Spontaneous Metastasis Mouse Models: A Platform to Study the Metastatic Potential of Orthotopically Injected Colorectal Cancer Cells

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

1. Dissection of CRC PDXs from Mice

Experimental procedures for dissection of CRC PDXs are outlined in Figure 1B (step 2).

1. Allow the tumor to grow subcutaneously to ~1 cm³ in size, which usually takes approximately 1-3 months.
2. Anesthetize the mouse with isoflurane inhalation using the small animal inhalation anesthesia device and disinfect the skin with 70% ethanol.
3. Place the mouse in a prone position on the laboratory bench. Incise the skin, expose the tumor, and carefully detach the skin from the tumor using sterile scissors. After removing the tumor, place it on a 10 cm Petri dish, remove any grossly necrotic regions from the tumor, and then rinse twice with 5 mL of PBS.
   NOTE: Necrotic areas are often whiter, softer, and more friable than the surrounding areas.
4. Place the tumor on ice and divide it equally into at least 4 parts for different applications, including 1) the CRC organoid culture, 2) transplantation into secondary mice, 3) histological examination, and 4) freezing and storage of the tumor fragments.
   1. For the generation of the CRC organoids, place the solid tumor tissue on a 6 cm petri dish containing 1 mL of sterile PBS. Keep the tissue on ice until processing.
   2. To establish the PDX model for human CRC, passage the fresh CRC PDXs into a secondary mouse. Cut the tumor tissue into small pieces (5 x 5 x 5 mm³ in size) using sterile razor blades and gently dip these tissue fragments into 50 µL of artificial extracellular matrix. Then, implant these fragments into the lower parts of the right and left subcutaneous pockets of the NOG mouse.
   3. Place the anesthetized mouse in a prone position on the laboratory bench. Make a small incision on the lower parts of the right and left flanks of the recipient mouse to generate subcutaneous pockets for tissue implantation using sterile scissors. Take care not to puncture the peritoneal membrane. There should be little bleeding with this procedure.
   4. Gently dip the above-prepared tumor fragments into 50 µL of artificial extracellular matrix and then implant them into the lower parts of the right and left subcutaneous pockets.
   5. Suture the incision with wound clips in the mice undergoing surgical procedures. Make sure that the implanted tumor tissues are located at some distance from the incision under the skin.
      NOTE: In accordance with minimizing surgical procedures, no treatments for post-surgical pain and infection were administered in this study.
   6. Place the mouse on a warm pad and maintain the sternal recumbent position until sufficient consciousness is restored. Once fully recovered and able to sit up on all four legs, the mice can be returned to their home cages. To prevent predation, do not allow breeding with non-operated animals in the same cage.
   7. For histological analysis, fix the samples in 5 mL of 10% neutral buffered formalin in a 15 mL conical tube at room temperature for 2 days.
   8. For cryopreservation, cut the tumor tissue into small pieces (5 x 5 x 5 mm³ in size) and gently dip the samples into 500 µL of cell-freezing solution in 1-1.5 mL cryovial tubes. Then, transfer the tube to a -80 °C freezer.

2. Extraction of PDXs into the Cell Suspension for the CRC Organoid Culture

Experimental procedures for dissociation of CRC PDXs are outlined in Figure 1B (step 3).

1. Place the above (step 1.4.1) prepared tumor fragments on a 10 cm Petri dish.
2. Mince the fragments using sterile razor blades and transfer them into a 15 mL conical tube containing 8 mL of 10% fetal calf serum (FCS)-Dulbecco's Modified Eagle's Medium (DMEM) with 80 µL of collagenase enzyme stock (see Table of Materials).
3. Close the tube tightly and wrap the cap with parafilm to prevent leakage. To dissociate the minced tumor fragments into the cell suspension, agitate it gently for 2 h at 37 °C. Note that there will still be many fragments of undigested tissue remaining in the tube.
4. Centrifuge the tube at 300 x g for 5 min at 4 °C and remove the supernatant. Resolve the cell pellet using 10 mL of 10% FCS-DMEM to make the cell suspension.
5. To eliminate undigested tissue debris, filter the cell suspension twice using the 40 µm cell strainer set on a 50 mL conical tube. Then, centrifuge the flow-through at 300 x g for 5 min at 4 °C. Remove the supernatant and keep the pellet on ice.

3. Generation of the CRC Organoids Cultured on Artificial Extracellular Matrix

Experimental procedures for the CRC organoid culture of the colon PDXs are outlined in Figure 1B (step 4).

