Tumor Enzymatic Digestion: A Procedure to Generate Tumor Cell Suspension from Patient-derived Xenograft Cancer Mouse Models

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Culturing cell lines

1. Grow HK cells in complete RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 nM glutamine, 100 U/mL penicillin G, and 100 mg/mL streptomycin at 37 °C in a 5% CO₂ humidified incubator.
   NOTE: HK cells are normal human follicular dendritic cells and can be grown and expanded for ≤ 15 passages in vitro.
2. To prepare for an experiment, trypsinize the cells.
   1. Remove media and add 2 mL of 1% trypsin in Hank's balanced salt solution (HBSS) to cells. Place cells back in the 5% CO₂ humidified incubator at 37 °C for 4 min.
   2. Collect cells from the dish into a 15 mL tube using a handheld pipet aid with a 10 mL serological pipet attached. Add 8 mL of complete RPMI-1640 medium.
   3. Combine 40 µL of cells and 40 µL of trypan blue in a single well of a 96 well plate. Add 10 µL of the mixture to a hemocytometer and count live cells. Add 1 million cells in 25 mL complete RPMI-1640 medium to a 150 mm sterile tissue culture-treated dish to continue growing the cells.
      NOTE: HK cell suspension prepared in this step must be used within an hour to mix with tumor cells for injection.

2. Patient specimen collection

1. Collect CRC tumors from consented patient 155 (CoCaPt155, T1 N0 M0) and 302 (CoCaPt302, T1 N0 M0) at resection surgery.

3. Expansion of patient tumor

1. Collect tumors at the surgery in cold sterile McCoy's medium containing penicillin G (500 U/mL) and streptomycin (500 mg/mL).
2. Implant tumors directly into the left and right flank of 6-8 week old female NOD/SCID mice.
   1. Mechanically mince tissues into small pieces (~1 mm³) using small surgical scissors.
   2. Implant tissue subcutaneously to the left and right flank using 13 G bone marrow aspiration biopsy needles.
      NOTE: Implant a total volume of 8 mm³ divided evenly to both sides of the flank.

4. Tagging and enrichment of luciferase-labeled tumors

1. Measure tumor growth bi-weekly using a digital caliper.
2. At 1 cm in diameter, transduce the tumor. Directly inject into the tumor a single dose of Luc/red fluorescent protein (RFP)-lentivirus (50 µL/tumor, 1:30 dilution from concentrated high titer lentivirus stock) using a 1 cc syringe with a 27 G needle.
   NOTE: The patient tumor typically reaches 1 cm in diameter in 1-2 months. However, the growth rate is highly variable and based on several factors, including tumor grade and type.
3. Monitor tumor weekly by bioluminescent imaging (BLI) in live animals.
   1. Weigh the mice. Inject conscious mouse with 150 mg/kg luciferin intraperitoneally and wait 5 min for the substrate to circulate in the mouse's body.
   2. Anesthetize the mouse with 2.5% isoflurane in 100% oxygen, 1 L/min in an induction chamber.
   3. Place the mouse on the stomach in a BLI imaging machine with isoflurane flowing and image. Take sequential images to confirm the presence of Luc/RFP positive tumor regions (false-color bio-luminescent image). Return mouse to the cage after imaging is complete.

5. Select the appropriate portion of the tumor for enzymatic digestion

1. On the day of the CRC procedure, image mouse with luciferase-tagged tumor as in steps 4.3.1-4.3.3.
   NOTE: The length of time for the subcutaneous tumor to grow depends on the speed of tumor growth and the planned number of animals to be injected in the experiment.
2. Harvest tumor from mouse flank and image.
   1. Euthanize the mouse by CO₂ inhalation after imaging. Place the mouse in the CO₂ chamber, turn on the gas at 1.4 L/min until the respiratory arrest and leave for 3 min. Follow this with cervical dislocation.
   2. Clean skin with 70% ethanol. Tent skin directly above the tumor. With surgical scissors, make a small incision in the skin. Separate skin from the tumor with scissors.
   3. Remove the tumor and place it in a sterile petri-dish. Image the entire dish in an imaging machine.
3. Use sterile scissors or scalpel to separate luciferase-negative sections from luciferase-positive sections in the tumor and re-image.
4. Repeat until only the most highly positive tumor pieces remain.

6. Enzymatic digestion of tumor

1. Under a laminar flow hood, mince luciferase-positive tumor pieces (step 5.4) into the smallest possible pieces using sterile surgical scissors and put them into a sterile 50 mL conical tube.
   NOTE: Mincing the tumor into the smallest possible pieces will yield more individual cells.
2. Prepare digest solution by adding 10 mL of collagenase IV (1.5 mg/mL), 80 µL of hyaluronidase (20 mg/mL), and 160 µL of deoxyribonuclease I (0.1 mg/mL) to 40 mL of HBSS. Mix solution by inverting.
3. Add 35-40 mL of the digest solution to the minced tumor. Incubate at 37 °C with continuous rotation for 2 h.
   NOTE: Vigorously shake the tube periodically throughout incubation to prevent tumor tissue from clumping.
4. Filter entire digestion through sterile 100 µm cell strainer followed by a 40 µm cell strainer to remove debris. Save the flow-through and discard debris.
5. Wash free cells by adding 20 mL of HBSS and centrifuge at 329 x g for 5 min. Aspirate the supernatant and resuspend pellet in 30 mL of HBSS.
6. Combine 10 µL of cell solution and 90 µL of trypan blue in a single well of a 96-well plate. Count live cells using a hemocytometer.
7. Transfer 1 x 10⁴ to 1 x 10⁶ tumor cells per mouse to a sterile 15 mL conical tube. Add 3 x 10⁵ HK cells from step 1.2.3 per mouse to the same tube with tumor cells.
   NOTE: Use a sterile 50 mL conical tube if the total volume exceeds 15 mL. Always calculate more doses for additional animals per study group to account for fluid loss during syringe use. For example, if a group contains 5 mice, make enough cells for 6 or 7 mice.
8. Centrifuge at 329 x g for 5 min. Discard supernatant either by aspirating or pipetting.
9. Resuspend cells in 50 µL per mouse for UCC model or 10 µL per mouse for CRC model in complete RPMI media. Keep cell suspension on ice until ready for use.