Tumor Implantation and Injection of a Tumor Vaccine in a Mouse Model

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

All mice used in the study were maintained and housed in the vivarium of the La Jolla Institute for Immunology (LJI) under specific pathogen-free conditions with controlled temperature and humidity. Animal experiments were performed with 8-14 weeks-old female C57BL/6 mice according to guidelines and protocols approved by the LJI Animal Care Committee.

1. Preparation of cultured tumor cells for implantation

1. Culture B16-F10 melanoma cells in Iscove's Modified Dulbecco's medium (IMDM) containing 10% heat-inactivated FBS, 2 mM glutamine, 1 mM sodium pyruvate, 1 mM minimum essential medium (MEM) nonessential amino acids, and 100 U/mL each of penicillin and streptomycin. Maintain the cell line at 37 °C under 5% CO₂.
2. Seed 1.5-2 x 10⁶ B16-F10 cells in a 175T flask and culture for 2 days. Harvest cells when they are 75%-80% confluent.
3. Remove the culture medium and wash the flask once with phosphate-buffered saline (PBS). Aspirate PBS and add 5 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) followed by harshly tapping the rim of the culture flask.
4. Add 15 mL of culture medium to neutralize the trypsin-EDTA and pour the contents of the flask into a 50 mL centrifuge tube. Wash the dish surface with 10 mL of PBS and pour into the same 50 mL tube.
5. Centrifuge the cells for 5 min at 200 x g. Discard the supernatant and break the cell pellet by finger-tapping the bottom of the tube.
6. Add cold 10 mL of PBS and gently pipet the cell suspension; then, manually count cells using a hemocytometer. Keep the cells on ice before injection.

2. Tumor implantation

1. Gas anesthetize mice with 5% isoflurane at gas flow rate of 1.0 L per min in the fume hood. Change the flow rate to the maintenance dose of 2% isoflurane once the mice are fully anesthetized. For this protocol, anesthetization was performed by the veterinarian following the institutional animal care and use guidelines.
2. Shave the hair on the left flank of the mice and sterilize the injection site using alcohol wipes. Intradermally (i.d.) implant B16-F10 tumor cells at 5 x 10⁵ cells in 50 µL of cold PBS in the left flank using a 30 G needle.
   NOTE: The dose of implanted B16-F10 tumor cells may need to be adjusted in the range of 0.5-5 x 10⁵ cells for successful tumor development.
3. After implantation, measure the tumor length and width three times a week using an electronic digital caliper. Calculate the tumor volume (mm³) using the formula:
   Tumor volume (mm³) = width² × length × 0.5
   Treat mice with tumor vaccine when tumors have reached a size of ≥2 mm.
   NOTE: Tumors usually can be measured on day 3 after implantation of 5 x 10⁵ tumor cells. Faster B16-F10 tumor growth rate was observed in male C57BL/6, Rag1^-/-, or Rag2^-/-γ_c^-/-mice. A similar observation was described in other studies. Keeping the gender of the mice consistent is recommended. However, note that the NIH places emphasis on sex as an important biological variable in biomedical research.

3. Vaccine preparation and injection of Flt3L-expressing B16-F10 (B16-Flt3L) cells

1. Maintain the B16-Flt3L cells in Dulbecco's modified Eagle medium (DMEM) containing 8% heat-inactivated FBS, 2 mM glutamine, and 100 U/mL each of penicillin and streptomycin at 37 °C under 5% CO₂.
2. Seed 1 x 10⁶ B16-Flt3L cells in a 175T flask and culture for 2 days. Harvest cells when they are 75%-80% confluent, as described in steps 1.3 to 1.6, and suspend in 1 mL of cold PBS.
3. Irradiate cells at 150 Gy dose of gamma rays using an X-ray Irradiator with 160 kV and 25 mA parameter setting. Count and check the cell viability by trypan blue staining before injection.
4. Gas anesthetize mice as described earlier and sterilize the injection site using alcohol wipes. Intradermally inject the mice with 1 x 10⁶ irradiated B16-Flt3L cells in 50 µL of cold PBS on the same flank as the original tumor implantation, ~1 cm away from the site of the primary tumor on days 3, 6, and 9 after the initial cell implantation.
5. Mark the vaccine injection sites with a colored pen to distinguish it from the primary tumor.
   NOTE: If 0.5 x 10⁵ B16-F10 cells are initially implanted, it is recommended to perform vaccine treatment on days 8, 11, and 14.