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. 2022 Oct 27;13(2):410–431. doi: 10.1158/2159-8290.CD-22-0523

Figure 3.

Figure 3. Senescent cancer cells efficiently activate DCs. A, Levels of extracellular ATP in the conditioned medium (CM) of 106 control (B16F10), ICD (5 μm doxorubicin; ICD-B16F10), and senescent B16F10 (0.1 μm doxorubicin, senB16F10). n = 2 independent experiments. **, P < 0.01; one-way ANOVA test compared with control B16F10. B, Immunoblot detection of CALR in the CM of 106 B16F10, ICD-B16F10, and senB16F10. Representative image (left) and quantification (right) of n = 3 independent experiments are shown. **, P < 0.01; one-way ANOVA test. C, Semiquantitative infiltration score of CD45+ cells within or closely surrounding the melanoma foci in skin sections of animals 7 days after subcutaneous injection of vehicle (no cells), B16F10, ICD-B16F10, or senB16F10 (n = 5 animals per group). Analysis performed by a histopathologist. ***, P < 0.001; *, P < 0.05; one-way ANOVA test. D, Immunochemistry staining of CD11b+ cells (purple) in skin sections of animals 7 days after subcutaneous injection of vehicle (no cells), B16F10, ICD-B16F10, or senB16F10. Representative images selected by a histopathologist of n = 5 animals per group are shown. Note that the brown pigmentation is due to melanin. Scale bars for each image are shown (100 μm). E, In vivo imaging detection of luciferase-expressing B16F10 (B16-Luc) in animals subcutaneously injected with vehicle, 106 control, ICD or senescent B16-Luc (n = 4 per group) at different time points after injection (as indicated). Representative images (left) and quantification (right) are shown. ***, P < 0.001; **, P < 0.01; two-way ANOVA test. F, Flow cytometry analysis of the DC activation markers CD80, CD86, and MHC-II (I-A/I-B) in CD11c+ FL-DCs upon coculture with RPMI medium either alone or with LPS, B16F10, or senB16F10. Representative histograms and quantification of n = 3 biological replicates are shown. ***, P < 0.001, **, P < 0.01; *, P < 0.05; one-way ANOVA test. G, Flow cytometry analysis of the uptake of CFSE (cytosolic dye) or WGA-Alexa647 (membrane dye) by BMDCs from labeled B16F10, ICD-B16F10, or senB10F10. Quantification after subtraction of autofluorescence from unstained BMDCs of n = 3 biological replicates. ***, P< 0.001; **, P < 0.01; *, P < 0.05; one-way ANOVA test. H, Flow cytometry analysis of OT-I CD8 T-cell activation, as measured by CD69 expression, upon coculture with RPMI medium either alone (naïve) or with PMA + I, control FL-DCs or FL-DCs previously cocultured with control (B16-OVA) or senescent (senB16OVA) B16-OVA cells, as indicated. Representative histograms and quantification of n = 3 biological replicates are shown. ***, P < 0.001; **, P < 0.01; *, P < 0.05; one-way ANOVA test.

Senescent cancer cells efficiently activate dendritic cells (DC). A, Levels of extracellular ATP in the CM of 106 control (B16F10), ICD B16F10 (5 μmol/L doxorubicin; ICD-B16F10), and senescent B16F10 (0.1 μmol/L doxorubicin; senB16F10). n = 2 independent experiments. **, P < 0.01; one-way ANOVA test compared with control B16F10. B, Immunoblot detection of CALR in the CM of 106 B16F10, ICD-B16F10, and senB16F10. Representative images (left) and quantification (right) of n = 3 independent experiments are shown. *, P < 0.05; one-way ANOVA test. C, Semiquantitative infiltration score of CD45+ cells within or closely surrounding the melanoma foci in skin sections of animals 7 days after subcutaneous injection of vehicle (no cells), B16F10, ICD-B16F10, or senB16F10 (n = 5 animals per group). Analysis performed by a histopathologist (N. Prats). ***, P < 0.001; *, P < 0.05; one-way ANOVA test. D, Immunochemistry staining of CD11b+ cells (purple) in skin sections of animals 7 days after subcutaneous injection of vehicle (no cells), B16F10, ICD-B16F10, or senB16F10. Representative images selected by a histopathologist (N. Prats) of n = 5 animals per group are shown. Note that the brown pigmentation is due to melanin. Scale bars for each image are shown (100 μm). E,In vivo imaging detection of luciferase-expressing B16F10 (B16-Luc) in animals subcutaneously injected with vehicle, 106 control, ICD B16-Luc, or senescent B16-Luc (n = 4 per group) at different time points after injection (as indicated). Representative images (left) and quantification (right) are shown. ***, P < 0.001; **, P < 0.01; two-way ANOVA test. F, Flow cytometry analysis of the DC activation markers CD80, CD86, and MHC-II (I-A/I-B) in CD11c+ FL-DCs upon coculture with RPMI medium either alone or with LPS, B16F10, or senB16F10. Representative histograms and quantification of n = 3 biological replicates are shown. ***, P < 0.001, **, P < 0.01; *, P < 0.05; one-way ANOVA test. G, Flow cytometry analysis of the uptake of CFSE (cytosolic dye) or WGA-Alexa647 (membrane dye) by BMDCs from labeled B16F10, ICD-B16F10, or senB16F10. Quantification after subtraction of autofluorescence from unstained BMDCs of n = 3 biological replicates. ***, P< 0.001; **, P < 0.01; *, P < 0.05; one-way ANOVA test. MFI, mean fluorescence intensity. H, Flow cytometry analysis of OT-I CD8 T-cell activation, as measured by CD69 expression, upon coculture with RPMI medium either alone (naïve) or with PMA + I, control FL-DCs or FL-DCs previously cocultured with control (B16-OVA) or senescent (senB16-OVA) B16-OVA cells, as indicated. Representative histograms and quantification of n = 3 biological replicates are shown. SSC-A, side scatter area. ***, P < 0.001; **, P < 0.01; *, P < 0.05; one-way ANOVA test.