Figure 5.
EnnA treatment triggers immunogenic cell death in vitro and in vivo
(A) EnnA-killed E0771 cells vaccinate mice against live cells. Three injections of E0771 cancer cells killed with EnnA (lower panel) are able to vaccinate mice against live cells (105) implanted in mammary fat pads of B6 mice (upper panel). Control mice receiving just culture media develop tumors, as expected.
(B) Electron microscopy (EM) analysis of E0771 cells treated with 5 μM EnnA for 24 h. Red arrows indicate autophagosomes. Blue arrows indicate vacuoles. The scale bar represents 1 μm.
(C) Western blot analysis of cell lysates from human Hs578T and MDA-MB-453 cells treated with indicated concentrations of EnnA. A specific antibody against total LC3B was used. β-actin was used as a loading control.
(D–G) Immunocytochemistry (IHC) analysis of E0771 cultured cells (D) and tumor samples (F), stained for autophagosomes (α-LC3BII) and the nucleus (DAPI). The scale bar represents 20 μm. E. 30 cells per field (n = 3) in control and EnnA-treated cells were analyzed for the presence of autophagic puncta. G. 70 cells per field (n = 3) in control and EnnA-treated tumors were analyzed for the presence of autophagic puncta. Data are represented in the percentage of mean ± S.E.M.
(H) Flow cytometry analysis of E0771 cells treated with 5 μM EnnA for 24 h. Cells were stained for surface expression of Hsp90β and calreticulin using specific antibodies. In each sample, a total of 10,000 cells were acquired. Gating strategies are shown in Figures S10–S12. Data are represented in the percentage of mean ± S.E.M.
(I) Flow cytometry analysis of E0771 tumor cell suspensions from control and EnnA-treated mice using indicated antibodies. In each sample, a total of 10,000 cells were acquired. Data are represented in the percentage of mean ± S.E.M. ∗∗p < 0.01; ∗∗∗∗p < 0.0001 by one-way ANOVA or unpaired tailed Student’s t test.