2.4. Induction of metastasis in the proper axillary LN (PALN) by injection of tumor cells into the subiliac LN (SiLN)

Mice were anesthetized (2% isoflurane in O₂), and the unilateral SiLN injected (under US guidance) with KM‐Luc/GFP cells (3.3 × 10⁵ cells/mL) or FM3A‐Luc cells (3.3 × 10⁵ cells/mL) suspended in 10 μL phosphate‐buffered saline plus 20 μL of 400 mg/mL Matrigel (Collaborative Biomedical Products, Bedford, MA). Tumor cells were injected into the SiLN to induce metastasis in the PALN. The day of tumor cell inoculation was defined as day 0.²³ Tumor growth in the PALN was assessed with an in vivo bioluminescence imaging system (IVIS; PerkinElmer, Waltham, MA, US). Luciferase activity was measured on day 3, day 6, and day 9 for KM‐Luc/GFP cells. Metastasis of the PALN was considered to have occurred when the luciferase activity exceeded the background level in controls (2 × 10⁵ photons/s) on day 6. The day on which PALN metastasis was confirmed was defined as day 0^T. For FM3A‐Luc cells, PALN tumor growth was assessed on day 7, day 14, and every 3 days after day 14. PALN metastasis was considered to have occurred when the luciferase activity exceeded the background level in controls (1 × 10⁶ photons/s). The day on which PALN metastasis was confirmed was defined as day –1^T.