The MC38 murine colon carcinoma model was prepared similarly to the B16F10 melanoma model as described previously (9). A total of 5 × 105 MC38 cells suspended in 50 μl of PBS were injected intradermally on the left side of the back of each C57BL/6 mouse. Mice were injected intravenously on days 6, 9, and 12 with aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg/kg). Mice were also treated intraperitoneally with 100 μg of anti–PD-1 (clone 29F.1A12, Bio X Cell) on days 10 and 13. Tumor growth was monitored as described above. Mice were euthanized when tumor volume had reached more than 600 mm3 or when active ulceration was observed. On day 60, naïve C57BL/6 mice or tumor-free survivors were rechallenged by intradermal injection of 5 × 105 MC38 cells.

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