Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

The most frequent sites for metastasis are the original lymph nodes. Somatids can be used to detect the adhesive affinity between tumor cells'lymph node sections in vitro. One advantage of this technique is that fresh lymph node sections provide a natural complexity of the original tissue, which wouldn't be possible to recreate using purified proteins.

This method could be useful for molecular oncology studies that seek to identify genes or therapies that interfere with tumor cells'adhesion at metastatic target organs, like lymph nodes. To harvest the lymph nodes within thirty minutes of sacrifice, place the adult Wistar rat in dorsal recumbency on a clean dissection board at room temperature and spray the fur with 70%isopropyl alcohol. Using sterilized instruments, lift the abdominal skin and make a medial skin incision to expose the abdominal viscera.

Extract the intestine until the thoracic and abdominal lymph nodes become visible. Using blunt-tip scissors, carefully excise the lymph nodes without injuring the underlying superior mesenteric artery, and place the lymph node into a 15 mL tube containing 5 mL of sterile PBS. When all of the lymph nodes have been harvested, place one lymph node per cryomold face down on the base of each mold and add just enough optimal cutting temperature medium to cover the tissues.

After snap freezing, use a cryostat to acquire 5 to 8 micrometer thick sections at 22 degrees Celsius and collect the sections onto glass microscope slides. Good quality cryosections from consecutive slices of the same lymph node are critical for minimizing differences between slices, and facilitating consistent cell adhesion rates. For tumor cell labeling, harvest the cells of interest from an appropriately staged cell culture and re-suspend the cells at a 1 x 10 to the 6 cells per mL concentration and serum-free medium.

Transfer 1 mL of cells into a new 15 mL conical tube and label the cells with 2 micrograms per mL DiI(C18)for ten minutes at 37 degrees Celsius, with gentle agitation at five minutes to avoid cell sedimentation. At the end of the incubation, pellet the cells by centrifugation and wash the labeled cells two times with 10 mL of fresh serum-free medium to remove any excess dye. After the second wash, re-suspend the cells at a 1 x 10 to the 6 cells per mL of serum-free medium, supplemented with 0.1%bovine serum albumin concentration.

Proceeding of the labeled tumor cells onto the harvested lymph nodes tissue sections, gently wash the sections two times in PBS, followed by re-hydration in fresh PBS for fifteen minutes at room temperature. At the end of the incubation, block any non-specific binding with 2.5%bovine serum albumin for thirty minutes at 37 degrees Celsius, before using a cotton swab to dry the slides. Use a hydrophobic pen to draw a barrier around each section and add 100 microliters of labeled cells onto each encircled lymph node.

After one to two hours at 37 degrees Celsius in a humidified chamber, remove any non-adherence cells with four gentle washes in PBS and fix the remaining adherent fluorescence cells with 3.7%formaldehyde in PBS for fifteen minutes at room temperature. For quantification of the adhesive index, use the 10x objective on a fluorescence microscope to obtain individual TIFF images of each section in the bright and red fluorescent fields. Then, name and save the images systematically before opening the images in Fiji.

After setting the scale, use the polygonal tool to select the region of interest to be quantified. Select to quantify lymph node area. The data will be expressed in square millimeters.

Next, select Plugins, Analyze, Cell Counter and click the photo to be quantified. Click Initialize and select Counter Type. Then, click on the cells in the photo.

The lymph node adhesion index will be expressed as the number of adherent tumor cells per lymph node covered area. To initialize the next photo, open the next photo and repeat the quantification as demonstrated. As observed, the morphology of the adherent cells is rounded in shape, and the cells are heterogeneously dispersed throughout the lymph node of interest.

The lymph node adhesion index is two to three fold higher in NDRG4 negative breast cancer cell lines, compared to that measured for corresponding NDRG4 positive cells. This procedure can be adapted to assist adhesion rates in other metastatic target tissues, like brain or lungs, to evaluate the secondary preferential sites of adhesion for inseminated tumor cells. Remember that both fresh and frozen tissues should be considered biohazardous and should be handled using appropriate bio-safety precautions.