Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

The overall goal of this procedure is to generate conditioned media using mouse lymph nodes, bones, lungs, and brains to identify and study organ derived soluble factors and their effects on the metastatic behavior of cancer cells. This method can help answer key questions in the metastasis field, such as, how do organ specific factors influence cancer cell growth, migration, and gene expression? This technique provides a straightforward ex vivo platform representative of organ specific in vivo soluble factors that influence the phenotype and behavior of cancer cells.

To isolate the lymph nodes, begin by placing a six to twelve week old mouse on a polystyrene foam pad in a sterile tissue culture hood, and secure the limbs in an outstretched position. Next, use forceps and scissors to make a midline incision in the abdominal skin from the genitalia to the mouth. Then, gently pull the skin from the abdominal muscles and pin it to the dissecting pad.

Using scissors, carefully remove the lymph nodes from the skin, fat, and vessels, placing the nodes in 30 milliliters of ice cold PBS as they are harvested. When all of the lymph nodes have been collected, use the forceps and scissors to carefully cut through the exposed abdominal wall in an upward motion through the sternum, exposing the thoracic cavity. Cut the diaphragm.

Lift the lungs from underneath, and cut the underlying tissue toward the trachea. Then, transfer the lungs into a new container of ice cold PBS. The heart can be removed from the lungs during this step or prior to organ weighing.

To harvest the leg bones, place the mouse in the prone position, and cut the skin across the lower back from flank to flank. Next, use a sterile piece of gauze to grasp the torso and the skin over the legs and feet. Using the same piece of gauze, hold the lower leg while carefully breaking the ankle joint of one foot, and peel the skin over the joint proximally towards the knee.

Then, using scissors, remove the tibia from the knee joint and place the leg bone in ice cold PBS. After harvesting the second tibia, hold the femur in place with forceps and use scissors to cut away the surrounding muscle tissue. Then, place the leg bone in ice cold PBS.

To harvest the brain, use a new piece of sterile gauze to grasp the head, and gently remove the skin with scissors and forceps to expose the skull. Next, use scissors to carefully the occipital bone from the top center of the skull in a straight and downward line to expose the posterior brain. Then, scooping underneath the tissue toward the anterior of the skull, transfer the whole brain into ice cold PBS.

To generate the lung conditioned medium, invert the tubes of lung tissue three times to remove any residual blood from the organs. Then, replace the wash with fresh cold PBS and repeat until the saline is blood free. Next, transfer the lungs into one 60 square millimeter glass Petri dish per mouse, and use two sterile scalpel blades to mince the tissues with repeated back and forth slicing.

When the tissue fragments are approximately one millimeter cubed in size, re-suspend the pieces in the appropriate volume of DMEM:F12 medium supplemented with antibiotics, and incubate the tissues in one well of a six well plate per mouse at 37 degrees Celsius and five percent CO2. After 24 hours, transfer the entire contents of each well into individual 50 milliliter conical tubes, and dilute the conditioned media with three equivalent volumes of fresh DMEM:F12 medium per tube. Centrifuge the tubes to remove any large pieces of tissue debris.

Then, pool the conditioned media through a 0.22 micron syringe strainer into a single 50 milliliter conical tube. To generate bone marrow conditioned medium, transfer the bones under a sterile tissue culture hood, and trim the excess tissue from the bone. Use scissors to remove the epiphyses.

Then, use a 27 and a half gauged needle to flush one milliliter of PBS through the medullary cavity of each bone into a tube of fresh PBS. When all of the bone marrow stromal cells have been collected, wash the cells three times in PBS. After the last wash, re-suspend the pellet in 20 millilters of bone stromal cell growth medium supplemented with 10%fetal bovine serum, and seed 10 milliliters of cells per T75 flask.

When the cells have reached approximately 70%confluency, wash the cultures two times with three milliliters of PBS. Then, detach the cells with three milliliters of 0.025%trypsin/EDTA solution, taking care that the trypsin covers the entire flask surface. After two to three minutes, arrest the enzymatic reaction with three milliliters of bone stromal cell growth medium, and pool the cells in a 50 milliliter tube.

Spin down the cells. Then, re-suspend the pellet in 10 milliliters of fresh medium. Now, pass into the cultures at a one to five ratio into three new T75 flasks until they reach 70%confluency.

Then, wash the cells three times with PBS as just demonstrated, and feed the cultures with serum free bone stromal cell collection medium supplemented with mitogen and antibiotics for 72 hours. After the third day of culture, pool the media through a 0.22 micron strainer. The stromal cells should be adherent in culture with an elongated and fibroblastic morphology observed in the lymph node stromal cell cultures, and a smaller and more rounded morphology exhibited by the bone marrow stromal cell cultures.

Flow cytometric analysis confirms that the lymph node stromal cells are largely CD45 negative, and gp38 positive. Bone marrow stromal cells should be positive for CD-44 and CD-29, weakly positive for CD-105 and Sca-1, and negative for CD-73. Human breast cancer cells with varying genetic backgrounds exhibit differential migration and growth patterns towards specific organ conditions.

For example, in this representative experiment, the bone-seeking 231-BoM variant preferentially migrates to the bone marrow conditioned medium over the brain and lung conditioned media. Similarly, the lung-seeking 231-LM2 variant preferentially migrates to lung conditioned medium over the bone marrow and brain conditioned media. Further, RT qPCR analysis demonstrates that the exposure of parental MDA-MB-231 breast cancer cells to bone or lung conditioned medium induces an up regulation of the genes associated with bone or lung specific metastases of the breast cancer cells in vivo.

Once mastered, the tissue harvesting and conditioned medium set ups can be completed in two to three hours if they are performed properly. The sub culturing steps on subsequent days take 15 to 30 minutes per day. While attempting these procedures, it is important to remember to keep everything sterile.

Following the conditioned medium generation, other methods like protein arrays, cell growth and migration assays, tumorsphere formation, and RT-PCR can be performed to answer additional questions about the effect of organ specific factors on cancer cell behavior. After its development, this technique paved the way for researchers in the field of breast cancer research to explore organ specific metastatic behavior in an ex vivo model system. After watching this video, you should have a good understanding of how to generate conditioned media using the bones, lungs, and brains of mice.

Don't forget that working with sharp dissection tools can be hazardous, and that precautions, such as using precise and careful dissection technique, should always be taken while performing this procedure.