Figure 1. DOX induces immunogenic cell death of murine breast cancer cells 4T1. (A) The surface exposure of CRT was determined by flow cytometry among viable (propidium iodine-negative) cells after treatment with different concentrations of DOX for 24 hours. DOX-treated cells were stained with propidium iodine and FITC-labeled anti-CRT antibodies according to the manufacturer's instructions. (B) The percentage of CRT-positive cells in PI-negative cells was quantified based on based on the results of flow cytometry. (C) S-HMGB1 of 4T1 cells treated with different concentrations of DOX was measured by western blotting, and BSA was used as the loading control. (D) Level of released ATP was determined by a chemiluminescent ATP Determination Kit. (E) Animal vaccination, using 2 rounds of s.c. injection of DOX-treated dying 4T1 cells at 7 days apart, followed by s.c. injection of live cells on the contralateral side. Two weeks later, the number of mice without tumor on contralateral side was counted. Successful tumor growth inhibition at the challenge site is suggestive of immune interference. (F) The number of mice without visible tumor on contralateral side 2 weeks post second injection.

Data represent means ± standard deviation.

DOX = doxorubicin; ATP = adenosine triphosphate; BSA = bovine serum albumin; DOX = doxorubicin; CRT = calreticulin; FITC = fluorescein isothiocyanate; PI = propidium iodide; SD = standard deviation; S-HMGB1 = supernatant high mobility group box 1; s.c. = subcutaneous.

^*p < 0.05; ^†p < 0.01; ^‡p < 0.001 (versus control group).