High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

This method can help answer key questions in the field of tumor biology about the micrometastases found in colon cancer patient. The main advantage of this technique is that it allows highly sensitive detection with micrometastases induced by GFP-labeled, patient-derived colon cancer organoids in the mice. Generally, individuals new to this method will struggle because it can be difficult to detect colon tumor organoid-derived micrometastases in distant organs in mice.

Demonstrating the procedure with Yu Okazawa will be Kosuke Mizukoshi and Yu Koyama, both PhD students and surgeons from my group. To generate human colorectal cancer cell organoids, first add 150 microliters of artificial extracellular matrix per well to a 12-well plate on ice. When all of the wells have been coated, incubate the plate for 30 to 60 minutes in a 37 degree Celsius and 5%CO2 incubator to solidify the gels.

Next, resuspend a patient-derived tumor xenograft cell pellet in fresh colorectal cancer organoid culture medium supplemented with 5%fetal calf serum, or FCS, to achieve a three times 10 to five cells per milliliter concentration, and seed one milliliter of cells onto each artificial extracellular matrix. Then, return the cells to the cell culture incubator. The next morning, carefully transfer the culture supernatants, including the floating cells, into 1.5-milliliter microcentrifuge tubes for their centrifugation, and resuspend the pellets in 70 microliters of fresh artificial extracellular matrix on ice.

To increase colorectal cancer cell viability, overlay the floating tumor cell containing artificial extracellular matrix back onto the artificial extracellular matrix-coated wells, and incubate the tumor cell cultures for 30 minutes in the cell culture incubator. When the new extracellular matrix gel has solidified, feed the cultures with one milliliter of fresh colorectal cancer organoid cell culture medium supplemented with 1%FCS, and return the cells to the cell culture incubator. After seven to 10 days of culture, detach the organoids mechanically with a sterile cell scraper, and transfer the organoids into 1.5-milliliter microcentrifuge tubes for their centrifugation.

Then, resuspend the pellets in 500 microliters of PBS with gentle tapping for another centrifugation. To dissociate the colorectal cancer organoids, add 500 microliters of proteolytic and collagenolytic enzyme solution to the tubes, and mix with gentle tapping. After 10 minutes at room temperature, add 500 microliters of medium supplemented with 1%FBS to the cells, and very gently pipette the cell suspension several times to break up the organoids.

Gentle pipetting of human CRC organoids during processing is critical to maintaining an intact spheroid formation in culture. Centrifuge the cells again, and resuspend the pellets in 100 microliters of 5X GFP lentivirus stock and 400 microliters of fresh colorectal cancer organoid culture medium. It is essential to infect the human CRC organoids with a high titer of GFP lentivirus, particularly to achieve a nearly 100%infection of CRC.

Adjust the volume in each tube to achieve a five times 10 to the five dissociated tumor cell per 500 microliters of medium concentration, and plate 500 microliters of cells into each well of an artificial extracellular matrix-coated 12-well plate as demonstrated. After 18 hours in the cell culture incubator, transfer the floating, cell-containing supernatants into individual 1.5-milliliter microcentrifuge tubes for centrifugation, followed by resuspension of the pellets in 70 microliters of artificial extracellular matrix per tube on ice. To enhance the colorectal cancer cell viability, overlay the tumor cell-containing artificial extracellular matrix onto the tumor organoids in the 12-well plate, and place the plate in the cell culture incubator for 30 minutes.

When the artificial extracellular matrix coating has solidified, feed the cultures with one milliliter of fresh CRC organoid culture medium supplemented with 1%FCS, and return the cells to the cell culture incubator for three days. Then, observe the infected cells by fluorescence microscopy to confirm a nearly 100%GFP positivity, and return the cells to the incubator for another four to six days. To establish a spontaneous metastasis mouse model of CRC patient-derived tumor xenografts, dissociate the GFP-labeled organoids as demonstrated, and dilute the single tumor cell suspensions at a five times 10 to the five tumor cells per 50 microliters of PBS supplemented with 50%artificial extracellular matrix concentration.

Load 50 microliters of cells per mouse into a syringe equipped with a 22-gauge needle, and place the tumor cells on ice. Next, confirm a lack of response to toe pinch, and place the first anesthetized NOG mouse in the supine position on the laboratory bench. Using sterile tweezers, gently pull the rectal mucosa out of the anus, and immediately inject the tumor cells into the rectal submucosa.

To establish an experimental metastasis model of CRC patient-derived tumor xenografts, dilute the single tumor cell suspensions at a four times 10 to the four cells per 50 microliters concentration, and load 50 microliters of cells per mouse into a syringe equipped with a 22-gauge needle. Place the first anesthetized NOG mouse in a prone position on the bench, and make a small incision in the lower region of the left flank. Use sterile scissors and tweezers to carefully cut the peritoneum and expose the spleen, and use the tweezers to grasp the fat attached to the spleen.

Then, slowly inject 50 microliters of the tumor cell suspension into the spleen, taking care that no leakage is observed. After all of the mice have been injected and sutured as necessary, return the animals to their home cages, and check for suture leakage and viability one day after the tumor cell delivery. Use calipers to measure the tumors weekly, harvesting the primary tumors and relevant tissues up to the appropriate experimental endpoints.

Transfer the dissected tumors and organs into five milliliters of ice-cold PBS in a 10-centimeter Petri dish on ice, and observe the dissected tissues under a stereo-fluorescence microscope to assess their GFP expression. CRC organoid infection with GFP lentivirus, as demonstrated, results in nearly all organoids expressing very bright GFP. Orthotopic and intrasplenic injection of the GFP-labeled organoids into secondary recipient NOG mice reveals the formation of GFP-positive primary tumors at the orthotopic site, as well as in the lungs of the majority of mice examined at 2.5 months after injection.

More, liver metastases are observed in recipient NOG animals at one month after intrasplenic injection. After watching this video, you should have a good understanding of how to detect micrometastases of GFP-labeled human colorectal cancer cell organoids after their introduction into recipient mice. While attempting this procedure, it's important to remember that the tumor and metastasis homing efficiency of human colon cancer cell organoids in mice is largely dependent on the interpatient variability of the original tumor cells.