1. Establish a culture medium suitable for the PDX-derived CRC organoids (see Table of Materials, "the CRC organoid culture medium") employing media previously described for human colon organoids.
2. Apply 150 µL of artificial extracellular matrix (see Table of Materials) per well on a 12-well plate on ice and then incubate it for 30-60 min in a 37 °C, 5% CO₂ incubator to solidify the gels.
3. Suspend the cell pellet from step 2.5 with the CRC organoid culture medium (from step 3.1) with 5% FCS to achieve adjustment to 3 x 10⁵ cells/mL. Then, seed 1 mL of the medium onto the artificial extracellular matrix-coated plate prepared in step 3.2. and incubate overnight at 37 °C under 5% CO₂. Use a hemocytometer for counting the number of tumor cells, including single cells and multicellular clusters (a group of adherent cells) in the cell suspension.
   NOTE: 5% FCS is used to quench the residual enzymatic activity of collagenase in this study.
4. Carefully collect the culture medium, including floating cells detached from the artificial extracellular matrix-coated plate, into a 1.5 mL centrifuge tube on the following day and centrifuge it at 1,400 x g for 5 min. Then, remove the supernatant and resuspend the pellet in 70 µl of artificial extracellular matrix on ice.
5. To increase CRC cell viability, overlay the tumor cell-containing artificial extracellular matrix onto the tumor organoid cells attached to the artificial extracellular matrix-coated plate and incubate for 30 min in a 37 °C, 5% CO₂ incubator solidify the artificial extracellular matrix coating. Then, incubate it with 1 mL of the CRC organoid cell culture medium with 1% FCS at 37 °C under 5% CO_2.
6. Change the culture medium every second day. As the CRC organoids fill the medium, it may become necessary to change the medium daily.

4. Generation and Enrichment of GFP Lentiviral Particles

Experimental procedures for the generation and enrichment of GFP lentiviral particles are outlined in Figure 1C (step 5).

1. Culture HEK293T cells with Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FCS and prepare six 10 cm dishes (2 x 10⁶ cells per dish) for the following transfection procedure.
2. For transfection per dish, prepare 500 µL of the mixture including 20 µl of the transfection reagent in DMEM with a PRRL-GFP vector (5 µg)¹² and two lentiviral packaging plasmids, such as pCMV-VSV-G (1 µg) and pCMV-dR8.2 dvpr (5 µg), in a sterile 1.5 mL microtube. Keep the mixture at room temperature for 20 min and then overlay it onto each of the 10-cm dishes and incubate them for 18 h.
3. Remove the medium, add 5 mL of fresh 10% FCS-RPMI 1640 medium to each dish, and leave standing for 24 h.
4. Collect the conditioned medium from each dish into a 50 mL centrifuge tube and store a total of 30 mL of the medium at 4 °C overnight. Add 5 ml of the fresh RPMI 1640 onto each dish and leave standing for 24 h.
5. Collect the conditioned medium from each dish into a 50 mL centrifuge tube and keep, in total, 30 mL of the medium on ice. Then, discard the cells.
6. Filter 60 mL of the medium (from steps 4.4-4.5) through a 0.45 µm filter to remove the cells and divide this quantity into twelve 5 mL polypropylene centrifuge tubes for ultracentrifugation.
7. To concentrate viruses, centrifuge them using a swinging bucket rotor (see Table of Materials) at 85,327 x g for 1.5 h at 4 °C.
8. Immediately remove the medium. To resolve the concentrated virus pellet, place 250 µL of Nutrient Ham's Mixture F-12 (F12)/DMEM medium without FCS in each of the twelve tubes and maintain it at 4 °C overnight. Note that the virus pellet is often invisible.
9. Gently pipette the medium to collect 3 mL of the concentrated 5x GFP lentivirus stock, in total, presumably including GFP lentiviral particles with 10⁸ transducing units per mL (TU/mL). Then, store the twelve 1.5 mL microtubes (including 250 µL per tube) under sterile conditions at -80 °C for up to a year. Prepare many small aliquots of the original solution to avoid multiple freeze–thaw cycles.

5. Labelling of CRC Organoid Cells with GFP Lentiviral Particles Cultured on Artificial Extracellular Matrix

Experimental procedures for labeling the CRC organoid cells with GFP lentivirus are outlined in Figure 1C (step 6).

