Ex Vivo Culture of Circulating Tumor Cells in the Cerebral Spinal Fluid from Melanoma Patients to Study Melanoma-Associated Leptomeningeal Disease

Leptomeningeal disease from melanoma, or M-LMD, is an aggressive form of metastasis of the meninges where circulating tumor cells, or CTCs, infiltrate into the cerebral spinal fluid. Sadly, there's no effective treatments for M-LMD so understanding its biology will help develop therapies to reduce mortality. Recent reports have shown how CTCs can colonize the nutrient scarce CSF environment, such as utilizing the iron scavenging ability to enhance their survival and complement C3 to promote the invasion into the cerebral spinal fluid via the choroid plexus.

Therefore, CTCs are very good tools to study M-LMD. The biggest challenge in studying CSF CTCs is the overall lack of preclinical model and the scarcity of these cells. Finding ways to grow these rare and fragile cells from patients and generating a xenograft model will greatly benefit researchers in understanding M-LMD pathology and designing rational therapies.

We have developed a method which successfully propagated CSF CTCs from M-LMD patients in vitro and in vivo, which provided us the ability to perform molecular and functional analyses of M-LMD cells, as well as efficacy study in xenograft models. Though the current methodologies is imperfect, but being able to culture and expand CSF CTCs from melanoma patients for the first time, gave us insights on the importance of understanding the CSF microenvironment. To begin pre-coat a T175 flask with poly-l-lysine.

Place the flask in an incubator at 37 degrees Celsius for one hour. Now use a sterile serological pipette to aspirate the lysine solution. Pipette 30 milliliters complete meningeal culture medium containing human meningeal cells into the flask.

Incubate the cells at 37 degrees Celsius under 5%carbon dioxide supplementation. When cells reach approximately 75%to 80%confluency, collect and save the HMC cultured media in a 50 milliliter conical tube. Then add complete meningeal culture medium to the tube in a one-to-one ratio.

Finally, pipette the growth factors into the tube to create the HMC conditioned media. To process the cerebral spinal fluid, first, place it in a 15 milliliter conical tube on ice to cool it. Centrifuge the fluid in a pre-cool centrifuge at 257 x g for five minutes at four degrees Celsius.

Pipette out the supernatant without disturbing the cell pellet and make aliquots of it in different tubes. Now add one milliliter of PBS to the cell pellet to resuspend it. Centrifuge the cells at 1, 500 x g for five minutes at four degrees Celsius.

Aspirate the PBS supernatant while leaving about 50 microliters of it at the bottom of the tube. Perform a cell count with the cell suspension before culturing the cells. Several growth factors were upregulated in the media cultured with human meningeal cells.

To begin, spin the thawed cultures of cryopreserved CSF CTCs at 257 x g for five minutes at four degrees Celsius. Aspirate the supernatant with a pipette. Add one milliliter of PBS to wash the cell pellet, aspirate the supernatant again.

Next, resuspend the cells in 450 microliters of HMC conditioned media in three separate wells of a 96-well plate. Pipette 150 microliters of the cell suspension into three different wells of a 96-well plate. Top up the HMC conditioned media with fresh media every three days.

When the cell culture reaches 90%confluency, transfer the trypsinized cells of a well into an empty well in a 24-well plate. During the continued culture of the tumor cells, select the clones of the cells that transform and expand exponentially for further experimentation. To begin, identify the female immunodeficient NSG mice having leptomeningeal disease or LMD.

Place an anesthetized mouse on the surgical table. Position the nose of the mouse in a modified L-shaped cone of a stereotactic apparatus, ensuring that the nostrils stay unobstructed. Pulling the skin gently forward across the ventral surfaces of both pinnae.

Then fix it to the nose cone with tape. Guide the surgical scissor tips downward from the pinnae across the occipital bone. Create a small midline incision measuring five to seven millimeters just above the palpated concavity.

With a pair of blunt tipped forceps having one to two millimeter tips, gently apply pressure on the cisterna magna. Introduce the tips in a closed position and open them while exerting downward pressure on the dura. Continue to perform blunt dissection until the dural membrane is clearly discernible and the associated blood vessels are visible within the exposed area.

Now keep the forceps open to retract the surrounding musculature. Then attach a 27 to 29-gauge needle to a one milliliter syringe. Insert the syringe beneath the dura to visualize the bevel.

Gradually retract the syringe plunger to collect as much cerebral spinal fluid as possible. Transfer the fluid into a micro centrifuge tube immediately. Place it on ice.

Centrifuge the sample at 257 x g for five minutes at four degrees Celsius. After aspirating the supernatant, pipette 500 microliters of sterile PBS to the cell pellet. Centrifuge the pellet at 257 x g for five minutes at room temperature.

Discard the supernatant, then transfer the pellet to a 96-well plate containing HMC conditioned media.