The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

This method can help answer key questions in the cancer metastasis field about the role of lung colonization in accomplishing cancer metastasis. The main advantage of this technique is that early extravasated cancer cells can be visualized in the lungs during cancer metastasis. Demonstrating the procedure will be Cheng-Han Yang, a PHD student from my laboratory.

When the tumor cell culture has reached 70 to 80 percent confluence, wash the culture two times in two milliliters of sterile PBS per wash, and add one milliliter of 0.5%Trypsin-EDTA to the culture dish. Immediately remove 800 microliters of the supernatant and place the culture dish at 37 degrees Celsius for 30 to 60 seconds until the majority of the cells have detached from the plate. Add one milliliter of fresh medium, supplemented with 10%FBS to stop the reaction and use a 1000 microliter pipette to vigorously mix the cell solution.

Transfer the resulting single-cell suspension into a 1.5 milliliter micro centrifuge tube and collect the cells by centrifugation. Then re-suspend the tumor cell pellet in 1.5 milliliters of medium supplemented with 20%FBS, and end-over end rotate the cells for two hours, at 37 degrees Celsius. At the end of the incubation, count the viable cells by trypan blue exclusion and collect them by centrifugation.

Re-suspend the pellet in one milliliter of sterile PBS for a second centrifugation and re-suspend the pellet in 500 microlitres of PBS supplemented with 10%FBS and 20 micro milliliter CFSE. After ten minutes at 37 degrees Celsius in the dark, centrifuge the cells, and re-suspend the pellet in four milliliters of medium supplemented with one percent FBS for another centrifugation. After the last wash, re-suspend the cells to a five times ten to the sixth tumor cells per milliliter of fresh medium without FBS concentration, and place the cells on ice.

Next, warm the tail of a four to six week old male C570 Black Six mouse for five to ten minutes to dilate the tail veins and use a one milliliter syringe equipped with a 26 and a half gauge needle to thoroughly mix the cells until a single cell suspension is achieved. Carefully load the syringe with 200 microliters of cells and place the mouse in a restrainer. Then inject the entire volume of cells into the lumen of a dilated tail vein.

At the appropriate post-injection time point, make a longitudinal skin and subcutaneous tissue incision from the abdomen to the chest and open the pleural cavity to expose the heart and lungs. Using sterile surgical sutures, ligate the superior and inferior vena cavas to prevent the backflow of the profusion solution and use scissors to make a two to four millimeter fissure in the left ventricle to facilitate draining of the perfusate from the lungs. Then use a three milliliter syringe to inject PBS into the right ventricle and use continuous suction to remove the drained solution until the lungs change from a reddish color to completely pale.

Take care to secure the superior and inferior vena cava properly for a successful lung perfusion. After harvesting the lungs, place the lobes in a custom-made lung holder in a six centimeter dish, and secure the lungs onto the reticulated texture of the holder. Cover and moisten the lobes with PBS, and place the culture dish on the imaging stage of a confocal microscope.

Select the 5x objective, and rotate the filter wheel to NIBA. Using a 488 nanometer laser, excite the CFSE to allow a clear visualization of the florescent blue labeled tumor cells within the lung lobes. Rotate the filter wheel to R690 to scan with a two microseconds per pixel scanning speed and optimize the high voltage gain and offset levels.

Then capture five 12 by five 12 pixel fluorescent images using a 10 microsecond per pixel scanning speed. Using the staining method as just demonstrated the tumor cells are effectively labeled with CFSE in a dose-dependent manner. With the fluorescence intensity of the labeling, almost reaching a plateau in six times 10 to the fifth Lewis lung carcinoma cells per milliliter at the 20 micromolar concentration.

Lung perfusion before harvest as just demonstrated clears the lung vasculature of non-specifically chopped circulating tumor cells prior to imaging analysis. Fluorescence confocal microscopy reveals the presence of the colonizing CFSE-labeled tumor cells within the harvested and perfused lung lobes. Using an appropriate image analysis software program to convert the fluorescent images to black and white, facilitates quantification of the lung colonization.

Intravenous delivery of fibronectin expressing or scrambled fibronectin expressing Lewis lung carcinoma cells demonstrates a significantly lower number of fibronectin expressing cells in lung tissue harvested 38 and 45 hours after injection, underscoring the role of fibronectin in lung lobe colonization and tumor development. While attempting this procedure, it's important to remember to carefully perfuse the lungs so that the unattached cells can be washed away. After its development, this technique paved the way for researchers in the field of cancer metastasis to explore lung colonization by circulating tumor cells in a mouse zero gram model.

After watching this video, you should have a good understanding of how to fluorescently label tumor cell suspensions, intravenously inoculate the tumor cells perfuse mouse lungs, and use confocal florescence microscopy to image the metastasized tumor cells.