FIGURE 1.

Development of experimental mouse models for the study of premetastatic to early metastatic phases. (A) Schematic representation of flow cytometric analysis to determine the timing of spontaneous metastasis to the lungs of tumor‐bearing mice. Mice were injected in the fourth mammary fat pad or the right back with tdTomato‐labeled E0771, tdTomato‐labeled LLC or EGFP‐labeled B16F10 cells, respectively, and the lungs were removed after perfusion at 7, 10, 14, 18, or 22 days. (B–D) Analysis of spontaneous metastasis for tdTomato‐labeled E0771 (B), tdTomato‐labeled LLC (C), or EGFP‐labeled B16F10 cells (D). Representative flow cytometric analysis of lung cells is shown, with cells corresponding to metastases (tdTomato⁺ or EGFP⁺ cells) being outlined by black boxes. (E) Summary of results for the timing of spontaneous metastasis. Day 5 after LLC or B16F10 cell transplantation or day 7 after E0771 cell transplantation is defined as the early premetastatic phase, day 7 after LLC or B16F10 cell transplantation or day 14 after E0771 cell transplantation as the late premetastatic phase, and day 14 after LLC or B16F10 cell transplantation or day 21 after E0771 cell transplantation as the micrometastatic phase.