The MMTV-PyMT model was prepared as described above. Mice were treated on day 7 with aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg/kg). Mice were euthanized on day 14. Cell suspensions were obtained from each tumor as described previously (9). Tumors were harvested and digested in DMEM supplemented with 2% FBS, collagenase D (2 mg/ml), and DNase I (40 μg/ml) (Roche) for 30 min at 37°C. Single-cell suspensions were obtained by gently disrupting the organs through a 70-μm cell strainer. Red blood cells were lysed with ACK lysing buffer (Quality Biological). Fixable live/dead cell discrimination was performed using Fixable Viability Dye eFluor 455 (eBioscience) according to the manufacturer’s instructions. Following a washing step, cells were stained with specific antibodies for 20 min on ice before fixation. The following antibodies were used to stain the cells: CD3 (145-2C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), CD8α (53-6.7, BD Biosciences), CD45 (30-F11, BD Biosciences), and NK1.1 (PK136, BD Biosciences). All flow cytometric analyses were done using a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

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