(A) Female MMTV/PyVmT female mice were subjected to thoracic irradiation at 8 weeks of age. At 13 weeks, blood was collected via heart puncture at the termination of the experiment, and its cellular fraction evaluated for its ability to form colonies ex vivo as described in Methods. CTCs, circulating tumor cells. Representative images of colonies arising from single circulating tumor cells harvested from mouse blood are shown below. Transgene-positive colonies were assessed under a fluorescence microscope using a PyVmT antibody and a fluorescent secondary antibody. (B) In same mice as in A, surface lung metastases were counted at 13 weeks of age. Data in A and B are representative of 3 independent experiments with 4 mice per group. Representative H&E-stained sections of lung tissues from control and irradiated transgenic mice obtained 5 weeks after thoracic radiation are shown below. Black arrows indicate lung metastases. (C) Eight-week-old tumor-bearing MMTV/PyVmT transgenic mice were treated 3 times with vehicle or doxorubicin (5 mg/kg i.p.) at 21-day intervals. The experiment was terminated on week 15, and surface lung metastases were counted. Representative H&E-stained lung sections containing metastatic foci are shown at right. Original magnification, ×100. *P < 0.05, **P < 0.01 versus control.