside the tissue culture hood for 30 minutes. Next, perfuse 200 microliters of warm qve growth medium over the micro slide and incubate for 20 minutes at room temperature. Slow dispensing of the liquid in the micro slide prevents bubble formation in the channel.

During the perfusion, prepare the vex suspension by rinsing vex with PBS and adding 0.1%collagenase plus 0.05%EDTA in PBS for one to two minutes at room temperature. Centrifuge the hve in two milliliters of growth medium at 180 times G for five minutes. Then measure the cell concentration using a hemo cytometer and prepare 10 million cells per 100 microliters of growth medium.

Next, carefully remove the medium from the inlet of the micro slide. Using a 200 microliter pipette tip, bring the micro slide to eye level and using a one milliliter lure lock syringe gently perfu 200 microliters of the prepared concentration of vex into the channel. Caution is required at this step to prevent bubble formation if the bubbles appear, keep perfusing for a slightly longer time until bubbles enter the outlet channel.

Next pipet an equal volume of approximately 80 microliters of VE medium into both the inlet and outlet of the micro slide. This prevents the flow of cells in either direction. Cover the slide and keep it in a 37 degrees Celsius incubator for 1.5 hours.

To begin flow chamber assembly, place a sterile 20 milliliter syringe, female and male lure connectors, a syringe pump and tubing in the incubator for 15 minutes for minimal dead volume. Used tubing with an inner diameter of 0.04 inches. Smaller tubing diameter prevents bubble formation.

Next, fill the 20 milliliter syringe with 12 milliliters of warm VE medium. Remove the bubble from the syringe completely. Fill the inlet of the micro slide with VE medium.

Connect this assembly to the micro slide. Gently attach the outlet connector to the micro slide. Bring the setup into the incubator and connect it to the syringe pump.

Then set the pump to a 10 microliter per minute shear rate before leaving the cells overnight in the incubator at 37 degrees Celsius. The next day disassemble the setup by removing the connector attached to the inlet of the micro slide. Then remove the outlet connector two to upregulate e selectin expression on endothelial cells.

Prepare fresh growth medium containing interleukin one beta. Aspirate the medium in a 10 milliliter syringe. Remove the leftover medium from the inlet of the slide, then add fresh, medium completely filling the inlet.

Connect the syringe to the slide. Set the syringe pump at a 10 microliter per minute shear rate for four hours in the incubator during interleukin one beta incubation of vex, prepare anti PSMA Alexa 4 88 antibody labeled prostate cancer cells. First, add 0.05%trypsin EDTA to the MDA prostate cancer cells for one minute.

Do not expose the cells to trypsin for longer times as it can affect the glycopeptides present on the cell surface. Centrifuge the cells at 200 times G for five minutes. Next, resuspend the MDA cell palate in one milliliter of Hank's Buffer and add the anti PSMA antibody incubate for 30 minutes at room temperature in a dark place, resus suspending the cells during the incubation.

After 30 minutes, centrifuge the cell solution at 800 times G for five minutes. Aspirate and resuspend the pellet in one milliliter of Hank's buffer. Count the labeled MDA cells and bring the final concentration to 1 million cells per milliliter.

Fill a five milliliter syringe with the labeled MDA cells and remove the bubbles. Turn on the inverted microscope and set the curler illumination at 10 x objective. Bring the syringe pump at the same level as the sample stage of the microscope and set it at one dime per centimeter square shear stress.

Next, open the Zeiss Axio vision software. Select the objective on the computer screen. Create a new folder under the tools option in the software.

Open the smart experiments option in Axio vision and adjust the settings to record short 32nd videos for 30 minutes For live fluorescent video, set the image options. These parameters help in attaining videos close to the video frame rate with the Zeiss MRM camera to visualize full width of the flow channel at 10 x objective on the computer screen, use a CM mount adapter switch on the mercury lamp before placing the syringe and the connector containing the cells onto the syringe pump. Next, attach the connector to the filled inlet channel of the micro slide containing interleukin one beta stimulated ax.

Connect the outlet channel with the connector attached to the tubing. Put the tubing into a dish or a 15 milliliter conical tube to collect the flow through. Start the infusion through the micro slide at 10 microliters per minute.

Observe the interactions between endothelial cells and labeled MDA cells or labeled CTCs derived from patients under 488 nanometer. Filter on the epi fluorescence microscope. Start recording the experiment as 32nd short videos.

During playback analysis, measure the rolling velocity. Rolling velocity is measured by dividing the distance traveled by the cells over time. Shown here is an overnight culture of a monolayer of endothelial cells.

On the micro slide. The scalings show that 100%of the micro slide is visible using a five x objective while 70%is visible using a 10 x objective for selected mediated interactions, cells rolling at the edges are not considered, which makes more than 70%of the micro slide available for video recording and playback analysis. The box plot shows the distribution of the rolling velocities of both unlabeled and anti PSMA, labeled MDA cells on interleukin one beta stimulated endothelial cells at different shear stress conditions.

No significant difference was observed in the rolling velocities at one and five dines centimeter square at higher sheer stress of 10 dimes per centimeter square. A statistically significant difference was observed between the rolling velocities of unlabeled and anti PSMA, labeled MDA cells, A time stitched video taken using the blood from a prostate cancer patient shows three types of interactions between labeled prostate CTCs and lectin expressing endothelial cells tethering where CTCs attach and reattach stable adhesion where CTCs did not detach even after 30 seconds and no interactions as non interacting CTCs enter the field of view. Micro slides can be easily immuno stain and fluorescently imaged the firm adhesion of anti PSMA, labeled MDA cells.

After profusion over interleukin one beta stimulated endothelial cells can be seen here. After watching this video, you should have a good understanding of how to examine the interactions between rare cells such as circulating prostate tumor cells and S selectin expressing endothelial cells following this procedure. Other methods such as immunofluorescence can also be performed in order to answer additional questions such as the presence of s selectin.Ligands.

Techniques like these can help the researchers to understand different steps involved in metastasis.