Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Well-differentiated SBNET cells are slow growing and hard to propagate. The few established cell lines are not readily available to researchers. To overcome these limitations, we developed this 3D culture protocol.

The advantage of this technique over the traditional method of establishing an SBNET cell line is that 3D cultures can be obtained and drug testing can be done within three weeks. This method can be applied to other neuroendocrine tumors such as neuroendocrine tumors from the pancreas. After obtaining a resected patient SBNET sample, cut it into five millimeter cubes and store it in 25 milliliters of DMEM F12 medium in a conical tube for transportation.

Incubate the tumors in wash medium prepared according to manuscript directions for 15 minutes. Then transfer them to a new dish and mince them to less than one millimeter pieces using sterile curved scissors. Transfer the minced tissues into a new 50 milliliter tube containing 25 milliliters of wash medium, then centrifuge the sample at 500 times g for 15 minutes at four degrees Celsius.

Discard the supernatant and resuspend the pellets in 10 milliliters of wash medium with collagenase and DNase. Allow the minced tumors to digest at 37 degrees Celsius with slow shaking for 90 minutes. It is important not to over digest the tumor samples.

Too much enzyme or longer incubation time will kill SBNET cells. After digestion is complete, centrifuge the tube at 500 times g for 15 minutes at four degrees Celsius. Discard the supernatant and resuspend the pellet in 15 milliliters of wash medium.

Place a 70 micrometer cell strainer on top of a new 50 milliliter tube and transfer 10 milliliters of the wash buffer through the strainer. Swirl the wash buffer inside the tube to prevent the cells from sticking to the sides. Then filter the cell suspension through the strainer.

Centrifuge the cells at 500 times g for 15 minutes at four degrees Celsius. Discard the supernatant and resuspend the pellet in 200 microliters of wash medium. Place the tube on ice and use five microliters of the cells for cell counting with a hemocytometer.

Calculate the number of cells, then repeat the centrifugation to remove the supernatant and resuspend the cells in liquid ECM at a concentration of one million cells per milliliter. Next, transfer five to 20 microliters of SBNET spheroids in ECM to a 96 well plate and place the plate in a 37 degrees Celsius incubator to allow the liquid ECM to solidify. Add 200 microliters of SBNET culture medium to each well containing spheroids or culture the organoids in stem cell media as previously described.

Use a P1000 pipette to transfer the organoids to a 1.5 milliliter tube and centrifuge the tube at 1, 500 times g for one minute to remove the supernatant. Wash the culture with one milliliter of PBS and centrifuge again to remove the supernatant. Then fix the organoids by adding 500 microliters of paraformaldehyde and incubating them for 15 minutes.

After the incubation, wash the culture twice with one milliliter of PBS. Add 500 microliters of PBS with 3%BSA and 0.1%Triton X-100 to permeabilize the culture. Incubate the tube for five minutes, then wash the organoids three times with one milliliter of PBS and 3%BSA.

Next, incubate the culture with primary antibodies for one hour then repeat the washes with PBS and BSA. Incubate with the secondary antibodies and repeat the washes making sure to aspirate and discard all supernatant. To image the spheroids, add five microliters of mounting medium containing DAPI and use a P20 pipette to transfer five microliters of the spheroids to a glass slide.

Seal the slide with a coverslip and image with a fluorescence microscope using the 10, 20, and 40X objectives. Mechanically break the ECM with a P1000 pipette and transfer it to a sterile 1.5 milliliter tube. Centrifuge the tube at 1, 000 times g at four degrees Celsius.

Then remove all supernatant and place the tube on ice. Add two to four times the volume of new ECM to the pellet and mix the contents of the tube by pipetting up and down 10 times making sure to avoid introducing air bubbles. Transfer five to 20 microliters of the ECM and SBNET mixture to a new 96 well plate and allow the ECM to solidify.

Then cover the solidified ECM with new SBNET medium and put the plate in the incubator. If shipping SBNET spheroids to another lab, transfer the spheroids with the new ECM to a T25 flask and allow the ECM to solidify. Fill the flask with SBNET spheroid medium, screw the cap on tightly, and prepare the shipping package.

Upon receiving the spheroid culture, remove the culture medium and repeat the previous steps to put the spheroids back in culture. SBNET spheroid growth rate was monitored with microscopic imaging. It takes approximately 14 days for SBNET spheroids to double in size when the culture media is changed once a week.

After 14 days in culture, SBNET spheroids do not increase in size. The spheroids were stained with antibodies specific against synaptophysin, chromogranin A, and SSTR2 and imaged using immunofluorescence microscopy. The data showed that these markers are localized in the cytoplasm and at the membrane of SBNET cells.

To ensure the specificity of the antibodies, the same staining procedures were performed on an organoid line from a pancreas tumor that does not express synaptophysin, chromogranin A and SSTR2 and no green signal was detected. The main advantage of immunofluorescence imaging is that it can be performed within four hours and give similar staining information as the immunohistochemistry. Culturing SBNET as spheroids is a valuable technique for identifying drugs that can inhibit SBNET growth.

The spheroids were treated with rapamycin and mTOR inhibitor. After five days, the rapamycin-treated spheroid formed a grape-like structure and became apoptotic or necrotic. When attempting this procedure, it is important to pre-wet the cell strainer and collection tube with wash medium.

This will prevent SBNET cells from sticking to the cell strainer and the plastic tube. With this protocol, scientists and clinicians can establish SBNET spheroid cultures from resected tumors, share them with other laboratories, and use them for testing FDA approved drugs.