Cordyceps militaris Induces Immunogenic Cell Death and Enhances Antitumor Immunogenic Response in Breast Cancer

Figure 4

CFSE T cell proliferation assay ((a)–(d)) and CTL activity assay ((e)–(g)). (a) Schematic illustration of T cell proliferation assay to evaluate the effects of CM-EE on immunogenic cell death, DC cross-presentation, and T cell proliferation ((b)–(d)). (b) HA expressing mouse breast cancer cells (4T1-neu-HA) were treated with CM-EE (0–50 µg/ml) for 72 h. Apoptotic and silent death control was prepared with CHX (2 ng/ml) + TNFα (10 µg/ml) for 72 h. Necrotic and immunogenic cell death control was prepared by repeating three times the freezing and thawing cycle (FT). Pretreated 4T1-neu-HA cells were fed to BMDCs for overnight. Ag-fed BMDCs were co-cultured with CFSE-labelled CL4 splenocytes for 72 h (c) Bone marrow-derived DCs were fed with FT-4T1-neu-HA cells and co-cultured with CFSE-labelled CL4 splenocytes for 72 h with or without CM-EE (10–50 μg/ml) or doxorubicin (0.5 μM). (d) CFSE-labelled CL4 splenocytes were stimulated with HA-peptide (0.2 μg/ml) with or without CM-EE (10–50 μg/ml). T cell proliferation was evaluated using flow cytometry. ((e)–(g)) CL4 splenocytes were activated with HA-peptide (0.2 μg/ml) for 72 h and co-cultured with CFSE-labelled 4T1-neu-HA or TUBO-HA cells for 8 h. Dead tumor cells stained with 7-AAD and analyzed with flowcytometry. (e) Representative histograms for 7-AAD staining of CFSE-positive cells. ((f)-(g)) Percentage of dead 4T1-neu-HA (f) and TUBO-HA (g) cells by activated HA-specific CL4 splenocytes. Data are presented as mean ± standard deviation (SD) for three independent experiments. The results are presented as mean ± standard deviation (SD) for triple replicates. The unpaired t test was used for statistical analysis. compared with control. DCs: dendritic cells.

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