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. 2016 Dec 14;3:16034. doi: 10.1038/mto.2016.34

Figure 4.

Figure 4

Virally-induced IFN-β expression augments DOX-induced apoptosis associated with increased surface exposure of surface CRT, phagocytosis of tumor cell debris by BM-derived DCs, and immunogenicity. Cell death in ID8-R tumor cells treated with OVV-Fc (MOI = 1), DOX (1 µmol/l) or OVV-Fc followed by DOX (12 hours after infection) was determined by staining with Annexin V-FITC and LIVE/DEAD fixable violet to measure the induction of early apoptosis (Annexin V+/LIVE/DEAD fixable violet−) and late apoptosis/necrosis (Annexin V+/−/LIVE/DEAD fixable violet +) by flow cytometry 24 hours later. (a) One representative experiment of three independent experiments performed is shown. (b) Results are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. (c) Culture supernatants were collected from OVV-Fc-infected ID8-R cells, filtered and treated with UV and psolaren (10 µg/ml). Culture supernatants collected from uninfected cells served as controls. The CVN media were added to uninfected ID8-R cultures alone or in combination with DOX and analyzed for induction of early apoptosis. The induction of early apoptosis in cultures treated with the CVN supernatant and DOX alone or in combination was inhibited by IFN-β blocking antibody (0.5 µg/ml). Data points represent mean ± SD of three independent experiments. *P < 0.05, **P < 0.01. (d) Surface exposure of CRT in ID8-R (left panel) and CAOV2-R (right panel) cultures untreated or treated with OVV-Fc, DOX, or OVV-Fc and DOX combination was determined by flow cytometry after staining with an anti-CRT Ab or an isotype control 24 hours after treatments. Results are presented as mean ± SD of four independent experiments. **P < 0.01. (e) Phagocytosis of cell-tracker-blue CMF2HC-labeled tumor cells treated with OVV-Fc, DOX, or OVV-Fc and DOX combination by DCs was measured after 12 hours by flow cytometry. All tumor cell cultures infected with vaccinia virus were treated with UV and psolaren to eliminate the virus before combining with DCs. Tumor cells receiving UV and psolaren treatment were included as additional controls. The percentages of CD11c-expressing DCs taking up tumor cells are indicated. One representative experiment of three independent experiments performed is shown. (f) In vivo anticancer vaccination. ID8-R cells cultured as described above were injected in one flank of five C57BL/6 mice per group. This was followed by injection of live tumor cells into the opposite flank 8 days later. Tumor growth was monitored by measuring s.c. tumor growth with microcaliper until control mice were killed due to extensive tumor burden. Results are presented as mean ± SD of five independent experiments. *P < 0.05, **P < 0.01. BM, bone marrow; CVN, OVV treatment-conditioned and virus-negative; DOX, doxorubicin; IFN, interferon; MOI, multiplicity of infection; OVV, oncolytic vaccinia virus; SD, standard deviation.