Tumor Cell Homing and Metastasis Assay

The experiments were conducted as the methods described in our prior studies^{5 (link),16 (link),17 (link)}. The period when the distant primary tumor grew was deemed the pre-metastatic phase. The metastatic phase was defined as the period after the intravenous (i.v.) injection of tumor cells into tumor-bearing mice. Synergistic tumor grafts were generated through subcutaneous (s.c.) or mammary fat pad (m.f.p.) implantation of 5 × 10⁶ tumor cells into 8 to 10-week-old mice. We injected tumor cells into mice with growing primary tumors of the same size (size-matched). For the tumor cell homing assays, 1–2 × 10⁴ fluorescent dye (PKH26, Sigma-Aldrich, St. Louis, MO, USA)-labeled metastatic cells were i.v. infused into the mice. At 24–48 h after tumor cell infusion, the lungs were perfused with phosphate-buffered saline (PBS) under physiological pressure to remove circulating tumor cells and then excised. Four to five randomly selected lung tissue fragments (3 mm in diameter) were chosen, and three 10 μm sections per fragment were evaluated using a confocal microscope (SP8 Leica, LSM-710, Carl Zeiss MicroImaging GmbH, Germany) or a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). The labeled tumor cell counts were normalized to the total tissue surface area. Age- and sex-matched mice were used for the experiments.