Tumor-initiating capability of primary tumor cells

Primary tumor or clonal cells were trypsinized, collected, washed with PBS (2×), counted using a hemocytometer, and resuspended at the appropriate concentration for a final injection volume of 50 μL (× cells/50 μL). Age matched male mice (2- to 3-month-old) were used for all experiments. Injection of all mice occurred at midday and in their home cage. Mice were wiped down with 70% ethanol saturated gauze to sterilize the injection site before receiving a subcutaneous injection with 50 μL of cell slurry (500,000 or 10,000 cells total). At the endpoint of experiments, mice were euthanized using a CO₂ chamber. CO₂ flow was maintained for at least 60 s after respiratory arrest and was followed by physical death verification by bilateral thoracotomy. The mass, length and width of tumors were measured before tumor tissues were processed for downstream analysis.