1. Allow the CRC organoids prepared in step 3.6 to grow without interference for 7-10 days, harvest them mechanically using a sterile cell scraper, and then transfer them into a 1.5 mL microtube.
   NOTE: The growth of CRC organoids in culture depends on the nature of the original tumors from patients. Avoid overgrowth of the CRC organoids by maintaining them at 60-70% confluence before transfer at a 1:2 split ratio onto a new artificial extracellular matrix-coated 12-well plate.
2. Centrifuge the microtube for 3 min at 1,400 x g, remove the culture medium and resolve the cell pellet in 500 µL of PBS by gentle tapping.
3. Centrifuge the microtube for 3 min at 1,400 x g and eliminate PBS.
4. To dissociate the adherent CRC organoids, add 500 µL of a cell detachment solution of proteolytic and collagenolytic enzymes to the cell pellet in the tube and mix it by gentle tapping. Then, leave the tubes to settle for 10 min at room temperature.
5. Very gently pipette the cell suspension with an additional 500 µL of 1% FCS-DMEM several times in a 1.5 mL microtube to roughly dissociate the cells. Generation of single cells from the CRC organoids by harsh pipetting markedly reduces cell viability. Thus, it is essential to leave the mass of cells visually detectable in the cell suspension by gently pipetting in a 1.5 mL microtube.
6. Centrifuge the cell suspension for 3 min at 1,400 x g and remove the supernatant.
7. Resolve the cell pellet with a mixture of the 100 µL of 5x GFP lentivirus stock (>10⁸ TU/mL) and 400 µl of the CRC organoid culture medium in a 1.5 mL microtube by gentle tapping to adjust to a concentration of 5 x 10⁵ dissociated tumor cells per 500 µl. Use a hemocytometer for counting the number of tumor cells, including single cells and groups of cells in the cell suspension, as described above (step 3.3).
8. Prepare an artificial extracellular matrix-coated 12-well plate, as described in step 3.2. Then, place 500 µl of the cell suspension prepared in step 5.7 on the plate and leave it for 18 h at 37 °C under 5% CO₂.
9. Collect the medium with the floating cells detached from the artificial extracellular matrix-coated plate into a 1.5 mL centrifugation tube. Centrifuge it at 1,400 x g and then remove the supernatant and re-suspend the pellet in 70 µl of artificial extracellular matrix on ice.
10. To increase CRC cell viability, overlay the tumor cell-containing artificial extracellular matrix onto the tumor organoids attached to the artificial extracellular matrix-coated 12-well plate, as described in step 3.5. Next, incubate the plate for 30 min in a 37 °C, 5% CO₂ incubator to solidify the artificial extracellular matrix coating and then culture it with 1 mL of the CRC organoid culture medium with 1% FCS at 37 °C under 5% CO₂.
11. Observe the cells at 3 days after infection under a fluorescence microscope to confirm nearly 100% GFP positivity due to using a high titer of lentiviral particles (see Figure 2A).
12. Culture the CRC organoids for 7-10 days to expand cell growth before injection into recipient mice.

6. Generation of Metastases by GFP-labeled CRC Organoids in Recipient Mice

Experimental procedures for the generation of metastases using the GFP-labelled CRC organoids are outlined in Figure 1C (step 7).

1. To dissociate the GFP-labeled CRC organoids into the cell suspension, harvest them mechanically using a sterile cell scraper and transfer them into a 1.5 mL microtube, as described above (step 5.1).
2. Centrifuge the microtube for 3 min at 1,400 x g, remove the culture medium, and resolve the cell pellet in 500 µL of PBS by gentle tapping.
3. Centrifuge the microtube for 3 min at 1,400 x g and eliminate PBS.
4. Overlay 500 µL of the cell detachment solution onto the cell pellet in the tube and mix it by gentle tapping. Then, leave the mixture standing for 10 min at room temperature to enzymatically dissociate the adherent CRC organoids, as described above (step 5.4).
5. Very gently pipette the cell suspension with an additional 500 µL of 1% FCS-DMEM several times in a 1.5 mL microtube to roughly dissociate the cells, as described above (step 5.5). Generation of single cells from the CRC organoids by harsh pipetting markedly reduces cell viability. Thus, leave the mass of cells visually detectable in the cell suspension by gently pipetting in a 1.5 mL microtube.
6. Centrifuge the cell suspension for 3 min at 1,400 x g and remove the supernatant.
7. To establish a spontaneous metastasis mouse model of CRC PDXs:
   1. Prepare a cell suspension including 5 x 10⁵ cells in 50 µL of PBS with 50% artificial extracellular matrix per mouse. Maintain the suspension on ice before use. Use a hemocytometer for counting the cancer cells, as indicated in step 3.3.
   2. Anesthetize a mouse with isoflurane inhalation using the small animal inhalation anesthesia device and disinfect the skin with 70% ethanol. Assure the normal rate and depth of respiration and the absence of a toe pinch reflex for proper anesthetization. Vet ointment on the eyes is an option to prevent ocular dryness while under anesthesia.
   3. Place the NOG mouse in a supine position on the laboratory bench. Grasp the rectal mucosa with sterile tweezers and gently pull it out of the anus. Then, immediately inject the cell suspension prepared in step 6.7.1 into the rectal submucosa of the mouse using a syringe (needle size: 22 G). Routinely, 4-6 mice per group are used to evaluate the results.